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(Circulation Research. 1996;78:431-442.)
© 1996 American Heart Association, Inc.

Articles

Differential Distribution of Electrophysiologically Distinct Myocytes in Conduit and Resistance Arteries Determines Their Response to Nitric Oxide and Hypoxia

Stephen L. Archer, James M.C. Huang, Helen L. Reeve, Václav Hampl, Simona Tolarová, Evangelos Michelakis, E. Kenneth Weir

From the Cardiovascular Section (111C), VA Medical Center and University of Minnesota, Minneapolis.

Correspondence to Dr Stephen L. Archer (111C), Associate Professor of Medicine, Minneapolis VA Medical Center, One Veterans Dr, Minneapolis, MN 55417. E-mail arche002@maroon.tc.umn.edu.

	Abstract

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Abstract The cellular mechanisms that determine differences in reactivity of arteries of varying size and origin are unknown. We evaluated the hypothesis that there is diversity in the distribution of K⁺ channels between vascular smooth muscle (VSM) cells within a single segment of the pulmonary arteries (PAs) and that there are differences in the prevalence of these cell types between conduit and resistance arteries, which contribute to segmental differences in the vascular response to NO and hypoxia. Three types of VSM cells can be identified in rat PAs on the basis of their whole-cell electrophysiological properties—current density and the pharmacological dissection of whole-cell K⁺ current (I_K)—and morphology. Cells are referred to as "K_Ca, K_DR, or mixed," acknowledging the type of K⁺ channel that dominates the I_K: the Ca²⁺-sensitive (K_Ca) channel, delayed rectifier (K_DR) channel, or a mixture of both. The three cell types were identified by light and electron microscopy. K_Ca cells are large and elongated, and they have low current density and currents that are inhibited by tetraethylammonium (5 mmol/L) or charybdotoxin (100 nmol/L). K_DR cells are smaller, with a perinuclear bulge, but have high current density and currents that are inhibited by 4-aminopyridine (5 mmol/L). Conduit arteries contain significant numbers of K_Ca cells, whereas resistance arteries have a majority of K_DR cells and few K_Ca cells. NO rapidly and reversibly increases I_K and hyperpolarizes K_Ca cells because of an increase in open probability of a 170-pS K_Ca channel. Hypoxia depolarizes K_DR cells by rapidly and reversibly inhibiting one or more of the tonically active K_DR channels (including a 37-pS channel) that control resting membrane potential. The effects of both hypoxia and NO on K⁺ channels are evident at negative membrane potentials, supporting their physiological relevance. The functional correlate of this electrophysiological diversity is that K_DR-enriched resistance vessels constrict to hypoxia, whereas conduit arteries have a biphasic response predominated by relaxation. Although effective in both segments, NO relaxes conduit more than resistance rings, in both cases by a cGMP-dependent mechanism. We conclude that regional electrophysiological diversity among smooth muscle cells is a major determinant of segmental differences in vascular reactivity.

Key Words: nitric oxide • hypoxia • pulmonary circulation • vascular smooth muscle • K⁺ channels

	Introduction

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Segmental differences in vasomotor reactivity are well documented in the pulmonary vasculature,¹ but the mechanism permitting local variations in response to common vasoactive stimuli, such as hypoxia and NO, are unknown. Hypoxia, for example, causes sustained constriction in resistance arteries^{2 3 4 5} while causing a biphasic response in conduit PAs, predominantly relaxation.^{6 7} HPV is caused in part by inhibition of a K⁺ channel in the PA VSM, resulting in membrane depolarization.^{8 9} The involvement of a K⁺ channel in HPV has been confirmed by several laboratories.^{10 11 12}

NO causes pulmonary vasodilatation,^{13 14} in part, by increasing the levels of cGMP, which activates a cGMP-dependent protein kinase, opening K_Ca channels.¹⁵ Segmental differences in PA VSM reactivity to NO have not yet been reported. Existing studies using inhaled NO¹⁶ are inconclusive in this respect because of the different availability of inhaled NO to resistance and conduit PAs.

Recent evidence suggests that there are three or four types of VSM cells in the conduit PA.¹⁷ The functional consequence of cell diversity is unknown, although data from systemic arteries suggest that there are several types of arterial VSM cells that respond differently to agonists, such as endothelin-1 and -thrombin.¹⁸

During previous experiments,¹⁵ we noted two types of whole-cell I_K in PA VSM cells. Some cells had I_K predominated by K_Ca channels. I_K in these cells increased in response to NO and was inhibited by TEA or the specific K_Ca channel blocker CTX.¹⁹ In other cells, I_K predominantly reflected the contribution of K_DR channels and was inhibited by low doses of 4-AP but not TEA or CTX.¹⁵ In these cells, NO did not increase I_K. We hypothesize that the regional differences in HPV and NO relaxation result from the existence of distinct populations of PA VSM cells in conduit and resistance vascular segments, with K_Ca- and K_DR-predominant cells being more prevalent in conduit PAs and resistance PAs, respectively. The present study was undertaken to characterize these PA VSM cell subtypes and examine the functional consequences of regional diversity in K⁺ channel prevalence to HPV and NO relaxation in PA rings. The reference to cells as "K_Ca, K_DR, or mixed" refers to the class of K⁺ channel predominating their whole-cell current and does not imply the cells have exclusively a single type of K⁺ channel.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study Design
Whole-cell electrophysiological characteristics (E_m, capacitance, and current density) were assessed in enzymatically dispersed PA VSM cells from rat conduit and resistance PAs. Conduit PAs were defined as the main, right, or left PA (external diameter, 500 to 600 µm), and resistance PAs were intraparenchymal, fourth- and fifth-division PAs (external diameter, <300 µm). The sensitivity of E_m and I_K to K⁺ channel inhibitors, TEA, 4-AP, and CTX was determined using whole-cell current and voltage-clamp techniques, respectively.²⁰ Morphological characteristics of cells from both arterial segments were determined by light and scanning electron microscopy. The prevalence of the three cell types was determined by a light microscopy survey of enzymatically dispersed cells from each segment. Single-channel characteristics of K_Ca and K_DR channels were measured in amphotericin-perforated vesicles and cell-attached patches, and the effects of NO and hypoxia on the channels were studied in these preparations. Conductances of K_Ca and K_DR channels were also calculated in excised patches, and the sensitivity of the K_Ca channel to Ca²⁺ was determined. To assess the functional significance of the electrophysiological diversity, we compared the effects of TEA, 4-AP, hypoxia, and NO on tension in conduit and resistance rings.

Electrophysiology
Cell Dispersion
PA VSM cells were freshly dissociated each day. Male Sprague-Dawley rats (body weight, 300 to 400 g) were anesthetized with 50 mg/kg sodium pentobarbital. The heart and lungs were removed en bloc and perfused with Ca²⁺-free Hanks' solution (see "Solutions"), as previously described.¹⁴ Conduit and resistance PAs were dissected free of adventitia, placed in Ca²⁺-free Hanks' solution for 10 minutes, and then transferred to a papain solution (see "Solutions") at 4°C for 20 minutes. Then the same papain solution was supplemented with dithiothreitol (0.85 mg/mL), and incubation was continued for 50 minutes at 4°C and then 15 minutes at 37°C (for all solutions, pH 7.4). Arteries were washed for 10 minutes and maintained in iced Hanks' solution supplemented with glucose (1 mg/mL). Gentle trituration produced a suspension of single cells, which was then pipetted into a perfusion chamber on the stage of an inverted microscope for patch-clamp studies.²⁰ After a brief period to allow partial adherence to the bottom of the recording chamber, cells were perfused via gravity with a bath solution (see "Solutions") at a rate of 2 mL/min. Conventional whole-cell patch-clamp experiments were performed as previously described.¹⁵ Cells were proven to be VSM cells at the beginning of the study by immunohistochemistry (factor VIII antigen negative, smooth muscle–specific actin positive). All drugs were supplied by Sigma Chemical Co except LY83583 (Calbiochem).

Whole-Cell Amphotericin-Perforated Patch-Clamp Studies
Whole-cell recordings were performed using the amphotericin-perforated patch-clamp technique.²¹ Amphotericin B was included in the pipette at a final concentration of 120 µg/mL. Experiments were performed in low light intensity because of the light sensitivity of amphotericin B. Pipette resistances ranged from 1 to 4 M after Sylgard coating and fire-polishing, with series resistance typically compensated by 90% to 95%. Membrane current was monitored on a digital oscilloscope. Cells were voltage-clamped at a holding potential of -70 mV, and currents were evoked by 10-mV steps to more positive potentials using test pulses of 200- to 400-millisecond duration at a rate of 0.033 to 0.1 Hz. Currents were filtered at 1 kHz and sampled at 4 kHz. For E_m experiments, cells were held in current clamp at their resting E_m, and control recordings were made for at least 1 minute before application of the drug, to ensure stability. All data were recorded and analyzed using pCLAMP 6.02 software (Axon Instruments). Whole-cell data are presented in the form of current-voltage plots, permitting a complete description of the channel activity over a wide range of E_ms.

Current Density
Whole-cell capacitance was measured using the manual whole-cell capacitance controls on the Axopatch amplifier. Whole-cell currents for each cell (at +70 mV) were divided by the cell's capacitance, giving a measure of current density (in picoamperes per picofarad).

Single-Channel Studies
Three single-channel configurations were used: amphotericin-perforated vesicles,²² excised patches, and cell-attached patches.²⁰ Bath and pipette solutions are given in the "Solutions" section. Pipette resistance for each configuration was 7 to 10 M.

Amphotericin-perforated vesicles and cell-attached patches were used to study the responses to NO and hypoxia, as these configurations preserve intracellular organelles and second messengers that are thought to be important to O₂ sensing⁹ and NO relaxation.¹⁵

The technique for amphotericin-perforated vesicles is similar to that for whole-cell perforated-patch recording, except that once a seal is formed on the cell and series resistance has been confirmed to decline slowly over 15 minutes, the pipette, containing the same solution as for the conventional whole cell, is withdrawn from the cell. This forms a perforated vesicle that envelops normal cytosolic and organellar contents.

Single-Channel Analysis
Mean single-channel amplitudes and open times were calculated using preset detection levels in the events list analysis program of pCLAMP 6.02 (Axon Instruments). Currents were filtered at 1 kHz and sampled at 4 kHz. Channels were characterized by measuring their slope conductance in symmetrical or near symmetrical K⁺ (see "Solutions"). Since intracellular K⁺ can only be estimated in the cell-attached configuration, the slope was not forced through the origin. All single-channel recordings contained at least 2500 openings. Effects of NO and hypoxia on P_o, NP_o, and channel open dwell time () were calculated using the following equations and pCLAMP 6.02 software:

(1)

where t_o and t_i are total open time and time interval, respectively, and N is the number of channels. Fit for dwell time is given as follows:

(2)

where P_n is the fractional proportion of the nth component to the area under the curve, is open dwell time, and t is time.

P_o and NP_o were calculated from latency analysis and subsequent P_o histogram generation for perforated vesicles, since single-channel data were obtained using Clampex. For cell-attached and excised patches, P_o and NP_o were calculated from continuous recordings obtained in Fetchex.

K⁺ Channel Pharmacology
To characterize the K⁺ channel subtypes, the cells/vesicles were treated with TEA (5 mmol/L, a preferential K_Ca inhibitor at this low dose), synthetic CTX (100 nmol/L, a relatively selective peptide K_Ca inhibitor¹⁹ ), or 4-AP (5 mmol/L, preferential K_DR inhibitor).^{12 23 24 25} Recent studies show that although high doses of TEA and 4-AP are not "specific" for K_Ca and K_DR channels, low doses do inhibit different components of the I_K in PA VSM.¹¹ Specifically, TEA (5 mmol/L) inhibits K_Ca channels, whereas 4-AP (5 mmol/L) inhibits CTX- and TEA-insensitive K⁺ channels.^{12 23 24 25} Although CTX can inhibit other K⁺ channels,²⁶ low doses (100 nmol/L) appear to be fairly specific for K_Ca channels. In PA VSM, CTX inhibits the portion of the I_K that is 4-AP insensitive.¹⁵

To study the effects of NO, 1 µL of a 2 mmol/L solution, prepared as previously described,²⁷ was administered close to the cell/vesicle. This dose was chosen on the basis of preliminary experiments, which established that it was the minimum that could be reliably delivered manually and reproducibly activate K⁺ channels. In experiments that examined the effects of hypoxia, pipette solutions were supplemented with various channel blockers (ie, niflumic acid, CTX, and/or TEA). The divalent cation concentration was also varied slightly to optimize detection of K_DR channels in these experiments (see "Solutions"). Experiments using NO were performed at room temperature.

Single-channel hypoxia experiments were performed on K_DR cells from resistance arteries. All experiments involving hypoxia were performed at 32°C, as the O₂-sensitive channel is relatively inactive at room temperature (data not shown).

Solutions
Hanks' Solution
Hanks' solution contained (mmol/L) NaCl 140, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, HEPES 10, and EGTA 0.1 (pH 7.4).

Papain Solution
Papain solution contained Hanks' solution without EGTA, papain (1 mg/mL), and bovine albumin (0.25 mg/mL).

Amphotericin B Perforated-Patch Whole-Cell and Vesicle Experiments
Pipette solution. The pipette solution contained (mmol/L) KCl 134, KH₂PO₄ 1.2, MgCl₂ 1.0, HEPES 5, and 120 µg/mL amphotericin B, pH 7.25.

Bath solutions. For hypoxia experiments, the bath solution contained (mmol/L) NaCl 115, NaHCO₃ 25, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 1.5, and HEPES 10, pH 7.4. For NO experiments, the bath solution contained (mmol/L) NaCl 140, KCl 4.2, KH₂PO₄ 1.2, MgCl₂ 0.5, CaCl₂ 1.5, and HEPES 10, pH 7.4.

Cell-Attached Single-Channel Experiments
Pipette solution for K_Ca channels. This solution contained (mmol/L) KCl 134, KH₂PO₄ 1.2, CaCl₂ 1.0, HEPES 5, and 4-AP 5, pH 7.30.

Pipette solution for K_DR channels. This solution contained (mmol/L) KCl 134, KH₂PO₄ 1.2, MgCl₂ 1.0, HEPES 5, pH 7.30, EGTA 10, TEA 7.5 or CTX 0.001, and niflumic acid 0.2.

Bath solution. This solution was the same as for amphotericin B perforated-patch vesicle experiments.

Excised-Patch Single-Channel Experiments
Pipette solution. This solution contained (mmol/L) KCl 134, KH₂PO₄ 1.2, CaCl₂ 1.0, and HEPES 5, pH 7.30.

Bath solution. This solution contained (mmol/L) KCl 134, KH₂PO₄ 1.2, CaCl₂ varied as described below, MgATP 4, sodium creatine phosphate 2, and EGTA 10, pH 7.30.

To vary bath Ca²⁺ to 50 nmol/L, 5.7 mmol/L Ca²⁺ was added to nominally Ca²⁺-free solution (yielding a 200 nmol/L solution¹² ) and then diluted 1:4.

Hypoxic Solutions
The effects of hypoxia were studied by switching between normoxic and hypoxic perfusate reservoirs. Normoxic and hypoxic perfusate reservoirs were bubbled with 20% and 0% O₂, respectively (plus 3.5% CO₂/balance N₂), producing PO₂s in the cell chamber of 162 and 27 mm Hg, respectively. PCO₂ was 40 mm Hg, and pH was 7.40 to 7.41 under both conditions.

Cell Morphology
Cell morphology was assessed by light microscopy in freshly dispersed cells immediately after trituration. In addition, identically handled cells were fixed in 4% gluteraldehyde for examination by scanning electron microscopy. Microscopic examination was performed by an independent electron microscopy service (Mark Sanders, PhD, University of Minnesota Imaging Center).

PA Rings
Rat conduit PA rings were isolated and mounted in 2-mL baths as previously described.²⁸ Resistance PA rings were harvested and treated identically. The optimal resting tension to maximize constriction to KCl for both conduit and resistance PA rings was 600 mg. Endothelium was left intact, as confirmed by a normal relaxation response to acetylcholine (10^-8 to 10^-5 mol/L). The baths housing the rings were bubbled with 20% O₂ (normoxia) and 0% O₂ (hypoxia) (plus 5% CO₂/balance N₂), resulting in PO₂ values of 124±2 and 35±3 mm Hg, respectively. PCO₂ was 26 to 30 mm Hg, and pH was 7.39 to 7.47. The comparative effect of K⁺ channel blockers and hypoxia on conduit and PA rings was assessed by adding TEA (10 mmol/L) or 4-AP (10 mmol/L) to the bath. Although the K_i values of TEA for K_Ca channels and 4-AP for K_DR channels are 0.25 and 0.7 mmol/L, respectively (in isolated renal VSM),^{29 30} vasoconstriction only occurs at doses of TEA and 4-AP above 1 mmol/L in isolated lungs³¹ and PA rings.¹⁵ This suggests that K_i in intact vascular tissue may be greater than that noted in dispersed cells.

To measure the relaxation response to NO (1 to 100 µL of 2 mmol/L solution), conduit and resistance PA rings were constricted with endothelin-1 (10^-7 mol/L) in normoxia in the presence or absence of the nonspecific guanylate cyclase inhibitor LY83583 (10^-4 mol/L).^{32 33}

Statistics
The data are mean±SEM. Changes in E_m were analyzed by a factorial ANOVA. The current-voltage and dose-response curves were analyzed using repeated measures ANOVA (StatView 4.02, Abacus Concepts). Data from the PA rings were electronically filtered, digitized at 12 samples per minute, and evaluated with repeated measures ANOVA. A value of P<.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
All currents were completely eliminated by substituting Cs⁺ (145 mmol/L) for K⁺ in the patch pipette, indicating they were conducted by K⁺ (n=3, data not shown). The resting E_m of PA VSM derived from resistance arteries was -38±4 mV. Outward K_DR currents (inhibited by 4-AP) were activated positive to -45 mV, whereas K_Ca currents (inhibited by TEA) were activated positive to -30 mV (Fig 1A and 1B).

View larger version (34K): [in this window] [in a new window]   Figure 1. Whole-cell electrophysiology demonstrates three cell types in the conduit PA. Three distinct cell types exist in the conduit PA. Their names reflect the type of K+ channel conducting the majority of the whole-cell current: the KCa channel, the KDR channel, and the mixed channel. The cells are characterized as follows (from left to right): A, Morphology of the macroscopic currents obtained by conventional whole-cell technique (example shown was obtained by stepped depolarization from -70 to +70 mV). Note that the small, noisy, spiky current in KCa cells is inhibited by TEA but not 4-AP. In contrast, the large smooth-profile current in KDR cells is much more sensitive to 4-AP than TEA. In mixed cells, 4-AP inhibits approximately half the total current, leaving the noisy TEA-inhibitable portion intact. B, Current-voltage curves (mean±SEM; n=7 KCa cells, n=5 mixed cells, and n=20 KDR cells, by conventional whole-cell technique) elicited by stepped depolarization from -70 to +70 mV. *P<.05 vs control. C, Cell capacitance and current density obtained in amphotericin-perforated cells. P<.01 vs all other groups.

Cell Diversity
VSM cells could be separated into three distinct populations based on current density (Fig 1C) and whole-cell current pharmacology (Fig 1B). In some cells, the whole-cell current was inhibited significantly by TEA with little effect from 4-AP; these cells were classified as K_Ca cells. In others, the K_DR cells, the reverse was true. There was a third group of mixed type cells in which TEA and 4-AP were approximately equally effective in inhibiting I_K. The average current densities were significantly different among K_DR, K_Ca, and mixed cell types (Fig 1C). Cell capacitance was highest in K_Ca cells (most abundant in the conduit PA) and lowest in the K_DR cells (found predominantly in the resistance PA), reflecting the larger size of the K_Ca cells.

Hypoxia (Fig 2C) and 4-AP (4 mmol/L, Fig 2B) both reversibly depolarized PA VSM cells obtained from resistance arteries, whereas TEA (5 and 10 mmol/L) and CTX (100 nmol/L) had no effect, indicating that resting E_m is largely controlled by K_DR channels (Fig 2A and 2C). The importance of the K_DR or 4-AP–sensitive K⁺ conductance was evident in studies of tone in resistance PA rings. Hypoxia and 4-AP both constricted resistance arteries (Fig 2D), whereas CTX (100 nmol/L) had no effect (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 2. Resting Em, basal tone, and response to hypoxia are controlled by 4-AP–sensitive K+ channels in the PA VSM of resistance arteries. A, Representative trace of Em. Note that 4-AP but not TEA or CTX depolarizes the membrane of this KDR cell. B, Mean±SEM of effects of TEA, CTX, 4-AP, and KCl on Em. P<.0001 vs control. C, Hypoxic depolarization of KDR cells (amphotericin-perforated whole-cell current-clamp technique). Top and bottom of panel show, respectively, mean±SEM for Em (n=5) and a representative recording. D, Effect of TEA, 4-AP, and hypoxia on tone in conduit and resistance PA rings. Hypoxia causes a monotonic increase in tension in resistance PA rings while exerting a biphasic effect in conduit rings, predominantly relaxation. Hypoxic constriction of resistance rings is enhanced by 4-AP (10 mmol/L) but not TEA (10 mmol/L). Mean±SEM are shown (n=35 for control and n=8 for TEA and 4-AP in both ring sizes). *P<.0001 vs control.

Cell phenotype was different among K_Ca, K_DR, and mixed cells (Fig 3). Phenotypic categorization by light microscopy, before approaching the cell with the patch pipette, predicted the cells' electrophysiological attributes when they were subsequently studied. At one end of the spectrum were K_Ca cells, which were elongated (72±5 µm) tapered cells (Fig 3) with a small I_K (440±11 pA at +70 mV, n=7). The marked inhibition of I_K by 5 mmol/L TEA and the minimal effect of 4-AP indicated that the predominant K⁺ channel was the K_Ca type. Furthermore, TEA removed the noisy spontaneously spiking component of the current in K_Ca cells, leaving behind a low-noise slowly inactivating current, consistent with inhibition of K_Ca channels (Fig 1A). At the other extreme were shorter cells (45±6 µm, P<.05 versus K_Ca cells) with a characteristic perinuclear bulge and significantly larger I_K (1747±11 pA at +70 mV, n=20, P<.0001 versus K_Ca cells) despite their smaller size (Fig 3). Inhibition of I_K with low-dose 4-AP suggested predominance of a K_DR-like channel (K_DR cells). Mixed cells were identified, which physically resembled K_DR cells (length, 50±4 µm) but lacked a distinct perinuclear bulge (Fig 3). I_K in these cells was of intermediate size (670±12 pA at +70 mV, n=5, P<.05 versus K_Ca or K_DR) and was inhibited similarly by both TEA and 4-AP (Fig 1A). Of note, 4-AP reduced currents 50%, leaving a noisy spiking current, consistent with residual K_Ca channel activity. Spherical cells were neither characterized morphologically nor used in electrophysiological studies, as they are judged likely to be injured.

View larger version (102K): [in this window] [in a new window]   Figure 3. Three PA VSM cell phenotypes are evident on microscopy. Three distinct cell phenotypes exist in the conduit PA, and these correspond to the cells identified by current density and whole-cell pharmacology as KCa, KDR, and mixed cells; see Fig 1. Note that the KCa cell is larger and that the KDR cell has a discrete perinuclear bulge. The scanning electron micrographs show more convolutions on the KDR than the KCa cell in the conduit PA. Surface convolutions and cytoplasmic projections are more common in cells from resistance PAs than cells from conduit PAs. The scale bars on light and scanning electron micrographs represent 25 and 5 µm, respectively. Cells shown in the scanning electron micrographs are slightly contracted because of ethanol dehydration.

On scanning electron micrographs, cells from conduit PAs had the same morphological appearance as seen on light microscopy. In general, their surfaces were smooth, with few cytoplasmic projections. K_DR cells tended to have more convoluted membranes than did K_Ca cells. In general, cells from the resistance PA (regardless of shape) were more convoluted than cells from the conduit PA, with prominent cytoplasmic projections, despite similar isolation protocols (Fig 3).

K_Ca cells were more prevalent in conduit than in resistance PAs (Fig 4). In contrast, the resistance PAs had very few K_Ca cells, and K_DR cells were most numerous (Fig 4). The mixed cell type is found throughout the vascular tree.

View larger version (14K): [in this window] [in a new window]   Figure 4. Conduit and resistance PAs have different prevalence percentages for the three smooth muscle types. The prevalence of the three cell types (identified by their morphology on light microscopy) was studied by light microscopy in 542 cells from conduit PAs of six rats and 386 cells from resistance PAs of five rats. Note the relatively greater occurrence of KCa cells in conduit arteries and greater prevalence of KDR cells in resistance arteries. Mixed cell types are found in great numbers throughout the circulation.

NO Effects on K_Ca Channels
NO increased I_K in K_Ca cells (Fig 5A) by activating K_Ca channels (Fig 5B). This caused reversible membrane hyperpolarization and relaxation of PA rings (Fig 5C and 5D). NO-induced K⁺ channel activation was inhibited by TEA or CTX, both in whole-cell and single-channel recordings (Figs 5 and 6).

View larger version (35K): [in this window] [in a new window]   Figure 5. NO activates KCa channels in KCa cells, causing hyperpolarization and enhancing relaxation in conduit PA rings. A, Whole-cell current-voltage plots from amphotericin-perforated cells (mean±SEM) are shown. In five KCa cells, IK is lower than in mixed cells. IK is increased by NO and then inhibited by TEA (5 mmol/L). *P<.005 vs control. In five mixed cells, NO tended to decrease IK (P=NS). B, Single-channel experiment with an amphotericin-perforated vesicle from a morphologically characterized KCa cell shows NO activating a KCa channel. Note the large amplitude of channel opening (20 pA at +70 mV). Frequency histograms are shown, along with examples of actual recordings (insets; pulse duration, 350 milliseconds at +70 mV). Po, increased by NO, was reduced by CTX (200 nmol/L). O indicates that the channel opened; C, the channel closed. C, NO reversibly hyperpolarized KCa cells from conduit PA (n=4 amphotericin-perforated cells). D, NO-induced relaxation, evident in both segments, is statistically greater in conduit than resistance PA rings constricted with endothelin-1 (10-7 mol/L). NO relaxation is prevented by the guanylate cyclase inhibitor LY83583 in both segments. *P<.05 vs conduit; P<.0001 vs absence of LY83583.

View larger version (37K): [in this window] [in a new window]   Figure 6. NO increases NPo of KCa channels in cell-attached single-channel studies in a mixed cell. A, Control data are shown in the left column, and the effects of NO are shown on the right. Traces are sequential. The bottom trace in each panel shows the identified segment in greater detail to illustrate the duration of opening. Note that the holding potential is -20 mV. The KCa channel was not active at -20 mV until NO was applied to the cell (right). These single-channel studies demonstrate the presence of KCa and KDR channels in a single cell type. This figure shows basal single-channel KDR activity (the smaller amplitude openings), with KCa activity (the larger amplitude openings) becoming evident with application of NO in a mixed type cell. B, Time course of open dwell time of KCa channel before and after NO application (indicated by arrow) is shown.

NO increased P_o of a CTX-sensitive channel, while having no effect on channel amplitude (Fig 5B). In single-channel cell-attached experiments, NO increased the NP_o of K_Ca channels at a holding potential of -20 mV. There was minimal K_Ca activity until NO was applied (Fig 6A). NO caused a rapid and reversible activation of K_Ca channels. The control value for open dwell time (1.6 milliseconds) was fitted with a single exponential curve. After application of NO, two channels appeared, and the open dwell time was fitted with a biexponential curve, resulting in open dwell times of 8.2 and 111.0 milliseconds.

In excised patches, increasing the bath Ca²⁺ concentration from nominally Ca²⁺-free to 50 nmol/L caused a proportionate increase in NP_o from 0.004 to 0.156 (Fig 7A). The slope conductance of the K_Ca channel in symmetrical K⁺ was similar in cell-attached and excised-patch configurations (170 and 176 pS, respectively; Fig 7B). The K_DR channel was also calculated to have similar conductance in these two configurations (37 versus 42 pS, respectively). The excised patch data were derived from mixed cells from the resistance PA, confirming the presence of both K_Ca and K_DR channels in such cells.

View larger version (30K): [in this window] [in a new window]   Figure 7. Characterization of the KCa channel. A, Representative traces from an excised-patch experiment in which the bath was either nominally Ca2+ free or contained 50 nmol/L Ca2+. Note the dramatic increase in opening with an increase in ambient Ca2+ concentration, confirming that this is a KCa channel. C indicates closed; 1 and 2, number of channels opened. B, The slope conductance () of the KCa channel activated by NO. Values are mean±SEM.

Hypoxic Effects on K⁺ Channels
Hypoxia rapidly inhibited I_K (Fig 8A) and caused membrane depolarization (Fig 2) in K_DR cells. Inhibition of K⁺ channels occurred within 1 to 2 minutes in whole-cell experiments (Fig 8A) and in <1 minute in single-channel experiments (Fig 8B). The hypoxic inhibition of I_K in whole cells was evident at physiologically relevant E_ms (-30 mV; inset, Fig 8A). In singlechannel studies in K_DR cells from resistance PAs, hypoxia reversibly decreased NP_o (Figs 8B and 9) and minimally shortened open dwell time (from 6.2 to 4.9 milliseconds in the example shown in Fig 9). In these cell-attached studies, there were several K_DR channels, judged by their different amplitudes, which could be measured during normoxia, despite the presence of TEA (or CTX) and niflumic acid in the patch pipette. The openings of these channels were inhibited by hypoxia. The channel most consistently inhibited was a 37-pS channel (Fig 9). Reversal of hypoxic K⁺ channel inhibition was almost universally present in single-channel experiments on reoxygenation but was only clearly demonstrated in two of seven whole-cell experiments. Hypoxia increased I_K in K_Ca cells at +70 mV (Fig 8).

View larger version (29K): [in this window] [in a new window]   Figure 8. Hypoxia inhibits K+ channels in KDR cells. A, Current-voltage plots from amphotericin-perforated cells are shown. Hypoxia increases whole-cell IK in morphologically identified KCa cells (n=6) and progressively reduces them in KDR cells (n=7). Currents are normalized relative to each cell's control (normoxic) current at +70 mV (100%).15 The maximal response to hypoxia (1 to 11 minutes after onset) is shown for KCa cells. *P<.0001 vs normoxia. Note the inset showing the effects of hypoxia at more negative Ems. This is an enlargement of the data in panel A from the KDR cells. Hypoxia inhibits currents at negative Ems (P<.05 vs normoxia). B, In an amphotericin-perforated vesicle from a morphologically characterized KDR cell obtained from the resistance PA, frequency histograms and examples of actual recordings show reversible K+ channel inhibition by hypoxia (pulse duration, 800 milliseconds at +70 mV). C indicates channel closed; 1, 2, and 3, number of channels opened.

View larger version (27K): [in this window] [in a new window]   Figure 9. Hypoxia rapidly and reversibly inhibits a KDR channel in KDR cells. Representative single-channel traces are from a cell-attached patch on a KDR cell. The pipette contains TEA and niflumic acid to inhibit KCa and chloride channels (holding potential, +20 mV). The dotted lines at the top indicate the amplitude of the opening of a 37-pS KDR channel. The activity of this channel decreases in hypoxia. Note that there are several smaller channel amplitudes evident in this patch that have not been fully characterized. Hypoxia decreases NPo for all channels within 1 minute, and this partially reverses within 3 minutes of the return to normoxia.

PA Rings
Basal Tone
TEA caused greater constriction in conduit PAs (+285±80 mg at 10 mmol/L, n=5) than in resistance PAs (+62±32 mg, n=5, P<.05), consistent with the greater prevalence of K_Ca cells in the former (Fig 4). Constriction to 4-AP was similar (P=.07) in conduit rings (+376±77 mg at 10 mmol/L, n=5) and resistance rings (+157±73 mg, n=5), consistent with the ubiquitous distribution of mixed type cells (Fig 4).

Response to NO
NO was an effective relaxant in both conduit and resistance PA rings. However, consistent with the greater prevalence of NO-activated K_Ca cells in the conduit PA, NO caused more relaxation in conduit than resistance PA rings (Fig 5D). NO-induced relaxation of both segments was attenuated by the guanylate cyclase inhibitor LY83583 (Fig 5D).

HPV
In control resistance rings (no K⁺ channel blocker present), hypoxic vasoconstriction was demonstrated without the use of pharmacological priming, which is commonly required to elicit hypoxic constriction in excised PAs (Fig 2D). This attests to the excellent condition of the rings. In resistance arteries, hypoxia caused monophasic constriction, which was sustained for the 12-minute exposure to hypoxia (Fig 2D). Hypoxic constriction of resistance rings was enhanced by pretreatment with TEA and to a greater extent by pretreatment with 4-AP (Fig 2D).

Hypoxic Pulmonary Relaxation
Hypoxia caused a biphasic response in conduit arteries, an initial small constriction followed by relaxation to tones below the baseline. Hypoxic relaxation was not abolished by 4-AP or TEA (Fig 2D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The primary finding of the present study is the great diversity in K⁺ channel electrophysiology (judged by current density and whole-cell K⁺ channel pharmacology) among cells taken from the same segment of the PA. Morphological criteria allow identification of three different cell types and also reveal that their distribution changes longitudinally along the pulmonary vasculature, from conduit to resistance segments. This diversity explains, in part, the segmental differences in the response of the pulmonary circulation to hypoxia and NO. In addition, the "side-by-side" existence of electrophysiologically distinct cells may explain the conflicting results investigators sometimes obtain when studying the effects of stimuli such as NO and hypoxia on randomly selected cells.

Three other important findings are discussed: (1) The E_m in PA VSM is primarily controlled by K_DR channels. (2) NO activates a K_Ca channel at physiological E_ms by increasing both P_o and open dwell time. (3) Authentic hypoxia causes a time-dependent inhibition of a 4-AP–sensitive whole-cell current. Hypoxia decreases the NP_o of one or more K_DR channels.

Cell Diversity
Arterial VSM is not homogeneous in terms of cell morphology¹⁷ and electrophysiology.^{15 18 34} The present study shows that there are three distinct cell types in the PA based on pharmacological, morphological, and electrophysiological criteria. Cells can be separated by electrophysiological criteria based on their current density and the class of K⁺ channels conducting the majority of their whole-cell current. The use of whole-cell current density provides an objective measure of net current, corrected for cell size. Using a similar technique, Albarwani et al³⁴ found that cells in resistance and conduit PAs had capacitances of 10 to 12 pF with current densities of 157±64 and 141±66 pA/pF, respectively, at a holding potential of +30 mV. The large standard error of their data and the fact that their values are similar to the mean obtained when the current densities of our three cell types are averaged (mean, 110 pA/pF; K_Ca, 15.6±2.2 pA/pF; K_DR, 265.3±36.6 pA/pF; and mixed, 51.7±10.8 pA/pF; Fig 1C) suggest that they may have had a similar, but unrecognized, diversity in cell types.

There are phenotypic similarities, on light and scanning electron microscopy, among cells of each electrophysiological type (Fig 3); consequently, morphology can be used to predict which channels are predominant in a given cell. It is important to emphasize that the morphology does not reflect variations in digestion, as all three cell types can be seen in a single dispersion from a single ring, in which all cells are treated identically. Furthermore, cells from conduit and resistance arteries have marked structural differences, despite identical harvesting and dispersion protocols. Overdigested cells are identified by their rounded or "humped-up" appearance and are not included in the electrophysiological or morphological survey.

The nomenclature "K_Ca, K_DR, or mixed cells" reflects the K⁺ channel type that contributes most to whole-cell current and is a convenient means of expressing the cell's identity. It does not mean that a cell has only one type of K⁺ channel. In fact, in both cell-attached and cell-excised patches, single-channel studies show the presence of both K_DR and K_Ca channels in a single K_DR cell (from a resistance artery). Nonetheless, the classification is quite useful, as the cells have characteristic responses to NO and hypoxia. Coupled with the finding that the three cell types (particularly K_Ca and K_DR cells) have a different frequency in different arterial segments, this suggests that K⁺ channel diversity may explain much of the segmental variation in the pulmonary vasculature's response to NO and hypoxia.

Conduit arteries are the main repository of K_Ca cells, where they reside interspersed with mixed and K_DR cell types (Fig 4). In contrast, the resistance arteries, the site at which vascular tone is primarily regulated in the lung,^{2 3} have a more homogeneous cell population composed mainly of mixed and K_DR cell types. NO increases I_K in the K_Ca cells but not in K_DR cells. Conversely, hypoxia inhibits I_K in K_DR but not K_Ca cells (Figs 8 and 9). This correlates well with the localization of HPV to the resistance PA and the somewhat greater relaxation caused by NO in the conduit PA (Fig 5D).

The existence of cell diversity in arteries is a recent concept. Frid et al¹⁷ found at least three populations of PA VSM, based on immunological and morphological criteria, within the media of the calf main PA. They noted diversity in cells within the PA, based on the distribution of contractile and cytoskeletal proteins (-actin, myosin, calponin, desmin, and metavinculin), as they surveyed from the lumen toward the adventitia. Furthermore, the prevalence of the cell types varied during development from fetus to adult. Unlike the present study, cells were studied in sections (rather than after isolation), and no data relating to cell function were acquired. Since our cells were dispersed, we do not know whether the three cell types in the main PA were arranged randomly or in a luminal to abluminal array.

In systemic arteries, there are two types of smooth muscle cells, based on myosin isoform distribution.³⁵ Furthermore, in the adult rat, cells appear in the neointima after vascular injury that differ in morphology from normal cells in the media, and these have an ability to produce matrix and growth factors, reminiscent of VSM from immature rats.^{36 37} Neylon et al¹⁸ found two cell types in cultures of adult rat aortic VSM. There were elongated cells that depolarized in response to vasoactive agonists (endothelin-1 and -thrombin), which elicited an influx of Na⁺ and Ca²⁺ and epithelium-like cells that hyperpolarized (in response to the same stimuli), probably as a result of the activation of K_Ca channels.¹⁸ Unlike the present study, they performed no patch-clamp studies and relied on the use of fluorescent dyes to measure E_m and cytosolic Ca²⁺. The cell morphology they described in nondispersed cultured cells is not comparable to the morphology in our dispersed isolated cells.

Neither the present study nor the studies of Frid et al¹⁷ and Neylon et al¹⁸ provide a clear explanation for the diversity of cell types within the vessel wall. We speculate that the disparity in the proportion of VSM cell subtypes between conduit and resistance PAs could be related to the different embryological origin of the conduit PA (sixth aortic arch) and resistance PA (lung bud).³⁸ Consequently, the conduit PA may have reactivity closer to that of systemic vessels (both dilate in response to hypoxia, whereas the resistance PAs constrict).

If regional differences in K⁺ channel distribution are of functional importance, one might expect that this would be evident when examining the segmental response of the pulmonary vasculature to hypoxia and NO, stimuli that accomplish vasoconstriction and dilatation in the pulmonary circulation, which are at least in part due to their effects on K⁺ channels.^{8 9 10 15}

HPV and 4-AP–Sensitive K_DR Channels
HPV is characterized by the rapid reversible constriction of intrapulmonary resistance arteries that, although modulated by many neurohumoral factors, is intrinsic to PA VSM^{4 5 39} (see Reference 40 for review). Our isolated vascular ring experiments confirm resistance PA as the site of more significant and sustained hypoxic constriction compared with conduit PA. This is in accord with previous reports from isolated PA VSM cells,⁴ PA rings,⁴¹ isolated lungs,⁴² and intact animals.^{2 43} The precise identity of the K⁺ channels involved in HPV has been uncertain, although whole-cell data suggest it may be a K_DR-type channel.^{9 10} In the present study, 4-AP, a preferential K_DR inhibitor, caused more constriction than did TEA, consistent with the finding that 4-AP, but not TEA or CTX, depolarizes PA VSM from resistance arteries (Fig 2B). The greater effect of 4-AP on tone in rings is consistent with results of a previous study that compared the effects of K⁺ channel blockers on tone in the isolated rat lung.³¹ Together, these findings support the contention of Yuan²⁵ and Post et al¹² that the resting E_m in PA VSM is controlled by the K_DR class of K⁺ channels. Our finding of several TEA-resistant, CTX-resistant, and niflumic acid–resistant hypoxia-inhibitable K⁺ channels suggests that the K_DR may not be a single channel but a family of channels (Fig 9). Further biophysical studies are required to comment on this possibility.

It is expected that the segment most enriched in such K_DR cells (the resistance PA, Fig 4) should have the best hypoxic constrictor response, which is in fact the case (Fig 2D). If resistance PAs constrict because of K_DR channels, why do conduit arteries relax during hypoxia? One possible explanation is that hypoxia increases I_K in K_Ca cells (Fig 8), much as it does in cerebral VSM.⁴⁴ It is possible that both K_Ca and K_ATP channels contribute to the hypoxic relaxation in conduit PA and systemic arteries.

The data in Figs 8 and 9 provide the first single-channel demonstration (using authentic hypoxia rather than dithionite) that hypoxia rapidly and reversibly inhibits K_DR channels. The fact that this occurs at negative E_ms (Fig 8, inset) and that hypoxia depolarizes the membrane (Fig 2C) confirms the physiological relevance of K⁺ channel inhibition to HPV. Dithionite, although a convenient way to reduce hemoproteins and lower PO₂, does not mimic hypoxia created by alveolar ventilation with hypoxic gas or N₂ bubbling of solutions.⁴⁵ In fact, dithionite only lowers PO₂ by means of an attendant and obligatory generation of O₂ radicals and peroxides, which actually results in vasodilatation, rather than constriction, in the isolated lung.⁴⁵

NO Activates K_Ca Channels
NO causes relaxation by several mechanisms, including sequestration of intracellular Ca²⁺,⁴⁶ inhibition of the voltage-gated Ca²⁺ channel, and desensitization of the contractile apparatus to Ca²⁺.⁴⁷ In addition, a variable portion of NO relaxation may relate to its ability to activate K_Ca channels, evident in coronary,⁴⁸ cerebral,⁴⁹ and pulmonary¹⁵ VSM. In the conduit PA, part of the dilator effect of NO is due to K_Ca activation, and relaxation is impaired by CTX.¹⁵ NO is an effective dilator in both conduit PAs and resistance PAs; thus, it might not appear that K⁺ channel distribution is important to the hemodynamic effects of NO. However, careful comparison of the relaxant effects of NO reveals a slight but significant increase in dilator efficacy in the conduit rings, relative to resistance rings (Fig 5D). The difference in the potency of NO between segments may reflect the greater prevalence in conduit PAs of K_Ca cells, which respond to NO with an increase in I_K and P_o and hyperpolarization (Figs 5 and 6). There is controversy concerning whether NO acts directly on the K_Ca channel⁵⁰ or via an intracellular pathway involving cGMP and cGMP-dependent protein kinase.^{15 48 49} In the present study, NO-induced relaxation was attenuated by the guanylate cyclase inhibitor LY83583, consistent with prior studies demonstrating that the effects of NO on K⁺ channels and vascular tone are mediated through cGMP.^{15 48 49} LY83583, like methylene blue, is a nonspecific inhibitor of guanylate cyclase and may have other unanticipated effects, although it does lower cGMP.^{32 33} Although direct cGMP-independent K⁺ channel activation by NO occurs in excised patch studies,⁵⁰ this effect does not appear essential to the dilator efficacy of NO in arterial rings.

The present study demonstrates that NO increases both the P_o and the open dwell time of the K_Ca channel. It is noteworthy that K_Ca channels were activated by NO at relatively negative E_ms (-20 mV, Fig 6A) and that NO causes membrane hyperpolarization, consistent with a physiological role for this effect.

We conclude that regional diversity in vascular reactivity to NO and hypoxia result, in large part, from differences in the prevalence of the three types of VSM cells, which possess electrophysiologically distinct types of K⁺ channels.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine CTX = charybdotoxin Em = membrane potential HPV = hypoxic pulmonary vasoconstriction IK = K+ current KCa channel (cell) = Ca2+-sensitive K+ channel (cell) KDR channel (cell) = delayed rectifier K+ channel (cell) NPo = number of channels multiplied by Po PA = pulmonary artery Po = channel open probability TEA = tetraethylammonium VSM = vascular smooth muscle

	Acknowledgments

 
This study was supported by the Department of Veterans Affairs, National Institutes of Health grant HL-45735 (Dr Archer), and the American Heart Association, Minnesota Affiliate, Inc (Drs Reeve and Hampl). We thank Dennis Knapp for immunochemical staining of VSM cells. We acknowledge the assistance of Mark Sanders, PhD, in performing the electron microscopy.

Received April 27, 1995; accepted November 21, 1995.

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				M. Diez, J. A. Barbera, E. Ferrer, R. Fernandez-Lloris, S. Pizarro, J. Roca, and V. I. Peinado Plasticity of CD133+ cells: Role in pulmonary vascular remodeling Cardiovasc Res, December 1, 2007; 76(3): 517 - 527. [Abstract] [Full Text] [PDF]

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				G. Zhao, A. Adebiyi, Q. Xi, and J. H. Jaggar Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2122 - C2128. [Abstract] [Full Text] [PDF]

				S. Hayoz, J.-L. Beny, and R. Bychkov Intracellular cAMP: the "switch" that triggers on "spontaneous transient outward currents" generation in freshly isolated myocytes from thoracic aorta Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1502 - C1509. [Abstract] [Full Text] [PDF]

				C. D. Fike, M. R. Kaplowitz, Y. Zhang, and J. A. Madden Voltage-gated K+ channels at an early stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets Am J Physiol Lung Cell Mol Physiol, December 1, 2006; 291(6): L1169 - L1176. [Abstract] [Full Text] [PDF]

				I. Fantozzi, O. Platoshyn, A. H. Wong, S. Zhang, C. V. Remillard, M. R. Furtado, O. V. Petrauskene, and J. X.-J. Yuan Bone morphogenetic protein-2 upregulates expression and function of voltage-gated K+ channels in human pulmonary artery smooth muscle cells Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L993 - L1004. [Abstract] [Full Text] [PDF]

				E. K. Weir and A. Olschewski Role of ion channels in acute and chronic responses of the pulmonary vasculature to hypoxia Cardiovasc Res, September 1, 2006; 71(4): 630 - 641. [Abstract] [Full Text] [PDF]

				R. Ma, J. Du, S. Sours, and M. Ding Store-Operated Ca2+ Channel in Renal Microcirculation and Glomeruli Experimental Biology and Medicine, February 1, 2006; 231(2): 145 - 153. [Abstract] [Full Text] [PDF]

				E. K. Weir, J. Lopez-Barneo, K. J. Buckler, and S. L. Archer Acute Oxygen-Sensing Mechanisms. N. Engl. J. Med., November 10, 2005; 353(19): 2042 - 2055. [Full Text] [PDF]

				X.-R. Yang, M.-J. Lin, K.-P. Yip, L. H. Jeyakumar, S. Fleischer, G. P. H. Leung, and J. S. K. Sham Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells Am J Physiol Lung Cell Mol Physiol, August 1, 2005; 289(2): L338 - L348. [Abstract] [Full Text] [PDF]

				S. L. Archer Pre-B-cell Colony-Enhancing Factor Regulates Vascular Smooth Muscle Maturation Through a NAD+-Dependent Mechanism: Recognition of a New Mechanism for Cell Diversity and Redox Regulation of Vascular Tone and Remodeling Circ. Res., July 8, 2005; 97(1): 4 - 7. [Full Text] [PDF]

				Y.-M. Zheng, Q.-S. Wang, R. Rathore, W.-H. Zhang, J. E. Mazurkiewicz, V. Sorrentino, H. A. Singer, M. I. Kotlikoff, and Y.-X. Wang Type-3 Ryanodine Receptors Mediate Hypoxia-, but Not Neurotransmitter-induced Calcium Release and Contraction in Pulmonary Artery Smooth Muscle Cells J. Gen. Physiol., March 28, 2005; 125(4): 427 - 440. [Abstract] [Full Text] [PDF]

				R. Moudgil, E. D. Michelakis, and S. L. Archer Hypoxic pulmonary vasoconstriction J Appl Physiol, January 1, 2005; 98(1): 390 - 403. [Abstract] [Full Text] [PDF]

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				M. S. McMurtry, S. Bonnet, X. Wu, J. R.B. Dyck, A. Haromy, K. Hashimoto, and E. D. Michelakis Dichloroacetate Prevents and Reverses Pulmonary Hypertension by Inducing Pulmonary Artery Smooth Muscle Cell Apoptosis Circ. Res., October 15, 2004; 95(8): 830 - 840. [Abstract] [Full Text] [PDF]

				Z. Hong, E. K. Weir, D. P. Nelson, and A. Olschewski Subacute Hypoxia Decreases Voltage-Activated Potassium Channel Expression and Function in Pulmonary Artery Myocytes Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 337 - 343. [Abstract] [Full Text] [PDF]

				S. L. Archer, X.-C. Wu, B. Thebaud, A. Nsair, S. Bonnet, B. Tyrrell, M. S. McMurtry, K. Hashimoto, G. Harry, and E. D. Michelakis Preferential Expression and Function of Voltage-Gated, O2-Sensitive K+ Channels in Resistance Pulmonary Arteries Explains Regional Heterogeneity in Hypoxic Pulmonary Vasoconstriction: Ionic Diversity in Smooth Muscle Cells Circ. Res., August 6, 2004; 95(3): 308 - 318. [Abstract] [Full Text] [PDF]

				S. J. Fountain, A. Cheong, R. Flemming, L. Mair, A. Sivaprasadarao, and D. J. Beech Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle J. Physiol., April 1, 2004; 556(1): 29 - 42. [Abstract] [Full Text] [PDF]

				J. Wang, L. A. Shimoda, and J. T. Sylvester Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L848 - L858. [Abstract] [Full Text] [PDF]

				A. Olschewski, Z. Hong, D. A. Peterson, D. P. Nelson, V. A. Porter, and E. K. Weir Opposite effects of redox status on membrane potential, cytosolic calcium, and tone in pulmonary arteries and ductus arteriosus Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L15 - L22. [Abstract] [Full Text] [PDF]

				A.M. Gurney, O.N. Osipenko, D. MacMillan, K.M. McFarlane, R.J. Tate, and F.E.J. Kempsill Two-Pore Domain K Channel, TASK-1, in Pulmonary Artery Smooth Muscle Cells Circ. Res., November 14, 2003; 93(10): 957 - 964. [Abstract] [Full Text] [PDF]

				Y. Liu, H. Zhao, H. Li, B. Kalyanaraman, A. C. Nicolosi, and D. D. Gutterman Mitochondrial Sources of H2O2 Generation Play a Key Role in Flow-Mediated Dilation in Human Coronary Resistance Arteries Circ. Res., September 19, 2003; 93(6): 573 - 580. [Abstract] [Full Text] [PDF]

				V. Hampl, J. Bibova, V. Povysilova, and J. Herget Dehydroepiandrosterone sulphate reduces chronic hypoxic pulmonary hypertension in rats Eur. Respir. J., May 1, 2003; 21(5): 862 - 865. [Abstract] [Full Text] [PDF]

				Z. I. Pozeg, E. D. Michelakis, M. S. McMurtry, B. Thebaud, X.-C. Wu, J. R.B. Dyck, K. Hashimoto, S. Wang, R. Moudgil, G. Harry, et al. In Vivo Gene Transfer of the O2-Sensitive Potassium Channel Kv1.5 Reduces Pulmonary Hypertension and Restores Hypoxic Pulmonary Vasoconstriction in Chronically Hypoxic Rats Circulation, April 22, 2003; 107(15): 2037 - 2044. [Abstract] [Full Text] [PDF]

				K. R. Stenmark and S. A. Gebb Lung Vascular Development: Breathing New Life Into An Old Problem Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 133 - 137. [Full Text] [PDF]

				L. Conforti, M. Petrovic, D. Mohammad, S. Lee, Q. Ma, S. Barone, and A. H. Filipovich Hypoxia Regulates Expression and Activity of Kv1.3 Channels in T Lymphocytes: A Possible Role in T Cell Proliferation J. Immunol., January 15, 2003; 170(2): 695 - 702. [Abstract] [Full Text] [PDF]

				V. Hampl, J. Bibova, Z. Stranak, X. Wu, E. D. Michelakis, K. Hashimoto, and S. L. Archer Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2440 - H2449. [Abstract] [Full Text] [PDF]

				A. Olschewski, Z. Hong, D. P. Nelson, and E. K. Weir Graded response of K+ current, membrane potential, and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1143 - L1150. [Abstract] [Full Text] [PDF]

				A. Yaghi, S. Mehta, and D. G. McCormack Delayed rectifier potassium channels contribute to the depressed pulmonary artery contractility in pneumonia J Appl Physiol, September 1, 2002; 93(3): 957 - 965. [Abstract] [Full Text] [PDF]

				S. Archer and E. Michelakis The Mechanism(s) of Hypoxic Pulmonary Vasoconstriction: Potassium Channels, Redox O2 Sensors, and Controversies Physiology, August 1, 2002; 17(4): 131 - 137. [Abstract] [Full Text] [PDF]

				J. L. Aschner, T. K. Smith, N. Kovacs, J. M. B. Pinheiro, and M. Fuloria Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L373 - L382. [Abstract] [Full Text] [PDF]

				C. V. Remillard, W.-M. Zhang, L. A. Shimoda, and J. S. K. Sham Physiological properties and functions of Ca2+ sparks in rat intrapulmonary arterial smooth muscle cells Am J Physiol Lung Cell Mol Physiol, August 1, 2002; 283(2): L433 - L444. [Abstract] [Full Text] [PDF]

				D. S Hogg, A. R.L Davies, G. McMurray, and R. Z Kozlowski KV2.1 channels mediate hypoxic inhibition of IKV in native pulmonary arterial smooth muscle cells of the rat Cardiovasc Res, August 1, 2002; 55(2): 349 - 360. [Abstract] [Full Text] [PDF]

				S. V Smirnov, R. Beck, P. Tammaro, T. Ishii, and P. I Aaronson Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries J. Physiol., February 1, 2002; 538(3): 867 - 878. [Abstract] [Full Text] [PDF]

				E. D. Michelakis, M. S. McMurtry, X.-C. Wu, J. R.B. Dyck, R. Moudgil, T. A. Hopkins, G. D. Lopaschuk, L. Puttagunta, R. Waite, and S. L. Archer Dichloroacetate, a Metabolic Modulator, Prevents and Reverses Chronic Hypoxic Pulmonary Hypertension in Rats: Role of Increased Expression and Activity of Voltage-Gated Potassium Channels Circulation, January 15, 2002; 105(2): 244 - 250. [Abstract] [Full Text] [PDF]

				A. Papapetropoulos, S. Andreopoulos, C. Y. Go, A. Hoque, L. C. Fuchs, and J. D. Catravas Regulation of the nitric oxide synthase-nitric oxide- cGMP pathway in rat mesenteric endothelial cells J Appl Physiol, December 1, 2001; 91(6): 2553 - 2560. [Abstract] [Full Text] [PDF]

				E. A. Coppock and M. M. Tamkun Differential expression of KV channel alpha - and beta -subunits in the bovine pulmonary arterial circulation Am J Physiol Lung Cell Mol Physiol, December 1, 2001; 281(6): L1350 - L1360. [Abstract] [Full Text] [PDF]

				L. A. Shimoda, J. T. Sylvester, G. M. Booth, T. H. Shimoda, S. Meeker, B. J. Undem, and J. S. K. Sham Inhibition of voltage-gated K+ currents by endothelin-1 in human pulmonary arterial myocytes Am J Physiol Lung Cell Mol Physiol, November 1, 2001; 281(5): L1115 - L1122. [Abstract] [Full Text] [PDF]

				S. Belohlavkova, J. Simak, A. Kokesova, O. Hnilickova, and V. Hampl Fenfluramine-induced pulmonary vasoconstriction: role of serotonin receptors and potassium channels J Appl Physiol, August 1, 2001; 91(2): 755 - 761. [Abstract] [Full Text] [PDF]

				E. A. Coppock, J. R. Martens, and M. M. Tamkun Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L1 - L12. [Abstract] [Full Text] [PDF]

				L. A. Shimoda, D. J. Manalo, J. S. K. Sham, G. L. Semenza, and J. T. Sylvester Partial HIF-1{alpha} deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L202 - L208. [Abstract] [Full Text] [PDF]

				P.J Boels, J Deutsch, B Gao, and S.G Haworth Perinatal development influences mechanisms of bradykinin-induced relaxations in pulmonary resistance and conduit arteries differently Cardiovasc Res, July 1, 2001; 51(1): 140 - 150. [Abstract] [Full Text] [PDF]

				H. L. Reeve, E. Michelakis, D. P. Nelson, E. K. Weir, and S. L. Archer Alterations in a redox oxygen sensing mechanism in chronic hypoxia J Appl Physiol, June 1, 2001; 90(6): 2249 - 2256. [Abstract] [Full Text] [PDF]

				E. D. Michelakis, E. K. Weir, X. Wu, A. Nsair, R. Waite, K. Hashimoto, L. Puttagunta, H. G. Knaus, and S. L. Archer Potassium channels regulate tone in rat pulmonary veins Am J Physiol Lung Cell Mol Physiol, June 1, 2001; 280(6): L1138 - L1147. [Abstract] [Full Text] [PDF]

				Z. Zhang, J. M. C. Huang, M. R. Turner, K. L. Rhinehart, and T. L. Pallone Role of chloride in constriction of descending vasa recta by angiotensin II Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2001; 280(6): R1878 - R1886. [Abstract] [Full Text] [PDF]

				H. L Reeve, S. Tolarova, D. P Nelson, S. Archer, and E K. Weir Redox control of oxygen sensing in the rabbit ductus arteriosus J. Physiol., May 15, 2001; 533(1): 253 - 261. [Abstract] [Full Text] [PDF]

				P. S. Andrew, Y. Deng, R. Sultanian, and S. Kaufman Nitric oxide increases fluid extravasation from the splenic circulation of the rat Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2001; 280(4): R959 - R967. [Abstract] [Full Text] [PDF]

				M. Nie, H. Kobayashi, M. Sugawara, T. Tomita, K. Ohara, and H. Yoshimura Helium inhalation enhances vasodilator effect of inhaled nitric oxide on pulmonary vessels in hypoxic dogs Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1875 - H1881. [Abstract] [Full Text] [PDF]

				M. A. Vander Heyden, T. R. Halla, J. A. Madden, and J. B. Gordon Multiple Ca2+-dependent modulators mediate alkalosis-induced vasodilation in newborn piglet lungs Am J Physiol Lung Cell Mol Physiol, March 1, 2001; 280(3): L519 - L526. [Abstract] [Full Text] [PDF]

				J. Marijic, Q. Li, M. Song, K. Nishimaru, E. Stefani, and L. Toro Decreased Expression of Voltage- and Ca2+-Activated K+ Channels in Coronary Smooth Muscle During Aging Circ. Res., February 2, 2001; 88(2): 210 - 216. [Abstract] [Full Text] [PDF]

				F. Chabot, F. Schrijen, and C. Saunier Role of NO pathway, calcium and potassium channels in the peripheral pulmonary vascular tone in dogs Eur. Respir. J., January 1, 2001; 17(1): 20 - 26. [Abstract] [Full Text] [PDF]

				A. J. Halayko and J. Solway Plasticity in Skeletal, Cardiac, and Smooth Muscle: Invited Review: Molecular mechanisms of phenotypic plasticity in smooth muscle cells J Appl Physiol, January 1, 2001; 90(1): 358 - 368. [Abstract] [Full Text] [PDF]

				S. Pluger, J. Faulhaber, M. Furstenau, M. Lohn, R. Waldschutz, M. Gollasch, H. Haller, F. C. Luft, H. Ehmke, and O. Pongs Mice With Disrupted BK Channel {beta}1 Subunit Gene Feature Abnormal Ca2+ Spark/STOC Coupling and Elevated Blood Pressure Circ. Res., November 24, 2000; 87 (11): e53 - e60. [Abstract] [Full Text] [PDF]

				D. N. Cornfield, E. R. Resnik, J. M. Herron, and S. H. Abman Chronic intrauterine pulmonary hypertension decreases calcium-sensitive potassium channel mRNA expression Am J Physiol Lung Cell Mol Physiol, November 1, 2000; 279(5): L857 - L862. [Abstract] [Full Text] [PDF]

				L. A. Shimoda, J. S. K. Sham, T. H. Shimoda, and J. T. Sylvester L-type Ca2+ channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes Am J Physiol Lung Cell Mol Physiol, November 1, 2000; 279(5): L884 - L894. [Abstract] [Full Text] [PDF]

				V. Hampl and J. Herget Role of Nitric Oxide in the Pathogenesis of Chronic Pulmonary Hypertension Physiol Rev, October 1, 2000; 80(4): 1337 - 1372. [Abstract] [Full Text] [PDF]

				K. M. Ito, M. Sato, K. Ushijima, M. Nakai, and K. Ito Alterations of endothelium and smooth muscle function in monocrotaline-induced pulmonary hypertensive arteries Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1786 - H1795. [Abstract] [Full Text] [PDF]

				Y.-X. WANG, P. K. DHULIPALA, and M. I. KOTLIKOFF Hypoxia inhibits the Na+/Ca2+ exchanger in pulmonary artery smooth muscle cells FASEB J, September 1, 2000; 14(12): 1731 - 1740. [Abstract] [Full Text]

				J. S. K. Sham, B. R. Crenshaw Jr., L.-H. Deng, L. A. Shimoda, and J. T. Sylvester Effects of hypoxia in porcine pulmonary arterial myocytes: roles of KV channel and endothelin-1 Am J Physiol Lung Cell Mol Physiol, August 1, 2000; 279(2): L262 - L272. [Abstract] [Full Text] [PDF]

				D. N. Cornfield, C. B. Saqueton, V. A. Porter, J. Herron, E. Resnik, I. Y. Haddad, and H. L. Reeve Voltage-gated K+-channel activity in ovine pulmonary vasculature is developmentally regulated Am J Physiol Lung Cell Mol Physiol, June 1, 2000; 278(6): L1297 - L1304. [Abstract] [Full Text] [PDF]

				L. Conforti, I. Bodi, J. W Nisbet, and D. E Millhorn O2-sensitive K+ channels: role of the Kv1.2 {alpha}-subunit in mediating the hypoxic response J. Physiol., May 1, 2000; 524(3): 783 - 793. [Abstract] [Full Text] [PDF]

				K. Sato, Y. Morio, K. G. Morris, D. M. Rodman, and I. F. McMurtry Mechanism of hypoxic pulmonary vasoconstriction involves ETA receptor-mediated inhibition of KATP channel Am J Physiol Lung Cell Mol Physiol, March 1, 2000; 278(3): L434 - L442. [Abstract] [Full Text] [PDF]

				C. Austin and S. Wray Interactions Between Ca2+ and H+ and Functional Consequences in Vascular Smooth Muscle Circ. Res., February 18, 2000; 86(3): 355 - 363. [Abstract] [Full Text] [PDF]

				P. Ghisdal, J.-P. Gomez, and N. Morel Action of a NO donor on the excitation-contraction pathway activated by noradrenaline in rat superior mesenteric artery J. Physiol., January 1, 2000; 522(1): 83 - 96. [Abstract] [Full Text] [PDF]

				E. D. Michelakis, E. K. Weir, D. P. Nelson, H. L. Reeve, S. Tolarova, and S. L. Archer Dexfenfluramine Elevates Systemic Blood Pressure by Inhibiting Potassium Currents in Vascular Smooth Muscle Cells J. Pharmacol. Exp. Ther., December 1, 1999; 291(3): 1143 - 1149. [Abstract] [Full Text]

				P.J. Boels, J. Deutsch, B. Gao, and S.G. Haworth Maturation of the response to bradykinin in resistance and conduit pulmonary arteries Cardiovasc Res, November 1, 1999; 44(2): 416 - 428. [Abstract] [Full Text] [PDF]

				C. B. Neylon, R. J. Lang, Y. Fu, A. Bobik, and P. H. Reinhart Molecular Cloning and Characterization of the Intermediate-Conductance Ca2+-Activated K+ Channel in Vascular Smooth Muscle : Relationship Between KCa Channel Diversity and Smooth Muscle Cell Function Circ. Res., October 29, 1999; 85 (9): e33 - e43. [Abstract] [Full Text] [PDF]

				J. T. Hulme, E. A. Coppock, A. Felipe, J. R. Martens, and M. M. Tamkun Oxygen Sensitivity of Cloned Voltage-Gated K+ Channels Expressed in the Pulmonary Vasculature Circ. Res., September 17, 1999; 85(6): 489 - 497. [Abstract] [Full Text] [PDF]

				L. A. Shimoda, J. T. Sylvester, and J. S. K. Sham Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes Am J Physiol Lung Cell Mol Physiol, September 1, 1999; 277(3): L431 - L439. [Abstract] [Full Text] [PDF]

				S. L. Archer, H. L. Reeve, E. Michelakis, L. Puttagunta, R. Waite, D. P. Nelson, M. C. Dinauer, and E. K. Weir O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase PNAS, July 6, 1999; 96(14): 7944 - 7949. [Abstract] [Full Text] [PDF]

				K.-X. Li, B. Fouty, I. F. McMurtry, and D. M. Rodman Enhanced ETA-receptor-mediated inhibition of Kv channels in hypoxic hypertensive rat pulmonary artery myocytes Am J Physiol Heart Circ Physiol, July 1, 1999; 277(1): H363 - H370. [Abstract] [Full Text] [PDF]

				S. S. Salvi {alpha}1-Adrenergic Hypothesis for Pulmonary Hypertension Chest, June 1, 1999; 115(6): 1708 - 1719. [Abstract] [Full Text] [PDF]

				N. F. Voelkel, J. D. Allard, S. M. Anderson, and T. J. Burke cGMP and cAMP cause pulmonary vasoconstriction in the presence of hemolysate J Appl Physiol, May 1, 1999; 86(5): 1715 - 1720. [Abstract] [Full Text] [PDF]

				O. Clement-Chomienne, K. Ishii, M. P Walsh, and W. C Cole Identification, cloning and expression of rabbit vascular smooth muscle Kv1.5 and comparison with native delayed rectifier K+ current J. Physiol., March 15, 1999; 515(3): 653 - 667. [Abstract] [Full Text] [PDF]

				H. L. Reeve, D. P. Nelson, S. L. Archer, and E. K. Weir Effects of fluoxetine, phentermine, and venlafaxine on pulmonary arterial pressure and electrophysiology Am J Physiol Lung Cell Mol Physiol, February 1, 1999; 276(2): L213 - L219. [Abstract] [Full Text] [PDF]

				H. L. Reeve, E. K. Weir, S. L. Archer, and D. N. Cornfield A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent Am J Physiol Lung Cell Mol Physiol, December 1, 1998; 275(6): L1019 - L1025. [Abstract] [Full Text] [PDF]

				S. O Brij and A. J Peacock Cellular responses to hypoxia in the pulmonary circulation Thorax, December 1, 1998; 53(12): 1075 - 1079. [Full Text]

				I. S. Anand, B. A. K. Prasad, S. S. Chugh, K. R. M. Rao, D. N. Cornfield, C. E. Milla, N. Singh, S. Singh, and W. Selvamurthy Effects of Inhaled Nitric Oxide and Oxygen in High-Altitude Pulmonary Edema Circulation, December 1, 1998; 98(22): 2441 - 2445. [Abstract] [Full Text] [PDF]

				M. G. Berger, C. Vandier, P. Bonnet, W. F. Jackson, and N. J. Rusch Intracellular acidosis differentially regulates KV channels in coronary and pulmonary vascular muscle Am J Physiol Heart Circ Physiol, October 1, 1998; 275(4): H1351 - H1359. [Abstract] [Full Text] [PDF]

				A. M. Evans, O. N. Osipenko, S. G. Haworth, and A. M. Gurney Resting potentials and potassium currents during development of pulmonary artery smooth muscle cells Am J Physiol Heart Circ Physiol, September 1, 1998; 275(3): H887 - H899. [Abstract] [Full Text] [PDF]

				H M Prior, M S Yates, and D J Beech Functions of large conductance Ca2+-activated (BKCa), delayed rectifier (KV) and background K+ channels in the control of membrane potential in rabbit renal arcuate artery J. Physiol., August 15, 1998; 511(1): 159 - 169. [Abstract] [Full Text] [PDF]

				J.-M. Hyvelin, C. Guibert, R. Marthan, and J.-P. Savineau Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes Am J Physiol Lung Cell Mol Physiol, August 1, 1998; 275(2): L269 - L282. [Abstract] [Full Text] [PDF]

				G. J. Waldron, S. B. Sigurdsson, E. A. Aiello, A. J. Halayko, N. L. Stephens, and W. C. Cole Delayed rectifier K+ current of dog bronchial myocytes: effect of pollen sensitization and PKC activation Am J Physiol Lung Cell Mol Physiol, August 1, 1998; 275(2): L336 - L347. [Abstract] [Full Text] [PDF]

				E. A. Aiello, A. T. Malcolm, M. P. Walsh, and W. C. Cole beta -Adrenoceptor activation and PKA regulate delayed rectifier K+ channels of vascular smooth muscle cells Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H448 - H459. [Abstract] [Full Text] [PDF]

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				L. A. Shimoda, J. T. Sylvester, and J. S. K. Sham Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1 Am J Physiol Lung Cell Mol Physiol, May 1, 1998; 274(5): L842 - L853. [Abstract] [Full Text] [PDF]

				A. J. Halayko, E. Rector, and N. L. Stephens Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition Am J Physiol Lung Cell Mol Physiol, January 1, 1998; 274(1): L17 - L25. [Abstract] [Full Text] [PDF]

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