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(Circulation Research. 2002;91:610.)
© 2002 American Heart Association, Inc.

Cellular Biology

Carbon Monoxide Dilates Cerebral Arterioles by Enhancing the Coupling of Ca²⁺ Sparks to Ca²⁺-Activated K⁺ Channels

Jonathan H. Jaggar, Charles W. Leffler, Serguei Y. Cheranov, Dilyara Tcheranova, Shuyu E, Xiaoyang Cheng

From the Department of Physiology, University of Tennessee Health Science Center, Memphis.

Correspondence to Jonathan H. Jaggar, Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163. E-mail jjaggar{at}physio1.utmem.edu

	Abstract

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Carbon monoxide (CO) is generated endogenously by the enzyme heme oxygenase. Although CO is a known vasodilator, cellular signaling mechanisms are poorly understood and are a source of controversy. The goal of the present study was to investigate mechanisms of CO dilation in porcine cerebral arterioles. Data indicate that exogenous or endogenously produced CO is a potent activator of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels and Ca²⁺ spark–induced transient K_Ca currents in arteriole smooth muscle cells. In contrast, CO is a relatively poor activator of Ca²⁺ sparks. To understand the apparent discrepancy between potent effects on transient K_Ca currents and weak effects on Ca²⁺ sparks, regulation of the coupling relationship between these events by CO was investigated. CO increased the percentage of Ca²⁺ sparks that activated a transient K_Ca current (ie, the coupling ratio) from 62% in the control condition to 100% and elevated the slope of the amplitude correlation between these events 2.6-fold, indicating that Ca²⁺ sparks induced larger amplitude transient K_Ca currents in the presence of CO. This signaling pathway for CO is physiologically relevant because ryanodine, a ryanodine-sensitive Ca²⁺ release channel blocker that inhibits Ca²⁺ sparks, abolished CO dilation of pial arterioles in vivo. Thus, CO dilates cerebral arterioles by priming K_Ca channels for activation by Ca²⁺ sparks. This study presents a novel dilatory signaling pathway for CO in the cerebral circulation and appears to be the first presents of a vasodilator that acts by increasing the effective coupling of Ca²⁺ sparks to K_Ca channels.

Key Words: ryanodine-sensitive Ca²⁺ release channels • Ca²⁺-sensitive K⁺ channels • heme oxygenase

	Introduction

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Carbon monoxide (CO) is an important paracrine and autocrine relaxing factor in the cerebral and systemic circulation (see review¹). However, mechanisms leading to the regulation of arterial diameter by CO are poorly understood and controversial.

CO is produced physiologically via the metabolism of heme by heme oxygenase.¹ Heme is found in virtually all cell types, and arterial smooth muscle and endothelial cells contain both heme oxygenase-1 and heme oxygenase-2.^2,3 Authentic CO or heme oxygenase substrates dilate vascular preparations from anatomically diverse locations.¹ Thus, CO may constitute a novel and relatively unexplored paracrine and autocrine gaseous vasodilator. In tail,⁴ gracilis muscle,⁵ renal,⁶ and cerebral arteries,⁷ CO-induced dilations are inhibited by blockers of the large-conductance Ca²⁺-activated K⁺ (K_Ca) channel. CO has also been reported to activate K_Ca channels in cultured arterial smooth muscle cells via chemical modification of an external histidine residue.⁸ These findings support an important role for K_Ca channels in CO dilations.

In arterial smooth muscle cells, localized intracellular Ca²⁺ transients, termed Ca²⁺ sparks, activate several sarcolemma K_Ca channels to induce a "spontaneous transient outward current."^9–11 Ca²⁺ sparks occur due to the opening of several ryanodine-sensitive Ca²⁺ release (RyR) channels located on the sarcoplasmic reticulum and elevate the intracellular Ca²⁺ concentration in the immediate vicinity of the release site by 10 µmol/L, but do not significantly contribute to global Ca²⁺ levels.^10,12 Ca²⁺ spark or K_Ca channel blockers depolarize arteries at physiological levels of pressure, leading to the activation of voltage-dependent Ca²⁺ channels, an elevation in the intracellular Ca²⁺ concentration, and constriction.^11,13,14 Genetic ablation of the associated ß₁ subunit reduces K_Ca channel Ca²⁺ sensitivity, attenuates Ca²⁺ spark coupling, and leads to an elevation in arterial tone and systemic blood pressure.^15,16 Conversely, activators of cAMP-dependent or cGMP-dependent protein kinases dilate cerebral arteries, in part, by activating Ca²⁺ sparks and K_Ca channels.¹⁷ To date, it is not clear whether vasodilators can act by elevating the coupling of Ca²⁺ sparks to K_Ca channels, but this mechanism seems to be a reasonable possibility. Furthermore, it is unknown whether Ca²⁺ sparks are important for the vasodilatory actions of CO, but because K_Ca channels appear to be required, this mechanism also deserves investigation.

The goal of the present study was to investigate potential mechanisms of CO-induced dilation using cerebral arterioles and arteriole smooth muscle cells of newborn pigs. Authentic CO was applied exogenously, or CO was elevated endogenously in smooth muscle cells with heme-L-lysinate, a heme oxygenase substrate. Exogenous or endogenous CO was a potent activator of single K_Ca channels. Although CO effectively elevated transient K_Ca current frequency and amplitude, CO only slightly increased Ca²⁺ spark frequency. To investigate this apparent discrepancy between potent activation of transient K_Ca currents and relatively poor activation of Ca²⁺ sparks, the effect of CO on the coupling of Ca²⁺ sparks to K_Ca channels was investigated. CO increased the effective coupling of Ca²⁺ sparks to K_Ca channels from 62% to 100% and elevated the slope of the amplitude correlation between Ca²⁺ sparks and transient K_Ca currents 2.6-fold. This dilatory signaling mechanism is physiologically relevant because ryanodine, an RyR blocker that inhibits Ca²⁺ sparks, abolished CO dilation in vivo.

	Materials and Methods

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Tissue Preparation
Anesthetized newborn pigs (1 to 3 days old, 1 to 2.5 kg) were maintained on -chloralose (50 mg/kg IV), and closed cranial windows were implanted over the left parietal cortices as previously described.^7,18 For removal of pial arterioles (50 to 200 µm), the brain was removed. Isolated arterioles were dissected from the brain, cleaned of basolateral connective tissue, and maintained in ice-cold HEPES-buffered PSS containing (in mmol/L) NaCl 134, KCl 6, CaCl₂ 2, MgCl₂ 1, HEPES 10, and glucose 10 (pH 7.4, NaOH). Smooth muscle cells were enzymatically dissociated from cerebral arterioles as previously described.¹³

Patch-Clamp Electrophysiology
K⁺ currents were measured in isolated smooth muscle cells by use of the perforated-patch configuration of the patch-clamp technique as previously described.²² The bathing solution was HEPES-buffered PSS (composition described above). The pipette solution contained (in mmol/L) potassium aspartate 110, KCl 30, NaCl 10, MgCl₂ 1, HEPES 10, and EGTA 0.05 (pH 7.2, KOH). In experiments in which the activities of single K_Ca channel currents were measured, Ca²⁺ sparks (and thus, transient K_Ca currents) were abolished with thapsigargin, a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor that depletes the sarcoplasmic reticulum of Ca²⁺.^11,13 In each patch under each condition, K_Ca channel activity (NP_o) was calculated from at least 60 seconds of continuous gap-free data by using Fetchan 6 (Axon Instruments). NP_o was calculated from the following equation: NP_o=(t₁+t₂. . .t_i), where t_i is the relative open time for each channel level. In the present study, the average number of events used for single NP_o analysis under each condition was as follows (mean±SEM): control, 896±169 (n=28 cells); CO, 3514±728 (n=7); heme-L-lysinate, 4980±11 (n=5); FeCl₃, 522±266 (n=5); bilirubin, 582±214 (n=5); and NADP, 586±192 (n=6). Transient K_Ca current analysis was performed offline as previously described.²² A transient K_Ca current was defined as the simultaneous opening of 3 K_Ca channels (threshold, 6 pA at -40 mV). Simultaneous openings of 3 K_Ca channels were not observed in the presence of thapsigargin (100 nmol/L, n=4 cells).

Confocal Ca²⁺ Imaging
Arteriole segments or isolated smooth muscle cells were loaded with the fluorescent Ca²⁺ indicator fluo 4-AM (10 µmol/L) as previously described.²² Arteriole segments were imaged in an extracellular solution containing 30 mmol/L K⁺, which depolarizes smooth muscle cells from -60 to -40 mV and elevates global intracellular Ca²⁺ concentration and Ca²⁺ spark frequency (see similar procedures^13,19). The 30 mmol/L K⁺ bath solution contained (in mmol/L) NaCl 110, KCl 30, HEPES 10, CaCl₂ 2, MgCl₂ 1, and glucose 10 (pH 7.4, NaOH). Smooth muscle cells were imaged by using a Noran Oz laser scanning confocal microscope as previously described.²² Fluorescent images of smooth muscle cells in arteriole segments were recorded every 16.7 ms (60 images/s). For isolated smooth muscle cells, images were acquired every 8.3 ms (120 images/s). In experiments in which confocal Ca²⁺ imaging was performed in combination with patch-clamp electrophysiology, current and fluorescence measurements were synchronized by using a light-emitting diode positioned above the recording chamber that was triggered during acquisition. Ca²⁺ sparks were detected in smooth muscle cells by using custom analysis software that was written with the use of IDL 5.0.2 (Research Systems Inc), kindly provided by Drs M.T. Nelson and A.D. Bonev (University of Vermont, Burlington). Detection of Ca²⁺ sparks was performed by dividing an area 1.54 µm (7 pixels)x1.54 µm (7 pixels) (ie, 2.37 µm²) in each image (F) by a baseline (F₀), which was determined by averaging 10 images without Ca²⁺ spark activity. Ca²⁺ spark amplitude was calculated as F/F₀. A Ca²⁺ spark was defined as a localized increase in F/F₀ that was >1.2. Calculations of changes in local (within the 2.37-µm² analysis area) or global Ca²⁺ concentration were performed by using methods previously described.¹⁹

In Vivo Arterial Diameter Measurement
Cranial windows were implanted as previously described.^7,20 The space under the window was filled with artificial cerebrospinal fluid, which was equilibrated with 6% CO₂/6% O₂/88% N₂, producing pH 7.33 to 7.40, PCO₂ 42 to 46 mm Hg, and PO₂ 43 to 50 mm Hg. Pial arterioles were observed with a dissection microscope, and diameter was measured with a calibrated video micrometer attached to a monitor. Artificial cerebrospinal fluid was exchanged topically to introduce treatments to the periarachnoid space.

Statistical Analysis
Values are expressed as mean±SEM. Differences among multiple groups were evaluated by ANOVA with the Tukey test. For comparison between two groups, paired and unpaired Student t tests were used as appropriate. First-order polynomial linear fits were used to calculate statistical correlation for Ca²⁺ spark and evoked transient K_Ca current amplitudes. A value of P<0.05 was considered significant and is illustrated as an asterisk where appropriate.

An expanded Materials and Methods section is available online at http://www.circresaha.org in the data supplement.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CO Activates K_Ca Channels
CO is generated endogenously from heme by the action of heme oxygenase.¹ To investigate whether exogenously applied CO or CO produced endogenously by heme oxygenase activates K_Ca channels in newborn cerebral arteriole smooth muscle cells, NP_o was measured in isolated arteriole smooth muscle cells by using the perforated-patch configuration of the patch-clamp technique.

Openings of large-conductance K_Ca channels were identified on the basis of the characteristic single-channel conductance (116±8 pS between -20 and 20 mV, n=6) and block by tetraethylammonium⁺ (1 mmol/L) (NP_o, control 0.025±0.016, tetraethylammonium⁺ 0.004±0.002; n=3). At 0 mV, authentic CO (100 nmol/L) increased mean NP_o 5.1-fold (Figure 1). Similarly, the heme oxygenase substrate, heme-L-lysinate (100 nmol/L), increased mean NP_o 6.9-fold (Figure 1). After administration, CO rapidly increased NP_o to a peak level, whereas heme-L-lysinate increased NP_o gradually to reach a plateau after 3 to 4 minutes, consistent with endogenous generation of an intracellular K_Ca channel activator via the enzymatic action of heme oxygenase. Additional byproducts of heme degradation that are produced in equimolar concentration, namely, FeCl₃ (100 nmol/L), bilirubin (100 nmol/L), and NADP (100 nmol/L), did not alter NP_o, suggesting that CO was the K_Ca channel activator generated endogenously (Figure 1). These data suggest that exogenous CO or CO produced endogenously by heme oxygenase is a potent activator of K_Ca channels in cerebral arteriole smooth muscle cells.

View larger version (23K): [in this window] [in a new window]   Figure 1. Exogenous or endogenous CO activates KCa channels in cerebral arteriole smooth muscle cells. A, Original recordings of single KCa channel currents in an isolated piglet cerebral arteriole smooth muscle cell at 0 mV before and after exogenous CO (100 nmol/L). C represents closed channel level, ie, zero current. B, Average effects of CO, heme-L-lysinate (HLL), FeCl3, bilirubin, or NADP (each at 100 nmol/L) on NPo. Mean NPo values were as follows: for CO studies, control 0.015±0.007, CO 0.071±0.032 (n=7); for HLL studies, control 0.024±0.010, HLL 0.154±0.049 (n=5); for FeCl3 studies, control 0.005±0.001, FeCl3 0.005±0.001 (n=5); for bilirubin studies, control 0.006±0.005, bilirubin 0.007±0.006 (n=5); and for NADP studies, control 0.004±0.003, NADP 0.003±0.002 (n=6).

CO Elevates Transient K_Ca Current Frequency and Amplitude
In smooth muscle cells, Ca²⁺ sparks activate several K_Ca channels, resulting in a transient K_Ca current.¹⁰ Inhibition of Ca²⁺ sparks or K_Ca channels leads to membrane depolarization, activation of voltage-dependent Ca²⁺ channels, elevated Ca²⁺ influx, and constriction.¹⁰ Conceivably, CO could dilate newborn cerebral arterioles via the activation of transient K_Ca currents. To examine the mechanisms of CO dilation, the regulation of transient K_Ca currents by exogenous CO or CO produced endogenously from heme-L-lysinate was investigated in isolated voltage-clamped (-40 mV) cerebral arteriole smooth muscle cells.

CO (100 nmol/L) increased mean transient K_Ca current frequency 2.4-fold and mean amplitude 1.6-fold (Figure 2). Heme-L-lysinate (100 nmol/L) increased mean transient K_Ca current frequency 2.9-fold and mean amplitude 1.9-fold (Figure 2). Similar to its effects on K_Ca channels, CO induced a rapid activation of transient K_Ca currents, whereas heme-L-lysinate gradually increased transient K_Ca current frequency and amplitude, consistent with endogenous generation of CO via heme oxygenase (Figure 2). These data suggest that exogenous CO or endogenous CO produced by cellular heme oxygenase activates transient K_Ca currents in cerebral arteriole smooth muscle cells.

View larger version (38K): [in this window] [in a new window]   Figure 2. CO (A) or HLL (B) elevates transient KCa current frequency and amplitude in cerebral arteriole smooth muscle cells voltage-clamped at -40 mV. C, Average effects of CO or HLL on transient K+ current frequency and amplitude. Mean frequency and amplitude of transient KCa currents were as follows: for CO (100 nmol/L) studies, control 0.13±0.017 Hz and 16.0±2.1 pA, CO 0.30±0.05 Hz and 21.6±3.4 pA (n=10); for HLL (100 nmol/L) studies, control 0.20±0.04 Hz and 12.6±1.4 pA, HLL 0.49±0.09 Hz and 22.6±2.7 pA (n=13).

Heme-L-Lysinate Activates Ca²⁺ Sparks
CO could increase transient K_Ca currents via the activation of Ca²⁺ sparks. To investigate this hypothesis, rapid changes in intracellular Ca²⁺ concentration were measured in the smooth muscle cells of cerebral arteriole segments by using a laser scanning confocal microscope and the fluorescent Ca²⁺ indicator fluo 4.

Heme-L-lysinate slightly increased mean Ca²⁺ spark frequency in the smooth muscle cells of cerebral arteriole segments from 0.7±0.08 to 0.99±0.11 Hz, or 1.4-fold (Figure 3). However, heme-L-lysinate did not significantly alter mean Ca²⁺ spark amplitude (for F/F₀, control 1.44±0.01 [n=188 sparks], heme-L-lysinate 1.46±0.01 [n=244 sparks]). Heme-L-lysinate reduced global F/F₀ in the same smooth muscle cells to 83±4% of control, suggesting that intracellular Ca²⁺ concentration decreased. Ryanodine (10 µmol/L), an RyR channel blocker, abolished Ca²⁺ sparks in smooth muscle cells (data not shown), suggesting that Ca²⁺ sparks occurred due to the opening of RyR channels, consistent with previous reports.¹⁰ These data suggest that CO elevates Ca²⁺ spark frequency and decreases global Ca²⁺ in arteriole smooth muscle cells.

View larger version (41K): [in this window] [in a new window]   Figure 3. HLL elevates Ca2+ spark frequency in smooth muscle cells of intact cerebral arterioles. A, Average fluorescence (100 of 600 images) over 10 seconds of two different 56.3-µmx52.8-µm areas of the same cerebral arteriole in control (30 mmol/L K+) and in HLL (100 nmol/L) conditions. The locations of Ca2+ sparks that occurred during 10 seconds are indicated by white boxes (1.54 µmx1.54 µm), and representative localized F/F0 changes with time are illustrated below respective images and labeled accordingly. B, Average effects of HLL on Ca2+ spark frequency (n=14 arteries).

CO Augments the Coupling Ratio and Amplitude Correlation Between Ca²⁺ Sparks and Transient K_Ca Currents
Heme-L-lysinate increased Ca²⁺ spark frequency only 1.4-fold in smooth muscle cells (Figure 3), which was considerably less than the 2- to 3-fold increase in transient K_Ca current frequency induced by heme-L-lysinate or exogenous CO (Figure 2). Furthermore, CO or heme-L-lysinate increased transient K_Ca current amplitude (1.6- to 1.9-fold), but heme-L-lysinate did not elevate Ca²⁺ spark amplitude (F/F₀). To investigate the apparent discrepancies between the effective activation of transient K_Ca currents and weak activation of Ca²⁺ sparks, regulation of the coupling relationship between Ca²⁺ sparks and transient K_Ca currents by CO was investigated in voltage-clamped (-40 mV) cerebral arteriole smooth muscle cells.

In the control condition, only 62% of Ca²⁺ sparks evoked a transient K_Ca current (Figures 4 and 5). The mean amplitude of uncoupled Ca²⁺ sparks (F/F₀ 1.68±0.11) was significantly smaller than the mean amplitude of coupled sparks (F/F₀ 2.0±0.07, P<0.05). Although the amplitude relationship between a coupled Ca²⁺ spark and the evoked transient K_Ca current was correlated, the slope was low (Figure 4). In the same cells, heme-L-lysinate (100 nmol/L) increased the percentage of Ca²⁺ sparks that evoked a transient K_Ca current (ie, the coupling ratio) to 100±0%. Mean Ca²⁺ spark amplitude was not altered by heme-L-lysinate (F/F₀, control 1.88±0.06 [n=52], heme-L-lysinate 1.80±0.05 [n=73]), suggesting that the increase in coupling was not due to a change in Ca²⁺ spark properties. Heme-L-lysinate also did not significantly alter global F/F₀ (105±6% of control, P<0.05). In the presence of heme-L-lysinate, the amplitude of a Ca²⁺ spark and that of the evoked transient K_Ca current were similarly correlated. However, heme-L-lysinate increased the slope of the amplitude correlation 2.6-fold, indicating that Ca²⁺ sparks of similar amplitude evoke significantly larger transient K_Ca currents after endogenous generation of CO. This finding explains the CO-induced increase in transient K_Ca current amplitude in the absence of a change in Ca²⁺ spark amplitude. These data suggest that heme-L-lysinate increases transient K_Ca current frequency and amplitude in newborn cerebral arteriole smooth muscle cells primarily by elevating the coupling ratio and amplitude relationship between Ca²⁺ sparks and transient K_Ca currents. The increase in effective coupling of Ca²⁺ sparks (>1.6-fold) contributes significantly to the increase in transient K_Ca current frequency (2- to 3-fold), particularly when in combination with an increase in spark frequency.

View larger version (17K): [in this window] [in a new window]   Figure 4. CO elevates the effective coupling of Ca2+ sparks to KCa channels in cerebral arteriole smooth muscle cells. A, Original simultaneous recordings of Ca2+ sparks that occurred in three different locations (red, green, and purple lines) and whole-cell current (black line) in the same voltage-clamped (-40 mV) smooth muscle cell in control condition and in the presence of HLL (100 nmol/L). Also illustrated below traces is the cell from which these data were obtained and the time course of the Ca2+ spark (illustrated by an asterisk). Colored boxes indicate the locations of the 1.54-µmx1.54-µm areas from which respective colored F/F0 traces were obtained. The vertical colored bar illustrates the F/F0 scale of the cell images. B, Scatterplot of Ca2+ spark and evoked transient KCa current amplitude in control condition (black) and in the presence of HLL (red, n=7 cells). In the control condition, the amplitude relationship between Ca2+ sparks and evoked transient KCa currents was significantly correlated (r=0.42, P<0.05), with a slope of 13.8±1.5. HLL increased the slope of the amplitude correlation to 35.2±2.4 (r=0.41, P<0.05).

View larger version (13K): [in this window] [in a new window]   Figure 5. A, In the control condition, the mean amplitude for uncoupled Ca2+ sparks (n=18) is smaller than that for coupled sparks (n=34). B, HLL elevates the percentage of Ca2+ sparks that elicit a transient KCa current.

Ryanodine, an RyR Channel Blocker, Abolishes CO Dilation of Cerebral Arterioles In Vivo
Dilations to CO are prevented by K_Ca channel blockers.^4–7 To investigate the importance of Ca²⁺ sparks for CO dilation, arteriole diameter was measured in vivo by using cranial windows implanted in anesthetized piglets. Dilations to exogenous CO were measured before and after the administration of ryanodine, an RyR channel blocker that blocked Ca²⁺ sparks (also see Jaggar et al¹⁰). Dilations to sodium nitroprusside, an NO donor, or isoproterenol, an adrenergic agonist, were also studied in the same arterioles before and after ryanodine administration.

CO (1 nmol/L), isoproterenol (1 µmol/L), or sodium nitroprusside (10 µmol/L) significantly dilated pial arterioles (Figure 6). Ryanodine (10 µmol/L) completely abolished dilations of the same arterioles to CO but only slightly attenuated dilations induced by isoproterenol or sodium nitroprusside. These data suggest that Ca²⁺ sparks are required for CO to induce arterial dilation in the newborn pig cerebral circulation and that isoproterenol and sodium nitroprusside cause dilation primarily via other mechanisms.

View larger version (21K): [in this window] [in a new window]   Figure 6. Ryanodine abolishes CO dilation of cerebral arterioles in vivo, whereas dilations to isoproterenol (ISO) or sodium nitroprusside (SNP) are only partially attenuated (n=7).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We show that CO dilates cerebral arterioles primarily by augmenting the effective coupling of Ca²⁺ sparks to K_Ca channels in smooth muscle cells. This leads to an increase in transient K_Ca current frequency and amplitude. A CO-induced increase in transient K_Ca current frequency and amplitude will lead to membrane hyperpolarization, a decrease in voltage-dependent Ca²⁺ channel activity, a reduction in intracellular Ca²⁺ concentration, and arteriole dilation. This pathway is physiologically relevant and dependent on Ca²⁺ sparks, because ryanodine, an RyR channel blocker, abolishes CO dilation of cerebral arterioles in vivo. This is the first time that a vasodilator has been reported to act by increasing the effective coupling between Ca²⁺ sparks and transient K_Ca currents. This finding is particularly relevant when the lack of knowledge regarding the vasodilatory actions of CO is considered.

CO Augments K_Ca Channel Activation by Ca²⁺ Sparks in Cerebral Arteriole Smooth Muscle Cells
Exogenous CO or heme-L-lysinate activated K_Ca channels, transient K_Ca currents, and Ca²⁺ sparks in cerebral arteriole smooth muscle cells. Heme oxygenase-2 is constitutively expressed in arterial smooth muscle cells,² and isolated arteries generate CO under basal conditions and in response to heme oxygenase substrate,¹⁸ suggesting that heme-L-lysinate activation of K_Ca channels is via CO. CO activation of K_Ca channels and transient K_Ca currents peaked almost immediately, whereas heme-L-lysinate activation took 3 to 4 minutes to reach a peak level, consistent with the generation of an endogenous secondary mediator. Furthermore, in control experiments, other products of heme degradation (Fe²⁺, bilirubin, and NADP) had no effect on K_Ca channels. At the same concentration, heme-L-lysinate was a more effective activator of K_Ca channels and transient K_Ca currents than was authentic CO, suggesting that heme oxygenase (and thus, CO production) may be localized near K_Ca channels.

Heme-L-lysinate decreased global Ca²⁺ concentration in smooth muscle cells of intact arteries to 83% of control, or from 195²¹ to 150 nmol/L, which would lead to dilation. The calculated peak elevation in local Ca²⁺ concentration caused by a Ca²⁺ spark would also decrease from 358 nmol/L in control to 265 nmol/L with heme-L-lysinate. This calculation suggests that that the micromolar Ca²⁺ concentration to which K_Ca channels located in the vicinity of the release site are exposed¹² should also be reduced. Membrane hyperpolarization in the intact arteriole would also decrease K_Ca channel Ca²⁺ sensitivity and the driving force for K⁺, which would also reduce the impact of Ca²⁺ sparks. However, heme-L-lysinate increased the slope of the amplitude relationship between Ca²⁺ sparks and transient K_Ca currents 2.6-fold. Thus, the decrease in spark amplitude, K_Ca channel Ca²⁺ sensitivity, and driving force for K⁺ that would occur in the intact arteriole because of membrane hyperpolarization would be compensated by an increase in the sensitivity of K_Ca channels to Ca²⁺ sparks.

CO or heme-L-lysinate increased transient K_Ca current frequency 2- to 3-fold in voltage-clamped cells, whereas heme-L-lysinate increased Ca²⁺ spark frequency only 1.4-fold in intact arteries. Because Ca²⁺ sparks are regulated by intracellular Ca²⁺ concentration,¹⁰ the concomitant decrease in global Ca²⁺ concentration induced by heme-L-lysinate would be expected to attenuate the elevation in Ca²⁺ spark frequency. However, in voltage-clamped cells in which global Ca²⁺ did not change, heme-L-lysinate would increase transient K_Ca current frequency only 1.5-fold if the coupling ratio of 60% was maintained, ie, (0.49 Hzx0.6)/0.2 Hz. Therefore, the majority of the heme-L-lysinate–induced elevation in transient K_Ca current frequency (2.9-fold) occurs because of an increase in the effective coupling of Ca²⁺ sparks to K_Ca channels rather than because of an increase in spark frequency.

Ryanodine, an RyR channel blocker, abolished CO-induced pial arteriole dilation, suggesting that Ca²⁺ sparks are critical for vasoregulatory actions of CO. CO increased NP_o 5- to 6-fold in the absence of Ca²⁺ sparks, but clearly, this was insufficient to induce dilation. Interestingly, in control conditions, ryanodine induced only a small arterial constriction, in contrast to effects in pressurized adult rat cerebral arteries.¹¹ The lack of effect of ryanodine is not surprising because transient K_Ca current frequency and amplitude (and thus, NP_o) were low in newborn piglet cerebral arteriole smooth muscle cells in the absence of CO. Because CO elevates the coupling ratio and amplitude relationship between Ca²⁺ sparks and K_Ca channels, and this ultimately leads to dilation, these data support the physiological role of Ca²⁺ sparks and K_Ca channels in negative-feedback regulation of arterial diameter with the use of an in vivo preparation.

Essentially 100% of Ca²⁺ sparks evoke a transient K_Ca current in cerebral artery smooth muscle cells of adult rats^22,23 and mice.¹⁵ Similar to the porcine preparation used in the present study, a significant proportion of Ca²⁺ sparks does not evoke a transient K_Ca current in human cerebral artery (28%)²⁴, esophageal (27%),²⁵ or Bufo marinus stomach (21%)²⁶ smooth muscle cells. In those tissues, the slope of the amplitude relationship between a Ca²⁺ spark and the evoked K_Ca transient is also low, similar to the slope obtained in the absence of CO in the present study. Thus, CO may be an effective relaxing factor in other smooth muscle preparations by increasing spark–K_Ca channel coupling. Conceivably, membrane depolarization, which also increases K_Ca channel Ca²⁺ sensitivity, may be an additional mechanism to increase the coupling of Ca²⁺ sparks to K_Ca channels. If this is the case, membrane depolarization may somewhat reduce the dilator efficacy of CO.

Signal Transduction Pathways for CO in Arteriole Smooth Muscle Cells
The intracellular signaling pathways affected by CO are unclear. CO may activate K_Ca channels via direct effects,⁸ via activation of soluble guanylyl cyclase,²⁷ via an associated membrane heme protein that acts as an oxygen sensor,²⁸ or via inhibition of formation of 20-hydroxyeicosatetraenoic acid, a K_Ca channel blocker.²⁹ Conceivably, CO could activate Ca²⁺ sparks via similar signaling mechanisms, although this would require further investigation. A major issue concerning signaling mechanisms for CO is whether the gas activates soluble guanylyl cyclase, which would increase cGMP and activate cGMP-dependent protein kinase. CO can activate soluble guanylyl cyclase but is only 1% as effective as NO. A saturated solution of CO (1 mmol/L) increases the activity of human soluble guanylyl cyclase only 4-fold.³⁰ In the present study, the highest concentration of CO used was 10 000 times lower than this concentration, suggesting that guanylyl cyclase activation would be extremely small. For comparison, sodium nitroprusside at micromolar concentrations elevates soluble guanylyl cyclase activity 300-fold, with an EC₅₀ of 2 µmol/L.³⁰ Further support for an insignificant role for guanylyl cyclase comes from previous studies demonstrating that CO or heme-L-lysinate does not increase cGMP in cranial window perfusate at concentrations that dilate cerebral arterioles.⁷ In contrast, in the same study, sodium nitroprusside induced a similar dilation and significantly elevated cGMP.

There are significant differences in the cellular signaling mechanisms for CO when they are compared with the actions of vasodilators that elevate cyclic nucleotides. Forskolin, which elevates cAMP, or sodium nitroprusside, which elevates cGMP, increases K_Ca current primarily by elevating Ca²⁺ spark frequency, combined with a small activation of K_Ca channels.¹⁷ In contrast, CO appears to increase K_Ca current primarily via effects on K_Ca channels, with a small effect on Ca²⁺ sparks. When compared with agents that increase cyclic nucleotides, CO was a far more effective K_Ca channel activator (5- to 6-fold [present study] versus 1.3-fold¹⁷) and a weak activator of Ca²⁺ sparks (1.4-fold [present study] versus 2- to 3-fold¹⁷). Functional evidence also supports the hypothesis that CO acts via a mechanism distinct from dilators that elevate cyclic nucleotides. In piglet arterioles, ryanodine abolished dilations to CO but only slightly reduced dilations to isoproterenol, an agonist that elevates cAMP, or sodium nitroprusside, an NO donor. Cellular targets for cyclic nucleotides may also be slightly different in smooth muscle cells of the newborn vasculature, particularly because the contribution of NO to arterial diameter regulation appears to be far less in the newborn compared with the adult vasculature.³¹

K_Ca channels can be composed of a pore-forming subunit and an associated ß subunit that increases intracellular Ca²⁺ sensitivity.³² Genetic ablation of the associated ß₁ subunit of the K_Ca channel significantly reduces the coupling of Ca²⁺ sparks to K_Ca channels, leading to membrane depolarization, enhanced pressure-induced constriction of cerebral arteries, and increased blood pressure.^15,16 Mechanisms that lead to the uncoupling of Ca²⁺ sparks from K_Ca channels in wild-type smooth muscle cells are unclear but may also involve a reduced Ca²⁺ sensitivity of K_Ca channels within the vicinity of the Ca²⁺ release site. Because some, but not all, Ca²⁺ sparks induce a transient K_Ca current, a heterogeneous population of K_Ca channels may exist in the sarcolemma: those that are composed of only an subunit and those that colocalize with a ß subunit. CO has been proposed to increase K_Ca channel Ca²⁺ sensitivity in cultured arterial smooth muscle cells.⁸ Our data suggest that CO increases Ca²⁺ spark coupling by shifting the Ca²⁺ sensitivity of the K_Ca channel to within the micromolar range of intracellular Ca²⁺ concentrations produced by a Ca²⁺ spark.¹² Conceivably, CO may act via the K_Ca channel ß subunit to enhance this coupling relationship.

In summary, we demonstrate that exogenous or endogenously produced CO dilates cerebral arterioles by activating K_Ca channels primarily by increasing the coupling ratio and amplitude relationship between Ca²⁺ sparks and K_Ca channels. Although CO was a potent and effective activator of K_Ca channels, CO did not dilate arterioles in the absence of Ca²⁺ sparks. Therefore, CO appears to act by priming K_Ca channels for activation by Ca²⁺ sparks, and this ultimately leads to arteriole dilation via membrane hyperpolarization.

	Acknowledgments

 
This study was supported by NIH grants NS-67061 (to Dr Jaggar), HL-042851 (to Dr Leffler), and HL-034059 (to Drs Leffler and Jaggar) and grants from the American Heart Association National Center and Southeast Affiliate (to Dr Jaggar). X. Cheng is a Predoctoral Fellow of the American Heart Association. We thank Liliya Balabanova for technical help with preliminary experiments and Alex Fedinec for in vivo measurements of arterial diameter.

Received July 10, 2002; revision received August 30, 2002; accepted August 30, 2002.

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