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(Circulation Research. 1995;76:53-63.)
© 1995 American Heart Association, Inc.

Articles

ATP-Sensitive K⁺ Channels Mediate _2D-Adrenergic Receptor Contraction of Arteriolar Smooth Muscle and Reversal of Contraction by Hypoxia

Jun Tateishi, James E. Faber

From the Department of Physiology, University of North Carolina, Chapel Hill.

	Abstract

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Abstract Evidence in rat skeletal muscle suggests that local metabolic control of blood flow is facilitated by the reliance on _2D-adrenergic receptors (ARs) for constriction of arterioles, together with the strong sensitivity of this constriction to inhibition by hypoxia. The present study examined the role of ATP-sensitive K⁺ (K_ATP) channels in the selective interaction between _2D-ARs and hypoxia. Arterioles from rat cremaster muscle that possess both _1D (_1A/D)- and _2D-AR subtypes were microcannulated, pressurized, and isolated in a tissue bath for measurement of changes in lumen diameter. Three studies first examined whether stimulation of _2D- and _1D-ARs involves inhibition of the K_ATP channel. Concentration-dependent constriction by the K_ATP antagonists glibenclamide (GLB, 0.01 to 10 µmol/L) and disopyramide (0.001 to 1 mmol/L) were abolished during _2D stimulation but unaffected during _1D stimulation. Activation of the K_ATP channel by cromakalim inhibited _2D constriction with greater potency than _1D (EC₅₀, 7.0±0.2 versus 6.3±0.1). Finally, GLB (0.5 µmol/L) abolished dose-dependent _2D constriction, whereas _1D was unaffected. These data suggest that _2D but not _1D stimulation is "coupled" with closure of the K_ATP channel, leading to depolarization and contraction of vascular smooth muscle. In a second series, hypoxic (PO₂, 6 mm Hg) inhibition of intrinsic smooth muscle tone was completely reversed by 0.1 µmol/L GLB, concentration-dependent GLB constriction was enhanced during hypoxia, and hypoxia reversed GLB constriction. These data confirm reports by others that hypoxia potentiates the activation of K_ATP channels, leading to hyperpolarization and relaxation. Finally, GLB constriction, which was abolished by concomitant _2D stimulation, was completely restored by simultaneous activation of K_ATP channels with hypoxia. These findings suggest that the sensitivity of _2D-AR constriction to inhibition by hypoxia arises through "antagonistic coupling" between these two stimuli, by which the _2D-AR inhibits and hypoxia activates K_ATP channels.

Key Words: -adrenergic receptor • vascular smooth muscle • microcirculation • receptor coupling • hypoxia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Metabolic regulation of blood flow is the major mechanism whereby oxygen delivery to tissues is adjusted to meet tissue metabolic demands. This is accomplished by changes in resistance of small arteries and arterioles of the microcirculation (resistance vessels) in response to increases in vasodilator tissue metabolites (eg, H⁺ and adenosine) and reductions in oxygen itself in the perivascular environment. Many aspects of how smooth muscle cells integrate these local signals with other neural, myogenic, humoral, and endothelial determinants of vascular tone remain unclear. The prevailing smooth muscle tone in resistance vessels of many vascular beds has a substantial dependence on -adrenergic receptor (AR)–mediated contraction, which is subject to inhibition by local metabolites and reduced oxygen. Recently, we have proposed that the type of -AR expressed by smooth muscle cells may be important for this neural-metabolic integration.^{1 2 3}

In general, constriction of most large arteries is mediated by ₁-ARs, whereas successively smaller resistance vessels become increasingly dependent on ₂-ARs (References 1 through 4^{1 2 3 4} and references therein). Constriction mediated by ₂- but not ₁-ARs in vivo is exquisitely sensitive to inhibition by reduced oxygen delivery, increased tissue metabolism, and reduced pH.^{2 3 4 5} These effects of acidosis and presumed hypoxia are also evident in isolated arterioles free of parenchymal cell influences, where ₂ but not ₁ constriction is inhibited by hypoxia and acidosis over the physiological range (IC₅₀, 24 mm Hg [PO₂] and 7.1 [pH], respectively).⁶ Recently, we pharmacologically determined that the specific -AR subtypes responsible for constriction of resistance vessels in the rat skeletal muscle model used in our studies are the _1D (_1A/D)- and _2D-ARs (_2A/D-ARs).⁷ Thus, the presence of _2D-ARs on resistance vessels may serve to optimize the capacity for metabolic adjustments of adrenergic tone in the control of blood flow. However, the cellular basis for the differential sensitivity of constriction induced by these -AR subtypes to oxygen (and other metabolites) is unknown.

Differences in second-messenger pathways activated by -AR subtypes may be important in conferring this selective sensitivity to inhibition by hypoxia. Although the pathways in smooth muscle cells that couple _1D- and _2D-ARs to Ca²⁺ channels and contractile protein activation have not been fully elucidated, ₁ contraction is generally dependent on release of intracellular Ca²⁺ and influx of extracellular Ca²⁺ primarily via dihydropyridine-insensitive and, to a lesser, variable degree, dihydropyridine-sensitive voltage-operated Ca²⁺ channels (VOCs).⁸ In contrast, ₂-ARs do not release intracellular Ca²⁺ but generally rely largely on influx via VOCs. Recently, we have used indirect approaches involving removal of extracellular Ca²⁺, depletion of intracellular stores, and use of organic VOC antagonists.⁹ Results from this preliminary report suggested that _1D constriction of rat skeletal muscle arterioles is coupled with intracellular Ca²⁺ release and activation of dihydropyridine-insensitive Ca²⁺ channels, whereas _2D constriction activates both VOCs and dihydropyridine-insensitive pathways but not intracellular release. Moreover, hypoxia selectively inhibits _2D constriction by interference with the prominent VOC component of the response.⁹ However, this inhibition does not appear to involve direct interference with Ca²⁺ channel activation by dihydropyridine agonists, depolarization, or _1D-AR stimulation.⁶

It is possible that the mechanism, currently unknown, by which G protein–coupled _2D-ARs activate dihydropyridine-sensitive VOCs is sensitive to inhibition by hypoxia. Recently, hypoxia^{10 11 12 13} has been shown to increase the activity of a specific type of K⁺ channel known to be activated by reduced cellular ATP (the K_ATP channel)^{14 15} and to be present on vascular smooth muscle cells.^{16 17 18 19 20} Evidence indicates that these K_ATP channels, which are selectively blocked by sulfonylureas such as glibenclamide (GLB) and activated by cromakalim and related compounds,^{20 21} may be tonically active.^{12 13 22 23} Moreover, increases and decreases in the activity of these channels result in relaxation and contraction of vascular smooth muscle in association with changes in membrane potential and VOC activity.^{16 17 20 24 25} The purpose of the present study was to test two hypotheses suggested by the above observations: (1) The _2D-AR but not the _1D-AR is coupled with inhibition of the K_ATP channel. (2) This action links _2D but not _1D constriction to inhibition by reductions in blood or tissue oxygen. Isolated arterioles were studied in vitro to eliminate hemodynamic, humoral, and local parenchymal cell influences.

	Materials and Methods

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General Procedures
Arterioles from 67 ten-week-old Sprague-Dawley rats (Dominion Labs, Dublin, Va; mean±SEM body weight, 303±2 g) were used. Techniques for isolation and cannulation of arterioles from cremaster muscle have been described previously.^{6 7 26} Briefly, a 2- to 3-mm length of the first-order arteriole was removed from the tissue in a 4°C dissection bath filled with a solution consisting of (mmol/L) NaCl 145, KCl 4.7, CaCl₂-2H₂O 2.0, MgSO₄-7H₂O 1.2, glucose 5.0, pyruvate 2.0, MOPS 3.0, EDTA 0.02, and NaH₂PO₄ 1.2 (pH 7.4), along with 10 pmol/L angiotensin II, 0.5 µmol/L verapamil, and 1% bovine serum albumin. The vessel was then transferred to a tissue bath mounted on the stage of a microscope and cannulated at both ends with glass micropipettes. The tissue bath contained a modified Krebs' solution composed of (mmol/L) NaCl 118.5, KCl 4.7, CaCl₂-2H₂O 2.55, MgSO₄-7H₂O 1.19, KH₂PO₄ 1.19, NaHCO₃ 19.9, dextrose 11.6, and EDTA 0.03, along with 10 pmol/L angiotensin II and 1 µmol/L propranolol to block ß-adrenergic receptors. The micropipettes and vessel were filled with the same solution plus 1% albumin. Angiotensin at 10 pmol/L is subthreshold for constriction and was included to maintain ₂-AR sensitivity and efficacy at in vivo values.²⁶ The pipettes were connected to reservoirs whose heights could be adjusted to produce changes in lumen pressure, which was measured with a pressure transducer. The "downstream" pipette was closed to eliminate the possibility of flow-related vasoactive stimuli, except during vessel perfusion "wash" periods (see below). Vessel lumen diameter was measured automatically at 15 Hz with a videomicroscopic image analysis system.²⁶ The 5-mL tissue bath was superfused at all times without recirculation at 4 mL/min from a Krebs' solution reservoir that was controlled with standard methods to maintain vessel bath pH at 7.4, PO₂ at 70 mm Hg, PCO₂ at 40 mm Hg, and temperature at 34°C, unless otherwise indicated. Bath and reservoir PO₂, PCO₂, and pH were monitored with polarographic electrodes (Radiometer). All test drugs were continuously infused into the suffusion line (4 to 12 µL/min) for the durations and bath concentrations indicated in the figures.

Preparation stability was assessed as described previously.^{7 26} During a 30-minute equilibration period (Fig 1), vessel length, lumen pressure, and bath temperature were gradually raised to the in vivo values of 70-mm Hg pressure and 34°C. Vessels with leaks or those that failed during a subsequent 30-minute interval to develop intrinsic tone sufficient to decrease their diameter by at least 20% below that measured during complete dilation at 28°C and 70-mm Hg pressure (Fig 1) were not included for analysis, because responses to constrictor stimuli in such vessels are often reduced or absent. Vessels were also excluded if intrinsic tone declined during the experiment. In addition, myogenic reactivity was used to judge viability and stability. After development of intrinsic tone, transmural pressure was increased within 1 second by 15 and 30 mm Hg for 3 to 4 minutes at each step until a stable diameter (ie, constant for at least 1 minute) was obtained (Fig 1). Myogenic reactivity was determined again at the end of the experiments. Vessels that dilated with increased pressure (ie, no response or weak myogenic response) were excluded from analysis. These exclusion criteria are required to ensure normal in vivo _2D reactivity^{6 7 26} and resulted in rejection of 48% of the preparations.

View larger version (31K): [in this window] [in a new window]   Figure 1. Graphs showing that stimulation of 2D-adrenergic receptors (ARs), but not 1D-ARs, abolishes the constrictor effect of blockade of ATP-sensitive K+ channels (KATP channels). KATP channels were antagonized with glibenclamide and disopyramide in the absence of -AR stimulation (control groups), during an intermediate level of stimulation of 1D-ARs with phenylephrine (1 µmol/L) or St-587 (3 µmol/L), or during 2D-AR stimulation with UK-14,304 (10 µmol/L). Rauwolscine (100 nmol/L) was present during 1D experiments, and prazosin (10 nmol/L) was present during 2D experiments to aid selectivity of agonists in these and all subsequent experimental groups and figures. PO2 was normal (70 mm Hg). Top, Results from a representative experiment showing protocol: intrinsic tone developed during equilibration, followed by evaluation of myogenic constriction during 15 and 30 mm Hg increases in transmural pressure, and then recovery (R) on return to baseline pressure (70 mm Hg). After lumen diameter was reduced by an -AR agonist (phenylephrine in the example) and restored (C2) to approximate control level (C1) by titration with nitroprusside (NP), a glibenclamide (or disopyramide) concentration-response curve was obtained (NP was replaced by adenosine in a different group; see "Results"). Exposure to all three agents was then sequentially stopped to determine reversibility. Myogenic reactivity (Myo) was retested, and maximal dilation was induced by 0.1 mmol/L NP. Bottom, Grouped data (mean±SEM) show that constriction produced by closure of KATP channels (glibenclamide or disopyramide), which is evident in control and 1D groups, is abolished during 2D stimulation. *P<.01 vs control for 10 µmol/L glibenclamide by two-tailed Bonferroni t test (disopyramide data not tested; n=2). Number of vessels (one per animal) is given in parentheses here and elsewhere. Baseline data are given in Table 1.

Experimental Protocol
Role of K_ATP Channels in -AR Constriction
Fig 1 shows the protocol used to examine the effect of activation of _1D- versus _2D-ARs on the response to concomitant blockade of K_ATP channels. After verification of intrinsic tone and myogenic reactivity, intermediate constriction was induced with the full ₁ agonist phenylephrine (PE, 1 µmol/L), the partial ₁ agonist St-587 (ST, 3 µmol/L) (both PE and ST plus 100 nmol/L rauwolscine, an ₂ antagonist), or the full ₂ agonist UK-14,304 (UK, 10 µmol/L) (plus 10 nmol/L prazosin, an ₁ antagonist). According to our previous experience^{6 7 26} and experiments herein (Fig 3), these concentrations produce 70% to 80% of the maximal constriction that can be achieved with each agonist. To restore diameter to control (C1, Fig 1) but retain -AR stimulation, -AR constriction was then reversed (C2, Fig 1) by titration with nitroprusside (NP, 0.016 to 0.156 µmol/L) while -AR agonist infusion was maintained. During continuous agonist and NP infusion, a cumulative concentration-response curve was then obtained for the K_ATP antagonist GLB (0.01 to 10 µmol/L). In all concentration-response curves here and below, drug concentrations were increased at 10-minute intervals or after attainment of a steady response (no change in diameter for 2 minutes). GLB, NP, and -AR agonist were then sequentially stopped, and myogenic reactivity was retested after an 20-minute wash interval. Wash periods here and elsewhere consisted of cessation of all drug infusions and perfusion of the vessel lumen by raising and lowering the pipette reservoirs by equivalent amounts to create a 5- to 15-mm Hg pressure drop and lumen flow of 3 to 4 nL/min (measured with an optical flowmeter). At the end of this and all protocols, myogenic reactivity was retested, and maximal diameter was obtained during full relaxation with 0.1 mmol/L NP to determine the level of intrinsic vessel tone during basal conditions. The control group received GLB but no -AR agonist or NP reversal. Other vessels were examined with this protocol by using a different K_ATP antagonist, disopyramide, and also by using adenosine (0.6 to 5 µmol/L) in place of NP to reverse -AR constriction. Prazosin or rauwolscine was present in the suffusion solution in this and subsequent ₂ or ₁ experiments, respectively, to minimize possible activation of the opposite receptor subtype by the selective agonists. The selectivity of the above -AR agonists and antagonists at the indicated concentrations have been determined previously for this vessel, as has maintenance for at least 50 minutes of stable intermediate levels of constriction produced by these agonists and the dilation produced by NP and adenosine^{1 2 3 6 26 27 28} ; verification of stable sustained agonist responses was also determined in this and subsequent protocols (see "Results"). Only one agonist was tested in each vessel.

View larger version (20K): [in this window] [in a new window]   Figure 3. Graph showing that treatment with an intermediate contractile concentration (see Fig 1) of glibenclamide (GLB, 0.5 µmol/L) has no effect on 1D constriction with St-587 (ST) relative to the vehicle-treated group but completely abolishes 2D constriction with UK-14,304 (UK). Mean±SEM values are given for three vessels per group. Vehicle alone (0.1% dimethyl sulfoxide) had no effect on intrinsic tone.

The protocol for a second experiment, which examined the potency of the K_ATP agonist cromakalim for inhibition of _1D- versus _2D-AR constriction, is shown in Fig 2. After establishment of an intermediate amount of constriction by 1 µmol/L PE or 10 µmol/L UK, a cumulative concentration-response curve was obtained for cromakalim. This was followed by sequential cessation of cromakalim and then AR agonist infusions to test for maintenance of, respectively, AR agonist constriction and baseline intrinsic tone. The sequence of AR agonists was randomized and separated by a 20-minute wash interval. Myogenic reactivity and intrinsic tone were determined as described above. A control group for this protocol was done to determine the sensitivity of intrinsic tone to cromakalim in the absence of -AR constriction. The design was identical to the preceding experiment, including the presence of prazosin (10 nmol/L) and rauwolscine (100 nmol/L) during the first versus second cromakalim concentration-response curve (randomized); these antagonists had no effect on intrinsic tone.

View larger version (28K): [in this window] [in a new window]   Figure 2. Graphs showing that 2D-adrenergic receptor (AR) constriction with UK-14,304 is more sensitive than 1D-AR constriction with phenylephrine to inhibition by the ATP-sensitive K+ (KATP) channel agonist cromakalim. Top, Representative graph showing protocol and results. After development of intrinsic tone and myogenic evaluation (see Fig 1 for explanation), an intermediate amount of 2D constriction was induced with UK-14,304, followed by dilation with cromakalim. Restoration of UK-14,304 constriction and then control diameter occurred after cessation of cromakalim and UK-14,304, respectively. The sequence was repeated during an intermediate amount of 1D constriction with phenylephrine, followed by myogenic (Myo) retest and maximal dilation with 0.1 mmol/L nitroprusside (NP). Rauwolscine was present during phenylephrine, and prazosin was present during UK-14,304 stimulation. PO2 was normal (70 mm Hg). Bottom, Mean±SEM data normalized to maximal dilation with 0.1 mmol/L NP. Compared with 1D constriction, 2D constriction was sevenfold more sensitive to inhibition by cromakalim (P<.01 by two-tailed paired t test).

A third experiment examined the effect of inhibition of K_ATP with an intermediate concentration of GLB on _1D versus _2D contractile sensitivity. After determining myogenic reactivity, the vehicle for GLB (0.1% dimethyl sulfoxide [DMSO], final bath concentration) was begun. During continuous vehicle infusion, which had no effect on intrinsic tone (diameter after 10 minutes of vehicle exposure was 97±2% of control), a cumulative concentration-response curve was generated with ST or UK. After a subsequent 20-minute wash period, 0.5 µmol/L GLB infusion was begun. Ten minutes later, a second curve for ST or UK was then obtained in the presence of GLB. A single agonist was examined in each experiment. Because of the resistance to reversal of GLB constriction during washing, which has been reported by others (eg, see References 13, 22, and 23^{13 22 23} ) and which was confirmed in the first set of experiments (Fig 1), GLB was always present during the second concentration-response curve.

Role of K_ATP Channels in Selective Inhibition of _2D-AR Constriction by Hypoxia
Previous studies of several vascular beds, including arterioles from cremaster studied herein,¹³ have suggested that hypoxic vasodilation is mediated by increased K_ATP activity. This hypothesis was evaluated in a fourth group. Gas mixtures of O₂, CO₂, and N₂ were used to reduce PO₂ in the closed tissue bath in steps of 150, 70, 30, and 6 mm Hg at 10-minute intervals, while holding PCO₂ and pH constant at 40 mm Hg and 7.4, respectively. During hypoxic dilation obtained at PO₂ of 6 mm Hg, a cumulative GLB concentration-response curve (0.01 to 10 µmol/L) was obtained to test for reversal of dilation. After a 10-minute wash interval at PO₂ of 6 mm Hg, oxygen was returned to 70 mm Hg (control), followed by myogenic retest and maximal dilation with NP. The ability of GLB to reverse hypoxic dilation and cause constriction was compared with its effect on baseline diameter during control PO₂ in a separate group.

A final experiment (see Fig 5 for protocol) was conducted to examine the central hypothesis: Hypoxia selectively inhibits _2D constriction because the _2D-AR but not _1D-AR is coupled with the closure of K_ATP channels, and reduced oxygen activates K_ATP channels, thereby inhibiting _2D constriction. After induction of an intermediate amount of UK constriction (10 µmol/L), PO₂ was lowered from 70 to 10 mm Hg to reverse the constriction. During combined _2D stimulation and hypoxia, a GLB concentration-response curve was then obtained. This was followed by sequential cessation of GLB, return of PO₂ to 70 mm Hg, and cessation of UK infusion. Sensitivity to GLB was compared with that obtained in a control group subjected to the same protocol but without UK infusion.

View larger version (26K): [in this window] [in a new window]   Figure 5. Graphs showing that hypoxia reverses 2D-adrenergic receptor–mediated inhibition of ATP-sensitive K+ (KATP) channels and restores sensitivity to glibenclamide (GLB). Top, Protocol and results are shown from one experiment. Induction of an intermediate level of 2D constriction with UK-14,304 (UK) was reversed (C2 vs C1) by hypoxia at a level (PO2, 10 mm Hg) that does not inhibit intrinsic tone (Table 2). This restored sensitivity to GLB. Hypoxic reversal of UK constriction was reestablished after GLB washout, UK constriction was restored after return to normoxia, and arteriole returned to control (C1) diameter after cessation of UK. Protocol was bracketed by myogenic evaluation and maximal dilation with 0.1 mmol/L nitroprusside (NP) for determination of intrinsic tone. Bottom, Data from Fig 1 are replotted (control: PO2, 70 mm Hg; UK: PO2, 70 mm Hg). Remaining groups were determined by protocol in top panel. Sensitivity to GLB, which was abolished during 2D constriction (UK: PO2, 70 mm Hg), was fully restored to control levels when 2D stimulation was combined with hypoxia (UK: PO2, 10 mm Hg). *P<.01 vs UK at PO2 of 10 mm Hg by one-tailed Bonferroni t test for 10 µmol/L GLB. Number of vessels is given in parentheses. Baseline data are given in Tables 1 and 2.

Data Analysis and Drugs
Average values are plotted in the figures at 1-minute intervals. Values given in figures and tables for diameters during control periods represent averages of the last 2 minutes during the control period. Myogenic responses represent the maximal steady-state constriction during the last 1-minute interval before a change in intraluminal pressure to a new value. All other responses are the average of diameter during the last 2 minutes of an intervention or change in condition. ST and UK concentration-response data are expressed as a percentage of maximum constriction: constriction=(D_c-D_x)/(D_c-D_mr)x100, where D_c is the control diameter, D_x is the diameter produced by x concentration of agonists, and D_mr is the steady-state diameter reached at the highest concentration of agonists in the absence of antagonists. Cromakalim dilation curves are expressed as a percentage of maximum response: response=(D_c-D_x)/(D_c-D_np)x100, where D_np is the maximum fully dilated diameter achieved with 0.1 mmol/L NP. The -logEC₅₀ values (concentration of agonist that produces 50% of the maximal response) were calculated as a measure of agonist sensitivity and were derived from nonlinear least-squares regression analysis. Data were analyzed with paired and grouped t tests and by ANOVA and the Dunn-Bonferroni procedure when data were compared among more than two groups. Results are expressed as mean±SEM for n vessels (one per animal), with P<.05 representing significance. Stock solutions of PE (Sigma Chemical Co) were prepared in 10^-3 mol/L ascorbate saline. Prazosin and UK (generously donated by Pfizer Pharmaceutical), rauwolscine (Atomergic Chemical Co), St-587 (Boehringer Ingelheim), and adenosine (Sigma) were dissolved in Krebs' solution. Phentolamine (CIBA-GEIGY), propranolol, NP, and disopyramide (Sigma) were dissolved in saline. Angiotensin II (Sigma) was dissolved in H₂O. GLB and cromakalim (Sigma) were dissolved in DMSO (0.1%, final bath concentration). These vehicles have no intrinsic effect. Concentrated stock solutions were stored at -20°C for no more than 6 weeks.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Baseline Data
For all experimental groups, spontaneous (intrinsic) tone developed during the initial equilibration period, resulting in a 35±1% (n=67) decrease in diameter from the fully dilated diameter that was obtained at the end of the experiments in the presence of NP (Tables 1 and 2; Figs 1, 2, 4, and 5). After cessation of the various interventions, vessels returned to baseline diameter (Tables 1 and 2; Figs 1, 2, 4, and 5), indicating preparation stability. Myogenic reactivity also remained constant during the experiments. For example, constrictor responses to elevation in transmural pressure at the beginning versus end of the protocols for the -AR agonist experiments were as follows (in percentage of control diameter): 92±1% versus 93±2% for a 15-mm Hg increase in pressure and 86±1% versus 88±1% for a 30-mm Hg increase (n=36). These values for intrinsic tone and myogenic reactivity agree with our previous studies.^{6 7 26}

View this table: [in this window] [in a new window]   Table 1. Baseline Diameters for Fig 1 Studies

View this table: [in this window] [in a new window]   Table 2. Baseline Diameters for Fig 5 Studies

View larger version (27K): [in this window] [in a new window]   Figure 4. Bar graph (top) and line graph (bottom) showing that hypoxic dilation is mediated by ATP-sensitive K+ (KATP) channel activation. Top, Protocol and mean±SEM responses are summarized for six arterioles. Dilation produced by step reduction of PO2 to 6 mm Hg (C2) was reversed by 0.1 µmol/L glibenclamide (GLB). Hypoxic dilation was restored 10 minutes after cessation of GLB (C3), and baseline tone was reestablished after return to normoxia. Myogenic reactivity was tested at the beginning and end of the protocol (see "Materials and Methods"), and maximal dilation with 0.1 mmol/L nitroprusside (NP) was obtained at the end of the protocol. tP<.01 vs C1; *P<.01 vs C2 (Bonferroni one-tailed paired t tests). Bottom, Concentration-dependent GLB constriction was enhanced by hypoxia when compared with responses during normoxia replotted from Fig 1 experiments. Data are normalized to percentage of control (C2, Figs 1 and 4). *P<.01 for 10 µmol/L GLB.

Role of K_ATP Channels in -AR Constriction
Three experiments were performed to examine the role of K_ATP channels in -AR constriction. The rationale for the first experiment was that if stimulation of either -AR subtype decreases the activity of (closes) K_ATP channels, then the action of a second agent (GLB or disopyramide), whose contraction is known to be mediated by closure of these channels, should be attenuated. GLB caused concentration-dependent constriction of arterioles under control conditions of intrinsic tone (Fig 1, control group). Phentolamine (10 µmol/L) had no effect on this constriction (n=2, not shown), indicating that the constriction was caused by blockade of K_ATP and depolarization of smooth muscle cells rather than nerve endings. Constriction by GLB was also observed in the presence of an intermediate (70% to 80% of maximum) amount of _1D-AR stimulation with PE and ST after their own constrictions were first reversed to restore baseline diameter (and aid comparisons among control and -AR agonist groups) (Fig 1). In contrast, during an intermediate amount of _2D stimulation with UK and reversal of constriction with NP, constriction in response to GLB and disopyramide was completely abolished. Similar results were obtained when adenosine was used to reverse PE and UK constriction. In the presence of adenosine (average concentration, 1.8 µmol/L) for full reversal of PE constriction, GLB (0.01 to 10 µmol/L) concentration-response constrictions decreased diameter to 98%, 93%, 88%, and 84% of control (means of two experiments). In contrast, in the presence of adenosine (average concentration, 4 µmol/L) for full reversal of UK constriction, the same concentration-response values were 98%, 99%, 107%, and 107% of control (means of two experiments).

Control data appear on Table 1. For the experiment shown in Fig 1, there were no significant differences among the four groups in control diameters before agonist administration (control₁ [C1]), in diameters after reversal of constriction with NP (control₂ [C2]), or in maximally dilated diameters with NP at the end of the protocol. This indicates that all groups had comparable intrinsic tone and that vessels were of similar size. The concentrations of PE, ST, and UK reduced diameters (in percentage of control) to 61±3%, 66±2%, and 80±1%, respectively. Based on our previous studies, these amounts of constriction represent 70% to 80% of the maximal response to each agonist, where the maximal response for ₂ agonists is 70% of the maximal response for ₁ agonists.^{1 2 6 8 26} This was the rationale for use of a concentration of UK that produced less absolute constriction than the ₁ agonists. Other data in Table 1 demonstrate (1) that the constrictor responses to GLB had not reversed when examined 10 minutes after cessation of infusion (maximal response of GLB versus after GLB) and (2) that 10 minutes after cessation of NP infusion, agonist constrictions returned to their initial values at the beginning of the protocol, suggesting maintenance of constant -AR sensitivity during this continuous activation protocol. The average concentrations of NP required to reverse PE, ST, and UK constrictions (control₂) were, respectively, as follows (nmol/L): 156±6, 22±6, and 16±5. This 10-fold greater sensitivity to NP of UK than PE is identical to that observed for this same vessel studied in vivo.²⁷ However, ST and UK sensitivities to NP were similar.

In the previous experiment the ability of GLB and disopyramide to close K_ATP channels and induce constriction (presumably due to depolarization) was abolished during _2D- but not _1D-AR stimulation. This suggests that _2D stimulation closes K_ATP. If this conclusion is correct, then an agonist of K_ATP (cromakalim) should exhibit greater inhibitory potency against _2D than _1D constriction. This prediction was supported in a second experiment, where _2D constriction was sevenfold more sensitive than _1D to antagonism by cromakalim (Fig 2). However, it is also possible that PE could modify membrane resistance and alter its sensitivity to cromakalim. Also, the estimation of inhibitory potency of cromakalim is unavoidably complicated, because it also includes the effect of cromakalim to inhibit intrinsic tone, which was, however, present in identical amounts (see below) in the two agonist groups.

Control diameters before _1D (PE) and _2D (UK) constrictions were, respectively, 104±8 and 108±4 µm, and maximal dilation with NP at the end of the protocol gave diameters of 177±6 µm; this maximal dilation was also achieved at cromakalim concentrations in excess of 0.5 µmol/L (Fig 2). PE (1 µmol/L) and UK (10 µmol/L) reduced diameters to 61±3% and 70±2% of control, respectively. After cessation of cromakalim infusion and reversal of its maximal dilation, baseline _1D and _2D constrictions were reestablished (63±3% and 71±2% of control for _1D and _2D groups, respectively), indicating maintenance of constant -AR sensitivity over the duration of the protocol (Fig 2, top). Likewise, cessation of -AR agonist infusions led to restoration of the original control diameters (105±9 and 107±11 µm for _1D and _2D groups, respectively), indicating that intrinsic tone remained constant (Fig 2, top). In control experiments (n=5) consisting of two cromakalim concentration-response curves generated in the absence of -AR agonists, slight sensitization was evidenced for the second curve (-log molar EC₅₀ was 7.3±0.1 versus 7.0±0.1 for the first curve; P<.05). Therefore, all EC₅₀ values obtained for second curves (for which PE and UK were randomized) were corrected by 0.3.

A third experiment served as an additional test of the hypothesis that _2D constriction is selectively coupled with the closure of K_ATP. In each experiment, a concentration-response curve was obtained with either UK or ST, first in the presence of the vehicle for GLB (0.1% DMSO) and then after a 30-minute wash period, in the presence of an intermediate contractile concentration (0.5 µmol/L) of GLB. GLB had no effect on sensitivity to the _1D agonist ST; -log molar EC₅₀ was 6.3±0.1 during vehicle and 6.4±0.2 during GLB (Fig 3). In contrast, _2D constriction with UK during vehicle (-log molar EC₅₀ was 5.5±0.2) was completely abolished in the presence of GLB. Control diameters for the UK and ST groups before the first agonist concentration-response curves were 93±10 and 105±3 µm, respectively, and were unaffected by GLB vehicle (91±12 and 103±4 µm, respectively). Control diameters before the second curves were 105±17 and 117±9 µm for the UK and ST groups, respectively; GLB decreased these diameters to 87±12 and 102±3 µm (83% and 87% of control for both groups). Maximal relaxation of vessels with 0.1 mmol/L NP at the end of the experiment dilated the respective groups to 168±10 and 165±2 µm. The maximal concentrations of UK and ST (both 30 µmol/L) reduced diameters to 67±7 and 55±3 µm, respectively, in the first curves; in the second curves in the presence of GLB, maximal constriction to ST was unaffected (56±4 µm), whereas no constriction occurred with UK (83±13 µm). As evident from the vehicle curves in Fig 3, ST acting at ₁-ARs is known to have a higher potency than UK acting at ₂-ARs. These data, which mirror the results from the complementary first experiment (Fig 1), suggest that prior closure of K_ATP selectively blocks _2D constriction, presumably by preventing additional closure, on which _2D-AR constriction depends.

Role of K_ATP Channels in Selective Inhibition of _2D-AR Constriction by Hypoxia
Before examining the role of K_ATP channels in selective inhibition of _2D-AR constriction by hypoxia, we tested the recent postulate that relaxation of vascular smooth muscle by hypoxia involves K_ATP activation. Hypoxia (PO₂, 6 mm Hg) induced a 36±2% dilation (absolute increase, 37±6 µm), which was completely reversed (absolute decrease, 34±7 µm) by 0.1 µmol/L GLB (Fig 4, top). By comparison, this same GLB concentration reduced baseline diameter under normoxia (70 mm Hg) by only 11±2% (absolute decrease, 13±2 µm) (Fig 4, bottom; control group). Compared with the sensitivity (position and slope of the concentration-response curve) to GLB alone during normoxia, sensitivity to GLB was greatly increased during hypoxia (Fig 4, bottom). Since absolute control (C2) diameter was larger in the hypoxic than in the control group (142±6 versus 109±9 µm, P<.01), the percentage of control changes plotted in Fig 4 actually underestimates the magnitude of the enhanced sensitivity to GLB during hypoxia. These results confirm that hypoxic dilation involves K_ATP activation.

Ten minutes after cessation of 10 µmol/L GLB infusion but during maintained hypoxia, diameter had returned to the same dilated diameter before GLB (Fig 4, C3), and this hypoxic dilation was completely reversible after return to normoxia (Fig 4, C4). This ability of hypoxia to rapidly reverse GLB constriction (confirmed in Fig 5, see below) contrasts with the resistance to reversal observed under normoxia (Fig 1, Table 1). Thus, these reversal data also support an association between hypoxic dilation and K_ATP activation.

A final experiment examined the central hypothesis of the present study, namely, that hypoxia selectively inhibits _2D constriction because this AR subtype is coupled with closure of the K_ATP channel and reduced oxygen activates the K_ATP channel, thereby inhibiting _2D constriction; since the _1D-AR is not coupled with the K_ATP channel, hypoxia does not inhibit its constriction. To examine this hypothesis (Fig 5, top), hypoxia at PO₂ of 10 mm Hg was used to reverse an intermediate level of UK constriction (Table 2, UK1 constriction value is 79% of C1 control diameter; C2 reversal value is 94±2% of C1). This reversal of _2D constriction by PO₂ of 10 mm Hg is in agreement with our previous studies.^{6 9} Also consistent with these reports, this level of hypoxia had no effect on intrinsic tone (baseline diameter) (Table 2, control [PO₂, 10 mm Hg] group, C2 versus C1). In the combined presence of UK stimulation (K_ATP "closed") and hypoxic reversal of UK constriction (K_ATP "reopened"), GLB produced concentration-dependent constriction, presumably because GLB closure of K_ATP was then possible because of the reopening by hypoxia (Fig 5, top; Fig 5, bottom, UK [PO₂, 10 mm Hg] group). This contrasts with the complete abolition of GLB constriction in the UK (PO₂, 70 mm Hg) group data replotted from Fig 1. In that group, closure of K_ATP by UK and the absence of hypoxia to reopen them presumably abolished any additional closure and constriction by GLB. As in Fig 4, GLB maximal constriction was rapidly reversed within 10 minutes of cessation of GLB infusion but with maintained hypoxia and UK infusion (Fig 5, top; Table 2, C3 versus C2). UK constriction was also restored on return to normoxia (Fig 5, top; Table 2, UK2 versus UK1). Finally, intrinsic tone was reestablished after cessation of UK (Fig 5, top; Table 2, C4 versus C1). These control data indicate that UK constriction, hypoxic inhibition of it, and intrinsic tone were sustained in these experiments and validate the experimental design. The results of this experiment support the hypothesis that selective inhibition of _2D constriction by hypoxia is mediated by "antagonistic coupling" of both stimuli to K_ATP channels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present results suggest two novel hypotheses: (1) The _2D-AR but not _1D-AR is coupled with closure (ie, reduced open-state probability) of the K_ATP channel. (2) This action underlies the effect of hypoxia to selectively inhibit _2D constriction. In the cremaster arteriole, as in resistance vessels of several other tissues studied in vivo, K_ATP channels appear to be active under conditions of basal tone.^{12 13 22 23} In these studies, attenuation of K_ATP channels (eg, with GLB) caused constriction, and augmentation (eg, with cromakalim) caused dilation. These actions have been associated with depolarization and hyperpolarization, respectively, of smooth muscle cells.^{16 17 24 25} Three findings in the present study (Figs 1 through 3) suggest that _2D constriction is mediated by closure of the K_ATP channel. First, intermediate _2D-AR stimulation (70% to 80% of maximum) abolished constriction by GLB and disopyramide; in contrast, during a comparable level of _1D stimulation or under basal conditions with no -AR tone, dose-dependent constriction by GLB and disopyramide was fully preserved. Second, _2D constriction was sevenfold more susceptible than _1D to inhibition by cromakalim. Third, closure of K_ATP channels with 0.5 µmol/L GLB eliminated subsequent dose-dependent _2D constriction, whereas _1D was unaffected. These data suggest that prior closure of K_ATP channels by _2D-AR stimulation prevents constriction to agents (eg, GLB) known to act by closing K_ATP channels. By the same reasoning, prior closure of K_ATP channels with GLB selectively abolishes subsequent closure and constriction by _2D. This proposed coupling of the _2D-AR with K_ATP closure and growing evidence that low oxygen opens (ie, augments open-state probability of) these channels^{10 11 12 29} suggested a mechanism for the effect of hypoxia to selectively inhibit _2D constriction that we observed previously.^{6 9} To test this premise, we first confirmed that hypoxia activates K_ATP channels (Fig 4). Three observations support this: (1) Hypoxic (PO₂, 6 mm Hg) dilation of arterioles was completely reversed by a low K_ATP-specific concentration of GLB (0.1 µmol/L; see below) that under normoxia produced a much smaller constrictor response. (2) Hypoxia markedly enhanced overall sensitivity to GLB constriction. (3) Hypoxia quickly reversed GLB constriction, which was resistant to removal by drug washout during normoxia, in agreement with earlier reports for this lipid-soluble agent.^{13 22 23} Having confirmed that hypoxia augments K_ATP channels, we then examined whether this action was responsible for inhibition of _2D constriction by hypoxia. Constriction by GLB, which under normoxia was completely abolished during the stimulation of _2D-ARs presumably due to K_ATP closure, was fully restored when _2D constriction was first reversed (ie, K_ATP first reopened) by hypoxia (Fig 5). To our knowledge, these findings represent the first evidence that _2D-ARs are coupled with the inhibition of K_ATP channels. They also present a novel hypothesis that links together a receptor and ion channel that have both been independently implicated^{3 5 6 10 11 12 13} in metabolic regulation of blood flow and tissue oxygen content.

A limitation of the present study is the reliance on the specificity of GLB and disopyramide for inhibition and cromakalim for activation of the K_ATP channel. Thus, these results require confirmation with direct ion channel analysis. However, several features of the experimental designs served to minimize nonspecific factors, in addition to the controls for time, drug reversal, agonist sensitization/desensitization, and constancy of intrinsic tone and myogenic reactivity. -AR subtypes were activated at submaximal levels with selective agonists in the presence of selective -AR antagonists. To prevent complications from changes in baseline tone produced by -AR stimulation in the experiments shown in Figs 1 and 5, AR constriction was reversed during maintained agonist infusion by simultaneous infusion of NP or adenosine. This enabled examination of -AR "stimulation" independent of constriction. NP dilation is mediated by activation of guanylate cyclase and is not affected by GLB,^{11 23} whereas adenosine increases cAMP. Although nitroprusside is more potent against _2D and adenosine against _1D constriction^{27 28} and although there is evidence that adenosine may also activate K_ATP channels (see below), identical results (abolition of GLB constriction during _2D stimulation) were obtained in experiments using either dilator to restore baseline diameter. Concentration-response relations were generated for GLB, disopyramide, and cromakalim, since these agents may affect the behavior of other K⁺ channels at higher concentrations. Several studies have demonstrated that concentrations of GLB 0.5 µmol/L are selective for K_ATP channels.^{17 18 20 21 25} In the first experiment, identical results were obtained with two structurally dissimilar K_ATP antagonists, the hypoglycemic sulfonylurea GLB and the class Ia antiarrythmic disopyramide. The latter agent, though possessing blocking activity at cholinergic receptors, has recently been shown to bind to a non-GLB site on the K_ATP channels of pancreatic ß cells.³⁰ In that study, disopyramide produced half-maximal inhibition of channel activity at 4 and 11 µmol/L when administered to the cytoplasmic and extracellular plasma membrane surfaces, respectively, in association with stimulation of insulin release.³⁰ However, there is as yet no direct evidence that disopyramide binds to the vascular smooth muscle K_ATP channel. In contrast to these antagonists, the selectivity of agonists such as cromakalim for K_ATP over other K⁺ channels may be less specific (see Reference 25²⁵ ), which could contribute to the smaller differential effect of cromakalim on _1D versus _2D constriction (Fig 2) than the striking effect of the K_ATP antagonists (Figs 1 and 3).

It is also possible that the presence of an intact endothelium in our preparation may have influenced the cromakalim results. Endothelial cells from some but not all vessels appear to possess K_ATP channels (see Reference 31³¹ ) that when activated may induce dilators such as nitric oxide (EDNO) and prostaglandins. However, dilation induced by cromakalim or related K_ATP agonists can be mediated by a direct action on smooth muscle cells.¹⁶ Endothelial cells were not removed in the present study, because we previously demonstrated that dilator levels of EDNO and prostaglandins are not released under basal conditions in these vessels studied in vitro in the absence of flow; endothelial cell factors also do not appear to be involved in the constrictor responses to _1D and _2D stimulation or inhibition of _2D contraction by hypoxia in our preparation.^{6 9 26} However, endothelial cells may modulate the effects of hypoxia in certain vessels.^{32 33 34 35} The absence of basal release of dilator levels of EDNO and prostaglandins mitigates against the possibility that GLB or disopyramide further reduced their release to influence the present results.

The involvement of depolarization versus pharmacomechanical coupling of -ARs appears to be vessel specific, and no generalization has emerged.⁸ Although the influences of -AR on membrane potential in the arterioles examined herein are unknown, the complementary designs of the experiments shown in Figs 1 and 3 make unlikely the possibility that differences in -AR coupling to changes in membrane potential, rather than _2D coupling to K_ATP channels, underlie the results. Other evidence does not support possible differences between the two -AR subtypes in membrane potential changes or in coupling with Ca²⁺ sources as explanations for the present results. We have demonstrated previously, using VOC antagonists, that _1D constriction mediated by the full agonists, PE and norepinephrine, does not involve activation of VOCs; in contrast, constrictions mediated by the partial _1D agonist ST and by _2D stimulation with UK or norepinephrine are highly VOC dependent in these and other vessels.^{6 8 9} The fact that ST and UK constriction have similar dependence on VOC and thus potentially similar effects on membrane potential, yet are completely opposite with regard to interaction with K_ATP channels (Figs 1 and 3), is discordant with the premise that the results of the present study extend from _2D but not _1D coupling with VOCs. It must also be mentioned that hypoxia with PO₂ at 10 mm Hg, which completely inhibits _2D constriction, has no effect on constriction mediated by KCl depolarization, ST, or a dihydropyridine VOC agonist.⁶

Jackson¹³ found that GLB constricted in vivo rat cremaster arterioles when PO₂ was <5 mm Hg but not 150 mm Hg, presumably because of the prior opening and closing of K_ATP channels by the respective O₂ states. As demonstrated in vivo for resistance vessels of several other tissues,^{12 22 23} the concentration-dependent effects of K_ATP agonists and antagonists in the present study suggest that K_ATP channels were partially active under conditions of basal tone, where arteriole PO₂ was maintained at the in vivo value of 70 mm Hg. It is interesting that this intrinsic tone is much less sensitive than _2D constriction to inhibition by hypoxia, requiring levels of PO₂ <10 mm Hg for inhibition.^{6 9} This was confirmed herein (Fig 4). In contrast, the IC₅₀ for hypoxic inhibition of _2D constriction is 24 mm Hg.⁶ Thus, in the present study, PO₂ of 10 mm Hg used in the experiment shown in Fig 5 identified a specific interaction between _2D and K_ATP channels independent of hypoxic effects on intrinsic tone. The basis for this difference in sensitivity of _2D and intrinsic tone to hypoxic inhibition is unclear but may relate to multiple mechanisms activated by different degrees of hypoxia or a resistance of intrinsic tone to the hyperpolarization favored by hypoxia.³⁵ More than one mechanism is in fact suggested by the absence of differences in sensitivity to GLB alone at PO₂ levels of 70 versus 10 mm Hg (Fig 5). However, the marked sensitivity (IC₅₀) of _2D constriction to hypoxia suggests that the threshold for activation of K_ATP channels by hypoxia could be well above a PO₂ of 6 mm Hg, in agreement with studies by others (see below).

The present studies provide the first evidence for the coupling of _2D-AR with the inhibition of K_ATP channels. This possibility is especially interesting for vascular smooth muscle, because the pathways coupling the _2D receptor–G protein complex to contraction have not been fully elucidated.^{8 36 37} Of related interest, in the pancreatic ß cell, where metabolism of glucose raises ATP and inhibits activation of K_ATP channels, resulting in depolarization, activation of VOCs, and insulin exocytosis, the _2D-AR also appears to be coupled with K_ATP channels, although other K⁺ channels may also be involved.^{30 38} However, in contrast to the present study, _2D stimulation increases K⁺ conductance, leading to hyperpolarization and inhibition of insulin exocytosis.³⁸

Although hypoxia directly decreases smooth muscle tone,^{35 39 40} the mechanisms remain unclear. In the past decade, a class of small conductance K⁺ channels (K_ATP channels) have been identified on certain cell types^{14 15} (including vascular smooth muscle cells^{16 17 18 19 20} ) that are voltage insensitive, inhibited by micromolar to millimolar ATP, activated by metabolic inhibition, and blocked by antidiabetic sulfonylureas, of which 1 µmol/L glibenclamide is prototypic (see References 20 and 21^{20 21} for reviews). Several in vivo studies suggest that these channels are activated by hypoxia in vascular smooth muscle, although the mechanisms of activation are unknown.^{10 11 12 13} It has been suggested that ATP levels are reduced by hypoxia, resulting in K_ATP activation.^{10 11 12 17 41} However, vascular smooth muscle of large arteries relies predominantly on glycolytic metabolism and evidences little change in ATP content during hypoxia.^{39 40} A recent study by Loutzenheiser and Parker²⁹ confirmed this for the rat afferent arteriole by use of the in vitro perfused hydronephrotic kidney model. Stepped reductions in perfusate PO₂ from 60 to 20 mm Hg produced progressive inhibition of myogenic constriction without any increase in arteriolar NADH or, presumably, any decrease in oxidative ATP production. Moreover, this hypoxic inhibition of myogenic constriction, where, for example, PO₂ of 30 mm Hg inhibited an intermediate level of myogenic constriction by >70% (similar sensitivity to our studies with _2D constriction^{6 9} ), was completely blocked by 1 µmol/L GLB. However, unlike the _2D-AR, myogenic constriction is not coupled with K_ATP closure, because GLB alone had no effect on myogenic reactivity.²⁹ Hypoxic dilation of rabbit cerebral arterioles over a similar physiological range of PO₂ has also been shown to be sensitive to GLB.⁴²

Although it remains unclear how hypoxia activates K_ATP channels, there is growing evidence that certain receptors may alter K_ATP activity, as postulated herein for the _2D-AR. This includes activation by the _2D-AR in pancreatic ß cells,³⁸ inhibition by the muscarinic receptor in urinary bladder smooth muscle,⁴³ and activation by certain but not all vasodilators, including receptors for calcitonin gene-related peptide,⁴⁴ adenosine,^{10 13 23 45} prostacyclin,^{13 23} and possibly vasoactive intestinal polypeptide¹⁶ in vascular smooth muscle.

In conclusion, these findings suggest that _2D-ARs but not _1D-ARs are coupled with closure of K_ATP channels, thus inducing constriction of rat cremaster arterioles. The results also suggest that this _2D-AR coupling and hypoxic activation of K_ATP channels underlies the effect of hypoxia to selectively inhibit _2D-AR but not _1D-AR constriction. Thus, a specific -AR subtype, previously shown to facilitate neurometabolic regulation of blood flow, may achieve this function through a close coupling with a unique K⁺ channel also implicated in metabolic vascular control. Selective expression and mode of coupling of the _2D-AR to constriction of resistance vessels may serve to optimize interactions between adrenergic and local metabolic vascular regulation.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-38783 and HL-02377.

	Footnotes

 
Reprint requests to James E. Faber, PhD, Department of Physiology 7545, 265 Medical Research, University of North Carolina, Chapel Hill, NC 27599-7545.

Received June 27, 1994; accepted October 3, 1994.

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