KATP channels contribute to beta - and adenosine receptor-mediated pulmonary vasorelaxation

Brett C. Sheridan1, Robert C. McIntyre Jr.1, Daniel R. Meldrum1, and David A. Fullerton2

1 Department of Surgery, University of Colorado, Denver, Colorado 80262; and 2 Department of Surgery, Northwestern University, Chicago, Illinois 60611

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

ATP-sensitive K+ (KATP) channels have been implicated in the regulation of vasomotor tone in aortic, mesenteric, and pulmonary vascular smooth muscle. Several investigators have described an association between KATP channels and isoproterenol (Iso)-stimulated relaxation responses. To study the relationship between receptor-dependent pulmonary vasorelaxation and KATP channels, we examined the response to agonists that generate adenosine 3',5'-cyclic monophosphate at two distinct levels of the signal transduction pathway after inhibition or activation of KATP channels in isolated rat pulmonary artery rings. Cumulative concentration responses to beta -adrenergic receptor stimulation (Iso), purinergic receptor stimulation [adenosine (Ado)], and direct stimulation of adenylate cyclase [forskolin (FSK)] were studied with and without concurrent inhibition of KATP channels (glibenclamide or tolbutamide). In addition, the effect of direct KATP channel activation (cromakalim) on the response to beta -adrenergic and purinergic receptor stimulation was determined. Last, we investigated the influence of KATP channel inhibition on endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation linked to guanosine 3',5'-cyclic monophosphate production. KATP channel inhibition impaired the response to Iso and Ado. Activation of KATP channels caused a leftward shift in the dose responses of Iso and Ado, with a significant decrease in the 50% effective concentration for each agent. KATP channel inhibition did not impair the pulmonary arterial vasorelaxation response to FSK, acetylcholine, or sodium nitroprusside. KATP channels appear to contribute to beta -adrenergic and purinergic receptor-stimulated vasorelaxation in rat pulmonary arteries.

glibenclamide; cromakalim; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate; isoproterenol; adenosine 5'-triphosphate-sensitive potassium channel

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

PULMONARY VASOMOTOR TONE reflects the balance of the mechanisms of pulmonary vasorelaxation and vasoconstriction. beta -Adrenergic and purinergic receptor stimulation results in activation of pulmonary vascular smooth muscle adenylate cyclase, an increase in adenosine 3',5'-cyclic monophosphate (cAMP), and, hence, vasorelaxation (9, 16). Recently, ATP-sensitive K+ (KATP) channels have been identified as mediators of systemic (22) and pulmonary (5) vasomotor tone. Several investigators have linked beta 1-adrenoreceptor-stimulated relaxation responses to KATP channel activation in dog coronaries (12, 19), the hamster cheek pouch microcirculation (11), the rat arterial mesenteric bed (23), and the rabbit pulmonary artery (4). There has been comparable interest and success linking purinergic receptor-induced vasodilation to KATP channels (1, 11, 15, 18, 23). It is currently unknown if KATP channels influence receptor-dependent vasorelaxation mechanisms in the pulmonary circulation that are associated with the generation of cAMP.

We hypothesized that 1) inhibition of KATP channels would impair beta -adrenergic and purinergic receptor-mediated pulmonary vasorelaxation and 2) activation of KATP channels would enhance receptor-dependent pulmonary vasorelaxation responses that are linked to the production of cAMP. To study this hypothesis, we inhibited KATP channels (glibenclamide or tolbutamide) in isolated rat pulmonary artery rings and investigated vascular relaxation responses to receptor-dependent [isoproterenol (Iso) and adenosine (Ado)] and receptor-independent [forskolin (FSK)] agonists that are associated with the generation of cAMP. We also examined the influence of KATP channel activation (cromakalim) on pulmonary vasorelaxation responses to beta -adrenoreceptor and purinoceptor stimulation. Last, we observed the influence of KATP channel inhibition on endothelium-dependent and -independent guanosine 3',5'-cyclic monophosphate (cGMP)-mediated mechanisms of pulmonary vasorelaxation.

The results of this study suggest that the response to beta -adrenergic and purinergic receptor stimulation is mediated, in part, by KATP channels. KATP channels contribute to receptor-dependent vasorelaxation mechanisms that are linked to the generation of cAMP but not to receptor-independent cAMP-mediated or cGMP-mediated vasorelaxation responses.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal housing and acclimation. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by National Institutes of Health. Male Sprague-Dawley rats (Sasco, Omaha, NE), weighing 300-350 g, were quarantined in quiet, humidified, light-cycled rooms for 2-3 wk before use.

Experimental protocol. Pulmonary vasomotor control mechanisms were studied in isolated pulmonary arterial rings (25). KATP channels were inhibited by incubating isolated pulmonary arterial rings with 1 mM glibenclamide or tolbutamide for 30 min before assessment of cumulative dose responses to vasodilatory agonists. Cromakalim was used to activate KATP channels in the isolated rings. We incubated rings with 10-7 M cromakalim after phenylephrine (PE) preconstriction and observed cumulative concentration responses to Iso and Ado. To determine if the effect of KATP channel inhibition was nonselective to mechanisms of pulmonary vascular smooth muscle relaxation, we also examined the influence of glibenclamide and tolbutamide on the vasorelaxation response to direct activation of adenylate cyclase with FSK and the influence of KATP channel inhibition on two cGMP-mediated mechanisms of pulmonary vasorelaxation with the endothelium-dependent agonist acetylcholine (ACh) and the endothelium-independent agonist sodium nitroprusside (SNP).

Isolated pulmonary arterial ring preparation. Rats were anesthetized with 50 mg/kg ip pentobarbital sodium. Median sternotomy was performed, and 500 USP heparin sulfate was injected into the right ventricular outflow tract. After removal of the heart and lungs en bloc, the main pulmonary artery with the right and left branches was dissected out and was placed in Earle's balanced salt solution (EBSS) at 4°C. Under dissecting microscope magnification, the surrounding tissue was excised from the pulmonary arteries. The right and left main branch pulmonary arteries were cut into rings 3- to 4-mm wide; two rings were obtained from each rat. Care was taken during this process to avoid endothelial injury. EBSS is a standard physiological salt solution and contains (in mM) 1.80 CaCl2, 0.83 MgSO4 (anhydrous), 5.36 KCl, 116.34 NaCl, 0.40 NaPO4 (dibasic), 5.50 D-glucose, 19.04 NaHCO3, and 0.03 phenol red sodium (as pH indicator).

The pulmonary artery rings were placed on 11-mil steel wires and were suspended in individual 10-ml organ chambers containing EBSS. The organ chambers were surrounded by water jackets and were continually warmed (37°C). Ring tension was determined by use of a force-displacement transducer (Grass FTO3; Grass Instruments, Quincy, MA) attached to each steel wire apparatus. Force displacement was recorded at 0.67 Hz using a MacLab Data Interface Module (ADI Instruments, Milford, MA) on a Macintosh Quadra 650 computer (Apple Computer, Cupertino, CA). Organ chambers had continuous bubbling gas flow at 40 ml/min of 21% O2, 5% CO2, and 74% N2. This produced a PO2 of 100-110 mmHg and a pH of 7.4.

Effect of inhibition of KATP channels on equilibration, vasomotor tone, and alpha 1-adrenergic-stimulated pulmonary vasoconstriction. The optimal resting mechanical tension (passive load) for pulmonary artery rings of this size was determined to be 750 mg in a prior study (10). Rings were suspended at 750 mg and were allowed to reach a steady state for 1 h, during which time the EBSS was changed every 15 min. Cumulative concentration responses to glibenclamide were observed in pulmonary artery rings equilibrated at 750 mg of tension and after preconstriction to 200-400 mg of tension with the alpha 1-adrenergic agonist PE.

Pulmonary vasorelaxation responses to agonists that generate cAMP. Cumulative concentration-response curves were generated for Iso, Ado, and FSK. These rings were equilibrated at 750 mg of tension for 1 h, with exchange of the oxygenated EBSS every 15 min. After equilibration, the rings were preconstricted between 200 and 400 mg of tension with PE. Cumulative concentration responses were generated over 10-9 to 10-6 M (Iso and FSK) and 10-9 to 10-3 M (Ado). For determination of the cumulative concentration-response curve, the ring was allowed to reach a steady state, usually requiring 2-3 min, before advancing to the next higher concentration. The ring tension remaining in the rings in response to each dose of vasorelaxing agent was expressed in milligrams of PE-induced tension.

Influence of KATP channels on pulmonary vasorelaxation mechanisms associated with the generation of cAMP. Cumulative concentration-response curves were generated for Iso, Ado, and FSK after in vitro incubation of isolated pulmonary artery rings with 1 mM glibenclamide or tolbutamide for 30 min before PE preconstriction. The physiological salt solution was changed before assessment of cumulative concentration-response curves to these receptor-dependent and -independent mechanisms of pulmonary vasorelaxation.

Cumulative concentration-response curves were generated for Iso and Ado after PE-induced preconstriction and in vitro incubation of isolated pulmonary artery rings with 10-7 M cromakalim.

Influence of KATP channel inhibition on pulmonary vasorelaxation by cGMP-mediated mechanisms. Cumulative concentration-response curves were generated over the concentration range of 10-9 to 10-6 M (ACh and SNP) after inhibition of KATP channels with glibenclamide.

Reagents. All reagents were obtained from Sigma Chemical (St. Louis, MO). Fresh solutions were prepared daily with either deionized water or normal saline as the diluent. Glibenclamide, tolbutamide, and cromakalim were dissolved in dimethyl sulfoxide and were further diluted in ethanol. Final concentrations of solvents were <0.5% and were without pharmacodynamic effect. The concentrations are expressed as final molar concentrations in the organ chambers.

Statistical analysis. Statistical analysis was performed with a MacIntosh Quadra 650 computer and StatView 4.01 software (Brain Power, Calabasas, CA). Data are presented as means ± SE of the number of pulmonary rings studied at each point of data collection. Similarly, the means ± SE were determined for the 50% effective concentration (EC50) for each agonist. Statistical evaluation utilized standard one-way analysis of variance with post hoc Bonferroni-Dunn test. P < 0.05 was accepted as statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of KATP channel inhibition on equilibration vasomotor tone and alpha 1-adrenergic-stimulated vasoconstriction. Cumulative concentration responses to 10-9 to 10-5 M glibenclamide on rings equilibrated at 750 mg of tension and after PE-induced contraction demonstrated no significant vasoactive effect (data not shown).

Influence of KATP channel inhibition on beta -adrenoreceptor- and purinoreceptor-stimulated pulmonary vasorelaxation. Inhibition of KATP channels with glibenclamide or tolbutamide impaired pulmonary vasorelaxation cumulative concentration responses to beta -adrenoreceptor stimulation with Iso. As illustrated in Fig. 1, control pulmonary artery rings were preconstricted to 293 ± 29 mg of PE-induced tension and were relaxed to 17 ± 4 mg of tension with 10-6 M Iso. Rings from glibenclamide-treated rings were preconstricted to 309 ± 17 mg of tension, and 94 ± 15 mg of PE-induced tension remained in response to 10-6 M Iso (P < 0.05 vs. control). Tolbutamide-treated rings were preconstricted with PE to 305 ± 16 mg of tension, with 108 ± 23 mg of tension remaining in response to 10-6 M Iso. Thus inhibition of KATP channels impaired the response to Iso compared with controls.


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Fig. 1.   Cumulative concentration responses to nonselective, beta -adrenergic receptor-stimulated pulmonary vasorelaxation [response to isoproterenol (Iso)] after inhibition of ATP-sensitive K+ (KATP) channels with 10-6 M glibenclamide or tolbutamide. Inhibition of KATP channels impaired the response to Iso compared with controls. Values are means ± SE. * P < 0.05 vs. control at same concentration of given agonist (n = 10 rings/5 rats for each group). PE, phenylephrine; Delta ring tension, change in ring tension. In all figures, inset is tension-time recording for an individual ring dose-response curve.

Inhibition of KATP channels with glibenclamide or tolbutamide impaired pulmonary vasorelaxation cumulative concentration responses to nonselective purinergic receptor stimulation with the agonist Ado. As demonstrated in Fig. 2, control pulmonary artery rings were preconstricted to 310 ± 10 mg of PE-induced tension and were relaxed to 101 ± 15 mg of tension with 10-3 M Ado. Glibenclamide-treated rings were preconstricted to 293 ± 17 mg of tension, and 199 ± 15 mg of PE-induced tension remained in response to 10-3 M Ado (P < 0.05 vs. control). Tolbutamide-treated rings were preconstricted with PE to 318 ± 18 mg of tension, with 147 ± 17 mg of tension remaining in response to 10-3 M Ado (P < 0.05 vs. control). Inhibition of KATP channels impaired the response to Ado as well as Iso compared with controls.


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Fig. 2.   Cumulative concentration responses to purinergic receptor-stimulated pulmonary vasorelaxation [response to adenosine (Ado)] after inhibition of KATP channels with 10-6 M glibenclamide or tolbutamide. Inhibition of KATP channels impaired the response to Ado compared with controls. Values are means ± SE. * P < 0.05 vs. control at same concentration of given agonist (n = 10 rings/5 rats for each group).

Effect of KATP channel activation on pulmonary vascular smooth muscle. Cromakalim, an activator of KATP channels, resulted in a dose-dependent relaxation response in PE-preconstricted pulmonary artery rings (Fig. 3). After PE preconstriction, the baseline tension of the rings was 277 ± 14 mg. Cromakalim relaxed the PE-preconstricted rings to 40 ± 9 mg of tension at 10-5 M. 


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Fig. 3.   Cumulative concentration responses to KATP channel activation with cromakalim after PE-induced pulmonary artery contraction. Values are means ± SE. * P < 0.05 vs. baseline ring tension (n = 6 rings/3 rats for each group).

KATP channel activation and cumulative dose responses to receptor-dependent vasorelaxation agonists Iso and Ado. Incubation of PE-preconstricted rings with 10-7 M cromakalim potentiated the vasorelaxation response to Iso (Fig. 4) and Ado (Fig. 5) as demonstrated by the leftward shift in the cumulative concentration response and decreased EC50 with KATP channel activation. The EC50 of control rings to a cumulative concentration response to Iso was 8.5 × 10-8 ± 1.0 × 10-8 M, which decreased to 1.9 × 10-9 ± 9.0 × 10-10 M after incubation with 10-7 M cromakalim. A potency shift of greater magnitude was observed in the cumulative concentration response to Ado in control rings with an EC50 of 4.3 × 10-4 ± 1.4 × 10-4 M that decreased to 5.5 × 10-8 ± 2.6 × 10-8 M after 10-7 M cromakalim incubation. Thus activation of KATP channels potentiates the pulmonary vascular smooth muscle relaxation response to receptor-dependent agonists associated with the production of cAMP.


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Fig. 4.   Cumulative concentration responses of PE-contracted pulmonary artery rings to the nonselective beta -adrenergic receptor agonist (response to Iso) after KATP channel activation with 10-7 M cromakalim. Activation of KATP channels with cromakalim was associated with a leftward shift in the response curve to Iso compared with control. Values are means ± SE. * P < 0.05 vs. control at same concentration of given agonist (n = 10 rings/5 rats for each group).


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Fig. 5.   Cumulative concentration responses of PE-contracted pulmonary artery rings to the purinergic receptor agonist (response to Ado) after KATP channel activation with 10-7 M cromakalim. Activation of KATP channels with cromakalim was associated with a leftward shift in the response curve to Ado compared with controls. Values are means ± SE. * P < 0.05 vs. control at same concentration of given agonist (n = 10 rings/5 rats for each group).

Effect of inhibition of KATP channels with a receptor-independent pulmonary vasorelaxation agonist, FSK. Inhibition of KATP channels with glibenclamide or tolbutamide did not influence receptor-independent pulmonary vasorelaxation responses as observed with direct stimulation of adenylate cyclase with FSK. As illustrated in Fig. 6, control pulmonary artery rings were preconstricted to 300 ± 14 mg of PE-induced tension and relaxed to 4 ± 3 mg of tension with 10-6 M FSK. Glibenclamide-treated rings were preconstricted to 309 ± 14 mg of tension, and 3 ± 1 mg of PE-induced tension remained in response to 10-6 M FSK. Tolbutamide-treated rings were preconstricted with PE to 307 ± 20 mg of tension, with 5 ± 2 mg of tension remaining in response to 10-6 M FSK. Thus the pulmonary cumulative concentration response to FSK was unchanged with inhibition of KATP channels compared with controls.


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Fig. 6.   Cumulative concentration responses to direct activation of adenylate cyclase [response to forskolin (FSK)] after inhibition of KATP channels with 10-6 M glibenclamide or tolbutamide. KATP channel inhibition did not influence the vasorelaxation response of PE-contracted pulmonary artery rings to FSK. Values are means ± SE (n = 10 rings/5 rats for each group).

Inhibition of KATP channels on endothelium-dependent and -independent cGMP-mediated pulmonary vasorelaxation. Unlike the observed impairment of vasorelaxation with glibenclamide on receptor-dependent cAMP-mediated vasorelaxation, KATP channel inhibition with glibenclamide did not impair the cumulative concentration responses to either endothelium-dependent or -independent mechanisms of pulmonary vasorelaxation that depend on the production of cGMP. As represented in Fig. 7, pulmonary artery rings from controls were preconstricted to 277 ± 15 mg of PE-induced tension and were relaxed to 11 ± 4 mg of tension with 10-6 M ACh. Glibenclamide-treated rings were preconstricted to 288 ± 10 mg of tension, and 16 ± 7 mg of PE-induced tension remained in response to 10-6 M ACh (P > 0.05 vs. control). Thus inhibition of KATP channels with glibenclamide did not impair this endothelium-dependent mechanism of pulmonary vasorelaxation.


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Fig. 7.   Cumulative concentration responses to endothelium-dependent cGMP-mediated pulmonary vasorelaxation (response to ACh) after inhibition of KATP channels with 10-6 M glibenclamide. Glibenclamide did not effect the vasorelaxation response to ACh of PE-contracted pulmonary artery rings compared with controls. Values are means ± SE (n = 8 rings/4 rats for each group).

Similar to the ACh cumulative concentration response, glibenclamide did not influence endothelium-independent vasorelaxation, as represented by the SNP cumulative concentration-response curve. As depicted in Fig. 8, controls were preconstricted to 298 ± 14 mg of PE-induce tension and were relaxed to 0 mg of tension with 10-6 M SNP. In glibenclamide-treated rings preconstricted to 316 ± 21 mg of tension, 0 mg of PE-induced tension remained in response to 10-6 M SNP (P > 0.05 vs. control). Neither endothelium-dependent nor -independent cGMP-mediated pulmonary vasorelaxation mechanisms were impaired by the inhibition of KATP channels with glibenclamide.


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Fig. 8.   Cumulative concentration responses to endothelium-independent cGMP-mediated pulmonary vasorelaxation [response to sodium nitroprusside (SNP)] after inhibition of KATP channels with 10-6 M glibenclamide. Glibenclamide did not effect the vasorelaxation response to SNP of PE-contracted pulmonary artery rings compared with controls. Values are means ± SE (n = 8 rings/4 rats for each group).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

These findings suggest that KATP channels contribute to receptor-dependent mechanisms of pulmonary vasorelaxation that are associated with the production of cAMP. KATP channel inhibitors glibenclamide and tolbutamide impaired the relaxation responses to Iso and Ado. Activation of KATP channels with cromakalim potentiated relaxation responses to Iso and Ado, as demonstrated by a leftward shift in the cumulative concentration-response curves and a decreased EC50. The influence of KATP channels on mechanisms of pulmonary vasorelaxation appears to be selective. Inhibition of KATP channels with glibenclamide or tolbutamide did not impair receptor-independent cAMP-mediated relaxation responses, nor did these agents impair endothelium-dependent or endothelium-independent mechanisms of pulmonary vasorelaxation that require the generation of cGMP. These findings challenge the current hypothesis that beta -adrenergic and purinergic vasorelaxation is entirely cAMP dependent.

Although these results are provocative, inherent limitations of this study design must be addressed. Large conduit vessels such as main branch pulmonary arteries may not accurately reflect the pulmonary resistance vessels such as arterioles. Despite this model limitation, isolated main branch arterial rings allow precise interrogation of endothelium-dependent and smooth muscle-dependent mechanisms of vasomotor function without confounding variables such as formed blood elements, neuromuscular interactions, and hormonal influences. A second, but potential, limitation of this study is that membrane potential data were not concurrently measured. Although direct measurement of membrane potential was beyond the scope of this physiological model, measurement of membrane potential in ex vivo intact vascular preparations has been accomplished with the KATP channel inhibitors glibenclamide and tolbutamide as well as with the often-used KATP channel activator cromakalim (3, 4, 26, 28). These investigations have confirmed the effects of glibenclamide, tolbutamide, and cromakalim with simultaneous physiological and membrane potential recordings, suggesting that these pharmacological tools have specific and reproducible effects on the KATP channel and membrane potential in vascular models. A third explanation to the observed results could reflect an interaction between the sulfonylurea compounds binding at the beta -adrenergic and purinergic receptors. This appears unlikely, however, because Randall and McCulloch (23) demonstrated that 100 mM glibenclamide did not influence the binding of [3H]dihydoalprenolol to rat mesenteric beta -adrenoreceptors. A nonselective inhibition of vasodilation by glibenclamide or tolbutamide may be ruled out as vasorelaxation responses to FSK, ACh, and SNP were not affected by these sulfonylurea compounds.

K+ channels inhibited by intracellular ATP concentration ([ATP]i) and opened as [ATP]i decreased were first described in the heart (20). Subsequently, these channels were also found in insulin-secreting cells and skeletal muscle (7). Standen and colleagues (26) initially described these channels in arterial smooth muscle. KATP channels in arterial smooth muscle are essentially Ca2+ (6) and voltage (21) independent, K+ selective, and half-maximally inhibited by [ATP]i in the range of ~30-40 mM in aortic smooth muscle in bilayers and portal vein (14, 21). Clapp and Gurney (5) inhibited whole cell KATP currents with larger concentrations of ATP (~1 mM). ADP activates single KATP channels in portal veins. A key pharmacological feature of KATP channels is their inhibition by antidiabetic sulfonylurea agents such as glibenclamide, tolbutamide, and external Ba2+ (22). A number of antihypertensive drugs appear to act through KATP channel activation. This class of antihypertensive drugs include older clinically used compounds such as minoxidil sulfate and diazoxide as well as newer drugs such as pinacidil, nicorandil, and cromakalim, to name only a few. Vasodilation to these compounds is blocked by glibenclamide (22).

Recent interest has focused on the regulation of vascular tone by KATP channels, and investigators have reported that these channels are important modulators of arterial tone. Clapp and colleagues (4) observed that KATP channel activation directly vasodilated rabbit pulmonary vascular smooth muscle. Narishige and co-workers (19) linked beta 1-adrenoreceptor vasodilation to KATP channels by blocking coronary vasorelaxation induced by Iso in dogs. Katsuda and colleagues (12) demonstrated that activation of KATP channels selectively augmented beta 1-adrenoreceptor-mediated coronary vasodilation. Jackson (11) found that inhibition of KATP channels also inhibited Ado- and Iso-induced vasorelaxation in hamster microcirculatory beds. In the isolated rat mesenteric arterial bed, Randall and McCulloch (23) likewise observed impairment of beta -adrenoreceptor-induced vasodilation by inhibition of KATP channels with glibenclamide. Recent electrophysiological evidence from cat ventricular myocytes has demonstrated that beta -adrenergic receptors are coupled to the activation of KATP channels (24). Similar to beta -adrenergic receptors, purinergic A1 receptors have been previously linked to K+ channels in cardiac tissue (2). More recently, Ado has been shown to activate KATP currents in single coronary artery smooth muscle cells, acting via an A1 receptor (8). It is plausable to suggest that Ado may open KATP channels independently of adenylate cyclase through a direct G protein pathway (8), as has been demonstrated in cardiac myocytes (13). Such findings, including the results of this study, challenge the traditional view that beta -adrenergic and purinergic receptors are solely coupled to adenylate cyclase and cAMP formation (27).

The data of the current study suggest that beta -adrenergic and purinergic receptors are associated with KATP channels, and this association appears to be proximal to the level of adenylate cyclase and protein kinase A. One possible mechanism of this interaction may be that G proteins link the beta -adrenergic and purinergic receptors to adenylate cyclase as well as KATP channels in pulmonary vascular smooth muscle. Several investigators have linked KATP channels to membrane-bound G proteins in pig coronary smooth muscle cells (8) and rat ventricular myocytes (13). In the present study, FSK vasorelaxant responses were not impaired by glibenclamide. Jackson (11) observed a similar response in that KATP channel blockade did not influence the vasodilator responses to dibutyryl cAMP. On the other hand, Miyoshi and Nakaya (17) observed that Iso, FSK, and dibutyryl cAMP activated KATP channels in an inside-out patch configuration with porcine coronary artery. The experiments of Miyoshi and Nakaya (17) required smooth muscle cell culture, whereas both Jackson (11) and the present study examined vascular responses with intact vascular rings. The findings of Jackson's work as well as those of the current study suggest that cAMP does not activate KATP channels. Although the precise mechanism by which receptors are linked to KATP channels in vascular smooth muscle remains unclear, it appears to be independent of cAMP in the hamster cheek pouch microcirculation and the rat pulmonary artery. Therefore, it is possible that beta -adrenergic and purinergic receptor-stimulated vasorelaxation triggers two parallel signal transduction mechanisms: 1) the primary signal through adenylate cyclase and protein kinase A and 2) a secondary signal through the KATP channel.

The findings of the current study support the hypothesis that the KATP channel contributes to beta -adrenergic and purinergic receptor-stimulated vasorelaxation mechanisms that also produce cAMP in rat pulmonary arteries. This role of the KATP channel seems to be limited to receptor-dependent cAMP-generating mechanisms of pulmonary vasorelaxation, independent of FSK-stimulated and cGMP-mediated vasorelaxation responses. These findings contribute to the growing body of evidence that beta -adrenergic and purinergic receptors are linked to mechanisms of pulmonary vasorelaxation other than adenylate cyclase and cAMP. The observation that these endogenous receptors are linked to clinically accessible KATP channels creates a potential therapeutic opportunity to modulate vasomotor tone in perturbed states such as pulmonary hypertension.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant R29 HL-49398 (to D. A. Fullerton) and an American College of Surgery Faculty Research Grant (to R. C. McIntyre, Jr.).

    FOOTNOTES

Address for reprint requests: R. C. McIntyre, Jr., Dept. of Surgery, 4200 East Ninth Ave., Box C-313, Univ. of Colorado Health Sciences Center, Denver, CO 80262.

Received 14 March 1997; accepted in final form 24 July 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(5):L950-L956
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