Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCa channels.

Background and Purpose. Caveolae act as signalling hubs in endothelial and smooth muscle cells. Caveolar disruption by the membrane cholesterol depleting agent methyl-ß-cyclodextrin (M-ß-CD) has various functional effects on arteries including (i) impairment of endothelium-dependent relaxation, and (ii) alteration of smooth muscle cell (SMC) contraction independently of the endothelium. The aim of this study was to explore the effects of M-ß-CD on rat femoral arteries. Methods. Isometric force was measured in rat femoral arteries stimulated to contract with a solution containing 20 mM K(+) and 200 nM Bay K 8644 (20 K/Bay K) or with one containing 80 mM K(+)(80 K). Results. Incubation of arteries with M-ß-CD (5 mM, 60 min) increased force in response to 20 K/Bay K but not that induced by 80 K. Application of cholesterol saturated M-ß-CD (Ch-MCD, 5 mM, 50 min) reversed the effects of M-ß-CD. After mechanical removal of endothelial cells M-ß-CD caused only a small enhancement of contractions to 20 K/Bay K. This result suggests M-ß-CD acts via altering release of an endothelial-derived vasodilator or vasoconstrictor. When nitric oxide synthase was blocked by pre-incubation of arteries with L-NAME (250 µM) the contraction of arteries to 20 K/Bay K was enhanced, and this effect was abolished by pre-treatment with M-ß-CD. This suggests M-ß-CD is inhibiting endothelial NO release. Inhibition of large conductance voltage- and Ca(2+)-activated (BKCa) channels with 2 mM TEA(+) or 100 nM Iberiotoxin (IbTX) enhanced 20 K/Bay K contractions. L-NAME attenuated the contractile effect of IbTX, as did endothelial removal. Conclusions. Our results suggest caveolar disruption results in decreased release of endothelial-derived nitric oxide in rat femoral artery, resulting in a reduced contribution of BKCa channels to the smooth muscle cell membrane potential, causing depolarisation and contraction.

Outcomes of surgery for chronic exertional compartment syndrome in a military population.

AIM: To determine the outcome following fasciectomy for chronic exertional compartment syndrome (CECS) in the UK military, and the association between presurgical intramuscular pressure (IMP) and outcome. METHODS: All patients who underwent fasciectomy for anterior CECS were identified between 2007 and 2010. Presurgery and postsurgery military medical grading for leg function was extracted from the medical records system. The Wilcoxon signed-rank test compared grades before and after surgery. Spearman's rank correlation examined the relationship between IMP and outcome. RESULTS: Presurgery and postsurgery grading was available for 63% of patients. These patients had significantly better leg function after surgery (Z=-3.63, p<0.001). Of these, 49% improved by at least one grade, 36% showed no improvement in grading and 15% had a poorer outcome. There were no significant correlations between IMP and outcome. CONCLUSIONS: A large proportion of patients do not return to full fitness following fasciectomy in the military population. This is in line with a recent study in the US military, but conflicts with most civilian reports. The reasons for these differences are not clear. Furthermore, the lack of a relationship between IMP and outcome questions the role of pressure in this condition. These results suggest that the role of postoperative rehabilitation protocols and other conservative options should be explored.

A pre-hospital technique for controlling haemorrhage from traumatic perineal and high amputation injuries.

Perineal trauma resulting from the adaptive use of improvised explosive devices (IEDs) has become an increasingly common problem during current operational conflicts in Afghanistan. Control of haemorrhage from the perineum and high amputations is a particular challenge due to the bony anatomy, rich pelvic vascular supply and the difficulty in achieving haemostasis by direct pressure. In this article, the authors describe a potential pre-hospital solution for controlling haemorrhage from perineal and high amputation injuries.

Caveolin-1 and caveolin-3 regulate Ca2+ homeostasis of single smooth muscle cells from rat cerebral resistance arteries.

The role of caveolins, signature proteins of caveolae, in arterial Ca(2+) regulation is unknown. We investigated modulation of Ca(2+) homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca(2+) transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca(2+) current and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). After repolarization, elevated [Ca(2+)](i) returned to the resting level. Ca(2+) removal rate was determined from the declining phase of the Ca(2+) transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82-101 of caveolin-1, significantly slowed Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM, with little effect at a measured [Ca(2+)](i) of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, also significantly slowed Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM, with little effect at a measured [Ca(2+)](i) of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca(2+)/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, also significantly inhibited Ca(2+) removal rate at a measured [Ca(2+)](i) of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca(2+) removal of resistance artery smooth muscle cells.

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes.

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.

Molecular analysis of the subtype-selective inhibition of cloned KATP channels by PNU-37883A.

1. In this study, we have used Kir6.1/Kir6.2 chimeric proteins and current recordings to investigate the molecular basis of PNU-37883A inhibition of cloned K(ATP) channels. 2. Rat Kir6.1, Kir6.2 and Kir6.1/Kir6.2 chimeras were co-expressed with either SUR2B or SUR1, following RNA injection into Xenopus oocytes, and fractional inhibition of K(ATP) currents by 10 microm PNU-37883A reported. 3. Channels containing Kir6.1/SUR2B were more sensitive to inhibition by PNU-37883A than those containing Kir6.2/SUR2B (mean fractional inhibition: 0.70, cf. 0.07). 4. On expression with SUR2B, a chimeric channel with the Kir6.1 pore and the Kir6.2 amino- and carboxy-terminal domains was PNU-37883A insensitive (0.06). A chimera with the Kir6.1 carboxy-terminus and Kir6.2 amino-terminus and pore was inhibited (0.48). These results, and those obtained with other chimeras, suggest that the C-terminus is an important determinant of PNU-37883A inhibition of Kir6.1. Similar results were seen when constructs were co-expressed with SUR1. Further chimeric constructs localised PNU-37883A sensitivity to an 81 amino-acid residue section in the Kir6.1 carboxy-terminus. 5. Our data show that structural differences between Kir6.1 and Kir6.2 are important in determining sensitivity to PNU-37883A. This compound may prove useful in probing the structural and functional differences between the two channel subtypes.

Ca2+-induced Ca2+ release in cardiac and smooth muscle cells.

Ca(2+) influx across plasma membranes may trigger Ca(2+) release by activating ryanodine-sensitive receptors in the sarcoplasmic reticulum. This process is called Ca(2+)-induced Ca(2+) release, and may be important in regulating cytosolic Ca(2+) concentration ([Ca(2+)](i)). In cardiac cells, the initial [Ca(2+)](i) increase, caused by L-type Ca(2+) current, is profoundly amplified with Ca(2+) release. The synchronized opening of several ryanodine-sensitive Ca(2+)-releasing channels was detected as discreet and highly localized Ca(2+) elevation, and termed as a 'Ca(2+) spark'. A Ca(2+) spark is under local control of an L-type Ca(2+) channel, and therefore a Ca(2+) spark does not normally trigger subsequent Ca(2+) sparks in the neighbouring area. In smooth muscle cells, the importance of Ca(2+)-induced Ca(2+) release in elevating [Ca(2+)](i) appears to differ among preparations and species. Significant elevation in [Ca(2+)](i) during depolarization was attributed to Ca(2+) release in some smooth muscle cells, but not in others. Ca(2+) sparks are also identified in smooth muscle cells, and may play a role as functional elementary events for Ca(2+)-induced Ca(2+) release. At rest, Ca(2+) sparks may be also important in regulating smooth muscle membrane potential. Ca(2+) sparks occurring at rest do not raise global [Ca(2+)](i), but trigger spontaneous transient outward currents (STOCs) or spontaneous transient inward currents (STICs), the former producing hyperpolarization; the latter, depolarization. Thus there may be multiple facets for Ca(2+)-induced Ca(2+) release in regulating the contractile status of smooth muscle cells.

Inhibition of vascular K(ATP) channels by U-37883A: a comparison with cardiac and skeletal muscle.

1 The aim of this study was to investigate the selectivity of the ATP-sensitive potassium (K(ATP)) channel inhibitor U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-1-cyclohexyl). Membrane currents through K(ATP) channels were recorded in single muscle cells enzymatically isolated from rat mesenteric artery, cardiac ventricle and skeletal muscle (flexor digitorum brevis). K(ATP) currents were induced either by cell dialysis with 0.1 mM ATP and 0.1 mM ADP, or by application of synthetic potassium channel openers (levcromakalim or pinacidil). 2 U-37883A inhibited K(ATP) currents in smooth muscle cells from rat mesenteric artery. Half inhibition of 10 microM levcromakalim-induced currents occurred at a concentration of 3.5 microM. 3 Relaxations of rat mesenteric vessels caused by levcromakalim were reversed by U-37883A. 1 microM levcromakalim-induced relaxations were inhibited at a similar concentration of U-37883A (half inhibition, 1.1 microM) to levcromakalim-induced KATP currents. 4 K(ATP) currents activated by 100 microM pinacidil were also studied in single myocytes from rat mesenteric artery, skeletal muscle and cardiac ventricle. 10 microM U-37883A substantially inhibited K(ATP) currents in vascular cells, but had little effect in skeletal or cardiac myocytes. Higher concentrations of U-37883A (100 microM) caused a modest decrease in K(ATP) currents in skeletal and cardiac muscle. The sulphonylurea K(ATP) channel antagonist glibenclamide (10 microM) abolished currents in all muscle types. 5 The effect of U-37883A on vascular inward rectifier (KIR) and voltage-dependent potassium (KV) currents was also examined. While 10 microM U-37883A had little effect on these currents, some inhibition was apparent at higher concentrations (100 microM) of the compound. 6 We conclude that U-37883A inhibits K(ATP) channels in arterial smooth muscle more effectively than in cardiac and skeletal muscle. Furthermore, this compound is selective for K(ATP) channels over KV and KIR channels in smooth muscle cells.

Fading and rebound of P2X2 currents at millimolar ATP concentrations caused by low pH.

When ATP is applied at high concentrations (above 1 mM) to PC12 cells, it produces a rapidly desensitizing peak current followed by a rebound of the current after termination of the ATP application. We expressed P2X2 receptors, which are thought to mediate the ATP currents of PC12 cells, in HEK293 cells and studied the effects of acidification on this 'fading and rebound' phenomenon. We found that the desensitization disappeared after adjusting the low pH (<5.0) of the millimolar ATP concentrations to a more physiological value (pH 7.3). Furthermore, the fading and rebound could also be induced at much lower ATP concentrations by decreasing the pH of the ATP containing application solutions. Thus, it appears this phenomenon is not caused directly by high concentrations of ATP, but is due to a concomitant acidification that occurs when high concentrations of ATP are dissolved in only moderately buffered application solutions.

ATP-sensitive K+ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle.

1. We used patch clamp to study whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries. 2. CGRP (50 nM) activated an inward current at -60 mV in symmetrical 140 mM K+ that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive potassium (KATP) channels. CGRP-induced currents were larger in cells dialysed with 0.1 mM ATP than with 3.0 mM ATP. 3. Forskolin (10 microM) activated a glibenclamide-sensitive current, as did intracellular dialysis with cAMP (100 microM). The catalytic subunit of cAMP-dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells. 4. CGRP-induced currents were reduced by the PKA inhibitors adenosine 3',5'-cyclic monophosphorothioate, RP-isomer, triethylammonium salt (Rp-cAMPS; 100 microM) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulphonamide+ ++ dihydrochloride (H-89; 1 microM), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution. 5. The beta-adrenergic agonist isoprenaline (10 microM) also activated a glibenclamide-sensitive K+ current. 6. CGRP-induced currents were unaffected by the inhibitor of cGMP-dependent protein kinase (PKG) KT5823 (1 microM). Sodium nitroprusside (10 microM) did not activate a glibenclamide-sensitive current in cells held at -60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nM iberiotoxin (an inhibitor of BKCa channels). 7. Our findings suggest that CGRP activates coronary KATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.

K+ channel modulation in arterial smooth muscle.

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.

ATP-sensitive and inwardly rectifying potassium channels in smooth muscle.

The properties and roles of ATP-sensitive (KATP) and inwardly rectifying (KIR) potassium channels are reviewed. Potassium channels regulate the membrane potential of smooth muscle, which controls calcium entry through voltage-dependent calcium channels, and thereby contractility through changes in intracellular calcium. The KATP channel is likely to be composed of members of the inward rectifier channel gene family (Kir6) and sulfonylurea receptor proteins. The KIR channels do not appear to be as widely distributed as KATP channels in smooth muscle and may provide a mechanism by which changes in extracellular K+ can alter smooth muscle membrane potential, and thereby arterial diameter. The KATP channels contribute to the resting membrane conductance of some types of smooth muscle and can open under situations of metabolic compromise. The KATP channels are targets of a wide variety of vasodilators and constrictors, which act, respectively, through adenosine 3',5'-cyclic monophosphate/protein kinase A and protein kinase C. The KATP channels are also activated by a number of synthetic vasodilators (e.g., diazoxide and pinacidil) and are inhibited by the oral hypoglycemic sulfonylurea drugs (e.g., glibenclamide). Together, KATP and KIR channels are important regulators of smooth muscle function and represent important therapeutic targets.

Angiotensin II inhibition of ATP-sensitive K+ currents in rat arterial smooth muscle cells through protein kinase C.

1. The effects of the vasoconstrictor angiotensin II (Ang II) on whole-cell ATP-sensitive K+ currents (IK,ATP) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique. 2. Ang II, at a physiological concentration (100 nM), reduced IK,ATP activated by 0.1 mM internal ATP and 10 microM levcromakalim by 36.4 +/- 2.3%. 3. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 microM) reduced IK,ATP by 44.1 +/- 2.7%. GDP beta S (1 mM), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected. 4. Pretreatment with the PKC inhibitors staurosporine (100 nM) or calphostin C (500 nM) prevented the Ang II-induced inhibition of IK,ATP. 5. Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 microM), and the PKA inhibitor Rp-cAMPS (100 microM) did not reduce IK,ATP. 6. Our results suggest that Ang II modulates KATP channels through activation of PKC but not through inhibition of PKA.

The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle.

1. Whole-cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at -140 mV it was -20.5 +/- 4.4 pA pF-1 (n = 23) in 4th order branches of the LAD, but -0.8 +/- 0.2 pA pF-1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage-dependent potassium current; densities at +30 mV were 12.8 +/- 1.3 pA pF-1 (n = 19) in 4th order branches and 17.4 +/- 3.1 pA pF-1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.

Evidence against the association of the sulphonylurea receptor with endogenous Kir family members other than KATP in coronary vascular smooth muscle.

We used whole-cell patch clamp to record inward rectifier (KIR) and ATP-sensitive (KATP) K+ currents from pig coronary arterial myocytes. KIR currents were blocked by Ba2+ ions with a KD around 3 microM, but were unaffected by 10 microM glibenclamide, and only reduced 16% by 100 microM of the sulphonlyurea (n=4). In contrast, pinacidil-activated KATP currents were over 1000 times more sensitive to glibenclamide, being inhibited with a KD close to 100 nM (n=5). Our findings suggest that the sulphonylurea receptor (SUR) in these cells associates with the appropriate subunits of the Kir family to form KATP channels, but does not show promiscuous association with subunits that form the strong inward rectifier KIR.

Pharmacology of ATP-sensitive K+ currents in smooth muscle cells from rabbit mesenteric artery.

The inference that ATP-sensitive K+ (KATP) channels are involved in arterial responses to the synthetic K+ channel openers, hypoxia, adenosine, and calcitonin gene-related peptide, has relied on the sensitivity of these responses to the sulfonylureas glibenclamide and tolbutamide and to tetraethylammonium (TEA+). The inhibition of KATP currents by glibenclamide, tolbutamide, and TEA+ was investigated in single smooth muscle cells from rabbit mesenteric artery by use of the whole cell patch-clamp technique. The synthetic K+ channel openers pinacidil (half-activation 0.6 microM), cromakalim (half-activation 1.9 microM), and diazoxide (half-activation 37.1 microM) activated K(+)-selective currents that were blocked by glibenclamide. Elevation of pipette (intracellular) ATP concentration decreased K+ currents induced by pinacidil. Half-inhibition of KATP currents by glibenclamide and tolbutamide occurred at 101 nM and 351 microM, respectively. KATP currents were also inhibited by external TEA+, with half-inhibition at 6.2 mM. The results indicate that glibenclamide is an effective inhibitor of KATP channels in arterial smooth muscle and that tolbutamide and TEA+ are much less effective. Furthermore, these results support numerous functional studies that have demonstrated that the vasorelaxations to K+ channel openers are inhibited by < 10 microM glibenclamide but not by < 1 mM TEA+.

Physiological roles and properties of potassium channels in arterial smooth muscle.

This review examines the properties and roles of the four types of K+ channels that have been identified in the cell membrane of arterial smooth muscle cells. 1) Voltage-dependent K+ (KV) channels increase their activity with membrane depolarization and are important regulators of smooth muscle membrane potential in response to depolarizing stimuli. 2) Ca(2+)-activated K+ (KCa) channels respond to changes in intracellular Ca2+ to regulate membrane potential and play an important role in the control of myogenic tone in small arteries. 3) Inward rectifier K+ (KIR) channels regulate membrane potential in smooth muscle cells from several types of resistance arteries and may be responsible for external K(+)-induced dilations. 4) ATP-sensitive K+ (KATP) channels respond to changes in cellular metabolism and are targets of a variety of vasodilating stimuli. The main conclusions of this review are: 1) regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries; 2) KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential; and 3) K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle.

KATP channels in vascular smooth muscle.

ATP sensitive potassium channels (KATP channels) appear widely distributed in the vascular system. At the single channel level, channels with both small and large conductance have been described, though the former appear to be activated by potassium channel openers or ATP depletion in whole cell studies. KATP channels are inhibited by cytoplasmic ATP, and may be activated by intracellular nucleotide diphosphates. Regulation by intracellular metabolites confers a degree of sensitivity of the channel to the metabolic status of the cell, and there is evidence that KATP currents are activated during metabolic inhibition. In general, activation of KATP channels will lead to membrane hyperpolarisation and so to vasorelaxation. The functional role of the channel is being intensively studied at present. The channel may form a target for a number of endogenous vasodilators, and may be inhibited by some vasoconstrictors. It may be involved in the vasodilator response to hypoxia, and may contribute to the resting membrane potential of smooth muscle in some blood vessels.

Calcitonin gene-related peptide activated ATP-sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

1. Whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole-cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide-sensitive currents. CGRP increased the glibenclamide-sensitive currents by 3- to 6-fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide-sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current-voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp-cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide-sensitive K+ currents 2.1-, 3.3- and 8.2-fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide-sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H-8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP-sensitive K+ channels.

Inward rectifier K+ currents in smooth muscle cells from rat resistance-sized cerebral arteries.

Inward rectifier K+ channels have been implicated in the control of membrane potential and external K(+)-induced dilations of small cerebral arteries. In the present study, whole cell K+ currents through the inward rectifier K+ channel were measured in single smooth muscle cells isolated from the posterior cerebral artery of Wistar-Kyoto rats. The whole cell K+ current-voltage relationship showed inward rectification. Inward currents were recorded negative to the K+ equilibrium potential, whereas outward currents were small. When extracellular K+ was elevated, the zero current potential shifted to the new K+ equilibrium potential, and the conductance of the inward current increased. Inward currents were reduced by external barium or cesium. Inhibition by barium and cesium increased with membrane hyperpolarization. The half-inhibition constant for barium was 2.2 microM at -60 mV, increasing e-fold for a 23-mV depolarization. We provide the first direct measurements of inward rectifier K+ currents in single smooth muscle cells and show that external barium ions are effective blockers of these currents.