BK Ca Blocker Research Articles

Matrix metalloproteinase 2–induced venous dilation via hyperpolarization and activation of K + channels: Relevance to varicose vein formation

Varicose veins are a common disorder of extensive venous dilation and remodeling with an as-yet unclear mechanism. Studies have shown increased plasma and tissue levels of matrix metalloproteinases (MMPs) in human varicose veins and animal models of venous hypertension. Although the effects of MMPs are generally attributed to extracellular matrix degradation, their effects on the mechanisms of venous contraction/relaxation are unclear. Our preliminary experiments have demonstrated that MMP-2 causes inhibition of phenylephrine-induced venous contraction. The purpose of this study was to determine whether MMP-induced inhibition of venous contraction involves an endothelium-dependent and/or -independent pathway. Circular segments of the inferior vena cava (IVC) were isolated from male Sprague-Dawley rats and suspended between two wire hooks in a tissue bath, and the effects of MMP-2 on phenylephrine- and KCl-induced contraction were measured. To study the role of endothelium-derived vasodilators, experiments were performed in the presence and absence of endothelium; N(G)-l-nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis; indomethacin, an inhibitor of prostacyclin synthesis; cromakalim, an activator of adenosine triphosphate-sensitive K+ channels (K(ATP)); and iberiotoxin, a blocker of large-conductance Ca2+-dependent K+ channels (BK(Ca)) and smooth muscle hyperpolarization. In endothelium-intact IVC segments, phenylephrine (10(-5) mol/L) caused significant contraction that slowly declined to 82.0% in 30 minutes. The addition of MMP-2 (1 microg/mL) caused a gradual decrease of phenylephrine contraction to 39.5% at 30 minutes. In endothelium-denuded IVC, MMP-2 induced a greater reduction of phenylephrine contraction, to 7.6%. In the presence of L-NAME (10(-4) mol/L), MMP-2 caused a marked decrease in phenylephrine contraction, to 4.4%. Large MMP-2-induced inhibition of phenylephrine contraction was also observed in IVC treated with L-NAME plus indomethacin. MMP-2 caused relaxation of phenylephrine contraction in IVC pretreated with cromakalim (10(-7) mol/L), an activator of K(ATP) channels. MMP-2-induced inhibition of phenylephrine contraction was abrogated in the presence of iberiotoxin (10(-8) mol/L), a blocker of BK(Ca). MMP-2 did not inhibit venous contraction during membrane depolarization by 96 mmol/L KCl, a condition that prevents outward K+ conductance and cell hyperpolarization. MMP-2 causes significant IVC relaxation that is potentiated in the absence of endothelium or during blockade of endothelium-mediated nitric oxide and prostacyclin synthesis. The lack of effects of MMP-2 on KCl contraction and in iberiotoxin-treated veins suggests MMP-2-induced smooth muscle hyperpolarization and activation of BK(Ca) channels--a novel effect of MMP that may play a role in the early stages of venous dilation and varicose vein formation.

Nitric Oxide does not suppress myogenic tone by activating ryanodine sensitive stores in rat middle cerebral artery

Inhibition of NOS in cerebral arteries results in vasoconstriction and the development of vasomotion, indicating that basal NO synthesis suppresses myogenic tone. Recent studies in rat posterior cerebral artery suggest that NO acts via ryanodine-sensitive calcium stores, evoking Ca2+ sparks, which activate smooth muscle BKCa. We investigated whether a similar action of NO was responsible for the suppression of myogenic tone in the rat middle cerebral artery. Segments of the middle cerebral artery from male Wistar rats were mounted in a wire myograph. SMC membrane potential (Em) was recorded with sharp glass microelectrodes and changes in [Ca2+]i measured using a confocal microscope after loading the SMCs with Oregon Green. Asynchronous oscillations in smooth muscle [Ca2+]i were slightly reduced by nifedipine, but not affected further by the addition of L-NAME. In the additional presence of ryanodine (10 μM) the oscillations were abolished. In the absence of nifedipine, L-NAME caused a significant vasoconstriction (~ 75% max) and vasomotion. Both the BKCa blocker iberiotoxin and ryanodine (alone) mimicked the effect of L-NAME. Application of nifedipine decreased tension and markedly reduced L-NAME induced constriction. These results indicate that in rat middle cerebral arteries, ryanodine-sensitive Ca2+ stores are essential for maintaining the asynchronous oscillations in smooth muscle [Ca2+]i observed under resting conditions, NO does not appear to be essential for initiation of these oscillations. Therefore, the effect of basal release of NO to suppress myogenic tone by stimulating BKCa is not due to an action on ryanodine-sensitive stores but may reflect a direct action of NO on these KCa channels.

The dietary flavonoid quercetin activates BKCa currents in coronary arteries via production of H2O2. Role in vasodilatation

Large conductance Ca(2+)-activated K(+) channels (BKCa) regulate coronary artery tone in vivo, play a key role in blood pressure regulation, and have been suggested as novel potential drug targets in hypertension. Quercetin exerts systemic and coronary vasodilator effects in vitro and reduces blood pressure in several rat models of hypertension, and its consumption is associated with a lower mortality rate from coronary heart disease in epidemiological studies. We hypothesized that quercetin might activate BKCa channel in isolated myocytes from rat coronary arteries and that this mechanism might be involved in its coronary artery relaxant effects. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Contractile tension was recorded in rat coronary artery rings mounted in a myograph. Quercetin (>0.1 muM) increased the outward currents in the whole range of test potentials, hyperpolarized cell membranes, and increased the frequency of spontaneous transient outward currents (STOCs) carried by BKCa channels. These effects were abolished by the selective BKCa blocker iberiotoxin and by catalase. Quercetin increased dichlorofluorescein fluorescence in coronary arteries in a polyethylenglycol-catalase-sensitive manner, indicating that it increased cytosolic H(2)O(2). The membrane-permeable analogue of H(2)O(2)t-butylhydroperoxide mimicked the effects of quercetin on outward currents. The vasodilator effect of quercetin in isolated rat coronary arteries was partially inhibited by iberiotoxin. Quercetin increased BKCa currents via production of intracellular H(2)O(2). This effect is involved, at least partly, in the coronary vasodilator effects of quercetin.

No major role of BKCa channels in the modulation of renal blood flow in rats

The aim of the present study was to investigate the role of large calcium activated potassium (BKCa) channels in the maintenance of basal renal vascular tone in vivo in anesthetized adult Sprague-Dawley rats. We also examined whether the vasoconstriction elicited by angiotensin II (Ang II) and norepinephrine (NE) was buffered by simultaneous activation of BKCa. Vasoactive agents, BKCa blockers or a stimulator were infused directly into the renal artery via a polyethylene (PE-10) catheter introduced into the left femoral artery and advanced through the abdominal aorta and approximately 1 mm into the left renal artery. Renal blood flow (RBF) was measured by using an electromagnetic flow sensor. Renal preinfusion of BKCa blockers tetraethylammonium chlorid (TEA) (3.0 μmol/min) for 1 (n = 8) or 5 min (n = 5) and iberiotoxin for 5 min (n = 6) (3.3 nmol/min) did not significantly affect the RBF baseline. Renal arterial injection of a 10 μL bolus of Ang II (n = 18) (1-4 ng/10 μL) or NE (n = 17) (10-40 ng/10 μL) produced a significant dose dependent temporary decrease in RBF. These responses were not significantly affected by 1 or 4 min preinfusion of TEA (n = 8) or a 4 min preinfusion of iberiotoxin (n = 6). Renal addition of the BKCa opener NS 1619 (n = 5) (30 and 90 μmol/min) did not significantly affect basal renal blood flow or the responses to Ang II and NE. The present results do not indicate any major role for BKCa channels in the control of basal renal tone in vivo. Furthermore, there is no support for the hypothesis that BKCa channels have a buffering effect on the responses to Ang II and NE in rat renal vasculature. (Supported by Danish Heart Foundation)

NO can suppress myogenic tone in rat middle cerebral arteries by activating BK Ca

Aim In rat middle cerebral arteries myogenic tone is normally suppressed by a basal release of NO as inhibition of nitric oxide synthase (NOS) causes both vasoconstriction and smooth muscle depolarization associated with oscillations in tension and membrane potential (vasomotion). We wished to characterise the cellular mechanisms which underpin the vasoconstrictor effect of inhibitors of NOS by simultaneously measuring smooth muscle cell (SMC) membrane potential and tension or SMC [Ca2+] and tension Methods Segments of the middle cerebral artery from male Wistar rats were mounted in a wire myograph. SMC membrane potential (Em) was recorded with sharp glass microelectrodes. SMC [Ca2+] was recorded on a confocal microscope with vessels loaded with fluo-4. Results Inhibition of NOS with L-NAME caused SMC depolarization associated with constriction and the development of vasomotion temporally linked to SMC [Ca2+]. This effect was mimicked by the blocker of guanylyl cyclase, ODQ and the BKCa blocker, iberiotoxin. Relaxation and hyperpolarization produced by the NO donor DEA-NONOate was inhibited by iberiotoxin. L-NAME-induced constriction and oscillations in SMC [Ca2+] were abolished by the L-type Ca channel inhibitor, nifedipine. Discussion In rat middle cerebral arteries inhibition of NOS with L-NAME induces vasoconstriction and depolarization involving the entry of Ca2+. L-NAME also induces vasomotion which is underpinned by oscillations in Em and SMC [Ca2+]. Blockade of BKCa mimics the effects of L-NAME suggesting that NO normally suppresses myogenic tone by activation of SMC BKCa channels. Supported by the British Heart Foundation

Nitroglycerin‐Induced Relaxation is Mediated by BKCa Channels in Nitrate Tolerant Arteries

Numerous studies have examined the cause(s) of nitrate tolerance, but remarkably little is known about possible adaptive processes that may occur in nitrate-tolerant blood vessels. We tested the hypothesis that nitrate tolerance induces a compensatory response involving K channels in nitroglycerin-induced smooth muscle relaxation. Rats were treated with placebo or NTG (0.6 mg/hr) patches for 3 days. Thoracic aortae were removed, cut into rings, and suspended in organ baths. Relaxation responses to NTG (10−9 to 10−5 M) were obtained in the absence and presence of several K channel blockers. Nitrate tolerance was evident by a >7-fold rightward shift in the NTG concentration-response curve in tissues from rats treated with NTG patches as compared with placebo. Iberiotoxin (10−7 M), a selective blocker of BKCa, had no effect on NTG-induced relaxation of rings from nontolerant rats, but caused a further rightward shift (>7-fold) in the concentration-response curve to NTG in rings from tolerant rats. Selective blockers of other K channels, including glyburide (10−6 M), apamin (10−6 M), and 4-aminopyridine (10−3 M) had no effect on responses to NTG in either nontolerant or tolerant rings. Whole cell voltage-clamp studies from placebo and NTG-treated rat aortic myocytes indicated that NTG (10 μM) significantly increased BKCa current density in tolerant aortic myocytes at +80 mV (22.6 ± 3.02 and 52.5 ± 5.52 pA/pF, control and tolerant respectively). The data suggests that in nontolerant arteries, NTG-induced relaxation is independent of BKCa channel activation. By contrast, prolonged in vivo treatment with NTG results in a major role for BKCa in NTG-induced relaxation of nitrate tolerant arteries.

Potassium channels at the beginnings of cell proliferation

In physiology it is common to think of the vascular smooth muscle cell in its contractile phenotype, orientated radially and contracting to regulate blood flow. However, in response to growth factors and other environmental signals these cells may lose their contractile traits in favour of a phenotype characterized by shape change, migration and proliferation. Such plasticity is important in physiology for new blood vessel development, but also contributes significantly to vascular diseases including atherosclerosis (Owens et al. 2004). The molecular machinery governing smooth muscle cell behaviour is of course complex and multifactorial. One important theme is ion channels, as is apparent in reviews within this issue of The Journal of Physiology. Ion channels are able to regulate different patterns of rhythmicity in smooth muscle tissues and control calcium influx, which is important for contraction but also profoundly influences many other cellular mechanisms. Probably the first directly observed ion channel of vascular smooth muscle cells – over two decades ago – was the large conductance calcium-activated potassium channel (commonly known as BKCa or maxiK; or in IUPHAR nomenclature KCa1.1). This channel is activated by elevated intracellular calcium and depolarization, and is reviewed in this issue by Lu et al. (2006). In contractile smooth muscle cells it provides hyperpolarizing counter balance against depolarization, limiting calcium-influx through L-type voltage gated calcium channels. When the cells switch to proliferative phenotype, BKCa is, however, down-regulated, along with the L-type calcium channel (Neylon et al. 1999). This does not mean that calcium and ion channels become unimportant but, for reasons not well understood, the proliferating cells use other ion channels. One of these is the intermediate conductance calcium-activated potassium channel (commonly known as IKCa, or in IUPHAR nomenclature KCa3.1). Unlike BKCa, IKCa does not depend on depolarization to facilitate its opening and thus can open at more hyperpolarized membrane potentials. It is suggested this is useful for cells having lost L-type calcium channels, i.e. that hyperpolarization driven by IKCa serves to enhance the electrochemical gradient for calcium entry through voltage-independent calcium channels. Consistent with this hypothesis, blockade of IKCa suppresses vascular smooth muscle cell proliferation and neointimal hyperplasia (Neylon et al. 1999; Cheong et al. 2005). Intriguingly, lymphocyte activation (Chandy et al. 2004) and proliferation in other cell types involves a similar mechanism, suggesting potassium channel activation and the consequent hyperpolarization of the membrane potential is a conserved and key mechanism enabling cell proliferation. In this issue of The Journal of Physiology, Ivanov et al. (2006) address an important question: how might a growth factor – in this case epidermal growth factor (EGF) – initiate the process of phenotypic modulation when the contractile smooth muscle cells express BKCa and not IKCa? In an elegant series of experiments including electrophysiology and injection into the cisterna magna they make progress towards answering the question, critically showing that EGF and related EGF-receptor ligands hyperpolarize smooth muscle cells of the basilar artery by activating BKCa, and that iberiotoxin – a highly specific blocker of BKCa– prevents EGF-evoked induction of a cell proliferation marker in vivo. Unanswered is how this BKCa-mediated hyperpolarization might lead to cell proliferation. With reference to a study of transient receptor potential channels in neuronal growth cones Ivanov et al. (2006) suggest EGF might concomitantly induce voltage-independent calcium entry channels. By implication, L-type calcium channels might also be expected to be down-regulated. Furthermore, there is the matter of EGF evoking hyperpolarization in the context of its observed role as a potent vasonconstrictor. These will be important matters to address in future studies. At this stage, Ivanov et al. provide good supporting data for the central importance of potassium channels in the early phases of cell proliferation, revealing that existing BKCa channels of contractile vascular smooth muscle cells can serve this function prior to the transition to IKCa.

Role of SKCa and IKCa in endothelium‐dependent hyperpolarizations of the guinea‐pig isolated carotid artery

1. This study was designed to determine whether the endothelium-dependent hyperpolarizations evoked by acetylcholine in guinea-pig carotid artery involve a cytochrome P450 metabolite and whether they are linked to the activation of two distinct populations of endothelial K(Ca) channels, SK(Ca) and IK(Ca.) 2. The membrane potential was recorded in the vascular smooth muscle cells of the guinea-pig isolated carotid artery. All the experiments were performed in the presence of N(omega)-L-nitro arginine (100 microM) and indomethacin (5 microM). 3. Under control conditions (Ca(2+): 2.5 mM), acetylcholine (10 nM to 10 muM) induced a concentration- and endothelium-dependent hyperpolarization of the vascular smooth muscle cells. Two structurally different specific blockers of SK(Ca), apamin (0.5 microM) or UCL 1684 (10 microM), produced a partial but significant inhibition of the hyperpolarization evoked by acetylcholine whereas charybdotoxin (0.1 microM) and TRAM-34 (10 microM), a nonpeptidic and specific blocker of IK(Ca), were ineffective. In contrast, the combinations of apamin plus charybdotoxin, apamin plus TRAM-34 (10 microM) or UCL 1684 (10 microM) plus TRAM-34 (10 microM) virtually abolished the acetylcholine-induced hyperpolarization. 4. In the presence of a combination of apamin and a subeffective dose of TRAM-34 (5 microM), the residual hyperpolarization produced by acetylcholine was not inhibited further by the addition of either an epoxyeicosatrienoic acid antagonist, 14,15-EEZE (10 microM) or the specific blocker of BK(Ca), iberiotoxin (0.1 microM). 5. In presence of 0.5 mM Ca(2+), the hyperpolarization in response to acetylcholine (1 microM) was significantly lower than in 2.5 mM Ca(2+). The EDHF-mediated responses became predominantly sensitive to charybdotoxin or TRAM-34 but resistant to apamin. 6. This investigation shows that the production of a cytochrome P450 metabolite, and the subsequent activation of BK(Ca), is unlikely to contribute to the EDHF-mediated responses in the guinea-pig carotid artery. Furthermore, the EDHF-mediated response involves the activation of both endothelial IK(Ca) and SK(Ca) channels, the activation of either one being able to produce a true hyperpolarization.

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.

Description and role in proliferation of iberiotoxin-sensitive currents in different human mammary epithelial normal and cancerous cells

Several studies suggested that potassium channels are involved in the proliferation of cancer cells but the involvement of the large conductance Ca 2+-activated K + channels (BK Ca) in the cancerous phenomenon is still controversial. In the present study, we used iberiotoxin, a specific blocker of BK Ca, and report the activity of an iberiotoxin-sensitive current in various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-435s) as well as in normal mammary epithelial cells (HME). Iberiotoxin and NS1619, an activator of BK Ca, did not interfere with either cell proliferation or with the invasive properties of the cells, under normal culture conditions. However, extracellular pulses of ATP, which induced transient increases in intracellular Ca 2+ concentration, revealed a significant reduction effect of iberiotoxin on cell proliferation. We conclude that the iberiotoxin-sensitive current is not involved in cell proliferation in basal conditions but participates when the intracellular Ca 2+ concentration is increased. These experiments also suggest that BK Ca channels are not involved in the cancerous transformation and are probably a relic from normal cells.

Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity.

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.

Potassium channels in human umbilical artery cells.

To identify K+ channels of smooth muscle of human umbilical artery using the patch-clamp technique and to study their effect on resting tone of umbilical artery rings. Whole-cell and single-channel patch-clamp recordings in enzymatically isolated smooth muscle cells were made. Measurements of developed isometric force were performed on intact tissue. Delayed rectifier K+ channels (KDR) and large-conductance Ca2+-activated K+ channels (BKCa) contribute to the whole-cell voltage- and time-dependent outward K+ current, as it was specifically inhibited by 5 mM 4-aminopyridine (4-AP; KDR blocker) (92 +/- 4% at 0 mV, n = 7), by 1 mM tetraethylammonium (TEA; BKCa blocker) (71 +/- 4% at +60 mV, n = 4), and by 200 nM iberiotoxin (BKCa blocker) (64 +/- 7% at +60 mV, n = 4). In outside-out patches, BKCa channels had a single-channel conductance of 132 +/- 4 pS (n = 24) in asymmetric K+ conditions and 216 +/- 4 pS (n = 4) in a symmetric K+ gradient. The activity of the BKCa channels was significantly augmented by 1 microM Ca2+ in the inside-out configuration. 4-AP had no effect on resting tone of intact arterial rings. TEA produced contraction of arterial rings whereas phloretin, an activator of BKCa, relaxed them, which means that BKCa channels are functional in intact tissue and are involved in the maintenance of resting tone in this human vessel. The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.

Large conductance Ca2+-activated K+ channels sense acute changes in oxygen tension in alveolar epithelial cells.

The rise in alveolar oxygen tension (PO(2)) that occurs as the newborn infant takes its first breaths induces removal of liquid from the lung lumen due to ion transport across the alveolar epithelium and the activity of alveolar Na(+) channel (ENaC). In the present study, we have aimed to identify an ion conductance in alveolar epithelial A549 cells that responds to acute changes in PO(2). Variation in PO(2) did not affect single-channel ENaC activity. However, in these cells we have detected single-channel conductance having properties similar to those of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Reverse transcriptase-polymerase chain reaction and Western blotting demonstrated presence of alpha-BKCa channel subunit and iberiotoxin, a blocker of BK(Ca) channels, inhibited whole cell K(+) current. Chronic changes in PO(2) did not affect expression, recruitment, or function of BK(Ca) channels in A549 cells. In contrast, acute changes of PO(2) regulated the BK(Ca) channel activity by controlling the channel mean open time. This effect of PO(2) was insensitive to inhibitor of flavoproteins, diphenylene iodinium. In addition, decrease in PO(2) and iberiotoxin induced membrane depolarization and Ca(2+) oscillations in A549 cells. We conclude that BK(Ca) channels serve as oxygen sensors in human alveolar A549 epithelial cells.

Identification and regulation of K + and Cl − channels in human parotid acinar cells

The properties of K + channels in these cells were studied using patch-clamp methods. Two channels, with conductances of 165±13 pS ( n=6) and 30±1 pS ( n=3), were identified in single-channel experiments. In cell-attached patches the reversal potentials were −67±8 and −74±2 mV for the large and small conductance channel, respectively, suggesting that both channels are K +-selective. The large conductance channel was also shown to be K +-selective in inside-out patches. The open probability ( P o) of this channel was increased at depolarizing potentials and by increasing intracellular Ca 2+ concentration ([Ca 2+] i). These properties suggest that the large conductance channel is a ‘maxi’ Ca 2+-activated K + channel (BK Ca). The small conductance channel was not observed in inside-out patches. Carbachol (CCh; 10 −5 M) activated the BK Ca channel, but not the small conductance channel, in cell-attached patches. CCh also caused a dose-dependent increase in [Ca 2+] i measured by fura-2 in microspectrofluorimetric studies, with a half-maximal response at approximately 3×10 −6 M. Neither isoproterenol (10 −5 M) nor substance P (10 −6 M) affected K +-channel activity or [Ca 2+] i. In whole-cell experiments, CCh caused an increase in outward current. Charybdotoxin (10 −7 M), a BK Ca blocker, inhibited a large component of the CCh-induced current. A large component of the charybdotoxin-insensitive current may be carried by Ca 2+-activated Cl − channels, which were also observed in human parotid acinar cells. The results indicate that BK Ca channels make a significant contribution to the whole-cell conductance in human parotid acinar cells.

BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy.

We investigated the influence of pregnancy on large-conductance calcium-activated potassium channel (BK(Ca)) activity (NP(o)) and on channel expression in membranes of isolated human myometrial smooth muscle cells. NP(o) in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and the half-maximal activation potential was shifted by 39 mV to more negative potentials. This effect was not due to an enhanced BK(Ca) channel expression. In the presence of cAMP kinase (PKA) or cGMP kinase (PKG), NP(o) increased in patches from PM but decreased in those from NPM. Western blot analysis and use of a specific PKG inhibitor (1 microM KT-5823) verified the existence of a partially active membrane-associated PKG. Inhibition of PKA by 100 nM PKI, the inhibitory peptide of PKA, had no effect on NP(o). 8-p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect was abolished by iberiotoxin, a specific blocker of BK(Ca) channels. It is concluded that an endogenous, membrane-bound PKG in myometrial cells specifically enhances BK(Ca) channel activity during pregnancy and thus may contribute to uterine quiescence during pregnancy.

Histamine increases [Ca(2+)](in) and activates Ca-K and nonselective cation currents in cultured human capillary endothelial cells.

We characterized the effects of histamine on intracellular Ca(2+) and activation of ionic currents in human capillary endothelial cells. Histamine produced both a transient and sustained increase in intracellular Ca(2+). The transient response was mediated largely through intracellular Ca(2+) release and the sustained response was due to extracellular Ca(2+) entry. The increase in intracellular Ca(2+) by histamine was not affected by the H2 blocker cimetidine. But was entirely blocked by the H1 antagonist diphenhydramine showing that the histamine response in these cells is mediated through the H1 receptor. A transient ionic current is coactivated with the histamine-induced increase in intracellular Ca(2+) and this current has several properties of a nonselective, Ca(2+) permeable, cation channel (NSC). The magnitude of the NSC current does not strictly correlate with intracellular Ca(2+) levels. A Ca(2+)-activated K(+) current (BKCA) is activated by the increase in intracellular Ca(2+) and this current is blocked by the selective BKCA blocker iberiotoxin.

Hydralazine-induced vasodilation involves opening of high conductance Ca 2+-activated K + channels

The purpose of this study was to investigate whether high conductance Ca 2+-activated K + channels (BK Ca) are mediating the vasodilator action of hydralazine. In isolated porcine coronary arteries, hydralazine (1–300 μM), like the K + channel opener levcromakalim, preferentially relaxed contractions induced by K + (20 mM) compared with K + (80 mM). In addition, concentration–relaxation curves for hydralazine (pD 2=5.38±0.06; E max=85.9±3.6%) were shifted 10-fold to the right by the BK Ca blockers tetraethylammonium (1 mM) and iberiotoxin (0.1 μM). In contrast, nimodipine (a Ca 2+-entry blocker), relaxed contractions induced by K + (20 mM) and K + (80 mM) equally and nimodipine-induced relaxations were neither antagonized by tetraethylammonium nor by iberiotoxin. In isolated perfused rat hearts, hydralazine (1 μM) increased coronary flow by 28.8±2.7%. Iberiotoxin (0.1 μM) suppressed this response by 82% ( P<0.05). In conscious, chronically catheterized rats the hypotensive response to hydralazine (0.6 mg kg −1 min −1) was significantly reduced by 41% during infusion of iberiotoxin (0.1 mg kg −1). It is concluded, that opening of BK Ca takes part in the mechanism whereby hydralazine produces vasodilation.

A K+ single channel and whole-cell clamp study on the effects of levcromakalim in guinea pig portal vein cells

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K+ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K+ in bath/pipette), 10 microM levcromakalim activated K+ channels with a chord conductance of 23.2 pS (K(KCO)), which were sensitive to the blocker of ATP-dependent K+ channels (K(ATP)), glibenclamide. Voltage steps from -80 mV to +20 mV activated 4-aminopyridine-sensitive K+ channels of 6.5 pS with properties of delayed rectifier K+ channels (Kv). In patches which upon a previous voltage step had revealed the existence of Kv, levcromakalim reduced the open-probability of Kv, but it did not concomitantly activate K(KCO). During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K+ channels (BK(Ca)) appeared which could be inhibited by iberiotoxin, a selective blocker of BK(Ca), and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to -100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 microM levcromakalim, the KCO-induced K+ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K+]i versus low [K+]o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of Kv to K(ATP) (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK(Ca) (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K(ATP) in other tissues.

Inhibitory effects of large Ca2+-activated K+ channel blockers on beta-adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles.

In human bronchi, relaxations to salbutamol and sodium nitroprusside were performed in the presence or absence of blockers of the large Ca2+-activated K+-channels (BKCa): charybdotoxin (Chtx), iberiotoxin (Ibtx) or tetraethylammonium (TEA). In bronchi under basal tone in presence of indomethacin (1 microM) or precontracted with acetylcholine (in presence or absence of indomethacin), the relaxations to salbutamol or sodium nitroprusside were unaffected or weakly inhibited by pretreatment with the BKca blockers (Chtx (100 nM), Ibtx (100 and 300 nM) and TEA (1 mM)). Significant inhibitions were mainly observed with TEA (1 mM) and iberiotoxin at high concentration (300 nM). These results contrasts with the potent inhibitory effects exerted by Chtx (100 nM) or Ibtx (100 nM) in guinea-pig trachea precontracted with acetylcholine in absence or presence of indomethacin indicating that human airways are less susceptible to BKCa blockade than guinea-pig airways. In addition, the BKCa blockers induced slowly developing contractions of human bronchi at basal tone. The contraction induced by TEA (1 mM) was abolished by verapamil (10 microM) suggesting that BKca blockade promotes an increase in membrane Ca2+-conductance through activation of voltage-gated Ca2+-channels. Verapamil also reversed the effects of TEA on salbutamol-induced relaxations in human bronchi as well as the effects of Ibtx on salbutamol- or sodium nitroprusside-induced relaxations in guinea-pig trachea. These data suggest that BKCa blockers induce activation of voltage-gated Ca2+-channels and therefore influx of Ca2+ which in turn cause a functional antagonism of beta2-adrenoceptor-agonist- and NO-donor-induced relaxations. Moreover, the BKCa opener, NS-1619, induced weak relaxations in human bronchi and guinea-pig trachea which were not blocked by TEA or Ibtx suggesting that BKCa opening is of minor significance for the relaxation of human airway smooth muscles. In conclusion, although a wealth of studies have demonstrated that beta-adrenoceptor agonists or NO-donors activate BKCa, the present study provides evidence that in human bronchi, as recently suggested in guinea-pig trachea, opening of BKCa does not appear to functionally participate in the relaxation to these relaxant agents.