Large-conductance KCa Channels Research Articles

Selective Blockade of the Intermediate-Conductance Ca 2+ -Activated K + Channel Suppresses Proliferation of Microvascular and Macrovascular Endothelial Cells and Angiogenesis In Vivo

Ca2+-activated K+ (K(Ca)) channels have been proposed to promote mitogenesis in several cell types. Here, we tested whether the intermediate-conductance K(Ca) channel (IKCa1) and the large-conductance K(Ca) channel (BK(Ca)) contribute to endothelial cell (EC) proliferation and angiogenesis. Function and expression of IKCa1 and BK(Ca)/Slo were investigated by patch-clamp analysis and real-time RT-PCR in human umbilical vein ECs (HUVECs) and in dermal human microvascular ECs 1 (HMEC-1). HMEC-1 expressed IKCa1 and BK(Ca)/Slo, whereas HUVECs expressed IKCa1. A 48-hour exposure to basic fibroblast growth factor (bFGF) augmented IKCa1 current amplitudes and induced a 3-fold increase in IKCa1 mRNA expression in HUVECs and HMEC-1. Vascular endothelial growth factor (VEGF) was also effective in upregulating IKCa1. BK(Ca)/Slo expression and current amplitudes in HMEC-1 were not altered by bFGF. bFGF- and VEGF-induced EC proliferation was suppressed by charybdotoxin, clotrimazole, or the selective IKCa1 blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), whereas inhibition of BK(Ca)/Slo by iberiotoxin was ineffective. In the Matrigel plug assay in mice, administration of TRAM-34 for 2 weeks significantly suppressed angiogenesis by approximately 85%. bFGF and VEGF upregulate expression of IKCa1 in human ECs. This upregulation of IKCa1 seems to be required for mitogen-induced EC proliferation and angiogenesis in vivo. Selective IKCa1 blocker might be of therapeutic value to prevent tumor angiogenesis.

Akt Activation Is Necessary for Growth Factor–Induced Trafficking of Functional KCaChannels in Developing Parasympathetic Neurons

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance K(Ca) channels in ciliary ganglion (CG) neurons evoked by beta-neuregulin-1 (NRG1) and transforming growth factor-beta1 (TGFbeta1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGF(beta)1. NRG1- and TGF(beta)1-evoked stimulation of K(Ca) is blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional K(Ca) channels. NRG1 and TGF(beta)1 evoked an Akt-dependent increase in cell-surface SLO alpha-subunits. These procedures have no effect on voltage-activated Ca2+ currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

Role of endothelial intermediate conductance KCa channels in cerebral EDHF-mediated dilations.

The present study evaluated the role of endothelial intermediate conductance calcium-sensitive potassium channels (IKCa) in the mechanism of endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in pressurized cerebral arteries. Male rat middle cerebral arteries (MCA) were mounted in an isolated vessel chamber, pressurized (85 mmHg), and luminally perfused (100 microl/min). Artery diameter was measured simultaneously with either endothelial intracellular Ca2+ concentration ([Ca2+]i; fura-2) or changes in endothelial membrane potential [4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS)]. Nitric oxide synthase and cyclooxygenase inhibitors were present throughout. Luminal application of UTP produced EDHF-mediated dilations that correlated with significant endothelial hyperpolarization. The dilation and endothelial hyperpolarization were virtually abolished by inhibitors of IKCa channels but not by selective inhibitors of small or large conductance KCa channels (apamin and iberiotoxin, respectively). Additionally, direct stimulation of endothelial IKCa channels with 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial hyperpolarization and vasodilatation that were blocked by inhibitors of IKCa channels. 1-EBIO hyperpolarized the endothelium but did not affect endothelial [Ca2+]i. We conclude that the mechanism of EDHF-mediated dilations in cerebral arteries requires stimulation of endothelial IKCa channels to promote endothelial hyperpolarization and subsequent vasodilatation.

Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function

The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.

Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function.

The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca2+ currents and Ca2+-activated K+ channels (KCa channels) is important for Purkinje cell activity, but how many different KCa channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca2+ channel and KCa channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca2+ entering through P-type voltage-gated Ca2+ channels activates both small-conductance (SK) and large-conductance (BK) KCa channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.

Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo.

Recent studies in vitro have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans. The aim of this study was to evaluate our hypothesis that endothelium-derived H2O2 is an EDHF in vivo and plays an important role in coronary autoregulation. To test this hypothesis, we evaluated vasodilator responses of canine (n=41) subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) with an intravital microscope in response to acetylcholine and to a stepwise reduction in coronary perfusion pressure (from 100 to 30 mm Hg) before and after inhibition of NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). After L-NMMA, the coronary vasodilator responses were attenuated primarily in small arteries, whereas combined infusion of L-NMMA plus catalase (an enzyme that selectively dismutates H2O2 into water and oxygen) or tetraethylammonium (TEA, an inhibitor of large-conductance K(Ca) channels) attenuated the vasodilator responses of coronary arteries of both sizes. Residual arteriolar dilation after L-NMMA plus catalase or TEA was largely attenuated by 8-sulfophenyltheophylline, an adenosine receptor inhibitor. These results suggest that H2O2 is an endogenous EDHF in vivo and plays an important role in coronary autoregulation in cooperation with NO and adenosine.

Fast, but not slow, effects of olivocochlear activation are resistant to apamin.

Olivocochlear (OC) efferent suppression of auditory-nerve responses comprises a fast effect lasting tens of milliseconds and a slow effect building and decaying over tens of seconds. Both fast and slow effects are mediated by activation of the same alpha 9 nicotinic receptor. We have hypothesized that fast effects are generated at the OC synapse, but that slow effects reflect activation of calcium-activated potassium (K(Ca)) channels by calcium release from the subsurface cisternae on the basolateral wall of the hair cells. We measured in vivo effects of apamin, a blocker of small-conductance (SK) K(Ca) channels, and charybdotoxin, a blocker of large-conductance K(Ca) channels, perfused through scala tympani, on fast and slow effects evoked by electrical stimulation of the OC bundle in anesthetized guinea pigs. Apamin selectively and reversibly reduced slow-effect amplitude without altering fast effects or baseline amplitude of the auditory-nerve response, but only when perfused at concentrations of 100 microM. In contrast, the effects of charybdotoxin were noted at 30 nM, but were not specific, reducing both afferent and efferent responses. The very high concentrations of apamin needed to block efferent effects contrasts with the high sensitivity of isolated hair cells to apamin's block of acetylcholine's effects. The results suggest that in vivo fast OC effects are dominated by a conductance that is not apamin sensitive.

BK-Type KCaChannels in Two Parasympathetic Cell Types: Differences in Kinetic Properties and Developmental Expression

The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca(2+)-activated K(+) channels (K(Ca)), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic K(Ca) carried by a kinetically homogeneous population of large-conductance (BK-type) K(Ca) channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic K(Ca) produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying K(Ca) channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of K(Ca) channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of K(Ca) channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance K(Ca) channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional K(Ca) channels appears to be regulated differently in the two cell types. Although both cell types acquire functional K(Ca) at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional K(Ca) channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain K(Ca) stimulatory activity. However, K(Ca) stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.

Expression and Function of Endothelial Ca 2+ -Activated K + Channels in Human Mesenteric Artery

Ca(2+)-activated K(+) (K(Ca)) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase-polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K(Ca) channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K(Ca) channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non-cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K(Ca) channel (hIK1) was detected in non-cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K(Ca) currents, whereas respective K(Ca) currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non-cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (P:<0.05). However, K(Ca) current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K(Ca) channel (hSlo) was detected in single ECs from CA patients. The increased K(Ca) channel expression in CA patients resulted in a 2. 7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (P:<0.05). This increased expression and function of K(Ca) channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.

Subplasmalemmal ryanodine-sensitive Ca2+ release contributes to Ca2+-dependent K+ channel activation in a human umbilical vein endothelial cell line.

The whole-cell configuration of the patch clamp technique was used to assess the involvement of ryanodine-sensitive Ca2+ release (RsCR) in histamine-activated Ca2+-dependent K+ (KCa) channels in the human umbilical vein endothelial cell line EA.hy926. Histamine (10 microM) induced a transient outward current that reached 18.9 +/- 5.5 pA pF-1 at +20 mV. This current was diminished by 1 mM tetraethylammonium or 50 nM iberiotoxin, by 90 % and 80 %, respectively, suggesting that this current results from the stimulation of large-conductance KCa (BKCa) channels. In about 50 % of the cells tested, stimulation of RsCR with 200 nM ryanodine initiated a small outward current that was also sensitive to iberiotoxin. Following the ryanodine-mediated RsCR, the potency of 10 microM histamine to activate KCa channels was reduced by about 60 %. In agreement, an inhibition of RsCR with 25 microM ryanodine diminished KCacurrent in response to histamine by about 70 %. The effect of 100 microM histamine on KCa channel activity was not reduced by previous RsCR with 200 nM ryanodine, or by an inhibition of RsCR by 25 microM ryanodine. Histamine (10 microM)-induced Ca2+ elevation was reduced by 30 % following ryanodine-mediated RsCR, whereas no inhibition occurred in the case of 100 microM histamine stimulation. In cells treated with 10 microM nocodazole for 16 h to collapse the superficial endoplasmic reticulum, 200 nM ryanodine failed to initiate any KCa current. Furthermore, the inhibitory effect of previous RsCR on 10 microM histamine-induced KCa current was not obtained in nocodazole-treated cells. Our data suggest that during moderate cell stimulation (10 microM histamine), subplasmalemmal RsCR greatly contributes to the activation of KCa channels in endothelial cells. Thus, the function of the subplasmalemmal Ca2+ control unit (SCCU) described previously must be extended as a regulator for KCa channels.

Molecular cloning and characterization of the intermediate-conductance Ca(2+)-activated K(+) channel in vascular smooth muscle: relationship between K(Ca) channel diversity and smooth muscle cell function.

Recent evidence suggests that functional diversity of vascular smooth muscle is produced in part by a differential expression of ion channels. The aim of the present study was to examine the role of Ca(2+)-activated K(+) channels (K(Ca) channels) in the expression of smooth muscle cell functional phenotype. We found that smooth muscle cells exhibiting a contractile function express predominantly large-conductance ( approximately 200 pS) K(Ca) (BK) channels. In contrast, proliferative smooth muscle cells express predominantly K(Ca) channels exhibiting a much smaller conductance ( approximately 32 pS). These channels are blocked by low concentrations of charybdotoxin (10 nmol/L) but, unlike BK channels, are insensitive to iberiotoxin (100 nmol/L). To determine the molecular identity of this K(+) channel, we cloned a 1.9-kb cDNA from an immature-phenotype smooth muscle cell cDNA library. The cDNA contains an open reading frame for a 425 amino acid protein exhibiting sequence homology to other K(Ca) channels, in particular with mIK1 and hIK1. Expression in oocytes gives rise to a K(+)-selective channel exhibiting intermediate-conductance (37 pS at -60 mV) and potent activation by Ca(2+) (K(d) 120 nmol/L). Thus, we have cloned and characterized the vascular smooth muscle intermediate-conductance K(Ca) channel (SMIK), which is markedly upregulated in proliferating smooth muscle cells. The differential expression of these K(Ca) channels in functionally distinct smooth muscle cell types suggests that K(Ca) channels play a role in defining the physiological properties of vascular smooth muscle.

Prostaglandin E1 activates the large-conductance KCa channel in human corporal smooth muscle cells.

The large conductance calcium-sensitive potassium channel (KCa or maxi-K) is an important modulator of human corporal smooth muscle tone, and therefore, erectile capacity. The goal of this investigation was to evaluate the actions of prostaglandin E1 (PGE1), the most widely used and effective drug for the treatment of impotence, on the activity of the KCa channel, a prominent K+ current present in human corporal smooth muscle. Whole-cell patch clamp studies conducted on short-term cultured and enzymatically dissociated human corporal smooth muscle cells, revealed mean resting potentials of -50.8 +/- 2.1 mV (n = 8) and -34 +/- 4 mV (n = 8), respectively. In the attached-patch configuration, the corresponding single-channel slope conductance values for the KCa channel subtype were 173 +/- 4 pS (n = 8) in cultured cells, and 190 +/- 13 pS (n = 3) in freshly isolated myocytes. Furthermore, voltage clamp experiments revealed that relative to control values, the application of PGE1 to cultured cells (3.3 or 33 microM) elicited an apparent increase in both the open probability (Po; ranging from 1.2-23 fold), and the mean open time (5-6 fold) of the KCa channel at membrane potentials of +90 mV and +110 mV. PGE1-induced alterations in KCa channel activity were also observed in freshly isolated corporal myocytes. In the whole cell-recording mode, statistically significant, Charybdotoxin-sensitive (100 nM) 2-3 fold increases in the outward K+ currents were observed in both cultured and freshly isolated corporal myocytes. The presence of a PKA inhibitor (fragment 6-22 amide; 10 microM) in the pipette tip was also associated with a nearly complete ablation of the observed PGE1-induced whole cell K+ currents. Taken together, these data confirm and extend our previous observations, and indicate that PGE1-induced relaxation of human corporal smooth muscle is related, at least in part, to activation of the KCa channel subtype resulting in cellular hyperpolarization.

Modulation of ion channels: a "current" view of AKAPs.

Modulation of ion channels: a "current" view of AKAPs.

Nitric oxide-independent activation of soluble guanylyl cyclase contributes to endotoxin shock in rats.

We investigated whether a complete inhibition of nitric oxide (NO) formation caused by bacterial endotoxin (lipopolysaccharide, LPS) in vivo prevents the hypotension and restores the vascular hyporeactivity to normal in vivo and ex vivo. The combination of dexamethasone (Dex; 3 mg/kg at 30 min before LPS) plus aminoguanidine (AG; 15 mg/kg at 2 h after LPS) inhibited the overproduction of nitrate (an indicator of NO) in the plasma and aortic smooth muscle and also prevented the development of the delayed hypotension in rats treated with LPS for 6 h. However, the vascular hyporeactivity to norepinephrine (NE) was only partially improved either in vivo or ex vivo in endotoxemic rats treated with Dex plus AG. Pretreatment of aortic rings with Nomega-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2, 4]oxidazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced the contraction to NE in rings obtained from LPS-treated rats, but not in those from Dex plus AG-treated endotoxemic rats. Methylene blue, an inhibitor of soluble guanylyl cyclase (GC), completely restored contractions to NE and aortic cGMP levels to normal either in LPS-treated rats or in Dex plus AG-treated endotoxemic rats, whereas the cGMP level was partially inhibited by ODQ in LPS-treated rats only. These results suggest that non-NO mediator(s) also activates soluble GC during endotoxemia. Interestingly, we found that in the presence of tetraethylammonium (an inhibitor of K+ channels) plus L-NAME or charybdotoxin [a specific inhibitor of large-conductance Ca2+-activated K+ (KCa) channels] plus ODQ, the vascular hyporeactivity to NE in the LPS-treated group was also completely restored to normal. In addition, in the presence of L-NAME or ODQ, the vascular hyporeactivity to high K+ was abolished in rings from the LPS-treated group. These results suggest that LPS causes the production of other mediator(s), in addition to NO, which also stimulates soluble GC (i.e., increases the formation of cGMP) and then activates the large-conductance KCa channels in the vascular smooth muscle causing vascular hyporeactivity.

Study on the Mechanism of Hypoxic Induced Vasodilatation and Vasoconstriction

Background：Although hypoxic pulmonary vasoconstriction (HPC and hypoxic coronary vasodilatation (HCD have been recognized by many researchers, the precise mechanism remains unknown. As isolated arteries will constrict or relax in vitro in response to hypoxia, the oxygen sensor/transduction mechanism must reside in the arterial smooth muscle, the endothelium, or both. Unfortunately, much of the current evidence is conflicting, especially concerning to the dependency of HPC and HCD on the endothelium and the role of the K + channel. Therefore, this experiment was attempted to clarify the dependency of HPC and HCD on the endothelium and the role of the K + channel on HPC and HCD. Methods：HPC was investigated in isolated main pulmonary arteries precontracted with norepinephrine (NE. HCD was investigated in isolated left circumflex coronary artery precontracted with prostaglandin F2α. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit solution. Hypoxia was induced by gassing the chamber with 95% N2＋5% CO2, which was maintained for 15-25 min. Results：1 Hypoxia elicited a vasoconstriction in NEprecontracted pulmonary arteries with endothelium, but a vasodilatation in PGF2α -precontracted coronary arteries with and without endothelium. There was no difference between the amplitude of the HPC and HCD induced by two consecutive hypoxic challenges and the effect of normoxic and hyperoxic control Krebs-Henseleit solution on subsequent response to hypoxia. 2 Inhibition of NO synthesis by the treatment with Nω w-nitro-L-arginine reduced HPC in pulmonary arteries, but inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HPC and HCD, respectively. 3 Blockades of the TEA-sensitive K + channel abolished HPC and HCD. 4 Apamin, a small conductance Ca2+-activated K + (KCa channel blocker, and iberiotoxin, a large conductance KCa channel blocker, had no effect on the HCD. 5 Glibenclamide, an ATP-sensitive K + (KATP channel blocker, reduced HCD. 6 Cromakalim, an KATP channel opener, relaxed the coronary artery precontracted with prostaglandin F2α . The degree of relaxation by cromakalim was similar to that by hypoxia and glibenclamide reduced both hypoxia- and cromakalim-induced vasodilations. 7 Verapamil, a Ca 2+ entry blocker, caffeine, a Ca 2+ emptying drug；and ryanodine, an inhibitor of Ca 2+ release from SR, reduced HPC, respectively. Conclusion：HPC is dependent on the endothelium and is considered to be induced by inhibition of the mechanisms of NO-dependent vasodilation while HCD is independent of the endothelium and is considered to be induced by activation of the K ATP channel. (Korean Circulation J 1998;28(12 :2011-2029

Mechanisms of relaxation of coronary artery by hypoxia.

This study was designed to clarify the dependency of hypoxic coronary vasodilation (HCD) on the endothelium and the role of the K+ channels on HCD in the rabbit coronary artery. HCD was investigated in an isolated left circumflex coronary artery precontracted with prostaglandin F2 alpha. Vascular rings were suspended for isometric tension recording in an organ chamber filled with Krebs-Henseleit (KH) solution. Hypoxia was induced by gassing the chamber with 95% N2 + 5% CO2 and was maintained for 15 approximately 25 min. Hypoxia elicited a vasodilation in the precontracted coronary artery with and without endothelium. There was no difference between the amplitude of the HCD induced by two consecutive hypoxic challenges and the effects of 20% O2 + 5% CO2 + 75% N2 and 95% O2 + 5% CO2 control K-H solution of subsequent responses to hypoxia. Inhibition of the cyclooxygenase pathway by treatment with indomethacin had no effect on HCD. Blockades of the tetraethylammonium chloride-sensitive K+ channel abolished HCD. Apamin, a blocker of the small conductance Ca(2+)-activated K+ (KCa) channel, and iberiotoxin, a blocker of the large conductance KCa channel had no effect on HCD, respectively. Glibenclamide, a blocker of the ATP-sensitive K+ (K+ATP) channel, reduced HCD. Cromakalim, an opener of the K+ATP channel, relaxed the coronary artery precontracted with prostaglandin F2 alpha. The degree of relaxation by cromakalim was similar to that by hypoxia while glibenclamide reduced both hypoxia- and cromakalim-induced vasodilatations. In conclusion, these results suggest that HCD is independent on endothelium and HCD is considered to be induced by activation of K+ATP channel.

Types of potassium channels involved in coronary reactive hyperemia depend on duration of preceding ischemia in rat hearts

This study was undertaken to clarify factors other than nitric oxide involved in reactive hyperemia after a short(30 sec) and a long(300 sec) coronary global no-flow ischemia in isolated rat hearts perfused at a constant pressure(90 mmHg) with special focuses on the contribution of various K channels including large and small conductance Ca-activated K(K Ca) channels as well as ATP-sensitive K(K ATP) channels. Reactive hyperemia was induced following 30 sec and 300 sec of no-flow ischemia of the heart. Coronary reactive hyperemia was observed even after the inhibition of nitric oxide synthase by N ω-nitro-L-arginine methylester(L-NAME). Selected K channel blockers, none of which affected the basal flow, were used to evaluate contribution of K channels to this L-NAME-resistant reactive hyperemia. After 30-sec ischemia, tetraethylammonium(TEA: a non-selective K channel blocker), glibenclamide(Gli: a K ATP channel blocker) and α,β-methylene adenosine 5t́-diphosphonate(AOPCP: an inhibitor of ecto 5t́-nucleotidase) all suppressed both peak flow/basal flow(%PF) and repayment of flow debt(%RFD); After 300-sec ischemia, TEA and charybdotoxin(ChTX: a large conductance KCa channel blocker) decreased %PF and %RFD; AOPCP decreased both %RFD and duration. 4-aminopyridine (a voltage-dependent K channel blocker) decreased only duration. Neither apamin(a small conductance K Ca channel blocker) nor indomethacin(a cy clooxygenase inhibitor) affected the both types of reactive hyperemia. These findings suggest that opening of K ATP channel contributes to coronary vasodilation in reactive hyperemia after short 30-sec ischemia, and that opening of KCa, but not K ATP, channel contributes to it after long 300-sec ischemia. These results also suggest that adenosine may partly be involved in both types of reactive hyperemia.

Spontaneous activity of guinea pig ileum longitudinal muscle regulated by Ca(2+)-activated K+ channel.

Isolated guinea pig ileum displayed spontaneous contraction and fluctuation of membrane potential originating from the longitudinal muscle. Transmural stimulation of the enteric nerve plexus evoked contractions that were followed by lowered muscle tone and decreased spontaneous activity. The Na+ channel blocker tetrodotoxin, N-type Ca2+ channel blockers omega-conotoxins GVIA and MVIIA, the muscarinic receptor antagonist atropine, and inhibitory mediators alpha, beta-methylene-ATP and sodium nitroprusside all inhibited stimulation-evoked contraction while restoring spontaneous motility. L-type Ca2+ channel blockers nifedipine and calciseptine completely suppressed evoked and spontaneous contractions. Blockade of the large-conductance Ca(2+)-activated K+ (Kca) channel with charybdotoxin (but not of small-conductance Kca channel by apamin or of ATP-regulated K+ channel by glybenclamide) induced spikelike depolarization, inhibited nerve stimulation-evoked membrane hyperpolarization, and increased spontaneous activity. The results suggest that the large-conductance Kca channel is constitutively activated for modulations of spontaneous activity, and that muscle excitation, through elevation of Ca2+ levels, stimulates the large-conductance Kca channel to suppress spontaneous activity.

The potentiation of nitroglycerin-induced relaxation by PKG inhibition in rat aortic rings

1. 1. In rat aortic rings precontracted by phenylephrine, H7 (10 −5M) and staurosporine (10 −7M), which inhibit PKA, PKG and PKC, and H-89 (10 −6M), which inhibits PKA and PKG, potentiated relaxations induced by nitroglycerin. Forskolin-induced relaxations were not affected by H7 (10 −5M). 2. 2. Nitroglycerin-induced relaxations were not affected by calphostin-C (10 −7M), which inhibits PKC, H-89 (10 −7M), which inhibits PKA, and staurosporine (2 × 10 −9M), which inhibits PKC. 3. 3. Iberiotoxin (3 × 10 −8M), an inhibitor of large conductance Kca channels, partly inhibited the relaxation induced by nitroglycerin and completely inhibited the potentiating effect of H7 on nitroglyc. erin-induced relaxations. 4. 4. The potentiating effect of zaprinast (10 −5M), an inhibitor of cGMP-phosphodiesterase, on nitroglycerin-induced relaxation was not affected by iberiotoxin. In the presence of methylene blue (10 −5M), an inhibitor of guanylate cyclase, the residual relaxing response to nitroglycerin was not affected by H7, but it was inhibited by iberiotoxin. 5. 5. These results suggest that the potentiation of nitroglycerin-induced relaxation by H7, staurosporine and H-89 may be due to inhibition of PKG.

Increased Ca2+ influx in the resting state maintains the myogenic tone and activates charybdotoxin-sensitive K+ channels in femoral arteries from young SHR.

1. To determine the possible role of voltage-dependent Ca2+ channels (VDCC) and Ca2+-activated K+ (KCa) channels in the regulation of resting tone of arteries from young spontaneously hypertensive rats (SHR), mechanical responses to the agents which interact with these channels were examined in endothelium-denuded strips of femoral arteries from 4 week old SHR and age-matched normotensive Wistar-Kyoto (WKY) rats. Systolic blood pressures at this age were not significantly different between SHR and WKY. 2. The strips from SHR, but not from WKY, maintained a myogenic tone; that is, the resting tone decreased when nifedipine was added. 3. Studies using 1 or 5 min pulse labelling of the strips with 45Ca showed that the basal Ca2+ influx was increased in SHR when compared with WKY, and this increase in SHR was abolished by nifedipine. Similar results were obtained when the cytoplasmic Ca2+ concentration ([Ca2+]i) in the resting state of the strips was measured by fura-PE3. 4. The addition of charybdotoxin (ChTX, a blocker of large conductance KCa channels) to the resting state caused a concentration-dependent contraction, which was much greater in SHR than in WKY. The ChTX-induced contraction in SHR was abolished by nifedipine. 5. In strips preloaded with 86Rb, the basal 86Rb efflux rate constant was significantly greater in SHR than in WKY. The increase in 86Rb efflux in SHR was abolished by nifedipine. 6. The results suggest that the Ca2+ influx via L-type VDCC was increased in the resting state of the femoral artery from 4 week old SHR, and therefore the myogenic tone was maintained and ChTX-sensitive K+ channels were highly activated.