Thyrotropin-releasing hormone activates KCa channels in gastric smooth muscle cells via intracellular Ca2+ release

Thyrotropin-releasing hormone (TRH) is released in high concentrations into gastric juice, but its direct effect on gastric smooth muscles has not been studied yet. We undertook studies on TRH effect on gastric smooth muscle using contraction and patch clamp methods. TRH was found to inhibit both acetylcholine- and BaCl2-induced contractions of gastric strips. TRH, applied to single cells, inhibited the voltage-dependent Ca2+ currents and activated the whole-cell K+ currents. The TRH-induced changes in K+ currents and membrane potential were effectively abolished by inhibitors of either intracellular Ca2+ release channels or phospholipase C. Neither activators, nor blockers of protein kinase C could affect the action of TRH on K+ currents. In conclusion, TRH activates K+ channels via inositol-1,4,5-trisphosphate-induced release of Ca2+ in the direction to the plasma membrane, which in turn leads to stimulation of the Ca2+-sensitive K+ conductance, membrane hyperpolarization and relaxation. The data imply that TRH may act physiologically as a local modulator of gastric smooth muscle tone.     

Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca(2+) channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba(2+) as the charge carrier. Maximal peak Ba(2+) current (I(Ba)) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 +/- 3%). The NO donor S-nitroso-N-acetylpenicillamine also increased I(Ba), and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in I(Ba) was blocked when cells were pretreated with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (10 microM) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 microM) and milrinone (3 microM), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on I(Ba). Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca(2+) channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO.     

KCa channel insensitivity to Ca2+ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells

In smooth muscle cells, localized intracellular Ca2+ transients, termed "Ca2+ sparks," activate several large-conductance Ca2+-activated K+ (KCa) channels, resulting in a transient KCa current. In some smooth muscle cell types, a significant proportion of Ca2+ sparks do not activate KCa channels. The goal of this study was to explore mechanisms that underlie fractional Ca2+ spark-KCa channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca2+ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of -40, 0, and +40 mV, mean transient KCa current frequency was approximately 0.18, 0.43, and 0.26 Hz and KCa channel activity [number of KCa channels activated by Ca2+ sparksxopen probability of KCa channels at peak of Ca2+ sparks (NPo)] at the transient KCa current peak was approximately 4, 12, and 24, respectively. Depolarization between -40 and +40 mV increased KCa channel sensitivity to Ca2+ sparks and elevated the percentage of Ca2+ sparks that activated a transient KCa current from 59 to 86%. In a Ca2+-free bath solution or in diltiazem, a voltage-dependent Ca2+ channel blocker, steady membrane depolarization between -40 and +40 mV increased transient KCa current frequency up to approximately 1.6-fold. In contrast, caffeine (10 microM), an RyR channel activator, increased mean transient KCa current frequency but did not alter Ca2+ spark-KCa channel coupling. These data indicate that coupling is increased by mechanisms that elevate KCa channel sensitivity to Ca2+ sparks, but not by RyR channel activation. Overall, KCa channel insensitivity to Ca2+ sparks is a prominent factor underlying fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes.     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

大鼠结肠平滑肌细胞的钙库操纵性通道

目的:探讨大鼠结肠平滑肌细胞是否存在钙库操纵性通道(SOC)。方法:荧光探针Fura-2/AM标记细胞内游离Ca2+后,用荧光分光光度计检测毒胡萝卜素(thapsigargin)和咖啡因(caffeine)耗竭胞内钙库后激活的SOC通道对酶解分离的大鼠结肠平滑肌细胞[Ca2+]i的影响。结果:在无Ca2+缓冲液中,thapsigargin(1μmol/L)以及caf-feine(10 mmol/L)分别使[Ca2+]i由静息时(68.32±3.43)nmol/L升高至(240.85±12.65)nmol/L(、481.25±34.77)nmol/L,继之,向细胞外液中引入两种浓度的Ca2+(1.5 mmol/L和3.0 mmol/L),导致[Ca2+]i进一步升高,分别为(457.55±19.80)nmol/L、(1005.93±54.62)nmol/L;(643.88±34.65)nmol/L、(920.16±43.25)nmol/L。且上述升高效应对维拉帕米(verapamil,5μmol/L)以及KCl引起的细胞膜去极化不敏感,但可被La3+(1 mmol/L)抑制。结论:在酶解分离的大鼠结肠平滑肌细胞上,存在胞内钙库耗竭激活的SOC通道,为支持在电兴奋性细胞上存在库容性Ca2+内流提供了实验和理论依据。     

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells

Arterial smooth muscle cell large-conductance Ca²⁺-activated potassium (K_Ca) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. K_Ca channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca²⁺ transients, termed Ca²⁺ sparks, but hypoxic regulation of Ca²⁺ sparks and K_Ca channel activation by Ca²⁺ sparks has not been investigated. We report here that in voltage-clamped (–40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O₂ partial pressure from 150 to 15 mmHg reversibly decreased Ca²⁺ spark-induced transient K_Ca current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca²⁺ spark frequency, amplitude, global intracellular Ca²⁺ concentration, or sarcoplasmic reticulum Ca²⁺ load. Hypoxia reduced transient K_Ca current frequency by decreasing the percentage of Ca²⁺ sparks that activated a transient K_Ca current from 89% to 63%. Hypoxia reduced transient K_Ca current amplitude by attenuating the amplitude relationship between Ca²⁺ sparks that remained coupled and the evoked transient K_Ca currents. Consistent with these data, in inside-out patches at –40 mV hypoxia reduced K_Ca channel apparent Ca²⁺ sensitivity and increased the K_d for Ca²⁺ from 17 to 32 µM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces K_Ca channel apparent Ca²⁺ sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of K_Ca channels from Ca²⁺ sparks. Transient K_Ca current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation. transient calcium-activated potassium current     

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

MCI-154 activates the Ca2+-activated K+ channel of vascular smooth muscle cells

The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca²⁺-activated K⁺ channel (K_Ca channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 μM MCI-154 activated the K_Ca channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100μM MCI-154 to the cytosolic side activated the K_Ca channel directly, suggesting that the Ca₂₊ sensitivity of the K_Ca channel itself is modulated. Though extracellular application of 100 μM amrinone, a phosphodiesterase inhibitor, activated the K_Ca channel in the cell-attached patch configurations, application of 100 μm amrinone to the cytosolic side could not activate the K_Ca channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca²⁺ sensitivity of the K_Ca channel in vascular smooth muscle cells.     

SNAP-25 inhibits L-type Ca2+ channels in feline esophagus smooth muscle cells

We recently reported that non-secretory gastrointestinal smooth muscle cells also possessed SNARE proteins, of which SNAP-25 regulated Ca(2+)-activated (K(Ca)) and delayed rectifier K(+) channels (K(V)). Voltage-gated, long lasting (L-type) calcium channels (L(Ca)) play an important role in excitation-contraction coupling of smooth muscle. Here, we show that SNAP-25 could also directly inhibit the L-type Ca(2+) channels in feline esophageal smooth muscle cells at the SNARE complex binding synprint site. SNARE proteins could therefore regulate additional cell actions other than membrane fusion and secretion, in particular, coordinated muscle membrane excitability and contraction, through their actions on membrane Ca(2+) and K(+) channels.     

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.     

Carbon monoxide and Ca2+-activated K+ channels in cerebral arteriolar responses to glutamate and hypoxia in newborn pigs

Large-conductance calcium-activated potassium (K(Ca)) channels regulate the physiological functions of many tissues, including cerebrovascular smooth muscle. l-Glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system, and oxygen tension is a dominant local regulator of vascular tone. In vivo, glutamate and hypoxia dilate newborn pig cerebral arterioles, and both dilations are blocked by inhibition of carbon monoxide (CO) production. CO dilates cerebral arterioles by activating K(Ca) channels. Therefore, the present study was designed to investigate the effects of glutamate and hypoxia on cerebral CO production and the role of K(Ca) channels in the cerebral arteriolar dilations to glutamate and hypoxia. In the presence of iberiotoxin or paxilline that block dilation to the K(Ca) channel opener, NS-1619, neither CO nor glutamate dilated pial arterioles. Conversely, neither paxilline nor iberiotoxin inhibited dilation to acute severe or moderate prolonged hypoxia. Both glutamate and hypoxia increased cerebrospinal fluid (CSF) CO concentration. Iberiotoxin that blocked dilation to glutamate did not attenuate the increase in CSF CO. The guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocked dilation to sodium nitroprusside, did not inhibit dilation to hypoxia. These data suggest that dilation of newborn pig pial arterioles to glutamate is mediated by activation of K(Ca) channels, consistent with the intermediary signal being CO. Surprisingly, although 1) heme oxygenase (HO) inhibition attenuates dilation to hypoxia, 2) hypoxia increases CSF CO concentration, and 3) K(Ca) channel antagonists block dilation to CO, neither K(Ca) channel blockers nor ODQ altered dilation to hypoxia, suggesting the contribution of the HO/CO system to hypoxia-induced dilation is not by stimulating vascular smooth muscle K(Ca) channels or guanylyl cyclase.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.