A close association of RyRs with highly dense clusters of Ca2+-activated Cl- channels underlies the activation of STICs by Ca2+ sparks in mouse airway smooth muscle

Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl((Ca)) channels in spark sites in airway myocytes from mouse. Ca(2+) sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) buffer, which was characterized by flash photolysis of NPEGTA. We found that I(Ca(spark)) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca(2+) sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I(Ca(spark)) by less than 3 ms, and its rising phase matches the duration of the I(Ca(spark)). We further determined that Cl((Ca)) channels on average are exposed to a [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) spark sites could account for all the Cl((Ca)) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl((Ca)) channels in Ca(2+) spark sites localize near to each other, and, moreover, Cl((Ca)) channels concentrate in an area with a radius of approximately 600 nm, where their density reaches as high as 300 channels/microm(2). This model reveals that Cl((Ca)) channels are tightly controlled by Ca(2+) sparks via local Ca(2+) signaling.     

猪冠状动脉平滑肌细胞的自发瞬时外向电流的特性

自发瞬时外向电流（spontaneous transient outward currents,STOCs）在小动脉的肌源性调节中起着非常重要的作用。本文应用穿孔膜片钳技术记录了猪冠状动脉平滑肌细胞上的STOCs,研究了其基本特性以及调节。结果显示：STOCs有明显的电压依赖性和钙依赖性,其频率和幅度具有变异性。STOCs可以随机叠加在阶跃刺激方案和斜坡刺激方案引出的全细胞钾电流上。STOCs可被大电导钙激活钾（large-conductance Ca^2＋-activated potassium,BKCa）通道的特异性阻断剂ChTX、螯合胞外钙离子和50μmol／L ryanodine完全抑制。钙离子载体A23187可以明显增加STOCs的幅度和频率;而L型钙通道阻断剂verapamil和CdCl2对STOCs的影响很小。咖啡因使STOCs瞬时爆发性增加,然后抑制。钠离子载体可明显增加STOCs的频率;钠钙交换体选择性抑制剂KB．R7943可明显抑制STOCs。由此可以认为STOCs是BKCa通道介导的。STOCs的产生和激活依赖于经钠钙交换的钙内流和经肌浆网ryanodine受体介导的钙释放,钠钙交换可能决定钙库重载,而细胞膜下肌浆网的胞内钙释放（钙火花）所致的局部钙浓度瞬时增加激活与其相邻的BKCa通道,产生STOCs。     

Role of IP(3) in modulation of spontaneous activity in pacemaker cells of rabbit urethra

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (<100 ms in duration) and "slow" (>1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store.     

Resting and activation-dependent ion channels in human mast cells

The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca(2+)-activated K(+) current exhibiting weak inward rectification, which polarized the cells to around -40 mV and a smaller outwardly rectifying Ca(2+)-independent Cl(-) conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca(2+)-activated K(+) currents and Ca(2+)-independent Cl(-) currents developing in approximately 50% of cells. In both cell types, the K(+) current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca(2+)-activated K(+) channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C(4) release, indicating that the Ca(2+)-activated K(+) current may enhance, but is not essential for, the release of these mediators.     

Presence of a calcium-activated chloride current in mouse ventricular myocytes

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.     

Interstitial cells of Cajal in the urethra

The smooth muscle layer of the urethra generates spontaneous myogenic tone that is thought to make a major contribution to urinary continence. The mechanisms underlying generation of tone remain unclear, however recent studies from our laboratory highlighted a role for a specialised population of pacemaker cells which we originally referred to as interstitial cells (IC) and now term ICC. Urethra ICC possess an electrical pacemaker mechanism characterised by rhythmic activation of Ca(2+)-activated Cl(-) channels leading to spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarisations (STDs) under current clamp conditions. Both STICS and STDs are now known to be associated with spontaneous Ca(2+) oscillations that result from a complex interplay between release of Ca(2+) from intracellular stores and Ca(2+) influx across the plasma membrane. In this review we will consider some of the precise mechanisms involved in the generation of pacemaker activity and discuss how these are modulated by excitatory and inhibitory neurotransmitters.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.     

Diabetic dyslipidemia and exercise affect coronary tone and differential regulation of conduit and microvessel K+ current

Spontaneous transient outward K(+) currents (STOCs) elicited by Ca(2+) sparks and steady-state K(+) currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K(+) currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca(2+) sparks were determined in pressurized microvessels, and perforated patch clamp assessed K(+) current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca(2+) spark activity was significantly lower in DD versus C and DDX (P < 0.05 vs. DDX). STOCs were pronounced in microvessel (approximately 35 STOCs/min) in sharp contrast to conduit cells ( approximately 2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K(+) current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K(+) current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K(+) current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.     

Calcium mobilization is required for peroxynitrite-mediated enhancement of spontaneous transient outward currents in arteriolar smooth muscle cells

Transiently local release of Ca(2+) from the sarcoplasmic reticulum (SR) activates nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO(-)) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca(2+) mobilization was involved in STOCs alteration by ONOO(-). STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca(2+); by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca(2+) channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO(-) donor, at concentrations of 20-200 microM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca(2+) (200 nM), ryanodine (10 microM), or nifedipine (10 microM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 microM) had little effect on ONOO(-) -induced STOCs activation. These findings suggest that complex Ca(2+)-mobilizing pathways, including external Ca2+ influx through VGCCs activation and subsequent internal Ca(2+) release through RyRs but not IP3Rs, are involved in ONOO(-)mediated STOCs enhancement in ASMCs.     

Selective downregulation of the BKbeta1 subunit in diabetic arteriolar myocytes

Diabetic retinopathy is an important cause of visual loss. Functional abnormalities including vasoconstriction precede structural changes. Using the streptozotocin-model of diabetes in rats, we have identified downregulation of the beta1 subunit of the BK channel in arteriole myocytes as a possible molecular mechanism underlying these early changes. BKbeta1 mRNA levels were reduced as early as one month after induction of diabetes, and BK Ca(2+)-sensitivity and caffeine-evoked BK currents were reduced at three months. This effect appears to be selective for the regulatory subunit, as BKalpha subunit expression was not altered at the mRNA level, and voltage-activated BK currents were unaltered. No changes were seen in voltage activated Ca(2+)-current, Ca(2+)-activated Cl(-)current, or A-type voltage activated K(+)-currents. Reduced Ca(2+)-activated BK activity may promote depolarization, Ca(2+)-channel activation and increased contraction under resting conditions or in response to Ca(2+)-mobilizing agonists.     

Whole cell current and membrane potential regulation by a human smooth muscle mechanosensitive calcium channel

Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

Synthesis and potassium channel opening activity of substituted 10H-benzo[4,5]furo[3,2-b]indole-and 5,10-dihydro-indeno[1,2-b]indole-1-carboxylic acids

Compounds in a structurally novel series of substituted 10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acids and related 5,10-dihydro-indeno[1,2-b]indole-1-carboxylic acids were prepared and shown to possess potent, bladder-selective smooth muscle relaxant properties and thus are potentially useful for the treatment of urge urinary incontinence. Electrophysiological studies using rat detrusor myocytes have demonstrated that prototype compound 7 produces a significant increase in hyperpolarizing current, which is iberiotoxin (IbTx)-reversed, thus consistent with activation of the large-conductance Ca(2+)-activated potassium channel (BK(Ca)).     

Cerebrosides of baifuzi, a novel potential blocker of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells

Calcium-activated chloride channels (CaCCs) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. However, the lack of a special blocker of CaCCs has limited the investigation of its functions for long time. Here, we report that CB is a novel potential blocker of I(Cl(Ca)) in rat pulmonary artery smooth muscle cells (PASMC). Cerebrosides (CB) were isolated from Baifuzi which is dried root tuber of the herb Typhonium giganteum Engl used for treatment of stroke in traditional medicine. Using the voltage-clamp technique, sustained Ca(2+)-activated Cl(-) current (I(Cl(Ca))) was evoked by a K(+)-free pipette solution containing 500nM Ca(2+) which exhibited typical outwardly rectifying and voltage-/time-dependence characterization. Data showed that CB played a distinct inhibitory role in modulating the CaCCs. Moreover, we investigated the kinetic effect of CB on I(Cl(Ca)) and found that it could slow the activation dynamics of the outward current, accelerate the decay of the inward tail current and change the time-dependence characterization. We conclude that CB is a novel potent blocker of CaCCs. The interaction between CB and CaCCs is discussed.     

Potassium channels regulate tone in rat pulmonary veins

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。     

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.     

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K(ATP)) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM-0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 microM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K(ATP) channel blocker glibenclamide (10 microM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K(+) channel (K(V)) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K(+) channel (K(Ca)) antagonist charybdotoxin (0.1 microM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM-0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K(ATP) channels function in rat corpus cavernosum.