Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.     

Effects of prostaglandin F2alpha on membrane currents in rabbit middle cerebral arterial smooth muscle cells

Although PGF(2alpha) affects contractility of vascular smooth muscles, no studies to date have addressed the electrophysiological mechanism of this effect. The purpose of our investigation was to examine the direct effects of PGF(2alpha) on membrane potentials, Ca(2+)-activated K(+) (K(Ca)) channels, delayed rectifier K(+) (K(V)) channels, and L-type Ca(2+) channels with the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells (SMCs). PGF(2alpha) significantly hyperpolarized membrane potentials and increased the amplitudes of total K(+) currents. PGF(2alpha) increased open-state probability but had little effect on the open and closed kinetics of K(Ca) channels. PGF(2alpha) increased the amplitudes of K(V) currents with a leftward shift of the activation and inactivation curves and a decrease in the activation time constant. PGF(2alpha) decreased the amplitudes of L-type Ca(2+) currents without any significant change in threshold or apparent reversal potentials. This study provides the first finding that the direct effects of PGF(2alpha) on middle cerebral arterial SMCs, at least in part, could attenuate vasoconstriction.     

Regional differences in cholinergic regulation of potassium current in feline esophageal circular smooth muscle

Potassium channels are important contributors to membrane excitability in smooth muscles. There are regional differences in resting membrane potential and K(+)-channel density along the length of the feline circular smooth muscle esophagus. The aim of this study was to assess responses of K(+)-channel currents to cholinergic (ACh) stimulation along the length of the feline circular smooth muscle esophageal body. Perforated patch-clamp technique assessed K(+)-channel responses to ACh stimulation in isolated smooth muscle cells from the circular muscle layer of the esophageal body at 2 (distal)- and 4-cm (proximal) sites above the lower esophageal sphincter. Western immunoblots assessed ion channel and receptor expression. ACh stimulation produced a transient increase in outward current followed by inhibition of spontaneous transient outward currents. These ACh-induced currents were abolished by blockers of large-conductance Ca(2+)-dependent K(+) channels (BK(Ca)). Distal cells demonstrated a greater peak current density in outward current than cells from the proximal region and a longer-lasting outward current increase. These responses were abolished by atropine and the specific M(3) receptor antagonist 4-DAMP but not the M(1) receptor antagonist pirenzipine or the M(2) receptor antagonist methoctramine. BK(Ca) expression along the smooth muscle esophagus was similar, but M(3) receptor expression was greater in the distal region. Therefore, ACh can differentially activate a potassium channel (BK(Ca)) current along the smooth muscle esophagus. This activation probably occurs through release of intracellular calcium via an M(3) pathway and has the potential to modulate the timing and amplitude of peristaltic contraction along the esophagus.     

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.     

Ca2+-activated Cl- channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection.

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca(2+)-activated K(+) current. The inward current elicited by PE was dependent on the Cl(-) gradient and was inhibited by niflumic acid, indicative of a Ca(2+)-activated Cl(-) (Cl(Ca)) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca(2+)-activated K(+) and Cl(Ca) channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time Cl(Ca) current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.     

Activating of ATP-dependent K+ channels comprised of K(ir) 6.2 and SUR 2B by PGE2 through EP2 receptor in cultured interstitial cells of Cajal from murine small intestine.

The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE(2) might affect the electrical properties of cultured ICC by activating ATP-dependent K(+) channels and, the EP receptor subtypes and the subunits of ATP-dependent K(+) channels involved in these activities were identified. In addition, the regulation of intracellular Ca(2+) ([Ca(2+)](i)) mobilization may be involved the action of PGE(2) on ICC. Treatments of ICC with PGE(2) inhibited electrical pacemaker activities in the same manner as pinacidil, an ATP-dependent K(+) channel opener and PGE(2) had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K(+) channels exist in ICC and that these are composed of K(ir) 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP(2) receptor agonist) showed the actions on pacemaker currents in the same manner as PGE(2). However sulprostone (a mixed EP(1) and EP(3) agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP(2) receptor in ICC. To investigate cAMP involvement in the effects of PGE(2) on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE(2) on pacemaker currents, and PGE(2) did not stimulate cAMP production. Also, we found PGE(2) inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. These observations indicate that PGE(2) alters pacemaker currents by activating the ATP-dependent K(+) channels comprised of K(ir) 6.2-SUR 2B in ICC and this action of PGE(2) are through EP(2) receptor subtype and also the activation of ATP-dependent K(+) channels involves intracellular Ca(2+) mobilization.     

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.     

Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal

The interstitial cells of Cajal (ICC), as pacemaker cells of the gut, contribute to rhythmic peristalsis and muscle excitability through initiation of slow-wave activity, which subsequently actively propagates into the musculature. An E-4031-sensitive K(+) current makes a critical contribution to membrane potential in ICC. This study provides novel features of this current in ICC in physiological solutions and seeks to identify the channel isoform. In situ hybridization and Kit immunohistochemistry were combined to assess ether-a-go-go-related gene (ERG) mRNA expression in ICC in mouse jejunum, while the translated message was assessed by immunofluorescence colocalization of ERG and Kit proteins. E-4031-sensitive currents in cultured ICC were studied by the whole cell patch-clamp method, with physiological K(+) concentration in the bath and the pipette. In situ hybridization combined with Kit immunohistochemistry detected m-erg1 and m-erg3, but not m-erg2, mRNA in ICC. ERG3 protein was colocalized with Kit-immunoreactive ICC in jejunum sections, but ERG1 protein was visualized only in the smooth muscle cells. At physiological K(+) concentration, the E-4031-sensitive outward current in ICC was different from its counterpart in cardiac and gut smooth muscle cells. In particular, inactivation upon depolarization and recovery from inactivation by hyperpolarization were modest in ICC. In summary, the E-4031-sensitive currents influence the kinetics of the pacemaker activity in ICC and contribute to maintenance of the resting membrane potential in smooth muscle cells, which together constitute a marked effect on tissue excitability. Whereas this current is mediated by ERG1 in smooth muscle, it is primarily mediated by ERG3 in ICC.     

NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli

In guinea pig taenia coli, the nitric oxide (NO) donor sodium nitroprusside (SNP, 1 microM) reduced the carbachol-stimulated increases in muscle force in parallel with a decrease in intracellular Ca(2+) concentration ([Ca(2+)](i)). A decrease in the myosin light chain phosphorylation was also observed that was closely correlated with the decrease in [Ca(2+)](i). With the patch-clamp technique, 10 microM SNP decreased the peak Ba(2+) current, and this effect was blocked by an inhibitor of soluble guanylate cyclase. Carbachol (10 microM) induced an inward current, and this effect was markedly inhibited by SNP. SNP markedly increased the depolarization-activated outward K(+) currents, and this current was completely blocked by 0.3 micorM iberiotoxin. SNP (1 microM) significantly increased cGMP content without changing cAMP content. Decreased Ca(2+) sensitivity by SNP of contractile elements was not prominent in the permeabilized taenia, which was consistent with the [Ca(2+)](i)-force relationship in the intact tissue. These results suggest that SNP inhibits myosin light chain phosphorylation and smooth muscle contraction stimulated by carbachol, mainly by decreasing [Ca(2+)](i), which resulted from the combination of the inhibition of voltage-dependent Ca(2+) channels, the inhibition of nonselective cation currents, and the activation of Ca(2+)-activated K(+) currents.     

Functional receptor-channel coupling compared in contractile and proliferative human vascular smooth muscle

We have previously identified a human vascular smooth muscle clone that can reversibly convert between proliferative and contractile phenotypes. Here we compared receptor-channel coupling in these cells using fura-2 to monitor [Ca(2+)](i) and patch-clamp to record currents. Histamine elevated [Ca(2+)](i) in all cells and caused contraction of cells exhibiting the contractile phenotype. The rise of [Ca(2+)](i) persisted in Ca(2+)-free solution and was abolished by thapsigargin, indicating involvement of stores. Whole cell electrophysiological recording revealed that histamine evoked transient outward K(+) current, indicating functional receptor-channel coupling. The time-course and amplitude of the histamine-activated current were similar in cells of the proliferative and contractile phenotypes. Moreover, a large conductance K(+) channel was recorded in cell-attached patches and was activated by histamine as well as the Ca(2+) ionophore A-23187, identifying it as the large conductance Ca(2+)-dependent K(+) channel. This K(+) channel showed similar characteristics and activation in both proliferative and contractile phenotypes, indicating that expression was independent of phenotype. In contrast, histamine also elicited an inward Cl(-) current in some contractile cells, suggesting differential regulation of this current depending on phenotype. These studies demonstrate the usefulness of this human vascular cell clone for studying functional plasticity of smooth muscle, while avoiding complications arising from extended times in culture.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.     

Carbon monoxide activates KCa channels in newborn arteriole smooth muscle cells by increasing apparent Ca2+ sensitivity of alpha-subunits

Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 microM) or CO released from dimanganese decacarbonyl (10 microM), a novel light-activated CO donor, increased K(Ca) channel activity 4.9- or 3.5-fold, respectively. K(Ca) channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 microM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca(2+) concentration-response curve for K(Ca) channels leftward and decreased the apparent dissociation constant for Ca(2+) from 31 to 24 microM. Western blotting data suggested that the low Ca(2+) sensitivity of newborn K(Ca) channels may be due to a reduced beta-subunit-to-alpha-subunit ratio. CO activation of K(Ca) channels was Ca(2+) dependent. CO increased open probability 3.7-fold with 10 microM free Ca(2+) at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca(2+). CO left shifted the current-voltage relationship of cslo-alpha currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity K(Ca) channels via a direct effect on the alpha-subunit that increases apparent Ca(2+) sensitivity. The optimal tuning by CO of the micromolar Ca(2+) sensitivity of K(Ca) channels will lead to preferential activation by signaling modalities, such as Ca(2+) sparks, which elevate the subsarcolemmal Ca(2+) concentration within this range.     

Large conductance Ca2+-activated K+ channels modulate endothelial cell outward currents and nitric oxide release in the intact rat superior mesenteric artery

Endothelial cells (EC) control vascular smooth muscle cell (VSMC) tone by release of paracrine factors. VSMC may also influence the EC layer, and therefore, the present study hypothesized that the opening of large-conductance Ca(2+) activated K(+) (BK(Ca)) channels may indirectly modulate EC hyperpolarization and nitric oxide (NO) release via myoendothelial gap junctions (MEGJ). To address this hypothesis 'in situ' EC ion current recordings, isolated VSMC patch clamp recordings, and simultaneous measurements of NO concentration and relaxation were conducted using segments of the rat superior mesenteric artery. In arteries constricted by α(1)-adrenoceptor activation, ACh (1 μM) evoked EC outward currents, vasorelaxation, and NO release. In contrast to preincubation with iberiotoxin (IbTx, 100nM) application of IbTx after ACh decreased EC outward currents, NO release and vasorelaxation. Furthermore, in phenylephrine (Phe)-contracted arteries treated with a gap junction uncoupler, cabenoxolone (CBX), IbTx failed to decrease ACh-evoked EC outward currents. In addition, CBX decreased EC outward currents, time constant of the capacitative transients, input capacitance, and increased input resistance. In isolated VSMC CBX did not affect BK(Ca) currents. Immunohistochemistry revealed only BK(Ca) channel positive staining in the VSMC layer. Therefore, the present results suggest that BK(Ca) channels are expressed in the VSMC, and that Phe by activation of VSMC BK(Ca) channels modulates ACh-evoked EC outward currents, NO release and vasorelaxation via MEGJ in rat superior mesenteric artery.     

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.     

Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).     

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.     

Gap junctional coupling between progenitor cells at the retinal margin of adult goldfish

We prepared living slice preparations of the peripheral retina of adult goldfish to examine electrical membrane properties of progenitor cells at the retinal margin. Cells were voltage-clamped near resting potential and then stepped to either hyperpolarizing or depolarizing test potentials using whole-cell voltage-clamp recordings. Electrophysiologically examined cells were morphologically identified by injecting both Lucifer Yellow (LY) and biocytin. All progenitor cells examined (n = 37) showed a large amount of passively flowing currents of either sign under suppression of the nonjunctional currents flowing through K(+) and Ca(2+) channels in the cell membrane. They did not exhibit any voltage-gated Na(+) currents. Cells identified by LY fills were typically slender. As the difference between the test potential and the resting potential increased, 13 out of 37 cells exhibited symmetrically voltage- and time-dependent current decline on either sign at the resting potential. The symmetric current profile suggests that the current may be driven and modulated by the junctional potential difference between the clamping cell and its neighbors. The remaining 24 cells did not exhibit voltage dependency. A gap junction channel blocker, halothane, suppressed the currents. A decrease in extracellular pH reduced coupling currents and its increase enhanced them. Dopamine, cAMP, and retinoic acid did not influence coupling currents. Injection of biocytin into single progenitor cells revealed strong tracer coupling, which was restricted in the marginal region. Immature ganglion cells closely located to the retinal margin exhibited voltage-gated Na(+) currents. They did not reveal apparent tracer coupling. These results demonstrate that the marginal progenitor cells couple with each other via gap junctions, and communicate biochemical molecules, which may subserve or interfere with cellular differentiation.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

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.