Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas

Nonselective cation channels have been identified and linked to important cell functions in rat hepatocytes. In this study, we characterized inward rectifying nonselective cation channels in detail by the patch clamp technique in human HepG2 cells. Channel properties were studied with high resistance borosilicate pipettes in cell-attached and inside-out configurations. With Ringer's solution and KCl as pipette solutions, the conductances were 19.7 +/- 2.1 and 22.2 +/- 0.0 picosiemens (pS), and reversal potentials were 30.9 +/- 3.5 and 31.3 +/- 4.6 mV, respectively. The channel was permeable to Ba2+, and the sequence of permeability ratios was Na+ > K+ > Cs+ > Ba2+. In the cell-attached configuration, the channel had a higher opening probability at depolarizing potential than at hyperpolarizing. In the inside-out patches with symmetric Ringer's solution, the current voltage curve was linear with conductance of 19.8 +/- 0.9 pS. Reversal potential shifted from -0.2 +/- 1.0 mV to 23.2 +/- 1.0 mV when the bath solution was replaced by dilute Ringer's solution. In the inside-out configuration, the gating was Ca(2+)-dependent, and the opening probability increased with increasing intracellular calcium concentration ([Ca2+]i). An outward rectifying channel appeared when [Ca2+]i was less than 1 mumol/L. The nonselective channel was reversibly blocked by 10 mumol/L internal flufenamic acid. We conclude that Ca(2+)- and voltage-dependent nonselective cation channels are present in human HepG2 cells. The channels might be involved in the regulation of Ca2+ influx and are associated with activation of other ion channels.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.     

The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca2+ release process in pancreatic acinar cells

In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca2+ ([Ca2+]i). The increases of [Ca2+]i evoked by TG was associated with significant changes of plasma membrane Ca2+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca2+ extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]i up to 1 microM Ca2+. In contrast, the activation of the Ca2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which reduces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]i evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca2+]i peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]i increase (from 12.3 +/- 1.0 to 6.1 +/- 0.6 nM Ca2+/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the continuous presence of TG evoked a further increase of [Ca2+]i. The amplitude of the [Ca2+]i increase upon caffeine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane Ca(2+)-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associated with an augmentation of the apparent rate of [Ca2+]i increase (from 12.3 +/- 1.2 to 16.1 +/- 1.9 nM Ca2+/s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells

1. We have studied the effects of hypoxia on membrane potential and [Ca2+]i in enzymically isolated type I cells of the neonatal rat carotid body (the principal respiratory O2 chemosensor). Isolated cells were maintained in short term culture (3-36 h) before use. [Ca2+]i was measured using the Ca(2+)-sensitive fluoroprobe indo-1. Indo-1 was loaded into cells using the esterified form indo-1 AM. Membrane potential was measured (and clamped) in single isolated type I cells using the perforated-patch (amphotericin B) whole-cell recording technique. 2. Graded reductions in PO2 from 160 Torr to 38, 19, 8, 5 and 0 Torr induced a graded rise of [Ca2+]i in both single and clumps of type I cells. 3. The rise of [Ca2+]i in response to anoxia was 98% inhibited by removal of external Ca2+ (+1 mM EGTA), indicating the probable involvement of Ca2+ influx from the external medium in mediating the anoxic [Ca2+]i response. 4. The L-type Ca2+ channel antagonist nicardipine (10 microM) inhibited the anoxic [Ca2+]i response by 67%, and the non-selective Ca2+ channel antagonist Ni2+ (2 mM) inhibited the response by 77%. 5. Under voltage recording conditions, anoxia induced a reversible membrane depolarization (or receptor potential) accompanied, in many cases, by trains of action potentials. These electrical events were coincident with a rapid rise of [Ca2+]i. When cells were voltage clamped close to their resting potential (-40 to -60 mV), the [Ca2+]i response to anoxia was greatly reduced and its onset was much slower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium entry activated by store depletion in human umbilical vein endothelial cells

We have used the patch clamp technique combined with simultaneous measurement of intracellular Ca2+ to record ionic currents activated by depletion of intracellular Ca(2+)-stores in endothelial cells from human umbilical veins. Two protocols were used to release Ca2+ from intracellular stores, i.e. loading of the cells via the patch pipette with Ins(1,4,5)P3, and extracellular application of thapsigargin. Ins(1,4,5)P3 (10 microM) evoked a transient increase in [Ca2+]i in cells exposed to Ca(2+)-free extracellular solutions. A subsequent reapplication of extracellular Ca2+ induced an elevation of [Ca2+]i. These changes in [Ca2+]i were very reproducible. The concomitant membrane currents were neither correlated in time nor in size with the changes in [Ca2+]i. Similar changes in [Ca2+]i and membrane currents were observed if the Ca(2+)-stores were depleted with thapsigargin. Activation of these currents was prevented and holding currents at -40 mV were small if store depletion was induced in the presence of 50 microM NPPB. This identifies the large currents, which are activated as a consequence of store-depletion, as mechanically activated Cl- currents, which have been described previously [1,2]. Loading the cells with Ins(1,4,5)P3 together with 10 mM BAPTA induced only a very short lasting Ca2+ transient, which was not accompanied by activation of a detectable current, even in a 10 mM Ca(2+)-containing extracellular solution. Also thapsigargin does not activate any membrane current if the pipette solution contains 10 mM BAPTA (ruptured patches). The contribution of Ca(2+)-influx to the membrane current during reapplication of 10 mM extracellular calcium to thapsigargin-pretreated cells was estimated from the first time derivative of the corresponding Ca2+ transients at different holding potentials. These current values showed strong inward rectification, with a maximal amplitude of 1.0 +/- 0.3 pA at -80 mV (n = 8; membrane capacitance 59 +/- 9 pF).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Effects of semotiadil fumarate (SD-3211) and its enantiomer, SD-3212, on the changes in cytosolic Ca2+ and tension caused by KCl and norepinephrine in isolated rat aortas

Semotiadil fumarate (SD-3211), a Ca2+ channel blocker of benzothiazine derivative and its (S)-(-)-enantiomer (SD-3212), inhibited K(+)- and norepinephrine (NE)-induced contractions in isolated rat aortas. Inhibition of NE contraction induced by both drugs was greater than that induced by diltiazem or bepridil, whereas inhibition of K(+)-contraction was similar to that induced by diltiazem or bepridil. Semotiadil and SD-3212 (10 microM) inhibited the increase in cytosolic Ca2+ ([Ca2+]i) induced by 65.4 mM K+ in fura-2-loaded preparations as well as diltiazem and bepridil (10 microM). On the other hand, semotiadil and SD-3212 (10 microM) inhibited only the early phase of increase in [Ca2+]i induced by 1 microM NE. After 5 min, no significant effect on [Ca2+]i was observed with these compounds despite the significant decrease in the contraction. In contrast to these compounds, diltiazem and bepridil 10 microM affected neither the increase in [Ca2+]i nor the contraction induced by NE. Semotiadil and SD-3212 inhibited the transient contraction induced by 1 microM NE in the absence of external Ca2+. Both compounds partially but significantly inhibited the NE-induced contraction in nifedipine-treated muscles. These results suggest that semotiadil and SD-3212 inhibit contractions of vascular smooth muscle (VSM) not only through blockade of voltage-dependent Ca2+ channels but also through other mechanisms, such as inhibition of Ca2+ release from Ca2+ stores or decrease in sensitivity of the contractile elements to Ca2+.     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302+/-15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum.     

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.     

Depletion of ryanodine-sensitive Ca2+ store activates Ca2+ entry in rat submandibular gland acinar cells

The existence of ryanodine-sensitive Ca2+ stores and their role in the Ca2+ entry mechanism were examined in the rat submandibular gland acinar cells, using the microfluorimetry of intracellular Ca2+ concentration ([Ca2+]i). In the presence of thapsigargin, a Ca(2+)-ATPase inhibitor of inositol (1, 4, 5) triphosphate (InsP3)-sensitive Ca2+ stores, caffeine caused an increase in [Ca2+]i, which was inhibited by treatment with ryanodine (a ligand to the Ca(2+)-induced Ca2+ release channels). In the cells treated with ryanodine, 1 mM Ca2+ addition to a Ca(2+)-free solution caused a marked increase in [Ca2+]i, which was eliminated by application of Ni2+ or SK & F 96365, suggesting a Ca2+ entry triggered by ryanodine. The maximal change in the net increase in [Ca2+]i caused by the ryanodine-coupled Ca2+ entry, was 104.0 +/- 16.0 nM, which intense was caused by 10 microM ryanodine. Emptying the InsP3-sensitive stores by treatment with thapsigargin also caused Ca2+ entry, which maximally changed [Ca2+]i by 349.6 +/- 15.1 nM. Ten mumol/liter ryanodine was confirmed to cause a release of 45Ca2+ from the parotidic microsomal fraction enriched in endopalsmic reticulum. We propose that ryanodine-sensitive Ca2+ stores are present in rat submandibular gland acinar cells. We further propose that release of Ca2+ from the ryanodine-sensitive stores, which means eventually depletion of the ryanodine-sensitive Ca2+ stores, can activate the Ca2+ entry. The ability for Ca2+ entry coupled with the ryanodine-sensitive Ca2+ stores seems to be about 30% of the ability for Ca2+ entry coupled with the thapsigargin-sensitive Ca2+ stores.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

Dual mechanisms of Mg2+ block of ryanodine receptor Ca2+ release channel from cardiac sarcoplasmic reticulum

We investigated the effects of cytosolic Mg2+ on ryanodine receptor Ca2+ release channel (RyR) of bovine cardiac sarcoplasmic reticulum incorporated into planar lipid bilayers recording single channel activities. Channels were activated by > or = 0.1 microM Ca2+ in the cis solution. At constant Ca2+, application of Mg2+ (0.1-1 mM) to cis side decreased channel activity in a concentration-dependent manner. A half maximal blocking concentration (Kd) was 35 microM and a complete block was obtained at 1 mM. In the presence of 1 mM free Mg2+ in cis solution, the relation between the channel open probability (Po) and concentration of free Ca2+ in cis solution ([Ca2+]cis) shifted to the right, indicating the competition of Mg2+ and Ca2+. Blocking effects of Mg2+ on RyR were antagonized by increasing [Ca2+]cis > or = 0.1 mM. In the presence of 1 m Mg2+ and 1 mM Ca2+ in cis solution, the channel conductance was markedly depressed to approximately 400 pS (n = 7) from 603 +/- 40 pS (mean +/- S.D., n = 22) in the absence of Mg2+, indicating the flickering block. These results show that Mg2+ causes a direct inhibition of RyR in cardiac SR and this inhibition may be mediated through two different mechanisms. A competition of Mg2+ and Ca2+ at a Ca2+ sensitive site on the RyR and a flickery block of the open channel by Mg2+.     

Spatial heterogeneity of caffeine- and inositol 1,4,5-trisphosphate-induced Ca2+ transients in isolated snail neurons

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.     

Intracellular calcium and pH alterations induced by tri-n-butyltin chloride in isolated rainbow trout hepatocytes: a flow cytometric analysis

The effects of tri-n-butyltin chloride (TBT) on ionic homeostasis on isolated trout hepatocytes were investigated by flow cytometry (FCM), using the Ca(2+)-sensitive and pH-sensitive fluorescent probes Indo-1 and SNARF-1, respectively. Cell viability was monitored concurrently. Treatment of hepatocytes with 1 and 5 microM TBT caused a rapid and sustained elevation of cytosolic free Ca2+ concentration [Ca2+]i and an important cytoplasmic acidification. These changes were dependent upon TBT concentration and were maintained over 60 min, the maximum exposure period investigated. At 0.5 microM TBT, there was a slight but not significant increase in [Ca2+]i and a significant reduction in intracellular pH (pHi) only after 60 min of exposure. A rise in [Ca2+]i and cytoplasmic acidification were observed before loss of viability was detectable. Experiments carried out in Ca(2+)-free medium suggest that TBT mainly mobilizes Ca2+ from intracellular stores in trout hepatocytes. The cytoplasmic acidification following TBT exposure seems to be caused by the combination of intracellular Ca2+ mobilization and by direct action of TBT. The present results suggest that ionic homeostasis perturbations could be early events in the mechanism of cell injury by TBT.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Effect of membrane hyperpolarization induced by a K+ channel opener on histamine-induced Ca2+ mobilization in rabbit arterial smooth muscle

1. The role of membrane hyperpolarization on agonist-induced contraction was investigated in intact and alpha-toxin-skinned smooth muscles of rabbit mesenteric artery by use of the ATP-sensitive K+ channel opener, (-)-(3S,4R)-4-(N-acetyl-N-hydroxyamino)-6-cyano-3,4-dihydro-2,2- dimethyl-2H-1-benzopyran-3-ol (Y-26763), and either histamine (Hist) or noradrenaline (NA). 2. Hist (3 microM) and NA (10 microM) both produced a phasic, followed by a tonic increase in intracellular Ca2+ concentration ([Ca2+]i) and force. Y-26763 (10 microM) potently inhibited the NA-induced phasic and tonic increase in [Ca2+]i and force. In contrast, Y-26763 attenuated the Hist-induced phasic increase in [Ca2+]i and force but had almost no effect on the tonic response. However, ryanodine-treatment of muscles in order to inhibit the function of intracellular Ca2+ storage sites altered the action of Y-26763 which now attenuated the Hist-induced tonic increase in [Ca2+]i and force in a concentration-dependent manner (at concentrations > 1 microM). Glibenclamide (10 microM) attenuated the inhibitory action of Y-26763. 3. Hist (3 microM) depolarized the smooth muscle cells to the same extent as NA (10 microM). In the absence of either agonist, Y-26763 (over 30 nM) hyperpolarized the membrane and glibenclamide inhibited this hyperpolarization. Y-26763 (10 microM) almost abolished the NA-induced membrane depolarization, but only slightly attenuated the Hist-induced membrane depolarization in which the delta (delta) value (the difference before and after application of Hist) was not modified by any concentration of Y-26763. In ryanodine-treated smooth muscle cells, Y-26763 hyperpolarized the membrane and potently inhibited the membrane depolarization induced by Hist. 4. In ryanodine-treated muscle, Y-26763 had no measurable effect on the Hist-induced [Ca2+]i-force relationship. Y-26763 also had no apparent effect on the myofilament Ca(2+)-sensitivity in the presence of Hist in alpha-toxin-skinned smooth muscles. 5. It is concluded that the membrane hyperpolarization induced by Y-26763 may not be enough to inhibit the Hist-activated Ca2+ influx. It is also suggested that Hist prevents the membrane hyperpolarization induced by Y-26763, activating an unknown mechanism which is thought to depend on the function of intracellular Ca2+ storage sites.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.