Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4-/- mice.

Agonist-induced Ca2+ entry into cells by both store-operated channels and channels activated independently of Ca2+-store depletion has been described in various cell types. The molecular structures of these channels are unknown as is, in most cases, their impact on various cellular functions. Here we describe a store-operated Ca2+ current in vascular endothelium and show that endothelial cells of mice deficient in TRP4 (also known as CCE1) lack this current. As a consequence, agonist-induced Ca2+ entry and vasorelaxation is reduced markedly, showing that TRP4 is an indispensable component of store-operated channels in native endothelial cells and that these channels directly provide an Ca2+-entry pathway essentially contributing to the regulation of blood vessel tone.

Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation.

In congenital bilateral absence of the vas deferens patients, the T5 allele at the polymorphic Tn locus in the CFTR (cystic fibrosis transmembrane conductance regulator) gene is a frequent disease mutation with incomplete penetrance. This T5 allele will result in a high proportion of CFTR transcripts that lack exon 9, whose translation products will not contribute to apical chloride channel activity. Besides the polymorphic Tn locus, more than 120 polymorphisms have been described in the CFTR gene. We hypothesized that the combination of particular alleles at several polymorphic loci might result in less functional or even insufficient CFTR protein. Analysis of three polymorphic loci with frequent alleles in the general population showed that, in addition to the known effect of the Tn locus, the quantity and quality of CFTR transcripts and/or proteins was affected by two other polymorphic loci: (TG)m and M470V. On a T7 background, the (TG)11 allele gave a 2.8-fold increase in the proportion of CFTR transcripts that lacked exon 9, and (TG)12 gave a sixfold increase, compared with the (TG)10 allele. T5 CFTR genes derived from patients were found to carry a high number of TG repeats, while T5 CFTR genes derived from healthy CF fathers harbored a low number of TG repeats. Moreover, it was found that M470 CFTR proteins matured more slowly, and that they had a 1.7-fold increased intrinsic chloride channel activity compared with V470 CFTR proteins, suggesting that the M470V locus might also play a role in the partial penetrance of T5 as a disease mutation. Such polyvariant mutant genes could explain why apparently normal CFTR genes cause disease. Moreover, they might be responsible for variation in the phenotypic expression of CFTR mutations, and be of relevance in other genetic diseases.

Amplitude modulation of Ca2+ signals induced by histamine in human endothelial cells.

We have addressed the problem of whether the agonist concentration sensed by endothelial cells is encoded by the sustained rise of the intracellular Ca2+ concentration ([Ca2+]i) or by the frequency of intracellular Ca2+ oscillations. Single or confluent endothelial cells from umbilical veins were stimulated for 15 min with histamine (0.03 to 100 mumol/l), and the concomitant changes in [Ca2+]i were measured with fura-2/AM. Application of histamine at concentrations above 0.1 mumol/l resulted always in a fast spike of [Ca2+]i, followed by a slow decline to a sustained plateau level, which depends on the presence of extracellular Ca2+. At the same time of the development of this plateau phase, quenching of the fura-2/AM signal occurred during agonist stimulation in a Ca(2+)-free, 1.5 mmol/l Mn2+ containing solution, indicating influx of divalent cations during this time. From 48 cells in 1.5 mmol/l [Ca2+]e we obtained a close relation between histamine concentration and time integral of [Ca2+]i taken over the 15 min recording of the plateau [Ca2+]i. The half-maximal increase in the integral of [Ca2+]i was at 0.7 mumol/l for solitary cells, 1.2 mumol/l for clustered cells and 1.2 mumol/l for the plateau Ca2+ level. Repetitive Ca2+ spikes or Ca2+ oscillations appeared only in 16 out of 48 cells, but their frequency was not correlated to the agonist concentration. Ca2+ oscillations were only observed in a concentration window between 0.1 and 1 mumol/l histamine, both in single and in clustered endothelial cells. Our results indicate that coding of the agonist concentration in endothelial cells is not related to the frequency of Ca2+ oscillations, but is closely correlated with the plateau level of intracellular Ca2+.

The conductance of single cardiac sodium channels from guinea pig depends on the intracellular sodium concentration.

Currents through DPI 201-106 modified single sodium channels have been measured in cell-free inside-out patches from guinea-pig ventricular myocytes. Single-channel conductance and reversal potential of the sodium channel have been calculated at different intracellular sodium concentrations [( Na+]i) from microscopic I-V curves, which were obtained by application of linear voltage ramps. The relation between the reversal potential and [Na+]i could be fitted with a modified Goldman-Hodgkin-Katz equation with a relative permeability for K+ over Na+ ions of 0.054. The zero-current conductance of the Na channel as a function of [Na+]i shows a plateau value at low Na concentrations, and increases in a sigmoidal manner at higher concentrations. It is concluded that the Na channel can carry outward currents and that its conductance depends on [Na+]i.

Ruthenium red and compound 48/80 inhibit the smooth-muscle plasma-membrane Ca2+ pump via interaction with associated polyphosphoinositides.

We will demonstrate the compound 48/80 and ruthenium red inhibit the smooth-muscle plasma-membrane Ca2+ pump by counteracting the stimulant effect of negatively charged phospholipids. Both substances did not affect the purified enzyme re-activated by pure phosphatidylcholine or phosphatidylinositol and measured in the absence of calmodulin, indicating that under these conditions they did not have a direct effect on the ATPase protein. Ruthenium red and compound 48/80 however inhibited the (Ca2(+) + Mg2+)-ATPase in the presence of phosphatidylinositol 4-phosphate and especially phosphatidylinositol 4,5-bisphosphate. The K0.5 for inhibition was 25 microM ruthenium red and 9 micrograms/ml of compound 48/80. The inhibition by ruthenium red developed slowly with half maximal inhibition occurring after about 75 s while that by compound 48/80 developed immediately within the time required for mixing. The efficacy of ruthenium red increased as the concentration of the acidic phospholipid increased, while no such cooperativity was observed for compound 48/80. Ruthenium red reduced the Vmax for Ca2+ without affecting the affinity for Ca2+, while compound 48/80 decreased both parameters. In conclusion, although ruthenium red and compound 48/80 affect the ATPase differently, both substances most likely inhibit the plasma-membrane Ca2+ pumping by counteracting the stimulation by negatively charged phospholipids.

Sodium and calcium interactions in vascular smooth muscle cells of the rabbit ear artery.

The effects of Na-free and of K-free solutions on the membrane potential, on tension development, and on 45Ca exchange have been investigated in rabbit ear artery. The contraction induced by Na-free solutions and the tension which develops in K-free solutions after a delay of about 1 h are both submaximal. Exposure for 4 h to K-free solutions does not affect the membrane potential, whereas Na-free solutions depolarize the cells by 10-20 mV, depending on the Na-substitute. Neither the amplitude nor the rate constant of the slowly exchanging 45Ca-fraction is affected by these experimental procedures. Substituting external Na by choline or TMA induces a transient increase of the 45Ca-efflux rate which does not occur in a Ca-free efflux medium, and which can be blocked with La. K readmission to Na-enriched tissues hyperpolarizes the cells up to -100 mV and induces a relaxation, without exerting any effect on the 45Ca efflux rate. The release of Ca from intracellular stores, induced by histamine and FCCP, and its subsequent extrusion through the plasma membrane produce a transient stimulation of the 45Ca efflux, which is not affected by the reduction of the Na gradient. The transient contraction induced by histamine in Ca-free solutions is affected in a different way by different Na substitutes. The results do not fit the Na-Ca exchange hypothesis but are consistent with an effect of the Na gradient on the passive Ca influx.

Modulation of voltage-dependent properties of a swelling-activated Cl- current.

We used the patch-clamp technique to study the voltage-dependent properties of the swelling-activated Cl- current (ICl,swell) in BC3H1 myoblasts. This Cl- current is outwardly rectifying and exhibits time-dependent inactivation at positive potentials (potential for half-maximal inactivation of +75 mV). Single-channel Cl- currents with similar voltage-dependent characteristics could be measured in outside-out patches pulled from swollen cells. The estimated single-channel slope conductance in the region between +60 and +140 mV was 47 pS. The time course of inactivation was well described by a double exponential function, with a voltage-independent fast time constant (approximately 60 ms) and a voltage-dependent slow time constant (>200 ms). Recovery from inactivation, which occurred over the physiological voltage range, was also well described by a double exponential function, with a voltage-dependent fast time constant (10-80 ms) and a voltage-dependent slow time constant (>100 ms). The inactivation process was significantly accelerated by reducing the pH, increasing the Mg2+ concentration or reducing the Cl- concentration of the extracellular solution. Replacing extracellular Cl- by other permeant anions shifted the inactivation curve in parallel with their relative permeabilities (SCN- > I- > NO3- > Cl- >> gluconate). A leftward shift of the inactivation curve could also be induced by channel blockers. Additionally, the permeant anion and the channel blockers, but not external pH or Mg2+, modulated the recovery from inactivation. In conclusion, our results show that the voltage-dependent properties of ICl,swell are strongly influenced by external pH, external divalent cations, and by the nature of the permeant anion.

Electro- and pharmacomechanical coupling in the smooth muscle cells of the rabbit ear artery.

A contraction of the rabbit ear artery can be induced by depolarizing the cells with a K-rich solution if Ca is present. 10(-9)-10(-6) M noradrenaline and 10(-8)-10(-7) M histamine cause a contraction of this tissue without modifying the membrane potential. If the histamine concentration exceeds 10(-7) M some depolarization of the membrane also occurs. Both noradrenaline and histamine also induce a contraction in Ca-free medium, even if La is present. None of these stimuli produces action potentials or fluctuations of the membrane potential. Besides these tonic contractions, the ear artery can also produce phasic contractions when 10 mM TEA is added to the medium. Such contractions are caused by the appearance of action potentials which are Ca dependent and which are similar to those appearing in visceral smooth muscle. A study of 45Ca fluxes has revealed that K depolarization and noradrenaline cause only a small increase in 45Ca uptake by the cells, while noradrenaline also releases cellular Ca, even in Ca-free medium. A comparison of tension development and 45Ca release induced by noradrenaline in Ca-free medium suggests that Ca extrusion could be very efficient in the rabbit ear artery and that it could play a direct role in its relaxation.

Activation of a Cl- current by hypotonic volume increase in human endothelial cells.

We have used whole-cell and perforated patches to study ionic currents induced by hypotonic extracellular solutions (HTS, 185 mOsm instead of 290 mOsm) in endothelial cells from human umbilical veins. These currents activated within 30-50 s after application of HTS, reached a maximum value after approximately 50-150 s and recovered completely after re-exposing the cells to normal osmolarity. They slowly inactivated at potentials positive to +50 mV. The same current was also activated by breaking into endothelial cells with a hypertonic pipette solution (377 mOsm instead of 290 mOsm). The reversal potential of these volume-induced currents using different extracellular and intracellular Cl- concentrations was always close to the Cl(-)-equilibrium potential. These currents are therefore mainly carried by Cl-. DIDS only weakly blocked the current (KI = 120 microM), while another Cl(-)-channel blocker, DCDPC (20 microM) was ineffective. We were unable to record single channel activity in cell-attached patches but we always observed an increased current variance during HTS. From the mean current-variance relation of the whole-cell current records, we determined a single channel conductance of 1.1 pS. The size and kinetics of the current were not correlated with the concomitant changes in intracellular calcium. Furthermore, the currents could still be activated in the presence of 10 mmol/liter intracellular EGTA and are thus Ca2+ independent. A similar current was also activated with iso-osmotic pipette solutions containing 300 mumol/liter GTP gamma S. Neomycin (1 mmol/liter), a blocker of PLC, did not prevent activation of this current. TPA (4 mumol/liter) was also ineffective in modulation of this current. The HTS-induced current was completely blocked by 10 mumol/liter pBPB, a PLA2 inhibitor. NDGA (4 mumol/liter) and indomethacin (5 mumol/liter), blockers of lipoxygenase and cyclo-oxygenase respectively, did however not affect the current induced by hypotonic solutions. The effects of arachidonic acid (10 mumol/liter) were variable. In 12 out of 40 cells it either directly activated a Cl- current or potentiated the current activated by HTS. The membrane current was decreased at all potentials in 18 cells, and was not affected in 10 cells. The HTS-induced currents may therefore be modulated by cleavage products of PLA2, but not by messengers downstream of arachidonic acid. Loading the cells with a segment of the heat stable protein kinase A inhibitor PKI (5-24) did not prevent activation of the HTS-induced current.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium ion homeostasis in smooth muscle.

Ca2+ plays an important role in the regulation of smooth-muscle contraction. In this review, we will focus on the various Ca(2+)-transport processes that contribute to the cytosolic Ca2+ concentration. Mainly the functional aspects will be covered. The smooth-muscle inositol 1,4,5-trisphosphate receptor and ryanodine receptor will be extensively discussed. Smooth-muscle contraction also depends on extracellular Ca2+ and both voltage- and Ca(2+)-release-activated plasma-membrane Ca2+ channels will be reviewed. We will finally discuss some functional properties of the Ca2+ pumps that remove Ca2+ from the cytoplasm and of the Ca2+ regulation of the nucleus.

Ca2+ extrusion across plasma membrane and Ca2+ uptake by intracellular stores.

The aim of this review is to summarize the various systems that remove Ca2+ from the cytoplasm. We will initially focus on the Ca2+ pump and the Na(+)-Ca2+ exchanger of the plasma membrane. We will review the functional regulation of these systems and the recent progress obtained with molecular-biology techniques, which pointed to the existence of different isoforms of the Ca2+ pump. The Ca2+ pumps of the sarco(endo)plasmic reticulum will be discussed next, by summarizing the discoveries obtained with molecular-biology techniques, and by reviewing the physiological regulation of these proteins. We will finally briefly review the mitochondrial Ca(2+)-uptake mechanism.

Thimerosal induced changes of intracellular calcium in human endothelial cells.

We have measured the effects of the -SH oxidizing agent thimerosal on the intracellular calcium concentration in single endothelial cells from human umbilical cord vein. Application of 1 microM thimerosal after a 10 s prepulse of 10 microM evoked oscillations of intracellular calcium. Concentrations higher than 10 microM induced a few oscillations which were followed by a long lasting increase in intracellular calcium between 120 and 980 nM at 10 microM thimerosal, between 250 and 1290 nM at 100 microM. The plateau level of the thimerosal induced increase in intracellular calcium depended on the extracellular calcium concentration, and was clearly decreased in calcium free solution. It was also reduced if the extracellular potassium concentration was increased to 140 mM. Nickel (5 mM) did not block the elevation of intracellular calcium. Thimerosal induced quenching of the Fura-2 fluorescence in Ca2+ free solutions containing 1 mM Mn2+. These effects indicate that thimerosal opens a pathway for Ca2+ entry from the extracellular side. The amount of calcium which could be released by histamine was drastically reduced after initiation of the thimerosal response. If refilling of Ca2+ stores was prevented by incubation of the cells in Ca2+ free solution, histamine still induced a transient, but not maintained, increase in [Ca2+]i. After application of thimerosal in Ca2+ free solutions to prevent refilling of the stores, a transient increase in [Ca2+]i could still be recorded but the histamine response on [Ca2+]i almost disappeared indicating a discharge of Ca2+ stores by thimerosal.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple effects of SK&F 96365 on ionic currents and intracellular calcium in human endothelial cells.

1. Multiple effects of the imidazole compound SK&F 96365 have been evaluated on endothelial cells from human umbilical vein using a combined patch clamp and Ca(2+)-microfluorimetric technique (Fura-2). 2. At concentrations of 100 mumol/l or higher of SK&F 96365, the block of the receptor-mediated Ca2+ entry overlaps with the activation of another Ca(2+)-entry mechanism, which is associated with a non selective cationic current. 3. This rise in [Ca2+]i depends on the extracellular Ca(2+)-concentration, and the entry pathway is in contrast with the receptor-mediated Ca(2+)-entry pathway permeable to Ni2+, as shown by quenching of the Fura-2 fluorescence signal. 4. The concentration of SK&F 96365 for half maximal increase in [Ca2+]i was 141 +/- 19 mumol/l (n = 16). 5. SK&F 96365 activated a current that reversed at +11.8 +/- 2.1 mV (n = 21) when measured using nystatin-perforated patches with either Cs+ or K+ in the pipette and 140 Na+, 1.5 Ca2+ in the bath (chloride equilibrium potential ECl = -36 mV). 6. SK&F 96365 (200 mumol/l) blocked an inwardly rectifying K+ current in endothelial cells independently of [Ca2+]i. This block caused depolarization of the endothelial cells from -55.3 +/- 2.57 mV (n = 33) to -10 +/- 5.5 mV (n = 6). This block was concentration-dependent, half maximal block occurred at a concentration of about 40 mumol/l SK&F 96365. 7. In cells which showed an outwardly rectifying current, this outward component was also completely blocked by 200 mumol/l SK&F 96365. 8. It is concluded that SK&F 96365 reversibly activates a non-selective cation channel at concentrations higher than 100 mumol/l, but also blocks K+ currents in endothelial cells independently of [Ca2+]i. These multiple effects overlap with the proposed block of receptor-mediated Ca2+ entry. The block of K(+)-channels may in unclamped cells reduce the driving force for Ca2+, and thereby interfere with the Ca(2+)-influx.

Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis.

The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.

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)

Immunological stimulation of single rat basophilic leukemia RBL-2H3 cells co-activates Ca(2+)-entry and K(+)-channels.

The relationship between type 1 Fc epsilon-receptor (Fc epsilon RI) mediated cell stimulation, Ca(2+)-signals and membrane currents was studied in rat mucosal mast cells, subline RBL-2H3 by combining patch-clamp, Fura-2, 45Ca(2+)-uptake and secretory response measurements. Cells were stimulated by Fc epsilon RI clustering either with IgE and antigen or by an IgE specific monoclonal antibody. Both stimuli induced a biphasic increase in the free intracellular Ca(2+)-concentration ([Ca2+]i). Fc epsilon RI clustering in Ca(2+)-free solution induces a transient increase in [Ca2+]i reflecting Ca2+ release from the Ins(1,4,5)P3 sensitive stores. Mn2+ applied to a nominally Ca(2+)-free solution, causes quenching of the Fura-2 emission during Fc epsilon RI clustering, indicating activation of a transmembrane pathway for the entry of extracellular calcium ions. Whole-cell current-voltage relationship of resting cells showed strong inward rectification. The inward current component at a potential of -100 mV is increased by 23 +/- 11% (n = 14) upon Fc epsilon RI clustering, whereas the outward component at +50 mV was enhanced by 45 +/- 6%. The Fc epsilon RI activated current was identified as due to K+ ions, because it reversed close to the K(+)-equilibrium potential, was blocked by Ba2+ or Cs+ containing or K(+)-free bath solutions. It was also inhibited by TEA and quinidine, while DIDS had no effect. Moreover, an inwardly rectifying K(+)-channel with a conductance of 28 pS was recorded in single channel measurements. The open probability of this channel increased by 39 +/- 16% (n = 8) upon Fc epsilon RI clustering. Superfusion of the cells with nominally K(+)-free solution also significantly inhibited both the Fc epsilon RI mediated 45Ca2+ uptake and the secretory response of the cells. We conclude that activation of K(+)-channels upon Fc epsilon RI clustering is functionally involved in the control and the maintenance of the secretory response of RBL-2H3 mast cells.

Calcium signalling through nucleotide receptor P2Y2 in cultured human vascular endothelium.

Microfluorometric measurements in Fura-2-loaded single cultured human vascular endothelial cells were used to characterize the intracellular calcium [Ca2+]i responses triggered by extracellular application of adenosine 5'-triphosphate (ATP) and other nucleotides. Application of ATP or uridine 5'-triphosphate (UTP) gave rise to dose-dependent elevations of [Ca2+]i in all the cells tested. At saturating concentrations of agonist, the [Ca2+]i response was biphasic, with an early peak and a sustained plateau. Unlike peak responses, the sustained Ca2+ plateau was sensitive to removal of Ca2+ from the external medium. Mn2+ quenching revealed the presence of Ca2+ influx during the agonist-induced calcium plateau. The agonist-evoked calcium plateau was inhibited in a dose-dependent manner by the Cl-channel blocker NPPB, by the divalent cation Ni2+ and by the imidazole antimycotic econazole. Previously, these compounds have been shown to block store-operated Ca2+ entry. The two phases of the agonist-evoked [Ca2+]i response were blocked by the specific phospholipase C inhibitor U-73122 and by intracellular injection of low molecular weight heparin, suggesting the involvement of IP3-sensitive intracellular Ca2+ stores. The pharmacological profile of the response, using different nucleotides and analogues, ATP = UTP > ADP = UDP, and no responses to P2X1 and P2Y1 agonists, suggested the involvement of P2Y2 receptors. The expression of mRNA for the P2Y2 receptor was detected by RT-PCR analysis. These results indicate that P2Y2 receptors linked to intracellular Ca2+ mobilization are present in human vascular endothelial cells. The initial [Ca2+]i mobilization is followed by a phase of elevated [Ca2+]i influx.

Calcium-activated potassium channels in cultured human endothelial cells are not directly modulated by nitric oxide.

Nitric oxide has been proposed to directly activated large conductance Ca(2+)-dependent K+ channels (BKCa) [Bolotina V.M., Najibi S., Palacino J.J., Pagano P.J., Cohen R.A. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 1994; 368: 850-853]. The nitric oxide (NO) donor S-nitrosocysteine (SNOC) was used to evaluate a possible direct modulation of BKCa by NO in EAhy926 (EA cells), a cultured human umbilical vein derived endothelial cell line, using the whole-cell, cell-attached and inside-out configuration of the patch-clamp technique, together with simultaneous amperometric measurement of NO and the concentration of free intracellular calcium [Ca2+]i. BKCa channels with a large conductance of approximately 190 pS, voltage-dependent activation and a reversal potential close to -80 mV have been identified in EA cells. Exposure of EA cells in the experimental chamber to 1 mM SNOC delivered approximately 5 microM NO, as recorded by an amperometric probe in situ. SNOC produced a modest increases in [Ca2+]i that was insufficient to activate BKCa channels. NO alone neither activated BKCa channels directly nor modulated preactivated BKCa channels in EA cells. These results do not support a direct modulatory effect of NO on large conductance BKCa channels in cultured endothelial cells.

Kinetic and pharmacological properties of the calcium-activated chloride-current in macrovascular endothelial cells.

We have studied the kinetic and pharmacological properties of the Ca(2+)-activated Cl- current (ICl,Ca) in cultured cell pulmonary artery endothelial (CPAE) cells by means of combined patch clamp and Fura-2 micro fluorescence measurements. The current was activated by loading the cells via the patch pipettes with Ca(2+)-buffered solutions. Currents activated slowly at positive potentials, and decayed rapidly at negative potentials. The time constant of activation decreased at more positive membrane potentials and more elevated intracellular Ca2+ concentrations ([Ca2+]i). The time constant of deactivation was Ca(2+)-independent and decreased at more negative potentials. Steady-state currents showed strong outward rectification, but the instantaneous current-voltage relationship was almost ohmic. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit ICl,Ca. Half maximal block for TFP occurred at 5.7 +/- 2.1 microM (n = 16). GTP gamma S did not activate ICl,Ca, but activated a Cl- current similar to the volume-activated Cl- current (ICl,vol). [Ca2+]i for half maximal activation of ICl,Ca was voltage-dependent, and suggests that the apparent binding constant for Ca2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the binding site is 12% within the electrical field from the cytoplasmic side. The Hill-coefficient, nH, of the binding was larger than 1 and increased with depolarization. The maximal Cl- conductance at saturating [Ca2+]i did not depend on the membrane potential. RT-PCR experiments did not provide any evidence that the endothelial Ca(2+)-activated Cl- channel might be identical with a recently cloned Ca(2+)-sensitive Cl- channel (CaCC).

Mibefradil (Ro 40-5967) blocks multiple types of voltage-gated calcium channels in cultured rat spinal motoneurones.

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.