C. Mironneau

Evidence for two distinct calcium channels in rat vascular smooth muscle cells in short-term primary culture

Smooth muscle cells were isolated from rat portal vein and studied during short-term primary culture using the whole-cell patch-clamp technique. Two distinct types of Ca channel could be separated by studying the inward currents in Ba solutions. The rapidly inactivating current was present when cells were held at very negative potentials (−80 mV). This current was prominent for relatively small depolarizations and was insensitive to nifedipine. A slowly inactivating current, corresponding to the slow Ca current previously reported in smooth muscles, was observed at less negative holding potentials (−50 mV), was prominent for positive depolarizations and was blocked by low concentrations of nifedipine. Both currents were unaffected by tetrodotoxin and both were blocked by Co.

Calcium-activated chloride current in rat vascular smooth muscle cells in short-term primary culture

Isolated cells from rat portal vein smooth muscle in short-term primary culture were studied using patch-clamp technique (whole-cell configuration). In order to study a calcium-activated chloride current, the potassium currents were blocked by intracellular cesium diffusion. Without EGTA in the pipette solution, depolarizing voltage pulses from a holding potential of −70 mV to positive potentials activated an early inward and a late outward current. The latter persisted as a long-lasting inward tail current when the membrane was repolarized to −70 mV. The outward current measured at the end of the pulse and the tail current were blocked by extracellular cobalt, after replacement of external calcium with barium, after removal of external calcium, and when the calcium concentration of the pipette solution was less than 0.5 μM, suggesting that they were calcium-dependent. The tail current decay was voltage sensitive, becoming faster with hyperpolarization. The reversal potential of the calcium-activated current was near the equilibrium potential for chloride ions, and was shifted as predicted by the Nernst equation when the extracellular or intracellular chloride concentration was changed. The calcium-activated current was blocked by adding micromolar concentrations of niflumic acid or millimolar concentrations of DIDS. This effect of compounds known to interfere with chloride channels together with the data on the equilibrium potential for chloride ions indicated above suggested the existence of a calcium-activated chloride current in vascular smooth muscle cells.

Noradrenaline activates a calcium‐activated chloride conductance and increases the voltage‐dependent calcium current in cultured single cells of rat portal vein

1 Membrane responses were recorded by a patch pipette technique in cultured cells isolated from rat portal vein. Using the whole‐cell mode, pressure ejections of noradrenaline evoked depolarization (current clamp) and inward current (voltage clamp) at membrane potentials of −60 to −70 mV. The noradrenaline‐induced response was reversibly blocked by prazosin indicating that the response was mediated by α1‐adrenoceptors. 2 The ionic mechanism of the noradrenaline‐induced inward current was investigated in potassium‐free caesium‐containing solutions. Alteration of the chloride equilibrium potential produced similar changes in the reversal potential of the noradrenaline‐induced current, indicating that noradrenaline opened chloride‐selective channels. There was no evidence implicating sodium or calcium as the charge‐carrying ion. 3 Caffeine applied in the bathing solution also induced a transient increase in chloride conductance but the noradrenaline‐induced response was lost after application of caffeine. This is interpreted to mean that the increase in chloride conductance induced by noradrenaline and caffeine can occur as a consequence of a rise in intracellular calcium concentration depending on release of calcium from the same intracellular stores. 4 In the presence of caffeine, noradrenaline increased both the voltage‐dependent calcium and chloride membrane conductances during application of repetitive depolarizing pulses. It is concluded that in isolated cells of the rat portal vein the depolarization in response to noradrenaline is mediated by an increase in chloride conductance depending on both the calcium release from intracellular stores and the increase of the voltage‐dependent calcium current.

Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein

Ca2+-activated Cl− current in freshly isolated smooth muscle cells from rat portal vein was studied using the whole-cell patch-clamp technique. Simultaneously, the free-cytosolic Ca2+ concentration (Cai) was estimated using emission from the dye Indo-1. Pretreatment of the cells with amytal and carbonyl-cyanide-m-chlorophenylhydrazone, which reduced the intracellular adenosine triphosphate concentration, was used to weaken the cellular Ca2+ homeostatic system. Cai of treated cells slowly increased during perfusion with an external Ca2+-containing solution. This rise in Cai gradually activated a Ca2+-dependent Cl− current which allowed the study of the relationship between activation of this current and Cai levels. The threshold Cai for activation of Cl− channels was around 180 nM and full activation occurred at 600 nM. The Cai dependence of the Cl− channels was not changed during application of noradrenaline and did not depend on the membrane potential. The gating of Ca2+-dependent Cl− channels of rat portal vein myocytes seems to be mainly controlled by intracellular Ca2+

Maintained contractions of rat uterine smooth muscle incubated in a Ca2+‐free solution

1 The effects of acetylcholine (10−4m), prostaglandin E2 (10−6m), vanadate (5 × 10−4m) and fluoride (10−2m) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+‐free EGTA‐containing solutions. 2 All four substances produced maintained contractions which could be initiated repeatedly after exposure to Ca2+‐free solution for more than 1 h, without a significant decrease. The largest contractions were obtained with vanadate and the smallest ones with acetylcholine. The tension was usually 7–30% of the control contraction triggered by an action potential in Ca2+ containing solution. 3 Maintained contractions induced by fluoride were unaffected by isoprenaline while those induced by acetylcholine, prostaglandin E2 and vanadate were completely relaxed. 4 Prostaglandin E2 ‐and vanadate‐induced contractions were slightly reduced by Na+ removal or by adding Ca2+ antagonists. In contrast, contractions induced by acetylcholine were suppressed in Na+‐free solution and largely inhibited in the presence of Ca2+ antagonists. 5 The depolarization induced by acetylcholine in Ca2+‐free solution was strongly dependent on the external Na+ concentration. The relationship between the size of the acetylcholine‐induced depolarization and the membrane potential (shifted by constant currents) was linear, giving an apparent reversal potential for acetylcholine close to zero potential. 6 In Ca‐free solutions and in the presence of atropine, Na+ action potentials of long duration can be evoked which produced contractions of the same order of magnitude as those initiated by acetylcholine‐induced depolarizations. 7 These results are consistent with the hypothesis that the maintained contractions in Ca2+‐free solutions induced by several stimulants could be related to Ca2+‐independent mechanisms (fluoride) or Ca2+ release from an intracellular store. This latter mechanism would include both pharmacomechanical (prostaglandin E2, vanadate) and electromechanical (acetylcholine) coupling.

Opposing effects of noradrenaline on the two classes of voltage-dependent calcium channels of single vascular smooth muscle cells in short-term primary culture

The effects of noradrenaline (NA) were investigated on both fast and slow Ca2+ currents in isolated vascular cells from rat portal vein in short-term primary culture using the whole-cell patch-clamp technique. NA (1 μM) stimulated the fast Ca2+ current when the slow Ca2+ current was inhibited either by application of a dihydropyridine derivative or by increasing the cytoplasmic Ca2+ concentration. Noradrenaline and caffeine greatly reduced the slow Ca2+ current. As this inhibitory effect is suppressed after depletion of the intracellular Ca2+ store, we propose that reduction of the slow Ca2+ current by noradrenaline is mediated by a Ca2+ release from the sarcoplasmic reticulum.

Two types of calcium currents in single smooth muscle cells from rat portal vein.

1. Using the whole‐cell recording mode of the patch‐clamp technique, we investigated the calcium currents in isolated cells from rat portal vein in short‐term primary culture. 2. From a holding potential of ‐70 mV the cells presented two types of calcium currents with 5 mM‐extracellular calcium: one type was activated by small depolarizations and inactivated quickly (fast calcium current), whereas the other required stronger depolarizations for activation and inactivated more slowly (slow calcium current). 3. Isradipine (PN 200‐110) blocked the slow calcium current at concentrations 300 times lower than those used to block the fast inward current. The isradipine‐induced inhibition was voltage‐dependent for the slow calcium current and voltage‐independent for the fast calcium current. 4. The slow calcium current was lost during internal perfusion with a 0.5 microM‐Ca2+ containing solution, and during stimulation of the cell at high frequencies (0.1‐0.2 Hz) within 5‐10 min. The fast calcium current was unchanged under these experimental conditions. 5. Steady‐state inactivation curves for both fast and slow calcium currents showed differences in their voltage dependence. Half‐maximal and complete inactivations of the fast calcium current were obtained at ‐50 and ‐30 mV while those of the slow calcium current were obtained at ‐20 and +10 mV. 6. Studied with the two‐pulse protocol, inactivation of the slow calcium current was dependent on both membrane potential and calcium influx while that of the fast calcium current appeared only dependent on membrane potential. 7. Two types of calcium currents, differing in potential dependence of inactivation, and in sensitivities to dihydropyridines, stimulation frequency and intracellular calcium concentration were identified in cultured smooth muscle cells isolated from portal vein.

Contractions of rat uterine smooth muscle induced by acetylcholine and angiotensin II in Ca2+‐free medium

1 The effects of acetylcholine (ACh, 10−4m) and angiotensin II (Ang II, 10−6 M) have been studied on the mechanical and electrical activities of rat myometrial strips perfused in Ca2+‐free EGTA‐containing solutions. 2 Both ACh and Ang II produced transient contractions, the amplitude of which can be taken as a measurement of the amount of Ca2+ present in a drug‐sensitive Ca2+ store. The degree of filling of this store depended on the external Ca2+ concentration, and on the presence of contractile responses during the Ca2+ loading period. The existence of two pathways (either direct or transcytoplasmic) is suggested for Ca2+ uptake into the internal Ca2+ store. 3 The rate of filling of the Ca2+ store in 2.1 mm‐Ca2+‐containing solution was faster (time to half‐maximal response, t1/2 = 29 ± 2.2s, n = 4) than the rate of depletion in Ca2+‐free solution (t1/2 = 3 ± 0.3 min, n = 3). The gradual depletion of this store was much slower at 18°C than at 35°C, and in the presence of vanadate which is known to inhibit Ca2+‐ATPases. 4 Methoxyverapamil (D600, 10−6‐10−5m) had no appreciable effect on the direct Ca2+ uptake or on the release of Ca2+ from the store by ACh and Ang II. Mn2+ (10−3m) completely inhibited the direct pathway to the internal Ca2+ store and also reduced the release of Ca2+. 5 ACh and Ang II induced repetitive depolarizations close to zero potential which did not parallel the transient contractions as a function of the time of perfusion in Ca2+‐free solution. Applications of 2 mm EGTA, 135 mm K+ or Ca2+ antagonists which suppressed or reduced the drug‐induced depolarizations did not affect appreciably the drug‐induced contractions. 6 These results suggest that myometrial cells have an intracellular Ca2+ store sensitive to different stimulus substances. This store is not affected by depolarization of the plasma membrane and is certainly different from that described in voltage‐clamp experiments.

Inositol 1,4,5-trisphosphatem and ryanodinemsensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes

Ca2+ signalling events were analyzed in single myocytes from rat portal vein by using a laser confocal microscope combined with the patch-clamp technique. Increase in inositol 1,4,5-trisphosphate (InsP3) concentration was obtained by photorelease from a caged precursor or intracellular dialysis of 3F-InsP3. Low InsP3 concentrations activated either small elevations of [Ca2+]i or localized Ca2+ transients whereas high InsP3 concentrations activated either homogeneous Ca2+ responses or propagated Ca2+ waves. The InsP3-evoked localized Ca2+ transients had spatio-temporal properties characteristic of Ca2+ sparks. In addition, compounds that blocked Ca2+ sparks and Ca2+ responses activated by Ca2+ jumps reduced the global InsP3-activated Ca2+ responses and suppressed the Ca2+ transients. In contrast, Ca2+ responses evoked by flash-photolytic Ca2+ jumps or caffeine were not affected by heparin (an InsP3 receptor antagonist). These results suggest that the absence of elementary Ca2+ events evoked by InsP3 may be related to the lack of clustered InsP3 receptor units in these cells, as confirmed by immunocytochemistry. Cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may represent a novel mechanism to amplify Ca2+ release from the same intracellular store and give rise to propagated Ca2+ waves.

Effects of calcium entry blockers on calcium-dependent contractions of rat portal vein

1 The effects of six calcium entry blockers belonging to the dihydropyridine (isradipine or PN 200‐110, nifedipine, nicardipine), verapamil (D888 or desmethoxyverapamil, D600 or gallopamil) and diltiazem classes were investigated on isometric spontaneous contractions and contractions induced by high‐K+ solutions, noradrenaline, acetylcholine and caffeine. 2 The rank order of potency was PN 200‐110>nicardipine = nifedipine = D888>D600>diltiazem from experiments on spontaneous contractions and high‐K+ induced contractions. With depolarized preparations, the concentration‐response curves for nicardipine, PN 200‐110, nifedipine and D600 were significantly shifted to the left indicating that the calcium entry blockers show voltage‐dependent inhibitory properties. This effect was not significant with D888 and diltiazem. 3 All the calcium entry blockers strongly reduced the noradrenaline (NA)‐ and acetylcholine (ACh)‐induced contractions at concentrations which produced complete inhibition of spontaneous contractions. They had a slight effect on caffeine‐induced contractions. 4 In Ca2+‐free, EGTA‐containing solutions, both ACh, NA and caffeine produced transient contractions, the amplitude of which could be taken as a measurement of the amount of internal calcium present in a drug‐sensitive calcium store. The filling of the calcium store was maximal after 10–12 min of calcium loading in 2.1 mm Ca2+, while the depletion was complete after 4–6 min of perfusion in Ca2+‐free solution. 5 At concentrations which abolished spontaneous contractions, PN 200‐110, nifedipine, D888 and D600 had no appreciable effect on contractions evoked in Ca2+‐free solutions by ACh, NA and caffeine. When added in Ca2+‐containing solutions diltiazem and, to a lesser extent, nicardipine strongly reduced the contractions evoked in Ca2+‐free solutions, suggesting that they inhibited the filling of the internal calcium store. 6 These results indicate that the six calcium entry blockers are potent inhibitors of calcium influx through voltage‐dependent calcium channels. Two of them (diltiazem and nicardipine) may exert an additional effect to depress contractions dependent on intracellular calcium release.