Gervaise Loirand

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.

NUCLEOTIDE RECEPTOR P2U PARTIALLY MEDIATES ATP-INDUCED CELL CYCLE PROGRESSION OF AORTIC SMOOTH MUSCLE CELLS

mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate‐early and delayed‐early cell cycle‐dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2‐methylthioATP, a preferential ligand for P2γ purinoceptors, induces only a significant increase of immediate‐early genes but not of delayed‐early genes. Moreover, the 2‐methylthioATP‐induced responses (c‐fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP‐or UTP‐induced genes code for chemotactic proteins (monocyte chemoattractant protein‐1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.

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+

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.

Pharmacological block of Ca2+-activated Cl− current in rat vascular smooth muscle cells in short-term primary culture

Ca2+-activated Cl− currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl− currents can be activated separately by Ca2+ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca2+ influx through voltage-dependent Ca2+ channels. The effects of several Cl− channel blockers and of spironolactone (a substance known to reduce internal Ca2+ loading) on both Cl− and Ca2+ currents were examined. Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2′-dicarboxylic acid (DPC) inhibited the Ca2+-activated Cl− current (IC50 values between 16.5 and 306 μM) with no effects on the inward Ca2+ current and on internal Ca2+ loading (tested by measuring the Ca2+-activated K+ current). These results indicate that the inhibition of Cl− current by these compounds is due to a direct interaction with the Cl− channel. In contrast, spironolactone inhibited both K+ and Cl− currents (IC50=7.6 μM) by reducing the amount of Ca2+ located in the internal stores, whereas the Cl− current activated by Ca2+ current through T-type Ca2+ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl− channels or intracellular Ca2+ stores.

Release of Ca2+ by noradrenaline and ATP from the same Ca2+ store sensitive to both InsP3 and Ca2+ in rat portal vein myocytes.

1. Changes in cytosolic free Ca2+ concentration ([Ca2+]i) induced by noradrenaline (NA) and ATP were investigated using indo‐1 microspectrofluorimetry in single smooth muscle cells of rat portal vein. 2. Activation of P2x‐purinoceptors by ATP (10 microM) increased [Ca2+]i from 92 +/‐ 7 nM (n = 18) to 557 +/‐ 30 nM (n = 11). In the presence of NA (10 microM), the ATP‐induced rise in [Ca2+]i was reduced to 23.6 +/‐ 1.5% (n = 7) of the control response (in the absence of NA). 3. Tetracaine (10 microM to 2 mM) inhibited in a concentration‐dependent manner the Ca(2+)‐induced Ca2+ release (CICR) evoked by 5 mM caffeine. In the presence of 1 mM tetracaine, the rise in [Ca2+]i induced by ATP (10(‐8)‐10(‐4) M) was strongly inhibited. A tetracaine‐resistant rise in [Ca2+]i, corresponding to 26.4 +/‐ 2.3% (n = 14) of control values, was recorded in response to 10 microM ATP. 4. The amplitude of the NA‐induced [Ca2+]i rise depended on NA concentrations (10(‐8)‐10(‐5) M) and was not modified by tetracaine (1 mM). 5. This study suggests that Ca2+ ions released through the InsP3 receptor‐channel upon NA application do not activate CICR and the InsP3‐ and Ca(2+)‐sensitive Ca2+ store appears to represent, at least functionally, a single releasable Ca2+ pool.

Spironolactone inhibition of contraction and calcium channels in rat portal vein

1 The effects of spironolactone have been studied on the mechanical activity of rat portal vein strips and the calcium channel currents of isolated cells using the patch clamp technique (whole‐cell configuration). 2 Spironolactone (50 nm to 0.1 mm) depressed both K+‐induced and twitch contractions within 5–6 min. This inhibitory effect was overcome by elevating the calcium concentration in the perfusing solution. 3 Spironolactone (60 μm) depressed the transient contractions induced in a Ca2+‐free, EGTA‐containing solution by either acetylcholine (0.1 mm) or noradrenaline (10 μm). The effect of spironolactone was dependent on a reduction in the filling of the internal calcium store. 4 Rapidly inactivating calcium channel current was maintained in the presence of spironolactone (60 μm), while slowly inactivating calcium channel current was blocked in a concentration‐dependent manner. Half‐inhibition of slow calcium channel current was obtained at concentrations between 5–7 μm. 5 Administration of spironolactone (10 μm) at rest reduced calcium channel current by about 70% (tonic inhibition). Repetitive depolarizations (300 ms long pulses to zero mV, applied between 0.05 and 0.5 Hz) had no further inhibitory effect on the inward current (absence of use‐dependence). 6 When cells were held at depolarized membrane potentials at which slow calcium current was inactivated by about 80%, the inhibitory effect of spironolactone (10 μm) was similar to that obtained with cells normally polarized. Spironolactone (10 μm) had no effect on the voltage‐dependence of inactivation of the calcium channel current. 7 Our results suggest that spironolactone acts primarily on the plasma membrane by depressing inward current through slow calcium channels. This effect may be explained by a preferential binding of the drug to the resting state of the slow calcium channel. In addition, spironolactone may depress contractions dependent on the release of calcium from the sarcoplasmic reticulum.