To examine the nature of inositol 1,4,5-trisphosphate (IP(3))-sensitive and ryanodine (Ryn)-sensitive Ca(2+) stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured using fura 2-AM fluorescence. Properties of elementary local Ca(2+) release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca(2+) stores with Ryn (300 microM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca(2+) transients but had little or no effect on the initial IP(3)-mediated intracellular Ca(2+) transient induced by ANG II (1 microM). Cyclopiazonic acid (CPA; 10 microM) abolished IP(3)-induced intracellular Ca(2+) transients but failed to attenuate the initial Caf-induced intracellular Ca(2+) transient. These results suggest that in canine PASMC, IP(3)-, and Ryn-sensitive Ca(2+) stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca(2+) stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca(2+) transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for "Ca(2+) sparks" in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca(2+) stores, ANG II (8 nM) induced slow, sustained [Ca(2+)](i) increases originating at sites near the cell surface, which were abolished by depleting IP(3) stores. Discrete quantal-like events expected due to the coordinated opening of IP(3) receptor clusters ("Ca(2+) puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca(2+) stores and elementary Ca(2+) release events in isolated PASMC.

Experiments were performed to determine whether capacitative Ca(2+) entry (CCE) can be activated in canine pulmonary and renal arterial smooth muscle cells (ASMCs) and whether activation of CCE parallels the different functional structure of the sarcoplasmic reticulum (SR) in these two cell types. The cytosolic [Ca(2+)] was measured by imaging fura-2-loaded individual cells. Increases in the cytosolic [Ca(2+)] due to store depletion in pulmonary ASMCs required simultaneous depletion of both the inositol 1,4,5-trisphosphate (InsP(3))- and ryanodine (RY)-sensitive SR Ca(2+) stores. In contrast, the cytosolic [Ca(2+)] rises in renal ASMCs occurred when the SR stores were depleted through either the InsP(3) or RY pathways. The increase in the cytosolic [Ca(2+)] due to store depletion in both pulmonary and renal ASMCs was present in cells that were voltage clamped and was abolished when cells were perfused with a Ca(2+)-free bathing solution. Rapid quenching of the fura-2 signal by 100 microM Mn(2+) following SR store depletion indicated that extracellular Ca(2+) entry increased in both cell types and also verified that activation of CCE in pulmonary ASMCs required the simultaneous depletion of the InsP(3)- and RY-sensitive SR Ca(2+) stores, while CCE could be activated in renal ASMCs by the depletion of either of the InsP(3)- or RY-sensitive SR stores. Store depletion Ca(2+) entry in both pulmonary and renal ASMCs was strongly inhibited by Ni(2+) (0.1-10 mM), slightly inhibited by Cd(2+) (200-500 microM), but was not significantly affected by the voltage-gated Ca(2+) channel (VGCC) blocker nisoldipine (10 microM). The non-selective cation channel blocker Gd(3+) (100 microM) inhibited a portion of the Ca(2+) entry in 6 of 18 renal but not pulmonary ASMCs. These results provide evidence that SR Ca(2+) store depletion activates CCE in parallel with the organization of intracellular Ca(2+) stores in canine pulmonary and renal ASMCs.

We examined the possibility of functional and molecular expression of volume‐regulated Cl− channels in vascular smooth muscle using the whole‐cell patch‐clamp technique and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) on cells from canine pulmonary and renal arteries. Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl−‐dependent outward‐rectifying membrane currents with an anion permeability sequence of SCN− > I− > Br− > Cl− > aspartate−. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. Experiments were performed to determine whether the molecular form of the volume‐regulated chloride channel (ClC‐3) is expressed in pulmonary and renal arteries. Quantitative RT‐PCR confirmed expression of ClC‐3 in both types of smooth muscle. ClC‐3 expression was 76.4 % of β‐actin in renal artery and 48.0 % of β‐actin in pulmonary artery. We conclude that volume‐regulated Cl− channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.

We compared the effects of thapsigargin (TG), a selective blocker of Ca(2+)-adenosinetriphosphatase of sarcoplasmic reticulum (SR), and ryanodine (Ry) in the single isolated myocytes of guinea pig ventricular myocardium loaded with indo 1 acetoxymethyl ester (AM). TG (2 x 10(-7) M) inhibited the rapid phase of Ca2+ transient, increased time to peak intracellular Ca2+ concentration ([Ca2+]i) from 158 +/- 12 to 391 +/- 60 ms and decreased the total amplitude of the transient to 89 +/- 4% of the pre-TG control. Time to peak of contractions increased from 350 +/- 47 to 410 +/- 37 ms and total duration from 666 +/- 62 to 850 +/- 198 ms. Total amplitude of contractions was hardly affected. In the cells not loaded with indo 1-AM TG decreased the amplitude of contractions to 71 +/- 3% of control. When the effects of TG were fully developed, the cells ceased to respond to 1 s of superfusion with 15.0 mM caffeine with transient elevation of [Ca2+]i and/or transient contracture. TG did not affect the amplitude or time course of Ca2+ current (ICa) or the current-voltage relation. We propose that Ca2+ transients and contractions in the cells treated with TG were initiated by sarcolemmal Ca2+ influx. Ry (1.0 microM) initiated similar changes in the time course of Ca2+ transients and contractions as TG; however, total amplitude of the transients and contractions was reduced to 78 +/- 5 and 55 +/- 7% of the control, respectively. The SR Ca2+ was also depleted by Ry. TG superfused over the cells pretreated with Ry increased the amplitude of Ca2+ transients and respective contractions to the pre-Ry level. TG did not affect the ICa in the cells pretreated with Ry nor did it change configuration of action potentials to increase the Ca2+ influx. We propose that the effect of Ry on amplitude of Ca2+ transients and contractions results from the trapping of a fraction of sarcolemmal Ca2+ influx by the SR and its rapid release into subsarcolemmal space. From there it is extruded out of the cell by Na(+)-Ca2+ exchange before ever reaching the contractile system.

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We investigated the effect of 10(-8) M noradrenaline (NA) on [Ca2+]i and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATs caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADs). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 microM BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by 70% and appearance of DATs. Also, the frequent multiple oscillations of Ca2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 microM Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 x 10(-7) M thapsigargin, a selective blocker of Ca(2+)-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance.

Binding of ligand to receptor, in various types of cells, results in changes in calcium concentration, which is an important factor in cellular signal transduction. Lymphocytes receive signals from the parasympathetic nervous system through the cholinergic receptors. Cholinergic receptors mediate response to stimuli through changes of the IP₃, or cAMP level and Ca²⁺mobilization in various types of cells. The aim of this work was to measure changes in calcium concentration in cytosol of lymphocytes stimulated with acetylcholine agonists - carbachol (analogue of acetylcholine) or nicotine. Human peripheral blood mononuclear cells (PMBC) and lymphocytes from the leukemic cell lines Jurkat and Raji were used. Immune cells were preactivated with mitogen phytohemagglutinin (PHA) for PBMC and Jurkat cells or lipopolysaccharide for Raji cells. Two methods of measurement of calcium concentration were used. With the first, calcium concentration was measured in the suspension of cells loaded with the fluorescent dye Fura-2AM. With the other method, calcium concentration was assessed in single cells loaded with the fluorescent dye Indo-1AM. Using the method of single cell investigation, we observed an increase in the level of calcium concentration induced by carbachol and nicotine. The method of measuring the Ca²⁺concentration in a cell suspension was found to be not sensitive enough for this purpose. The increase in calcium concentration resulted both from the stimulation of Ca²⁺influx and from the release of Ca²⁺from intracellular stores, most likely due to the increase in the concentration of IP₃. In conclusion, we suggest that the lymphocytes activated with PHA respond to cholinergic stimulation with an increase in their free cytoplasmic Ca²⁺concentration.