Stoichiometry of contraction and Ca2+ mobilization by inositol 1,4,5-trisphosphate in isolated gastric smooth muscle cells

Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach and permeabilized by brief exposure to saponin. Both permeabilized and intact muscle cells contracted in response to cholecystokinin octapeptide (CCK-8) and acetylcholine, but only permeabilized muscle cells contracted in response to inositol 1,4,5-trisphosphate (InsP3). The contractile response to InsP3 was prompt (peak less than 5 s), concentration-dependent (EC50-0.3 microM), and insensitive to antimycin or oligomycin. Contraction induced by either InsP3 or CCK-8 was accompanied by a concentration-dependent increase in free Ca2+ that was directly correlated with the magnitude of contraction. Both InsP3 and CCK-8 caused rapid net efflux of Ca2+ from cells preloaded with 45Ca2+. Contraction, increase in free Ca2+ concentration, and net 45Ca2+ efflux elicited by a combination of maximal concentrations of InsP3 and CCK-8 were not significantly different from those elicited by maximal concentrations of either agent alone. Repeated stimulation of single muscle cells with either InsP3 or CCK-8 in Ca2+-free medium caused eventual loss of the contractile response to all agents. The response to all agents was restored upon re-exposure of the cell to a cytosol-like concentration of Ca2+, implying equal access of InsP3 and receptor-linked agonists to the same intracellular Ca2+ store. The results demonstrate that InsP3 mimics the effects of receptor-linked agonists on contraction and mobilization of intracellular Ca2+ in permeabilized smooth muscle cells that retain the functional properties of intact smooth muscle cells and support a role for InsP3 as membrane-derived messenger responsible for mobilization of intracellular Ca2+ in smooth muscle cells.     

Cyclic AMP enhances inositol trisphosphate-induced mobilization of intracellular Ca2+ in cultured aortic smooth muscle cells

The effect of cAMP on ATP-induced intracellular Ca+ mobilization in cultured rat aortic smooth muscle cells was investigated. Treatment of cells for 3 min at 37 degrees C with dibutyryl cAMP, a membrane-permeable analogue of cAMP, at concentration up to 500 microM resulted in 1.5- to 1.7-fold increase in the peak cytosolic Ca2+ concentration when cells were stimulated with 3 to 200 microM ATP either in the presence or absence of extracellular Ca2+. Similar results were obtained when 0.5 mM 8-Br-cAMP or 10 microM forskolin was used instead of dibutyryl cAMP. In contrast to the Ca2+ response, dibutyryl cAMP did not affect ATP-induced formation of inositol trisphosphate (IP3). Furthermore, the dibutyryl cAMP treatment did not affect the size of the Ca2+ response elicited by 10 microM ionomycin. These results suggest that intracellular cAMP potentiates the ATP-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ store(s), rather than by increasing the ATP-induced production of IP3 or by increasing the size of the intracellular Ca2+ store. Using saponin-permeabilized cells, we have shown directly that cAMP enhances Ca2+ mobilization by potentiating the Ca2+-releasing effect of IP3 from the intracellular Ca2+ store.     

Hydrogen peroxide-induced Ca2+ mobilization in pulmonary arterial smooth muscle cells

Reactive oxygen species (ROS) generated from NADPH oxidases and mitochondria have been implicated as key messengers for pulmonary vasoconstriction and vascular remodeling induced by agonists and hypoxia. Since Ca(2+) mobilization is essential for vasoconstriction and cell proliferation, we sought to characterize the Ca(2+) response and to delineate the Ca(2+) pathways activated by hydrogen peroxide (H(2)O(2)) in rat intralobar pulmonary arterial smooth muscle cells (PASMCs). Exogenous application of 10 microM to 1 mM H(2)O(2) elicited concentration-dependent increase in intracellular Ca(2+) concentration in PASMCs, with an initial rise followed by a plateau or slow secondary increase. The initial phase was related to intracellular release. It was attenuated by the inositol trisphosphate (IP(3)) receptor antagonist 2-aminoethyl diphenylborate, ryanodine, or thapsigargin, but was unaffected by the removal of Ca(2+) in external solution. The secondary phase was dependent on extracellular Ca(2+) influx. It was unaffected by the voltage-gated Ca(2+) channel blocker nifedipine or the nonselective cation channel blockers SKF-96365 and La(3+), but inhibited concentration dependently by millimolar Ni(2+), and potentiated by the Na(+)/Ca(2+) exchange inhibitor KB-R 7943. H(2)O(2) did not alter the rate of Mn(2+) quenching of fura 2, suggesting store- and receptor-operated Ca(2+) channels were not involved. By contrast, H(2)O(2) elicited a sustained inward current carried by Na(+) at -70 mV, and the current was inhibited by Ni(2+). These results suggest that H(2)O(2) mobilizes intracellular Ca(2+) through multiple pathways, including the IP(3)- and ryanodine receptor-gated Ca(2+) stores, and Ni(2+)-sensitive cation channels. Activation of these Ca(2+) pathways may play important roles in ROS signaling in PASMCs.     

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.     

Inhibition of energy-dependent Ca2+ accumulation in fibroblasts by smooth muscle antibodies.

An antibody prepared against smooth muscle myosin interferes with active Ca2+ accumulation of fibroblasts. This provides further evidence for the existence of myosin at the cell surface.     

Regulation of inositol trisphosphate receptors by luminal Ca2+ contributes to quantal Ca2+ mobilization.

The quantal behaviour of inositol trisphosphate (InsP3) receptors allows rapid graded release of Ca2+ from intracellular stores, but the mechanisms are unknown. In Ca2+-depleted stores loaded with Fura 2, InsP3 caused concentration dependent increases in the rates of fluorescence quench by Mn2+ that were unaffected by prior incubation with InsP3, indicating that InsP3 binding did not cause desensitization. When Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP3 caused rapid, partial decreases in fluorescence ratios. Subsequent addition of a maximal InsP3 concentration caused the fluorescence to fall to within 5% of that recorded after ionomycin. Addition of all but the lowest concentrations of InsP3 to stores loaded with the lower affinity indicator, Calcium Green-5N, caused almost complete emptying of the stores at rates that increased with InsP3 concentration. The lowest concentration of InsP3 (10 nM) slowly emptied approximately 80% of the stores, but within 3 min the rate of Ca2+ release slowed leaving approximately 7 microM Ca2+ within the stores, which was then rapidly released by a maximal InsP3 concentration. In stores co-loaded with both indicators, InsP3-evoked Ca2+ release appeared quantal with Fura 2 and largely non-quantal with Calcium Green-5N; the discrepancy is not, therefore, a direct effect of the indicators. The fall in luminal [Ca2+] after activation of InsP3 receptors may, therefore, cause their inactivation, but only after the Ca2+ content of the stores has fallen by approximately 95% to < or = 10 microM.     

Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle

We examined the effects of Rho kinase on contraction and intracellular Ca2+ concentration ([Ca2+](i)) in guinea pig trachealis by measuring isometric force and the fura 2 signal [340- to 380-nm fluorescence ratio (F340/F380)]. A Rho kinase inhibitor, Y-27632 (1-1,000 microM), inhibited methacholine (MCh)-induced contraction, with a reduction in F340/F380 in a concentration-dependent manner. The values of EC(50) for contraction and F340/F380 induced by 1 microM MCh with Y-27632 were 27.3 +/- 5.1 and 524.1 +/- 31.0 microM, respectively. With 0.1 microM MCh, the values for these parameters were decreased to 1.0 +/- 0.1 and 98.2 +/- 6.2 microM, respectively. Tension-F340/F380 curves for MCh indicated that Y-27632 caused an ~50% inhibition of MCh-induced contraction, without a reduction in F340/F380. These effects of Y-27632 were not inhibited by a protein kinase C inhibitor, GF-109203X. Our results indicate that inhibition of Rho kinase attenuates both Ca2+ sensitization and [Ca2+](i).     

Uncoupling of ATP-induced inositol phosphate formation and Ca(2+) mobilization by phorbol ester in canine cultured tracheal epithelial cells

The regulation of the increase in inositol phosphates (IPs) production and intracellular Ca(2+) concentration ([Ca(2+)](i)) by protein kinase C (PKC) was investigated in canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca(2+) mobilization. The concentrations of PMA that gave half-maximal (EC(50)) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca(2+)](i) were 5-10 and 4-12 nM, respectively. Prior treatment of TECs with staurosporine (1 microM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, -theta, and -zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, -betaI, -betaII, -gamma, -delta, -epsilon, and -theta from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-zeta was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca(2+)](i) increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-alpha, -betaI, -betaII, -delta, -epsilon, -gamma, and -theta induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca(2+) mobilization in TECs.     

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.     

Ca2+ microdomains in smooth muscle

In smooth muscle, Ca(2+) controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca(2+) to perform these multiple functions is the cell's ability to localize Ca(2+) signals to certain regions by creating high local concentrations of Ca(2+) (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca(2+) influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca(2+) store. A single Ca(2+) channel can create a microdomain of several micromolar near (approximately 200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca(2+)] and the rapid rates of decline target Ca(2+) signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca(2+) by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca(2+). In this review, the generation of microdomains arising from Ca(2+) influx across the plasma membrane and the release of the ion from the SR Ca(2+) store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

Inhibition of inositol 1,4,5-trisphosphate-mediated Ca2+ release by Ca2+ in cells from peripheral tissues

Permeabilized cells attached to culture plates were used to evaluate the inhibition of inositol 1,4,5-trisphosphate-mediated release (IPMCR) by Ca2+. In AR42J cells, a pancreatic acinar cell line, when permeabilization and Ca2+ uptake were carried out at low ionized Ca2+ (0.06 microM), Ca2+ had little effect on IPMCR. On the other hand, when permeabilization and Ca2+ uptake were performed at 5 microM Ca2+, IPMCR was inhibited by Ca2+ with an apparent affinity of 0.24 microM. This inhibition could be modified by exposing the cytosol of permeabilized cells to low Ca2+. Hence, permeabilizing the cells in the presence of 5 microM Ca2+ and then exposing them to Ca2+ concentrations between 0.01 and 5 microM before washing and Ca2+ uptake in the presence of 5 microM Ca2+ resulted in a Ca2(+)-dependent loss of inhibitory activity. The loss of inhibitory activity occurred with an apparent affinity for Ca2+ of 0.21 microM. A similar phenomenon with a comparable apparent dissociation constant for Ca2+ was found with three other cell types from peripheral tissues: the osteosarcoma cell line UMR-106-01, the kidney inner medullary cell line IMCD, and primary culture of urinary bladder smooth muscle cells. The properties of inhibition of IPMCR by Ca2+ in cells from peripheral tissues differ from those previously described in neuronal tissues and suggest that a different factor(s) mediates the inhibition of IPMCR by Ca2+ in cells from peripheral and neuronal tissues.     

A pertussis/cholera toxin sensitive G-protein may mediate vasopressin-induced inositol phosphate formation in smooth muscle cell

Arg-vasopressin (AVP) stimulates the production of inositol-1,4,5-triphosphate, inositol-1,4-bisphosphate and inositol-1-phosphate in A10 smooth muscle cell line. The AVP stimulation is rapid, time and dose dependent with an ED50 value of 5 nM. Protein kinase C activator, phorbol ester blocks the AVP effect on the production of inositol phosphates, suggesting that AVP induced phospholipase C (PLC) activation is under the negative feedback regulation by diacylglycerol production. Prolonged overnight treatment with either pertussis toxin and cholera toxin resulted partial inhibition of AVP-induced production of inositol phosphates. This result suggests that a novel G-protein similar to transducin might be involved in the AVP-induced PLC activation.