Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells

To clarify interactions between the cytoskeleton and activity of L-type Ca2+ (CaL) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca2+ current [ I Ba(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-α-actin and anti-α-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I Ba(L) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I Ba(L) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I Ba(L) due to another unknown mechanism, e.g., a direct block of CaLchannels. In summary, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not.

Receptors linked to polyphosphoinositide hydrolysis stimulate Ca2+ extrusion by a phospholipase C-independent mechanism

In A7r5 cells with empty intracellular Ca2+ stores in which the cytosolic free Ca2+ concentration ([Ca2+]i) had been increased by capacitative Ca2+ entry, stimulation of receptors linked to phospholipase C (PLC), including those for Arg8-vasopressin (AVP) and platelet-derived growth factor (PDGF), caused a decrease in [Ca2+]i. This effect was further examined in a stable variant of the A7r5 cell line in which the usual ability of hormones to stimulate non-capacitative Ca2+ entry is not expresssed. In thapsigargin-treated cells, neither AVP nor PDGF affected capacitative Mn2+ or Ba2+ entry, but both stimulated the rate of Ca2+ extrusion, and their abilities to decrease [Ca2+]i were only partially inhibited by removal of extracellular Na+. These results suggest that receptors linked to PLC also stimulate plasma membrane Ca2+ pumps. Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 1 μM) also caused a decrease in [Ca2+]i by accelerating Ca2+ removal from the cytosol; the effect was again only partially inhibited by removal of extracellular Na+. An inhibitor of PKC, Ro31-8220 (10 μM), abolished the ability of PDBu to decrease [Ca2+]i,without affecting the response to maximal or submaximal concentrations of AVP. Similar experiments with PDGF were impracticable because Ro31-8220, presumably by inhibiting the tyrosine kinase activity of the PDGF receptor, abolished all responses to PDGF. U73122 (10 μM), an inhibitor of PLC, completely inhibited PDGF- or AVP-evoked Ca2+ mobilization, without preventing either stimulus from causing a decrease in [Ca2+]i. We conclude that receptors coupled to PLC, whether via G-proteins or protein tyrosine kinase activity, also share an ability to stimulate the plasma membrane Ca2+ pump via a mechanism that does not require PLC activity.

Depletion and filling of intracellular calcium stores in vascular smooth muscle

In vascular smooth muscle, binding of vasoactive substances to surface membrane receptors leads to a rise of intracellular cytoplasmic Ca2+ and to contraction. Cytoplasmic free Ca2+ concentration ([Ca2+]i) increases through release of Ca2+ from intracellular stores and Ca2+ entry through surface membrane ion channels. Membrane-permeant and membrane-impermeant forms of fura 2 were used to distinguish changes in intracellularly stored Ca2+ ([Ca2+]s) from changes in [Ca2+]i. The spatiotemporal patterns of the movement of Ca2+ between these two cellular compartments in cultured vascular smooth muscle cells (A7r5 cell line) were visualized with digital imaging fluorescence microscopy. The Ca2+ stores were localized by double staining with a fluorescent organelle-specific dye and the Ca2+ indicator. [Ca2+]s was measured after accumulation of the membrane-permeant form of fura 2 inside the stores and quenching of the fura 2 fluorescence in the cytoplasmic compartment with manganese. Stimulation with vasopressin led to a transient increase of [Ca2+]i and a concomitant decrease of [Ca2+]s. After stimulation with vasopressin, [Ca2+]i returned rapidly to normal resting levels, whereas the recovery of [Ca2+]s occurred on a much slower time scale. The refilling pathway of depleted stores involved Ca2+ entry into the bulk cytoplasmic compartment before uptake into the stores.

Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for calcium channel activation and dihydropyridine agonist action.

We have investigated the gating kinetics of calcium channels in the A7r5 cell line at the level of single channels and whole cell currents, in the absence and presence of dihydropyridine (DHP) calcium channel agonists. Although latencies to first opening and macroscopic currents are strongly voltage dependent, analysis of amplitude histograms indicates that the primary open-closed transition is voltage independent. This suggests that the molecular mechanisms for voltage sensing and channel opening are distinct, but coupled. We propose a modified Monod-Wyman-Changeux (MWC) model for channel activation, where movement of a voltage sensor is analogous to ligand binding, and the closed and open channels correspond to inactive (T) and active (R) states. This model can account for the activation kinetics of the calcium channel, and is consistent with the existence of four homologous domains in the main subunit of the calcium channel protein. DHP agonists slow deactivation kinetics, shift the activation curve to more negative potentials with an increase in slope, induce intermingled fast and slow channel openings, and reduce the latency to first opening. These effects are predicted by the MWC model if we make the simple assumption that DHP agonists act as allosteric effectors to stabilize the open states of the channel.