h-89 and 1-4-dihydropyridine

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Calcium Channel Blockers; Calcium Channels; Calcium Channels, L-Type; Cesium; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dihydropyridines; Drosophila; Enzyme Inhibitors; Isoquinolines; Larva; Membrane Potentials; Muscles; Mutagenesis; Nervous System; Phosphodiesterase Inhibitors; Sulfonamides

This study examines the role of the cAMP signaling pathway in the regulation of 45Ca influx in cultured vascular smooth muscle cells from the rat aorta. K+o-induced depolarization of smooth muscle cells increased the rate of 45Ca uptake by twofold to threefold. This effect was completely abolished by the dihydropyridine derivatives nifedipine and nicardipine, with a Ki of 3 and 10 nmol/L, respectively. Activators of cAMP signaling (isoproterenol, forskolin, cholera toxin) increased cAMP content by 50- to 100-fold and decreased voltage-dependent 45Ca uptake by 50% to 70%. Neither the dihydropyridines nor the cAMP activators affected basal 45Ca influx. Direct addition of the protein kinase inhibitor H-89 to the incubation medium in the 1- to 10-micromol/L range did not alter basal 45Ca uptake but completely abolished voltage-dependent Ca2+ transport. Preincubation of cells for 1 hour with 10 micromol/L H-89 did not modify basal and depolarization-induced 45Ca uptake in H-89-free medium but prevented forskolin-induced inhibition of voltage-dependent Ca2+ influx. The addition of cytoskeleton-active compounds reduced voltage-dependent Ca2+ transport and completely abolished its regulation by cAMP. Activation of cAMP signaling decreased the volume of smooth muscle cells by 12% to 15%. The same cell volume diminution in hyperosmotic medium did not alter voltage-dependent 45Ca uptake. Thus, data obtained in this study show that in contrast to cardiac and skeletal myocytes, in vascular smooth muscle cells, 45Ca influx, putatively due to L-type channels, is inhibited by cAMP. This regulatory pathway appears to be mediated via protein kinase A activation and cytoskeleton reorganization.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Size; Cells, Cultured; Cyclic AMP; Dihydropyridines; Enzyme Inhibitors; Ion Transport; Isoquinolines; Muscle, Smooth, Vascular; Rats; Rats, Inbred BN; Signal Transduction; Sulfonamides