dibutyryl-cyclic-gmp and 1-4-dihydropyridine

A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 microM - 1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 microM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre-incubation of cells with 1 microM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 microM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores.

Topics: Animals; Calcium; Calcium Channels; Cells, Cultured; Dibutyryl Cyclic GMP; Dihydropyridines; Nucleotides, Cyclic; Phosphodiesterase Inhibitors; Pituitary Gland; Rats; Terpenes; Thapsigargin

During neuromuscular synapse development, the degradation rate of ACh receptors (AChRs) accumulated in the synaptic portion of the muscle membrane is drastically reduced under neural control, their half-life t1/2 increasing from 1 d to about 12 d. Recent evidence suggests that the metabolic stability of synaptic AChRs is mediated by the muscle activity induced by the nerve. We have now investigated the pathway linking muscle activity and metabolic stabilization of synaptic AChRs in organ cultured rat muscle. Soleus and diaphragm muscles were denervated for 14-40 d, a procedure leading to the destabilization of synaptic AChRs, and conditions required to restabilize synaptic AChRs in the denervated muscle were analyzed. The activity-dependent stabilization of synaptic AChRs in chronically denervated endplates required calcium entry through dihydropyridine-sensitive Ca2+ channels activated by high-frequency stimulation for approximately 6 hr and was specific for synaptic AChRs. As in vivo, extrasynaptic AChRs were not stabilized, and their t1/2 remained 1 d. The stabilization process was not dependent on de novo protein synthesis, and it could also be brought about by elevated cAMP levels. Furthermore, it required shorter stimulation periods in the presence of the phosphatase inhibitors okadaic acid and calyculin A, whereas blockade of protein kinases with high doses of staurosporine blocked the stabilization. Activity-dependent, dihydropyridine-sensitive as well as cAMP-dependent phosphorylation of myosin light chain was observed. These findings are consistent with the notion that muscle activity initiates AChR stabilization via the activation of calcium-dependent protein phosphorylation reactions.

Topics: Alkaloids; Animals; Bucladesine; Calcium; Calcium Channels; Cyclic AMP; Dibutyryl Cyclic GMP; Dihydropyridines; Electric Stimulation; Ethers, Cyclic; Kinetics; Male; Marine Toxins; Motor Endplate; Muscle Denervation; Muscles; Okadaic Acid; Organ Culture Techniques; Oxazoles; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Potassium Chloride; Protein Kinase Inhibitors; Protein Kinases; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Staurosporine; Synapses; Tetrodotoxin; Time Factors