calcimycin and 5-dimethylamiloride

Topics: Adenosine Diphosphate; Amiloride; Aniline Compounds; Blood Platelets; Calcimycin; Calcium; Cytosol; Fluorescent Dyes; Humans; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Spectrometry, Fluorescence; Thrombin; Xanthenes

GnRH stimulates LH release from gonadotropes in a Ca2(+)-dependent manner. Because of the apparent relationship between cellular Ca2+ metabolism and Na(+)-driven antiports, we investigated their influence on GnRH action. We also assessed the influence of bicarbonate, because its transport may alter effects of Na+/H+ exchange on intracellular pH. In pituitary cell cultures without bicarbonate, GnRH-stimulated LH release was reduced by Na+ omission, by amiloride, and by amiloride analogs that selectively block Na+/H+ exchange. The Na+ dependence of amiloride action (EC50, 14 and 100 microM in medium with 20 and 135 mM NaCl, respectively, and no effect in Na(+)-free medium) and the order of potency of these analogs, indicated specific inhibition of Na+/H+ exchange. 5-(N,N-Di-methyl)amiloride (DMA; a potent Na+/H+ exchange inhibitor) reduced GnRH-stimulated LH release but not GnRH receptor binding or Ca2+ ionophore (A23187)-stimulated LH release, suggesting inhibition at a locus beyond receptor occupancy but before exocytosis. Amiloride analogs that selectively inhibit Na+/Ca2+ exchange also modestly reduced GnRH-stimulated LH release. Bicarbonate (10 mM) reduced the inhibitory effects of DMA and Na+ omission (but not the effects of the Na+/Ca2+ exchange inhibitors or of a Ca2+ channel antagonist), and the effect of bicarbonate was inhibited by a blocker of bicarbonate-dependent antiports. These observations reveal the Na+ dependence of GnRH action and that gonadotropes possess a Na+/H+ exchanger. The Na+ dependence of GnRH-stimulated LH release appears to reflect at least in part dependence upon this antiport. Prevention of the Na+/H+ exchange inhibitor effects by bicarbonate supports the specificity of their action, but suggests regulation of this antiport as an unlikely means of controlling LH release in vivo.

Topics: Amiloride; Animals; Basophils; Bicarbonates; Buserelin; Calcimycin; Carrier Proteins; Female; Gonadotropin-Releasing Hormone; Gonadotropins; Luteinizing Hormone; Pituitary Gland, Anterior; Rats; Sodium; Sodium-Hydrogen Exchangers

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.

Topics: Adenosine Triphosphate; Alkaloids; Amiloride; Arachidonic Acids; Aspirin; Blood Platelets; Calcimycin; Carrier Proteins; Enzyme Activation; Humans; Indoles; Ionomycin; Ionophores; Kinetics; Lactams; Phospholipases; Phospholipases A; Phospholipases A2; Phosphorylation; Protein Kinase C; Sodium-Hydrogen Exchangers; Staurosporine; Tetradecanoylphorbol Acetate; Thrombin; Type C Phospholipases

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.

Topics: Amiloride; Blood Platelets; Body Fluids; Calcimycin; Carrier Proteins; Ethers; Humans; Hydrogen-Ion Concentration; Intracellular Fluid; Ionomycin; Nigericin; Prostaglandin D2; Prostaglandins D; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Thrombin; Time Factors