calcimycin and 3--4--dichlorobenzamil

The aim of this study was to analyze the relationship of the Na+/Ca2+ exchanger, cytosolic calcium, and chloride to the transepithelial transport of sodium in isolated frog skin. Sodium transport was measured as amiloride-inhibitable short circuit current (SCC). We studied the effect of variations in the concentrations of external chloride and of the manipulation of calcium on sensitive amiloride SCC. Modifications in the movement of Ca2+ were induced by an ionophore, A23187, and a Ca2+ channel blocker, nifedipine. Calcium ionophore A23187 (5 and 20 microM), in a normal Ringer's solution, increased SCC and transepithelial potential difference (PD). In contrast, nifedipine (20 microM) reduced SCC and PD. The role of the Na+/Ca2+ exchanger was studied using dichlorobenzamil (DCB, 50 microM) and quinacrine (1 mM), inhibitors of this exchanger. They selectively increased SCC and PD on the mucosal side of the skin, with no effect on the serosal side. This response occurred only in the presence of extracellular calcium. Replacement of NaCl by sodium methanesulfonate or the addition of furosemide (1 mM) at the serosal compartment, decreased basal SCC and PD and blocked the response to A23187 and the mucosal effect of DCB and quinacrine. These results suggest the presence of an Na+/Ca2+ exchanger located on the mucosal side of the frog skin, which participates in the transepithelial sodium transport. The action of this exchanger may be modulated by external chloride and calcium. J. Exp. Zool. 289:23-32, 2001.

Topics: Amiloride; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Chlorides; Electric Conductivity; Enzyme Inhibitors; Epithelium; Ion Transport; Ionophores; Membrane Potentials; Nifedipine; Quinacrine; Rana pipiens; Skin Physiological Phenomena; Sodium; Sodium-Calcium Exchanger; Time Factors

The platelet integrin IIbbeta3 has become a new target for the treatment of pathological thrombosis. It becomes apparent that the affinity of IIbbeta3 for its ligands is dynamically regulated by inside-out signaling. However, the components that couple diverse intracellular signals to the cytoplasmic domains of IIbbeta3 remain obscure. Employing a chymotrypsin-induced IIbbeta3 activation model, we previously proposed the hypothesis that Na+/Ca2 + exchanger (NCX) may be involved in inside-out signaling (Shiraga et al: Blood 88:2594, 1996). In the present study, employing two unrelated Na+/Ca2+ exchange inhibitors, 3',4'-dichlorobenzamil (DCB) and bepridil, we investigated the role of NCX in platelet activation induced by various agonists in detail. Both inhibitors abolished platelet aggregation induced by all agonists examined via the inhibition of IIbbeta3 activation. Moreover, these inhibitors abolished IIbbeta3 activation induced by phorbol 12-myristate 13-acetate or A23187. On the other hand, neither of these inhibitors showed apparent inhibitory effects on protein phosphorylation of pleckstrin or myosin light chain, or an increase in intracellular calcium ion concentrations evoked by 0.1 U/mL thrombin. These effects of the NCX inhibitors are in striking contrast to those of protein kinase C inhibitor, Ro31-8220. Biochemical and ultrastructural analyses showed that NCX inhibitors, particularly DCB, made platelets "thrombasthenic". These findings suggest that the NCX is involved in the common steps of inside-out signaling through integrin IIbbeta3.

Topics: Amiloride; Bepridil; Blood Platelets; Blood Proteins; Calcimycin; Calcium; Chymotrypsin; Cytoplasmic Granules; Fibrinogen; Humans; Ion Transport; Myosin Light Chains; P-Selectin; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Processing, Post-Translational; Serotonin; Signal Transduction; Sodium; Sodium-Calcium Exchanger; Tetradecanoylphorbol Acetate

Contractions of intact rat aortic rings induced by endothelin were totally inhibited by the amiloride analogue dichlorobenzamil (DCB) at concentrations known to block Na-Ca exchange (IC50 = 30 microM). Amiloride (100 microM) was ineffective. Ca-channel blockers and a K-channel opener elicited only partial inhibition. These results could indicate that the Na-Ca exchanger plays an important role in endothelin-induced contractions. Endothelin, however, had no effect on the kinetics of the exchanger, and, in addition, contractions also occurred in Na-depleted vessels. The endothelin-induced contractions produced by Ca release from intracellular pools were also completely inhibited by DCB. In fact, the latter compound was found to block contractions induced by Ca itself in the presence of Ca ionophore or detergent. We conclude that DCB acts directly on Ca-induced activation of myofilaments in smooth muscle.

Topics: Amiloride; Animals; Aorta, Thoracic; Calcimycin; Calcium; Calcium Channel Blockers; Carrier Proteins; Egtazic Acid; Endothelins; In Vitro Techniques; Membrane Proteins; Muscle, Smooth, Vascular; Peptides; Rats; Sodium-Calcium Exchanger; Vasoconstriction

The importance of extracellular calcium for the expression of endothelium-dependent relaxation was examined in isolated rat aortic rings contracted by methoxamine. The endothelium-dependent relaxation generated by acetylcholine or the calcium ionophore A23187 was eliminated when rings were placed in physiological buffer to which calcium had not been added. The endothelium-independent relaxation to sodium nitroprusside was still elicited in the presence of this "low calcium" buffer. Pretreatment of aortic rings with high concentrations of nifedipine (5 X 10(-7) M) or verapamil (10(-5) M) caused a comparable displacement to the right (2-3 times) in the relaxant dose-response curve for acetylcholine, A23187 and sodium nitroprusside with little or no changes in the maximal relaxation obtained with these vasodilators. Increasing concentrations of dichlorobenzamil, an analog of amiloride and a recently described inhibitor of calcium influx via sodium-calcium exchange, functionally antagonized and abolished the relaxations elicited by acetylcholine and A23187, but had no appreciable effect on the relaxations to sodium nitroprusside or atrial natriuretic factor (an endothelium-independent vasodilator). Similar results were obtained using isolated rabbit aortic rings. Thus, although the presence of extracellular calcium is critically required for the expression of endothelium-dependent relaxation, the associated calcium translocation is not blocked by the organic calcium entry blockers. The results with dichlorobenzamil suggest that sodium-calcium exchange may be an important mechanistic step in the release of endothelium-derived relaxant factor.

Topics: Acetylcholine; Amiloride; Animals; Calcimycin; Calcium; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Nifedipine; Nitric Oxide; Nitroprusside; Rabbits; Rats; Rats, Inbred Strains; Sodium; Vasodilation; Vasodilator Agents; Verapamil