kb-r7943 and benzamil

The role of Na(+)-Ca(2+) exchanger (NCX) in vascular endothelium is still matter of debate. Depending on both the endothelial cell (EC) type and the extracellular ligand, NCX has been shown to operate in either the forward (Ca(2+) out)- or the reverse (Ca(2+) in)-mode. In particular, acetylcholine (Ach) has been shown to promote Ca(2+) inflow in the intact endothelium of excised rat aorta. Herein, we assessed the involvement of NCX into the Ca(2+) signals elicited by ATP in such preparation. Removal of extracellular Na(+) (0Na(+)) causes the NCX to switch into the reverse-mode and induced an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which disappeared in the absence of extracellular Ca(2+), and in the presence of benzamil, which blocks both modes of NCX, and KB-R 7943, a selective inhibitor of the reverse-mode. ATP induced a transient Ca(2+) signal, whose decay was significantly prolonged by 0Na(+), benzamil, DCB, and monensin while it was unaffected by KB-R 7943. Notably, lowering extracellular Na(+) concentration increased the sensibility to lower doses of ATP. These date suggest that, unlike Ach-stimulated ECs, NCX promotes Ca(2+) extrusion when the stimulus is provided by ATP in intact endothelium of rat aorta. These data show that, within the same preparation, NCX operates in both modes, depending on the chemical nature of the extracellular stimulus.

Topics: Adenosine Triphosphate; Amiloride; Animals; Aorta; Calcium; Endothelium, Vascular; In Vitro Techniques; Rats; Rats, Wistar; Sodium-Calcium Exchanger; Thiourea

1. The role of sodium-calcium exchanger in acetylcholine (Ach)-induced hyperpolarization of intact endothelial cells was studied in excised rat aorta. The membrane potential was recorded using perforated patch-clamp technique. 2. The mean resting potential of endothelial cells was -44.1+/-1.4 mV. A selective inhibitor of sodium-calcium exchanger benzamil (100 microm) had no significant effect on resting membrane potential, but reversibly decreased the amplitude of sustained Ach-induced endothelial hyperpolarization from 20.9+/-1.4 to 5.7+/-1.1 mV when applied during the plateau phase. 3. The blocker of reversed mode of the exchanger KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate, 20 microm) reversibly decreased the amplitude of sustained Ach-induced hyperpolarization from 20.5+/-2.9 to 7.5+/-1.8 mV. 4. Introduction of tetraethylammonium (10 mm) in the continuous presence of Ach decreased the sustained phase of hyperpolarization from 17.9+/-1.5 by 12.9+/-0.9 mV. Subsequent addition of 20 microm KB-R7943 further depolarized endothelial cells by 4.8+/-1.1 mV. 5. Substituting external sodium with N-methyl d-glucamine during the plateau phase of Ach-evoked hyperpolarization reversibly decreased the hyperpolarization from -61.8+/-2.7 to -54.2+/-1.9 mV. In the majority of preparations, the initial response to removal of external sodium was a transient further rise in the membrane potential of several mV. Sodium ionophore monensin hyperpolarized endothelium by 10.3+/-0.7 mV. 6. The inhibitory effect of benzamil on Ach-induced endothelial sustained hyperpolarization was observed in endothelium mechanically isolated from smooth muscle. 7. These results suggest that the sodium-calcium exchanger of intact endothelial cells is able to operate in reverse following stimulation by Ach, contributing to sustained hyperpolarization. Myoendothelial electrical communications do not mediate the effect of blockers of sodium-calcium exchanger.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Amiloride; Animals; Aorta, Thoracic; Calcium; Cell Membrane; Diuretics; Endothelial Cells; Female; Gap Junctions; In Vitro Techniques; Ionophores; Male; Membrane Potentials; Monensin; Muscle, Smooth, Vascular; Phenylephrine; Rats; Sodium; Sodium-Calcium Exchanger; Stimulation, Chemical; Thiourea

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.

Topics: Acetylcholine; Amiloride; Animals; Aorta, Thoracic; Cells, Cultured; Drug Synergism; Endothelium, Vascular; In Vitro Techniques; Ionophores; Male; Monensin; Nitric Oxide; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger; Swine; Thiourea; Vasodilation; Vasodilator Agents

Our study aimed to assess a possible functional role of the Na(+)/Ca(2+) exchanger in the regulation of renal vascular resistance (RVR). Therefore, we investigated the effects of an inhibition of the Na(+)/Ca(2+) exchanger either by lowering the extracellular sodium concentration ([Na(+)](e)) or, pharmacologically on RVR, by using isolated perfused rat kidneys. Graded decreases in [Na(+)](e) led to dose-dependent increases in RVR to 4.3-fold (35 mM Na(+)). This vasoconstriction was markedly attenuated by lowering the extracellular calcium concentration, by the L-type calcium channel blocker amlodipine or by the chloride channel blocker niflumic acid. Further lowering of [Na(+)](e) to 7 mM led to an increase in RVR to 7.5-fold. In this setting, amlodipine did not influence the magnitude but did influence the velocity of vasoconstriction. Pharmacological blockade of the Na(+)/Ca(2+) exchanger with KB-R7943, benzamil, or nickel resulted in significant vasoconstriction (RVR 2.5-, 1.8-, and 4.2-fold of control, respectively). Our data suggest a functional role of the Na(+)/Ca(2+) exchanger in the renal vascular bed. In conditions of partial replacement of [Na(+)](e), vasoconstriction is dependent on chloride and L-type calcium channels. A total replacement of [Na(+)](e) leads to a vasoconstriction that is nearly independent of L-type calcium channels. This might be due to an active calcium transport into the cell by the Na(+)/Ca(2+) exchanger.

Topics: Amiloride; Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Egtazic Acid; In Vitro Techniques; Kidney; Kinetics; Male; Nickel; Niflumic Acid; Perfusion; Rats; Rats, Sprague-Dawley; Renal Circulation; Sodium; Sodium-Calcium Exchanger; Thiourea; Vascular Resistance; Vasoconstriction

The intracellular free calcium concentration ([Ca2+]i) is a central regulator of renin secretion and the contractility of vascular smooth muscle cells. As [Ca2+]i results from calcium influx and calcium extrusion, we were interested in the role of the Na+/Ca2+-exchanger as an important calcium-extrusion pathway in the regulation of renin secretion. Therefore, we investigated the effects of inhibiting the Na+/Ca2+-exchanger, either by reducing the extracellular sodium concentration ([Na+]e) or using pharmacological tools, on renin secretion and vascular resistance in the isolated perfused rat kidney model. Stepwise reductions of [Na+]e led to progressive (up to sevenfold) increases in renal vascular resistance ([Na+]e 7 mM) whilst renin secretion rates were not altered significantly. Similarly, pharmacological blockade of the Na+/Ca2+-exchanger by benzamil (100 microM) or KB-R7943 (30 microM) resulted in significant vasoconstrictions without altering basal renin secretion rates. Also renin secretion that was pre-stimulated by isoproterenol (10 nM), blockade of macula densa salt transport by bumetanide (100 microM) or lowering the perfusion pressure to 40 mmHg was not attenuated by Na+/Ca2+-exchanger inhibition, although the vascular resistance increased significantly. In contrast, angiotensin II (100 pM) reduced pre-stimulated renin secretion values by 50%. The subsequent lowering of the [Na+]e however did not augment the inhibition of renin secretion, although the renal vascular resistance increased markedly. We conclude that the Na+/Ca2+-exchanger has no functional role in the regulation of [Ca2+]i in juxtaglomerular cells controlling renin secretion, whereas it markedly affects the preglomerular vascular smooth muscle cells of the renal vasculature.

Topics: Adrenergic beta-Agonists; Amiloride; Angiotensin II; Animals; Anti-Arrhythmia Agents; Bumetanide; Diuretics; In Vitro Techniques; Isoproterenol; Kidney; Male; Pressoreceptors; Rats; Rats, Sprague-Dawley; Renin; Sodium; Sodium-Calcium Exchanger; Thiourea; Vascular Resistance; Vasoconstrictor Agents