monensin and benzamil

A dysregulation of the hypothalamic-pituitary-adrenocortical (HPA) axis represents a prominent finding in major depression, possibly related to a dysfunction of the corticosteroid receptor system. Antidepressants are involved in the restoration of the altered feed-back mechanism of the HPA-axis, probably via normalization of corticosteroid receptor functions. Since Hypericum perforatum has antidepressive properties, we here examined its putative actions on glucocorticosteroid receptor mRNA levels in human blood cells as a peripheral model for neuroendocrine effects in human brain cells. Our data show that Hypericum (LI 160) affects the cellular mRNA levels of both, the glucocorticoid receptor (GR)-α and its inhibitory counterpart, the GR-β, at clinically-relevant concentrations. Under these conditions, a bimodal effect was observed. Dose-response studies suggest a rather small effective concentration range and time-effect data show a primary and transient up-regulation of GR-α mRNA levels and a down-regulation of GR-β mRNA levels after 16 h of treatment. The sodium channel blocker benzamil neutralized the effects of Hypericum, pointing to an at least partial mechanism of action via this pathway. In conclusion, Hypericum treatment differentially affects GR-mRNA levels in the human system. Our data suggest a bimodal effect on GR, resulting in a time-and dose-related modification of GR-mediated cellular effects. Such a mechanism has been alleged as an important way of action for a number of antidepressants. It is the first time that a specific effect on both receptors, especially on the subtype of GR-β, is shown under antidepressive treatment in a human system under in vitro conditions.

Topics: Amiloride; Cell Line, Transformed; Coccidiostats; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Hypericum; Monensin; Monocytes; Plant Extracts; Receptors, Steroid; RNA, Messenger; Sodium Channel Blockers

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

Angiotensin II (AngII) helps to regulate overall renal tubular reabsorption of salt and water, yet its effects in the distal nephron have not been well studied. The purpose of these studies was to determine whether AngII stimulates luminal Na(+) transport in the cortical collecting duct (CCD). Intracellular Na(+) concentration ([Na(+)](i)), as a reflection of Na(+) transport across the apical membrane, was measured with fluorescence microscopy using sodium-binding benzofuran isophthalate (SBFI) in isolated, perfused CCD segments dissected from rabbit kidneys. Control [Na(+)](i), during perfusion with 25 mM NaCl and a Na(+)-free solution in the bath containing the Na(+)-ionophore monensin (10 microM, to eliminate basolateral membrane Na(+) transport) averaged 19.3 +/- 5.2 mM (n = 16). Increasing luminal [NaCl] to 150 mM elevated [Na(+)](i) by 9.87 +/- 1.5 mM (n = 7; P < 0.05). AngII (10(-9) M) added to the lumen significantly elevated baseline [Na(+)](i) by 6.3 +/- 1.0 mM and increased the magnitude (Delta = 25.2 +/- 3.7 mM) and initial rate ( approximately 5 fold) of change in [Na(+)](i) to increased luminal [NaCl]. AngII when added to the bath had similar stimulatory effects; however, AngII was much more effective from the lumen. Thus, AngII significantly increased the apical entry of Na(+) in the CCD. To determine if this apical entry step occurred via the epithelial Na(+) channel (ENaC), studies were performed using the specific ENaC blocker, benzamil hydrochloride (10(-6) M). When added to the perfusate, benzamil almost completely inhibited the elevations in [Na(+)](i) to increased luminal [NaCl] in both the presence and absence of AngII. These results suggest that AngII directly stimulates Na(+) channel activity in the CCD. AT(1) receptor blockade with candesartan or losartan (10(-6) M) prevented the stimulatory effects of AngII. Regulation of ENaC activity by AngII may play an important role in distal Na(+) reabsorption in health and disease.

Topics: Amiloride; Analysis of Variance; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Antihypertensive Agents; Benzimidazoles; Benzofurans; Biphenyl Compounds; Hydrogen-Ion Concentration; Intracellular Membranes; Ionophores; Kidney Tubules, Collecting; Losartan; Mice; Microscopy, Fluorescence; Monensin; Rabbits; Receptor, Angiotensin, Type 1; Receptors, Angiotensin; Signal Transduction; Sodium Chloride; Tetrazoles

The purpose of this study was to characterize the role of ions other than Ca2+ in hepatic responses to alpha 1-adrenergic stimulation. We report that the alpha 1-adrenoreceptor activation of hepatic functions is accompanied by extracellular acidification and an increase in intracellular pH. These effects are dependent on extracellular Na+ concentration and are inhibited by the Na+/H+ antiporter blocker 5-(N-ethyl-N-isopropyl) amiloride under conditions that preclude antagonistic effects on agonist binding. Thus, the activation of plasma membrane Na+/H+ exchange is an essential feature of the hepatic alpha-adrenoreceptor-coupled signaling pathway. The following observations indicate that the sustained hepatic alpha 1-adrenergic actions rely on a functional coupling between the plasma membrane Na+/H+ and Na+/Ca2+ exchangers, resulting in the stimulation of Ca2+ influx. 1) Inhibition of the Na+/K(+)-ATPase does not prevent the alpha 1-adrenergic effects. However, alpha 1-adrenoreceptor stimulation fails to induce intracellular alkalinization and to acidify the extracellular medium in the absence of extracellular Ca2+. 2) A non-receptor-induced increase in intracellular Na+ concentration, caused by the ionophore monensin, stimulates Ca2+ influx and increases vascular resistance. 3) Inhibition of Na+/Ca2+ exchange prevents, in a concentration-dependent manner, most of the alpha 1-agonist-induced responses. 4) The actions of Ca(2+)-mobilizing vasoactive peptide receptors or alpha 2-adrenoreceptors, which produce neither sustained extracellular acidification nor release of Ca2+, are insensitive to Na+/H+ exchange blockers.

Topics: Amiloride; Animals; Calcium; Carrier Proteins; Hydrogen-Ion Concentration; Liver; Membrane Potentials; Monensin; Ouabain; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Sodium; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Vasopressins