eplerenone and benzamil

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. During the control period, the concentration of NaCl that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone 10(-8) mol/l to the CNT lumen caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma).

Topics: Aldosterone; Amiloride; Animals; Arterioles; Cycloheximide; Dactinomycin; Epithelial Sodium Channels; Eplerenone; Feedback; Kidney Tubules; Male; Rabbits; Receptors, G-Protein-Coupled; Receptors, Mineralocorticoid; Spironolactone

The effects of aldosterone and mineralocorticoid receptor (MR) blockers on presympathetic neurons in the rostral ventrolateral medulla (RVLM) are well studied. To directly investigate whether aldosterone, eplerenone (an MR blocker), FAD286 (an aldosterone synthase inhibitor) and benzamil (an epithelial sodium channel (ENaC) blocker) affect RVLM neurons, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole-cell patch-clamp technique during superfusion with these drugs to brainstem-spinal cord preparations. Aldosterone superfusion (0.1 μmol/l) depolarized the RVLM neurons. In contrast, eplerenone superfusion (1 μmol/l) hyperpolarized them. To evaluate the existence of aldosterone, FAD286 superfusion (10 μmol/l) was performed, and the RVLM neurons became hyperpolarized during FAD superfusion. These data suggest that MRs exist and that aldosterone is synthesized in the brainstem. Benzamil superfusion (1 μmol/l) hyperpolarized the RVLM neurons. To clarify whether aldosterone, eplerenone, FAD286 and benzamil acted directly on the RVLM neurons, a low-Ca(2+), high-Mg(2+) solution was used to block the synaptic input to the RVLM neurons, and the above-mentioned drugs were added during the low-Ca(2+) superfusion. During the aldosterone superfusion, the RVLM neurons became depolarized, and they became hyperpolarized during eplerenone, FAD286 or benzamil superfusion. Importantly, when aldosterone was superfused after the benzamil solution, the MPs of the RVLM neurons did not depolarize. These results suggest that MRs are present in the RVLM neurons and that aldosterone is synthesized in the RVLM. The RVLM neurons themselves possess ENaCs, and ENaCs are the underlying mechanism by which aldosterone activates RVLM neurons.

Topics: Aldosterone; Amiloride; Animals; Aromatase Inhibitors; Calcium; Diuretics; Epithelial Sodium Channels; Eplerenone; Excitatory Postsynaptic Potentials; Fadrozole; Magnesium; Medulla Oblongata; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, Mineralocorticoid; Spinal Cord; Spironolactone; Tissue Fixation

Pregnancy-mediated sodium (Na) retention is required to provide an increase in plasma volume for the growing fetus. The mechanisms responsible for this Na retention are not clear. We first used a targeted proteomics approach and found that there were no changes in the protein abundance compared with virgin rats of the β or γ ENaC, type 3 Na(+)/H(+) exchanger (NHE3), bumetanide-sensitive cotransporter (NKCC2), or NaCl cotransporter (NCC) in mid- or late pregnancy. In contrast, we observed marked increases in the abundance of the α-ENaC subunit. The plasma volume increased progressively during pregnancy with the greatest plasma volume being evident in late pregnancy. ENaC inhibition abolished the difference in plasma volume status between virgin and pregnant rats. To determine the in vivo activity of ENaC, we conducted in vivo studies of rats in late pregnancy (days 18-20) and virgin rats to measure the natriuretic response to ENaC blockade (with benzamil). The in vivo activity of ENaC (U(Na)V postbenzamil-U(Na)V postvehicle) was markedly increased in late pregnancy, and this difference was abolished by pretreatment with the mineralocorticoid receptor antagonist, eplerenone. These findings demonstrate that the increased α-ENaC subunit of pregnancy is associated with an mineralocorticoid-dependent increase in ENaC activity. Further, we show that ENaC activity is a major contributor of plasma volume status in late pregnancy. These changes are likely to contribute to the renal sodium retention and plasma volume expansion required for an optimal pregnancy.

Topics: Amiloride; Animals; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels; Eplerenone; Female; Immunoblotting; Kidney; Mineralocorticoid Receptor Antagonists; Mineralocorticoids; Natriuresis; Plasma Volume; Pregnancy; Proteomics; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Spironolactone; Up-Regulation

Sympathoexcitatory and hypertensive responses to central infusion of Na(+)-rich artificial cerebrospinal fluid (aCSF) are enhanced by aldosterone and mediated by mineralocorticoid receptors (MRs) and benzamil-blockable Na(+) influx, leading to "ouabain" release and ANG II type 1 (AT(1)) receptor stimulation. The present study evaluated the functional role of these mechanisms in the paraventricular nucleus (PVN). In conscious Wistar rats, Na(+)-rich aCSF was infused either directly into the PVN or intracerebroventricularly preceded by aldosterone and blockers. Infusion of Na(+)-rich aCSF in the PVN caused gradual increases in blood pressure (BP) and heart rate (HR). Aldosterone and a subpressor dose of ouabain in the PVN alone did not affect BP and HR but enhanced responses to Na(+). Eplerenone, benzamil, and "ouabain"-binding Fab fragments only blocked the enhancement by aldosterone, whereas losartan blocked all responses to Na(+)-rich aCSF in the PVN. Increases in BP and HR by intracerebroventricular infusion of Na(+)-rich aCSF were enhanced by aldosterone infused intracerebroventricularly, but not in the PVN. Telmisartan in the PVN again blocked all responses. In contrast, both eplerenone and benzamil in the PVN did not change the pressor responses to intracerebroventricular infusion of aldosterone and Na(+)-rich aCSF. These findings indicate that AT(1) receptors in the PVN mediate the responses to Na(+)-rich aCSF and their enhancement by aldosterone, both locally in the PVN or in the general CSF. MRs, benzamil-blockable Na(+) channels or transporters, and "ouabain" can be functionally active in the PVN, but in Wistar rats appear not to contribute to the pressor responses to short-term increases in CSF [Na(+)].

Topics: Aldosterone; Amiloride; Angiotensins; Animals; Antihypertensive Agents; Blood Pressure; Cardiotonic Agents; Cerebrospinal Fluid; Diuretics; Dose-Response Relationship, Drug; Eplerenone; Heart Rate; Hypertension; Injections, Intraventricular; Losartan; Ouabain; Paraventricular Hypothalamic Nucleus; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Receptors, Mineralocorticoid; Sodium; Spironolactone