endothelin-1 and Alkalosis

Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).

Topics: Adenosine Triphosphate; Alkalosis; Animals; Antibodies, Blocking; Antibody Specificity; Antiporters; Buffers; Cross Reactions; Endothelin-1; Glutathione; Hydrogen-Ion Concentration; Male; Membranes; Myocardium; Rabbits; Rats; Rats, Wistar; Stimulation, Chemical

This study tested whether hypocapnic constriction of the rabbit basilar artery in vitro can be triggered by a nitric oxide (NO) synthase inhibitor, and whether the resulting constriction is (1) due to the alkaline pH associated with hypocapnia, and (2) endothelin-1 mediated. Hypocapnic (25 mM NaHCO(3); pH 7.76; pCO(2) 14.2) or isocapnic alkaline solution (50 mM NaHCO(3); pH 7.73; pCO(2) 35.0) rarely altered basal tension. N(G)-monomethyl-L-arginine monoacetate (L-NMMA; 0.1 mM) challenge in hypocapnic or isocapnic alkaline solution resulted in near maximal tension that was maintained for 2-2.5 h even following L-NMMA washout. L-NMMA challenge in normal solution (25 mM NaHCO(3); pH 7. 42; pCO(2) 36.9) also induced near maximal tension, although the tension was maintained for only 25 min (mean). Ac-D-Bhg-L-Leu-Asp-L-Ile-L-Ile-L-Trp (PD145065), homopiperidinyl-CO-Leu-D-Trp(CHO)-D-Trp (BQ610), and N-cis-2, 6-dimethyl-piperidinocarbonyl L-gamma-MeLeu-D-Trp (COOCH(3))-Nle (BQ788; 1-3 microM), endothelin ET(A)/ET(B), endothelin ET(A), and endothelin ET(B) receptor antagonists, respectively, completely relaxed the tension that resulted from L-NMMA challenge in hypocapnic or isocapnic alkaline solution. These results demonstrate that constriction due to hypocapnia in vitro can be triggered by an NO synthase inhibitor and is endothelin-1 mediated. Additionally, alkaline pH in the absence of decreased pCO(2) is sufficient to elicit the constriction.

Topics: Acetylcholine; Alkalies; Alkalosis; Animals; Basilar Artery; Carbon Dioxide; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Hypocapnia; In Vitro Techniques; Male; Oligopeptides; omega-N-Methylarginine; Papaverine; Piperidines; Rabbits; Receptor, Endothelin A; Receptor, Endothelin B; Solutions; Vasoconstriction; Vasodilation; Vasodilator Agents

This study tested whether hypocapnic constriction of the rabbit basilar artery in vitro can be triggered by serotonin, and whether the resulting constriction is (1) due to the alkaline pH associated with hypocapnia, and (2) endothelin-1 mediated. Hypocapnic alkaline solution (25 mM NaHCO(3); pH 7.76; pCO(2) 14.2) or isocapnic alkaline solution (50 mM NaHCO(3); pH 7.73; pCO(2) 35.0) rarely altered basal tension. Serotonin (3 microM) challenge in hypocapnic or isocapnic alkaline solution resulted in near maximal tension. Washout of the serotonin did not decrease tension in 54% of the tissues, as plateau tension was maintained for 2-2.5 h. The plateau tension of washed tissues was relaxed by 1-3 microM PD145065 (Ac-D-Bhg-L-Leu-Asp-L-Ile-L-Ile-L-Trp), BQ610 (homopiperidinyl-CO-Leu-D-Trp(CHO)-D-Trp), and BQ788 (N-cis-2, 6-dimethyl-piperidinocarbonyl-L-gamma-MeLeu-D-Trp (COOCH(3))-Nle), endothelin ET(A)/ET(B), endothelin ET(A), and endothelin ET(B) receptor antagonists, respectively. In contrast, serotonin-induced tension in normal solution (25 mM NaHCO(3); pH 7.42; pCO(2) 36.9) was maintained for only 40 min (mean). These results demonstrate that (1) constriction due to hypocapnia in vitro can be triggered by serotonin and is endothelin-1 mediated and (2) alkaline pH in the absence of decreased pCO(2) is sufficient to elicit the constriction triggered by serotonin.

Topics: Acetylcholine; Alkalosis; Animals; Basilar Artery; Endothelin Receptor Antagonists; Endothelin-1; Free Radical Scavengers; Hypocapnia; Male; Rabbits; Serotonin; Vasoconstriction; Vasodilation; Vasodilator Agents