calcimycin and Acidosis

Bax Inhibitor-1 (BI-1) is an evolutionarily conserved six-transmembrane domain endoplasmic reticulum (ER)-localized protein that protects against ER stress-induced apoptotic cell death. This function is closely connected to its ability to lower steady-state ER Ca2+ levels. Recently, we elucidated BI-1's Ca(2+)-channel pore in the C-terminal part of the protein and identified the critical amino acids of its pore. Based on these insights, a Ca(2+)-channel pore-dead mutant BI-1 (BI-1(D213R)) was developed. We determined whether BI-1 behaves as a bona fide H+/Ca2+ antiporter or as an ER Ca(2+)-leak channel by investigating the effect of pH on unidirectional Ca(2+)-efflux rates. At pH 6.8, wild-type BI-1 expression in BI-1(-/-) cells increased the ER Ca(2+)-leak rate, correlating with its localization in the ER compartment. In contrast, BI-1(D231R) expression in BI-1(-/-), despite its ER localization, did not increase the ER Ca(2+)-leak rate. However, at pH < 6.8, the BI-1-mediated ER Ca2+ leak was blocked. Finally, a peptide representing the Ca(2+)-channel pore of BI-1 promoting Ca2+ flux from the ER was used. Lowering the pH from 6.8 to 6.0 completely abolished the ability of the BI-1 peptide to mediate Ca2+ flux from the ER. We propose that this pH dependence is due to two aspartic acid residues critical for the function of the Ca(2+)-channel pore and located in the ER membrane-dipping domain, which facilitates the protonation of these residues.

Topics: Acidosis; Animals; Apoptosis; Apoptosis Regulatory Proteins; Calcimycin; Calcium; Cell Line; Endoplasmic Reticulum; HeLa Cells; Humans; Hydrogen-Ion Concentration; Membrane Proteins; Mice; Peptides; Protein Structure, Tertiary

Acidosis induces vasodilation both in vivo and in vitro. Although it is commonly surmised that acidosis alters contractility by affecting contractile proteins and calcium entry, the exact role of these mechanisms in acidosis-induced vasodilation has not been determined. In the present study, we demonstrated that a novel mechanism, involving increased calcium sequestration into intracellular sites sensitive to norepinephrine, mediates the vasodilation associated with relatively modest decreases in pH. The effects of changing pH from 7.4 to 7.0 on tension development, 45Ca fluxes, and the norepinephrine-releasable intracellular calcium stores were studied in isolated rat aorta. Acute acidification produced marked endothelium-independent dilations of aortic rings that had been precontracted with norepinephrine. In contrast, this maneuver had only modest effects on contractions elicited by 80 mM KCl or phorbol ester. Acidification in this range did not alter basal or norepinephrine-stimulated undirectional 45Ca influx, nor did it reduce the norepinephrine-induced net gain in 45Ca content. Furthermore, neither norepinephrine-stimulated 45Ca efflux nor the peak contractile response to norepinephrine in calcium-free buffer was affected, although in this setting, the duration of the phasic contractile response was shortened. When calcium was restored to tissues exposed to norepinephrine in calcium-free buffer, acidification slowed the rate of tension development without altering 45Ca uptake, thus changing the relation between tension development and calcium entry. These effects of acidification were shown to be associated with an increase in the amount of calcium sequestered into the norepinephrine-sensitive intracellular calcium store. These findings clearly indicate that acidification, within a range that has no effect on other aspects of smooth muscle activation, elicits vasodilation by stimulating intracellular calcium sequestration. This action may represent a predominant mechanism whereby acidosis alters vascular smooth muscle contractility.

Topics: Acetylcholine; Acidosis; Animals; Aorta, Thoracic; Calcimycin; Calcium; Egtazic Acid; Endothelium, Vascular; Hydrogen-Ion Concentration; In Vitro Techniques; Models, Biological; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Potassium Chloride; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate; Vasodilation