Masamichi Kozuka

Endothelin induces two types of contractions of rat uterus: Phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels

Effects of endothelin on nonvascular smooth muscle have been examined using rat uterine horns and two modes of endothelin action have been revealed. Endothelin (0.3 nM) caused rhythmic contractions of isolated uterus in the presence of extracellular calcium. The rhythmic contractions were completely inhibited by calcium channel antagonists. These characteristics of endothelin-induced contractions were very similar to those induced by oxytocin. Binding assays using 125I-endothelin showed that endothelin and the calcium channel blockers did not compete for the binding sites. However, endothelin was unique in that it caused, in addition to rhythmic contractions, a slowly developing monophasic contraction that was insensitive to calcium channel blockers. This developing contraction became dominant at higher concentrations of endothelin and was also calcium dependent.

Immunohistochemical localization of atrial natriuretic peptide receptor in bovine kidney and lung.

Receptors for atrial natriuretic peptide (ANP) were localized in the alveoli and bronchiolar smooth muscle cells of bovine lung and in podocytes of the kidney by immunofluorescence and immunoperoxidase methods. Two specific antisera were raised against the ANP receptor purified from bovine lung plasma membranes: anti-Rc 140 and anti-Rc 70. Anti-Rc 140 was raised against the 140 KD native receptor having a homodimeric structure, and anti-Rc 70 was elicited by immunizing a rabbit with the 70 KD reduced subunits. Essentially identical staining patterns were obtained with both antisera. Identification of ANP receptor sites would provide useful information in understanding the pulmonary and renal actions of ANP.

Solubilization of endothelin receptors from bovine lung plasma membranes in a non-aggregated state and estimation of their minimal functional sizes

Bovine lung endothelin receptors were solubilized in a non-aggregated state and characterized in terms of their minimal functional size and chemical nature of the ligand-binding site. A variety of detergents and their combinations were tested for their efficiency to solubilize endothelin receptors from bovine lung plasma membranes, and a combination of 0.4% digitonin and 0.25% CHAPS was found to be very effective in obtaining highly dispersed receptor solution. Gel filtration of the CHAPS/digitonin-solubilized receptors revealed the presence of two receptor species eluting at the positions corresponding to 34 and 52 kDa. These values were in good agreement with those estimated by affinity labeling and SDS-polyacrylamide gel electrophoresis, establishing that they represent minimal functional units. Chemical modification of ligand-occupied and free receptors with p-chloromercuriphenylsulfonic acid revealed that both 34 and 52 kDa receptors have SH group(s) essential for the receptor activity in their ligand-binding sites.

Stimulation of Na-K-Cl cotransport in cultured vascular endothelial cells by atrial natriuretic peptide.

Vascular endothelial cells have been shown to contain atrial natriuretic peptide (ANP)-sensitive Na-K-Cl cotransport system whose activity is regulated by intracellular cGMP levels. Addition of ANP to culture medium stimulated 86Rb+ uptake in bovine endothelial cells with a concomitant increase in cGMP contents. This action of ANP was mimicked by 8-bromo-cGMP and completely diminished by furosemide. These results indicate that ANP selectively activates the Na-K-Cl cotransporter in vascular endothelial cells via cGMP and offer new insight into the physiological significance of endothelial ANP receptors.

Affinity chromatographic purification of bovine lung endothelin receptor using biotinylated endothelin and avidin-agarose

Endothelin receptor was purified from bovine lung by affinity chromatography using biotinylated endothelin and avidin-agarose. Endothelin was biotinylated with sulphosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate, a reactive form of biotin with a cleavable spacer arm containing a disulphide bond designed for a simple elution by thiols. Starting from 3.5 kg of bovine lung, about 200 micrograms of pure receptor were obtained.

Separation and purification of 34- and 52-kDa species of bovine lung endothelin receptors and identification of the 34-kDa species as a degradation product.

We solubilized bovine lung endothelin receptors in a nonaggregated state and identified 34- and 52-kDa species of endothelin receptor by affinity labeling. The ratio of 34-kDa species to 52-kDa species varied widely from sample to sample, suggesting that the 34-kDa species is a proteolytic product; we therefore examined the effect of various proteinase inhibitors and found that EDTA (greater than 50 mM) was very effective in preventing the conversion. These results indicate that the lower Mr species arose from the 52-kDa receptor as a result of limited proteolysis by metal proteinase(s). The resulting 34-kDa species had endothelin-binding activity, was very stable, and was not processed further even in the absence of proteinase inhibitors. We therefore first tried to purify the 34-kDa species by affinity chromatography. Bovine lung extracts were mixed with biotinylated endothelin and the ligand-receptor complexes formed were isolated with avidin-agarose. Starting from 3.5 kg of bovine lung, approximately 200 micrograms of the pure receptor was obtained. Microsequencing of two well-separated tryptic fragments yielded the following partial amino acid sequences: Ala-Asn-Asp-His-Gly-Tyr-Asp-Asn-Phe and Ser-Gly-Met-Gln-Ile-Ala-Leu-Asn-Asp-His-Leu-Lys.