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Endothelin-1 from pulmonary artery and microvessels acts on vascular and airway smooth muscle
The conditioned medium (CM) from microvascular and macrovascular endothelial cell (EC) cultures was tested for constrictor activity. Sheep coronary artery rings, under 1 g tension, and sheep tracheal smooth muscle strips, under 2 g tension, were hung in organ baths containing Krebs-Henseleit solution (39 degrees C) and were equilibrated with 95% O2 and 5% CO2. Isometric force was measured in response to 80 mM KCl and constrictor responses to 100% CM were expressed as a percentage of maximum KCl response. Serum-free CM from confluent microvascular endothelial cells caused a sustained, slow-onset contraction of the coronary rings similar to that obtained with CM from macrovascular ECs. Indomethacin (5 microM) added to either the microvascular or macrovascular CM enhanced the constrictor responses by 1.6- and 1.8-fold, respectively, and the constrictor effects of both media were reduced by the calcium antagonist gallopamil (10 nM). CM from macrovascular EC caused a sustained contraction of tracheal strips similar to microvascular CM. In both cases, constriction was preceded by a brief relaxation as noted in vascular smooth muscle. Unconditioned medium had no constrictor activity on either vascular or airway smooth muscle. Microvascular-derived constrictor activity was heat stable. There was a slight loss of activity in the heat-treated CM from the macrovascular cells compared with control CM. Constrictor effects on tracheal smooth muscle were partially reversed by 1 microM gallopamil
Microvascular and macrovascular endothelial cells produce different constrictor substances
The media from cultured microvascular and macrovascular endothelial cells (conditioned media, CM) were collected and tested for constrictor activity in sheep coronary artery rings and tracheal smooth muscle strips in vitro (isometric force), expressed as percentage of contraction produced by 80 mM KCl. Both microvascular (micro) and macrovascular (macro) CM caused a sustained slow-onset contraction (P less than 0.05) of the coronary artery rings by 71 +/- 10% (micro; n = 7) and 67 +/- 8% (macro; n = 6) and tracheal smooth muscle strips by 33 +/- 14% (micro; n = 6) and 34 +/- 6% (macro; n = 11); the calcium antagonist gallopamil (10(-7) M) attenuated these effects by 25–55%. Unconditioned medium and medium conditioned by cultured tracheal smooth muscle cells had no constrictor activity on coronary artery rings or tracheal smooth muscle strips. Synthetic endothelin (ET-1) also produced contraction of coronary artery rings and tracheal smooth muscle strips. The mean levels of ET-1 measured by radioimmunoassay were 1,200 pg/ml in the macro CM and 33 pg/ml in the micro CM. Depleting macro CM of ET-1 by affinity columns constructed with protein A agarose and anti-ET-1 antibody removed the contractile activity for coronary artery rings and tracheal smooth muscle strips. Thus ET-1 did not appear to be the contractile substance in the micro CM. Preliminary characterization of the contractile substance in micro CM revealed that it was heat stable, had a molecular weight of less than 10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze-thawing