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 < 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 <10,000, was inactivated by trypsin, and retained its activity after two cycles of freeze- thawing. These observations suggest that 1) macrovascular and microvascular endothelial cells release different contractile substances that have a greater activity on sheep vascular than airway smooth muscle, 2) the contractile substance released from macrovascular endothelial cells is ET-1, and 3) the contractile responses involve calcium influx.