Further isolation of endogenous factors in canine and human plasma that inhibit [(3)H]norepinephrine accumulation

We have previously shown that both homologous canine plasma and a crude extract of this plasma contain substances that inhibit accumulation of [(3)H]norepinephrine ([(3)H]NE) by the canine saphenous vein. The purpose of this study was to further purify these substances and to determine if similar factors were present in human plasma. Crude extracts of plasma were purified with a Folch extraction in which most of the biological activity was recovered in the bottom or organic phase. This phase significantly inhibited [(3)H]NE uptake by the canine saphenous vein (23.5 +/- 7.6% by concentrate from 9.1 ml of original plasma/ml incubate solution) and increased development of tension following transmural electrical stimulation by 91.5 +/- 23.3% (extract from 1 ml of plasma/ml bath solution). The Folch extracts obtained from 100ml of plasma were purified by column and thin layer (TLC) chromatography. Samples were applied to a silicic acid column and eluted with chloroform, acetone, and methanol. The [(3)H]NE uptake inhibitory activity was primarily recovered in the methanol fraction. TLC of the methanol fraction of canine plasma on silica gel G plates (with pre-absorbent) resulted in five zones which were then assayed for their ability to inhibit [(3)H]NE accumulation by the saphenous vein. In the first zone (concentrate from 27.5 ml plasma/ml bath solution) there was significantly greater inhibitory activity (55.4 +/- 8.3%), than in the corresponding zone obtained from solvent blanks (20.7 +/- 4.1%). These results indicate that there is a factor or possibly factors in canine and human plasma that have thin layer chromatographic properties of a polar lipid, which inhibit [(3)H]NE accumulation and enhance the contractile response of vascular smooth muscle to transmural electrical stimulation.