Effect of Morphine Sulfate (MS) on Endothelin Production and Receptor Responses in Newborn Piglet Brain Endothelial Cells † 349

We have shown that high dose MS infusion, in-vivo, resulted in increased cerebrovascular production of endothelin-1 (ET-1). In the present study, we determined the effect of MS on endothelin production and receptor (ET_A and ET_B) gene expression in cultured brain endothelial cells from newborn piglet cerebral microvessels; grown to confluence and treated with MS(100 ng/ml media) or naloxone (100 ng/ml media). The control cells were treated with an equivalent volume of saline. The cells were exposed to drug or saline for 6, 24, 48 & 96 hrs. At the end of each time interval (n=6 flasks), media was analyzed for ET-1 and big ET-1 levels (ng/ml) by enzyme immunoassay. The cells were examined for ET_A and ET_B mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR). Exposure to MS resulted in increased production of ET-1 at 6 hrs(0.69±0.07, p<0.05 ng/ml) to 96 hrs (0.68±0.06,p<0.05) compared to time 0 (0.43±0.09). ET_A receptor expression increased by 2-fold (p<0.01) at 6 hrs and 4-fold by 96 hrs (p<0.001). In contrast, MS exposure significantly decreased big ET-1 levels by 24 hrs (0.24±0.05,p<0.001) through 96 hrs (0.18±0.03, p<0.001) compared to time 0 (0.51±0.01). ET_B receptor mRNA expression decreased at 48 hrs (55%, p<0.01) and 96 hrs (82%,p<0.01). These responses were reversed with exposure to naloxone. ET-1 levels decreased at 6 hrs (0.15±0.09, p<0.05) to 96 hrs (0.23±0.03, p<0.05) versus time 0 (0.41±0.07). Big ET-1 levels increased at 6 hrs (0.99±0.09, p<0.01) to 96 hrs (1.0±0.07, p<0.01) versus time 0(0.55±0.04). ET_A receptor mRNA expression decreased by 40%(p<0.01) at 6 hrs and by 94% (p<0.001) at 96 hrs. ET_B receptor mRNA expression increased 2-fold (p<0.05) by 24 hours and 3-fold by 96 hours (p<0.001). No changes in control ET-1, big ET-1, ET_A or ET_B were detected. These data show that MS alter endothelin production and mRNA expression of its receptor subtypes in brain endothelial cells. Cardiovascular and cerebrovascular effects of MS may be partly due to altered production and expression of vasoactive agents such as endothelins.