With in vivo television microscopy, changes in arteriolar diameter to topical administration of various vasoactive agents were examined in the absence or in the presence of NG-monomethyl-L-arginine (L-NMMA, topical 100 microM) or NG-nitro-L-arginine (L-NNA, 2.5 microM, 20 microliters/min ia), specific inhibitors of endothelium-derived relaxing factor (EDRF) biosynthesis. In cremaster muscle arterioles (15-22 microns) of rats (n = 6-11), dilations to acetylcholine (1-100 ng) were significantly inhibited (60-70%) by either of the arginine analogues. This inhibition was reversed by subsequent administration of 1 mM L-arginine. Dose-dependent constriction to norepinephrine was enhanced by L-NMMA. Indomethacin treatment reduced arteriolar dilation to bradykinin (BK, 1-100 ng), which was significantly inhibited by additional administration of L-NNA. Application of L-NNA first, followed by additional indomethacin, elicited similar results. Dilations to sodium nitroprusside and adenosine were not reduced in the presence of the inhibitors. L-NMMA or L-NNA caused no change in systemic blood pressure but elicited a significant reduction in arteriolar diameter; this effect was not reversed by 1 mM L-arginine. These data demonstrate the presence of an L-arginine pathway to produce EDRF (nitric oxide) in skeletal muscle microcirculation that mediates and/or modulates arteriolar responses to vasoactive agents and could contribute to the regulation of basal vascular tone.

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Pharmacological probes were used to assess the possible roles of guanosine 3',5'-cyclic monophosphate (cGMP)-associated endothelium-derived relaxing factor (EDRF) in mediating microvascular responses to endogenous and exogenous agents in vivo. Pentobarbital-anesthetized rats (Wistar, 6 wk old) were prepared for in vivo microscopic observation and quantification of changes in diameter of third-order arterioles (15-25 microns) in the cremaster muscle to topical application of all agents. In indomethacin-pretreated preparations, cremasteric arteriolar dilator responses to acetylcholine, bradykinin, or ATP, but not to adenosine, histamine, or prostaglandin E2, were inhibited by hydroquinone (50 microM). Vasodilation to acetylcholine was also inhibited by methylene blue (5 microM), a blocker of guanylate cyclase activation. Constrictor responses to norepinephrine were not affected by hydroquinone or methylene blue. The inhibition of acetylcholine-induced vasodilation by hydroquinone and methylene blue was reversed by superoxide dismutase, suggesting that superoxide anion antagonized the response. On the other hand, basal arteriolar diameters or responses to acetylcholine were not affected by oxygen metabolite scavengers. Unlike in isolated arteries, vasodilator responses to the calcium ionophore A23187 or arachidonic acid were completely antagonized by cyclooxygenase inhibition. These data suggest that EDRF could be involved in the control of microvascular tone; however, significant differences exist in the stimuli that elicit dilation through this mediator in small and large blood vessels.

Some preliminary experiments have been carried out in feeding large doses of exogenous cortisol to subjects in each of the 3 groups studied and measuring % of dose recovered as 6P-OH-F. There was 13.5 and 16% conversion to 6P-OH-F in 2 toxemics and 13.5 and 22% in 2 non-toxemic pregnant women, while 2 non-pregnant women excreted 3.8 and 676 as 6P-OH-F. These observations indicate that there is an increased conversion of cortisol to the 6-beta derivative in both toxemia and normal pregnancy.The biological effects of 6P-OH-F have not been fully studied, but at present there is no evidence which would assign tot it a causative role in toxemia. The likelihood is that 6Phydroxylation represents an available pathway for cortisol degradation, rendering the steroid more water soluble and suitable for excretion without the necessity for A-ring reduction and glucuronide conjugation. If the enzymatic steps concerned with either of these 2 reactiolns are interfered with, as might occur in some conditions, then 6-beta-hydroxylation could function as an important alternate pathway of cortisol metabolism.Summary. 6p-OH-cortisol has been found to be the most abundant unconjugated corticoid in human urine. Normal pregnancy is accompanied by elevated levels which are further increased in toxemia. The evidence to date suggests that in these conditions an altered metabolism of cortisol takes place in which 6p-hydroxylation becomes of greater quantitative significance.