Phoebe Tsai

Participation of cytocheome P-450 in the steroid 17α-hydroxylase of testis microsomes

Abstract The effect of metyrapone on the activity of the steroid 17α-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition vas observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17α-hydroxylase reaction.

Inhibition of vasopressin action in vascular smooth muscle by the V1 antagonist OPC-21268.

In vascular smooth muscle cells arginine vasopressin acting through the V1 receptor increases intracellular Ca2+, leading to vasoconstriction. Recent studies have also shown that vasopressin activates mitogen-activated protein kinase (MAP kinase), which may contribute to vasopressin-induced hypertrophy of vascular smooth muscle cells. We examined the ability of an orally active, nonpeptide selective V1 antagonist (OPC-21268) to block vasopressin binding and postreceptor signaling in these cells. [3H]Vasopressin binding at 2 x 10(-9) mol/L was half-maximally blocked at 10(-9) mol/L OPC-21268. To compare effects of OPC-21268 on binding and postreceptor signaling, we stimulated cells with 10(-8) mol/L vasopressin. At this vasopressin concentration, half-maximal inhibition of binding occurred at 5 x 10(-9) mol/L OPC-21268. Half-maximal inhibition of Ca2+ efflux or increases in intracellular free Ca2+ required higher concentrations of antagonist (10(-7) mol/L), and half-maximal inhibition of vasopressin-stimulated MAP kinase was observed only at 10(-6) mol/L OPC-21268. These results indicate that this agent selectively blocks both vasopressin binding and postreceptor signaling in vascular smooth muscle cells. The requirement of higher concentrations of OPC-21268 for blocking increases in intracellular Ca2+ and activation of MAP kinase suggests that binding to a fraction of V1 receptors generates maximal levels of second messengers or the existence of subtypes of the V1 receptor with differential affinity for this antagonist. These data have implications for the clinical use of this compound.

Mechanisms of the vascular effect of pressor hormones

There exists a considerable gap between the recognition of the effects of pressor hormones and the understanding of their mechanisms. Studies are necessary to provide a more accurate basis for potential therapeutic interventions on the hormonal mechanisms of hyper- and hypotensive states. From recent and previous experimental results, a modified model of vascular smooth muscle cell activation is emerging. In this model, contraction is a complex process involving Ca2+ release, protein kinase C and ionic channel activation and modification in pH and energy metabolism. The combined action of these processes is necessary for the full expression of the contractile response.

Stimulatory effect of soluble supernatant on hydroxylase activity of rat testis microsomes

Addition of soluble supernatant to testis microsomes results in 42% increase in steroid 17,20-lyase activity and a 65% increase in 17alpha-hydroxylase activity. This stimulatory activity could be partially purified by salt fractionation. The activating factor(s) was not removed by dialysis nor did it appear to be lipid. It was destroyed by trypsin. Differential effects of heat were observed with the hydroxylase and lyase activators. The activation did not affect Km but only increased Vmax. The supernatant could be added to each enzyme to the point of maturation. No binding of steroids by the supernatant could be detected. Corpus luteum and placental supernatant did not stimulate enzymic activity, but supernatant from an adrenal adenoma was active.