An evaluation of the role of hypothalamic enzymes in the metabolism of oxytocin and vasopressin

In order to evaluate fully the physiological significance of a hormone it is essential to know the secretory rates and the circulating levels of that hormone over a wide range of physiological situations. In the case of the neurohypophysial hormones these can only be measured in a very limited number of situations. The work reported below was undertaken to evaluate the suggestion that two hypothalamic enzymes, one in the supernatant and the other in the mitochondrial fraction, reflect the synthesis and/or the secretion of oxytocin (Hooper, 1966 a, b). Two methods of approach were used; (a) the enzyme activity in additional physiological and experimental states was measured, and (b) preparatory to testing the hypothesis directly a method of extracting and purifying the hormones from fresh glands was attempted. The enzyme-containing fractions were prepared as described by Hooper (1966a). When the enzyme activity was measured using oxytocin and vasopressin separately it was found that the supernatant enzyme inactivated both hormones to approximately the same extent, whereas the particulate enzyme inactivated vasopressin much more effectively than oxytocin. Following mating, the particulate enzyme showed an increase in activity at 9 and 12 hrs. The supernatant enzyme showed an apparent slight increase at 12 hrs followed by a decerase in activity at 36 and 48 hrs. During dehydration induced by administration of 3% NaC1 for 3 days the supernatant enzyme activity was doubled when tissue concentrations up to 70 mug/ml were used. Above this concentration there was a progressive decrease in activity. In animals that had been overhydrated to i.p. administration of 8% glucose corresponding to 10% bodyweight every 3 hrs for 3 days the supernatant enzyme was doubled while the particulate was halved. In animals that were overhydrated but stressed due to gut damage both the enzymes were increased, the former five-fold the later only slightly. Enzyme activity was tested for in one dog diagnosed as suffering from diabetes insipidus. Activity was only found in the supernatant fraction. The pattern of enzyme activity throughout pregnancy is known (Hooper, 1966 a, b). The body water/kg body weight and body water turnover were measured to see whether there was any correlation with enzyme activity. A correlation was found between body water turnover and enzyme activity. With the exception of days 10-20 of pregnancy the body water turnover was increased when the ratio of supernatant/particulate enzyme was increased above the control value, and decreased when the ratio was decreased. A method suitable for the partial purification of neurohypophysial hormones from small numbers of fresh glands has been devised. Hemogonization in 0.5 ml of 0.1 N HC1/gland followed by salt precipitation and chromatography on a long Sophadox G-25 Column results in separation of the hormones from the high and much of the low molecular weight material with a 70% recovery of hormone. The finding that the enzymes respond to the transient physiological stimuli of mating suggests a physiological role for them. The time course of the change in the supernatant enzyme shows that this enzyme does not, as has been suggested (Hooper, 1958), reflect luteinizing hormone section. The fact that particulate enzyme inactivates vasopressin more effectively than oxytocin, that its activity reflects vasopressin secretion during dehydration and overhydration and that no activity was found in the diabetic dog suggests that this enzyme is involved in vasopressin and not oxytocin metabolism. Since the supernatant enzyme inactivates both hormones to the same extent it seems probable that it is not involved in the metabolism of oxytocin itself, but rather a substance similar to it. A correlation has emerged between the ratio of the enzyme activities and water metabolism. A significant finding is that in overhydrated animals the ratio supernatant/particulate enzyme is increased, a result of an increase in the former and a decrease in the latter; in overhydrated stressed animals in diuresis the enzyme ratio was similar. In this case the particulate enzyme appeared to be increased, the ratio being maintained by a very large increase in the supernatant enzyme. Vasopressin secretion in the latter case would be expected to be increased due to the stress. This suggests that diuresis is achieved by a balance of actions of vasopressin and a diuretic agent, the latter being an oxytocic-like substance or even oxytocin itself. This possibility is at present being investigated further