Explorer Oxytocinase in the Female Rat Hypothalamus : a Novel Mechanism Controlling Oxytocin Neurons During Lactation

In addition to its peripheral actions, oxytocin released within the brain is important for birth and essential for milk ejection. The oxytocinase enzyme (placental leucine aminopeptidase, P-LAP) is expressed both peripherally and centrally. P-LAP controls oxytocin degradation in the uterus, placenta and plasma during pregnancy, but its role in the hypothalamus is unclear. We investigated P-LAP expression and activity in the hypothalamus in virgin, pregnant and lactating rats, and its role in vivo during the milk-ejection reflex. P-LAP mRNA and protein were expressed in magnocellular neurons of the supraoptic (SON) and paraventricular (PVN) nuclei. Oxytocin neurons co-expressed P-LAP without strong subcellular co-localisation of oxytocin and P-LAP, indicating that they are packaged in separate vesicles. Examination of the intracellular distribution of oxytocin and P-LAP showed a redistribution of P-LAP to within 1 μm of the plasma membrane in the somata of oxytocin neurons during lactation. Both P-LAP mRNA expression and hypothalamic leucyl/cystinyl aminopeptidase activity in the soluble fraction were higher during lactation than in late pregnant or virgin states. Inhibition of central enzyme activity by i.c. v. injection of amastatin in anaesthetized suckling mothers increased the frequency of reflex milk ejections. As hypothalamic P-LAP expression and activity increase in lactation, and prevention of its action mimics central oxytocin administration, we conclude that P-LAP regulates auto-excitatory oxytocin actions during the suckling-induced milk-ejection reflex. Introduction Oxytocin is synthesized in the somata of magnocellular neurons in four hypothalamic nuclei; the paired supraoptic (SON) and paraventricular nuclei (PVN). It is packaged in large dense-cored vesicles which are transported to axon terminals in the neurohypophysis: from these terminals, oxytocin is secreted into the general circulation. In the rodent, during both parturition and the milk-ejection reflex, the magnocellular oxytocin neurons exhibit intermittent, quasi-synchronous bursts of electrical activity, typically every 5-10 min, which trigger pulsatile oxytocin secretion from the terminals. This pulsatile mode of oxytocin secretion is important for the progress of parturition and is essential for milk transfer to the young during lactation (1-4), and it critically depends on release of oxytocin from the somata and dendrites of oxytocin neurons (5). Increased somato-dendritic release of oxytocin precedes, and is required for, the co-ordination of bursting activity: the frequency of these bursts can be accelerated by i.c. v. injection of oxytocin or delayed by i.c. v. injection of an oxytocin antagonist (1, 6, 7). The availability of hormones and transmitters is regulated not only by the amount released but also by the rate of degradation. In the periphery, serum levels and uterine and placental expression of the oxytocin degrading enzyme, placental leucine aminopeptidase (PLAP), increase during midto late pregnancy (8, 9). This increased expression of P-LAP is thought to be important for preventing premature uterine contractions, thereby helping to maintain normal pregnancy to term (10, 11), and deletion of P-LAP in a transgenic mouse model advances the onset of birth (12). However, P-LAP also degrades vasopressin, angiotensin III, met-enkephalin and dynorphin (13), and central injections of the P-LAP inhibitor amastatin have been shown to modify drinking behavior (14, 15). In peripheral tissues, the enzyme is found in the same intracellular vesicles as the glutamate transporter GLUT4, and is activated upon translocation from the cytosol to the plasma membrane (16-18). Inserted into the plasma membrane, P-LAP is a membranespanning protein (19), which is active in the extracellular domain. In addition, the enzyme can be freed from the plasma membrane into the extracellular space by sheddases (20). Although P-LAP is thought to be active physiologically only after insertion into the plasma membrane, P-LAP activity is measurable in both membrane-bound and cell associatedsoluble fractions of tissue homogenates (21). P-LAP was recently identified in the hypothalamus (22) and inhibition of P-LAP activity with amastatin has been shown to mimic the effect of exogenous oxytocin administration on SON neuronal activity in a hypothalamic slice preparation (23). There have been two conflicting reports of the effect of knocking out the P-LAP gene on parturition, one without effect (24), the other showing a significant shortening of pregnancy duration (12). In this latter study, wild-type mice given continuous subcutaneous infusion of recombinant PLAP showed a delay in the onset of labour. However, there have been no studies to determine if central P-LAP plays a role in the control of oxytocin-dependent activity in vivo. In this study, we first visualized P-LAP mRNA and protein expression in the hypothalamus, and investigated its intracellular distribution and co-localization with oxytocin-neurophysin using immunofluorescence. Pregnancyand lactation-associated changes in hypothalamic P-LAP mRNA expression were analyzed by in situ hybridization, and changes in cystinyl/leucyl aminopeptidase activity in both membrane-bound and soluble fractions from samples of hypothalamus and posterior pituitary were assayed in vitro. Finally, we investigated the effect of inhibiting central P-LAP enzyme activity in vivo on the milk-ejection reflex.