Role of neuronal nitric oxide in the regulation of vasopressin expression and release in response to inhibition of catecholamine synthesis and dehydration

Abstract

We used neuronal nitric oxide synthase (nNOS) gene knockout mice to study the effects of catecholamines and neuronal nitric oxide on vasopressin expression in the hypothalamic neurosecretory centers. nNOS gene deletion did not change the level of vasopressin mRNA in the supraoptic or paraventricular nuclei. In contrast, vasopressin immunoreactivity was lower in nNOS deficient mice than in wild-type animals. Dehydration increased vasopressin mRNA levels and decreased vasopressin immunoreactivity in both wild-type and nNOS knockout mice, but these responses were more marked in the nNOS knockout mice. Treatment with alpha-mpt, a pharmacologic inhibitor of catecholamine synthesis, resulted in increased vasopressin mRNA levels in wild-type mice and in reduced vasopressin immunoreactivity in both wild-type and nNOS knockout mice. From these results, we conclude: (1) neuronal nitric oxide suppresses vasopressin expression under basal conditions and during activation of the vasopressinergic system by dehydration; (2) catecholamines limit vasopressin expression; (3) nNOS is required for the effects of catecholamines on vasopressin expression.

Figure 1

α-mpt treatment, dehydration, and α-mpt treatment during dehydration increase VP mRNA content and diminish VP immunoreactivity in VP magnocellular neurons of hypothalamic SON. A. The content of VP RNA; B. VP-immunoreactivity in the SON in WT (white columns) and nNOS knockout mice (black columns). X axis: control – intact mice; α-mpt – injection of catecholamine synthesis blockator α-mpt (100 mg/kg); DH –dehydrated mice; DH+α-mpt - α-mpt injection (100 mg/kg) during dehydration. Y axis: optical density (conventional units/μm²) * - p<0.05 compared to control; **- p<0.05 compared to control and *; #- p<0.05 as compared with WT mice.

Figure 2

Effects of α-mpt treatment, dehydration, and α-mpt treatment during dehydration on VP expression in PVN of hypothalamus. A. The content of VP RNA; B. VP-immunoreactivity in the PVN in WT (white columns) and nNOS knockout mice (black columns). X and Y axes, *, **, # are same as that for Figure 1.

Figure 3

In situ hybridization of VP mRNA with digoxigenin-labeled antisense VP-RNA probe in the SON of nNOS deficient and WT mice. A, C – digoxigenin-positive VP mRNA in control group of WT and nNOS knockout mice respectively; and in dehydrated animals: B – WT mice, D – nNOS knockout mice. Hybridization with sense VP-RNA probe was negative (not shown). Arrows indicate localization of the SON. OT is optical tract. Scale bar is 100 μm.

Figure 4

Effect of dehydration on functional activity of VP-ergic cells in the SON. Cytoplasmic stain of VP in neurons of the SON in control WT (A) and nNOS knockout mice (C); and dehydrated animals: B – WT, and D – nNOS knockout mice. Arrows indicate location of SON. Detection of VP was performed without additional counterstaining. OT is optical tract. Scale bar is 100 μm.

Figure 5

nNOS deficiency leads to decrease of TH protein content in ZI of mouse hypothalamus. Data for optical density is presented as arbitrary units per μm². Axis X: WT (white columns) – intact WT mice; nNOS-/- (black columns) – nNOS knockout intact mice. * - p<0.05 compared to WT mice.