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Atrial (ANP) and C-type (CNP) natriuretic peptide generate physiological effects via selective activation of two closely related membrane receptors with guanylyl cyclase (GC) activity, known as GC-A and GC-B. As yet, however, the discrete roles for ANP/GC-A vs. CNP/GC-B signaling in many mammalian tissues are still poorly understood. We here used receptor affinity labeling and GC assays to characterize comparatively GC-A/GC-B expression and functional activity during rat brain development. The study revealed that GC-B predominates in the developing and GC-A in the adult brain, with regional differences each between cerebral cortex, cerebellum, and brain stem. Whereas GC-A levels nearly continuously increase between embryonal d 18 and adult, GC-B expression in brain is highest and widely distributed around postnatal d 1. The striking perinatal GC-B peak coincides with elevated expression of nestin, a marker protein for neural stem/progenitor cells. Immunohistochemical investigations revealed a cell body-restricted subcellular localization of GC-B and perinatal abundance of GC-B-expressing cells in regions high in nestin-expressing cells. However, and supported by examination of nestin-GFP transgenic mice, GC-B and nestin are not coexpressed in the same cells. Rather, GC-B(+) cells are distinguished by expression of NeuN, an early marker of differentiating neurons. These findings suggest that GC-B(+) cells represent neuronal fate-specific progeny of nestin(+) progenitors and raise the attention to specific and pronounced activities of CNP/GC-B signaling during perinatal brain maturation. The absence of this activity may cause the neurological disorders observed in GC-B-deficient mice.

The cardiac hormone atrial natriuretic peptide (ANP) signals via interaction with a plasma membrane receptor, which has guanylyl cyclase (GC) activity and is referred to as GC-A. Desensitization of GC-A is thought to represent a physiologically important regulatory mechanism, but the signaling pathways implicated and cell type-specific effects are still poorly understood. Here we demonstrate that sustained exposure to either ANP itself or the bioactive lipid lysophosphatidic acid (LPA) elicits GC-A desensitization in MA-10 Leydig cells. Both reactions show similar kinetics and evoke equal decreases (by 40%) in GC-A hormone responsiveness. Homologous (ANP induced) desensitization, in which cGMP is generated as second messenger, is blocked by distinct cAMP-dependent protein kinase [protein kinase A (PKA)] inhibitors, H 89, and Rp-8-CPT-cAMPs, providing evidence that PKA mediates the reaction. Accordingly, the ANP/cGMP-elicited effects are mimicked by a cAMP analog, 8-bromo-cAMP. The LPA-induced (heterologous) desensitization is not blocked by PKA inhibition, indicating a different signaling pathway. LPA, but not ANP, enhances ERK phosphorylation and induces cell rounding together with a dramatic reorganization of actin filaments. Consistent with the identification of LPA receptor (LPA2 and LPA3) gene expression, the findings are indicative of LPA receptor-mediated reactions. This study demonstrates for the first time coexistence of homologous and heterologous desensitization of GC-A in the same cell type, reveals that these reactions are mediated by different pathways, and identifies a novel cross talk between phospholipid and natriuretic peptide signaling. The morphoregulatory activities exerted by LPA suggest a crucial role for Leydig cell physiology.

Neuropeptide Y (NPY) conjugated to saporin (NPY-SAP), a ribosomal inactivating toxin, is a newly developed compound designed to selectively target and lesion NPY receptor-expressing cells. We injected NPY-SAP into the basomedial hypothalamus (BMH), just dorsal to the arcuate nucleus (ARC), to investigate its neurotoxicity and to determine whether ARC NPY neurons are required for glucoprivic feeding. We found that NPY-SAP profoundly reduced NPY Y1 receptor and alpha MSH immunoreactivity, as well as NPY, Agouti gene-related protein (AGRP), and cocaine and amphetamine-related transcript mRNA expression in the BMH. NPY-SAP lesions were localized to the injection site with no evidence of retrograde transport by hindbrain NPY neurons with BMH terminals. These lesions impaired responses to intracerebroventricular (icv) leptin (5 microg/5 microl x d) and ghrelin (2 microg/5 microl), which are thought to alter feeding primarily by actions on ARC NPY/AGRP and proopiomelanocortin/cocaine and amphetamine-related transcript neurons. However, the hypothesis that NPY/AGRP neurons are required downstream mediators of glucoprivic feeding was not supported. Although NPY/AGRP neurons were destroyed by NPY-SAP, the lesion did not impair either the feeding or the hyperglycemic response to 2-deoxy-D-glucose-induced blockade of glycolysis use. Similarly, responses to glucagon-like peptide-1 (GLP-1, 5 microg/3 microl icv), NPY (5 microg/3 microl icv), cholecystokinin octapeptide (4 microg/kg ip), and beta-mercaptoacetate (68 mg/kg ip) were not altered by the NPY-SAP lesion. Thus, NPY-SAP destroyed NPY receptor-expressing neurons in the ARC and selectively disrupted controls of feeding dependent on those neurons but did not disrupt peptidergic or metabolic controls dependent upon circuitry outside the BMH.

By interacting with a guanylyl cyclase (GC) activity-containing receptor, termed GC-A, atrial natriuretic peptide (ANP) acts as a regulator of blood pressure and fluid volume homeostasis. High expression levels of GC-A in the testis and reported effects of ANP on testosterone secretion by Leydig cells are indicative of important local functions in this organ. Here we show, based on radioligand receptor labeling and immunological approaches, that seminiferous tubules rather than Leydig cells are the predominant GC-A expression sites in the rat testis. Functional activity was proved by ANP- induced cGMP accumulation in isolated seminiferous tubules. Although ontogenetic studies revealed a massive increase in GC-A levels during sexual maturation, the so-called natriuretic peptide clearance receptor, another type of ANP receptor proposed to locally control the availability of natriuretic peptides, was found to be expressed predominantly before puberty, exceeding the level of GC-A expression at this time. Natriuretic peptide clearance receptor also shows a distinct distribution pattern surrounding the seminiferous tubules. These findings raise the possibility of novel physiological roles for ANP and cGMP in the testis related to germ cell maturation and/or the regulation of the onset of puberty and suggest that the two ANP receptors function in a coordinated manner at this target organ.

This study evaluated whether renal escape from vasopressin-induced antidiuresis is associated with alterations of vasopressin V2 receptor binding in the kidney inner medulla. A radioligand binding assay was developed using a novel iodinated vasopressin V2 receptor antagonist to analyze vasopressin V2 receptor binding in kidney inner medullary tissue from three groups of rats: normal rats maintained on ad libitum water intake, rats treated with 1-deamino-[8-D-arginine]vasopressin (DDAVP), and rats treated with DDAVP that were also water loaded to induce renal escape from antidiuresis. Analysis of the binding data showed that DDAVP treatment reduced vasopressin V2 receptor binding to 72% of normal levels. Water loading induced a marked further down-regulation of vasopressin V2 receptor binding. This receptor down-regulation began by day 2 of water loading, which correlated with the initiation of renal vasopressin escape; by day 3 of water loading, vasopressin V2 receptor expression fell to 43% of DDAVP-treated levels. No differences in vasopressin V2 receptor binding affinities were found among the three groups. This study demonstrates that vasopressin V2 receptor binding capacity is down-regulated during renal escape from vasopressin-induced antidiuresis and suggests that both vasopressin-dependent mechanisms as well as vasopressin-independent mechanisms associated with water loading are involved in this receptor down-regulation.

To investigate the hypothesis that the release of angiotensin-II (AII) in the rat brain increases on the day of proestrus, samples of cerebrospinal fluid (CSF) and interstitial fluid from the general region of the bed nucleus of the stria terminalis pars ventralis in the preoptic-anterior hypothalamic area were monitored for AII-immunoreactive material (AII-ir) using push-pull cannulas. Samples of CSF were obtained on the day of proestrus and diestrus day 1 at 30-min intervals from 1200-1600 h. Samples of interstitial fluid were obtained at 25-min intervals from 0930-1600 h. The rate of release of AII-ir into CSF was significantly greater on proestrus compared to diestrus day 1, and in the early afternoon of proestrus compared to the late afternoon. In five of seven rats and in the overall comparison of AII-ir release from the preoptic-anterior hypothalamic area, significantly more AII-ir was released on the day of proestrus vs. diestrus day 1. These observations are consistent with previous studies suggesting that brain AII may play a role in the regulation of LH release on the day of proestrus.

Specific angiotensin II (Ang II) binding and functional responses have been studied in a number of male and female reproductive structures, but to date have not been characterized in the epididymis. Some epididymal functions, including smooth muscle contraction and regulation of fluid and electrolyte balance, are mediated by Ang II in other tissues. This study demonstrates specific, saturable (63 fmol/mg protein), high affinity (Kd, less than 1 nM) Ang II-binding sites in the epididymis. These binding sites, localized in the circumference of the epididymal tubule and most concentrated within the proximal cauda, are present throughout the caput, corpus, and remaining cauda epididymis. Ang II caused powerful expulsions of spermatozoa in segments of epididymis in situ. These results suggest Ang II involvement in epididymal function.