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It is well accepted that neuropeptide Y (NPY) plays a pivotal role in the regulation of food intake and energy homeostasis in the rodent, with NPY neurons in the arcuate nucleus (ARH) being thought of as the major contributor to the complex central feeding circuitry. Recent data from our group also indicate that NPY is important in the regulation of energy homeostasis in the nonhuman primate (NHP); exogenous NPY administration into the 3rd ventricle is a potent stimulator of food intake in the male rhesus macaque. The purpose of this study was to determine if NPY neurons in the rhesus macaque respond to a metabolic challenge, induced by 48 h of fasting, in a manner similar to that seen in the rodent. NPY mRNA was detected in hypothalamic sections from 48-h fasted or fed rhesus monkeys by in situ hybridization, using a [35S]UTP-labeled riboprobe specific for human NPY. Not surprisingly, NPY mRNA was abundant in the ARH of the NHP; however, of great interest was the expression of NPY mRNA in neurons within the paraventricular nucleus of the hypothalamus (PVH) and the supraoptic nucleus (SON). This raised the question as to whether all of these populations of NPY neurons are sensitive to changes in energy availability. Indeed, NPY expression in the ARH and PVH was significantly elevated in response to fasting; however, no significant change was detected in the SON. These data indicate that the NPY neurocircuitry involved in the regulation of food intake is more complex in the NHP than in rodents.

The orexins are produced in neurons of the lateral hypothalamic area and implicated in the regulation of both feeding and reproductive function. Orexins stimulate LH secretion in steroid-primed ovariectomized female rats and suppress LH secretion in nonprimed ovariectomized rats. The aim of the present study was to characterize the neuroanatomical pathway by which orexin might modulate LH secretion in the rat. Using double- and triple-label immunofluorescence coupled with confocal microscopy, we found that 75-85% of GnRH neurons were contacted by orexin fibers, and triple labeling with synaptophysin provided additional confirmation of close contacts. Furthermore, about 85% of GnRH neurons were colocalized with the orexin receptor 1 (OX-R1), and the OX-R1-expressing GnRH neurons were contacted by orexin terminals, providing the basis for a functional neuroanatomical pathway. GnRH nerve terminals in the median eminence, however, do not express OX-R1. An additional study investigated the coexpression of neuropeptide Y Y4-like receptors and orexin fibers in relation to GnRH neurons. There is evidence that Y4 receptor stimulation results in LH release, and studies from our laboratory show Y4-like immunoreactivity in the majority of orexin cell bodies in the lateral hypothalamic area and some orexin fibers scattered throughout the hypothalamus. The present study found that, although Y4-positive orexin fibers are in present in the area of GnRH neurons, they never come in close contact with GnRH neurons. Together, these data suggest that Y4 receptor modulation of LH release is likely to be indirect through orexin cell bodies and that orexin modulates GnRH neurons directly via OX-R1.

The gastrointestinal peptide hormone ghrelin stimulates appetite in rodents and humans via hypothalamic actions. We discovered expression of ghrelin in a previously uncharacterized group of neurons adjacent to the third ventricle between the dorsal, ventral, paraventricular, and arcuate hypothalamic nuclei. These neurons send efferents onto key hypothalamic circuits, including those producing neuropeptide Y (NPY), Agouti-related protein (AGRP), proopiomelanocortin (POMC) products, and corticotropin-releasing hormone (CRH). Within the hypothalamus, ghrelin bound mostly on presynaptic terminals of NPY neurons. Using electrophysiological recordings, we found that ghrelin stimulated the activity of arcuate NPY neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus (PVH). We propose that at these sites, release of ghrelin may stimulate the release of orexigenic peptides and neurotransmitters, thus representing a novel regulatory circuit controlling energy homeostasis.

The receptor subtypes that mediate the effects of neuropeptide Y (NPY) on food intake have not been clearly defined. The NPY Y4 receptor has been identified recently as a potential mediator of the regulation of food intake. The purpose of the present study was to characterize the central site of action of the Y4 receptor using a combination of neuroanatomical and physiological approaches. Using immunocytochemistry, Y4-like immunoreactivity was found to be colocalized with orexin cell bodies in the lateral hypothalamic area (LHA) and orexin fibers throughout the brain. In situ hybridization confirmed the expression of Y4 mRNA in orexin neurons. To determine the functional interaction between Y4 receptors and orexin neurons, we examined the effects of rat pancreatic polypeptide (rPP), a Y4-selective ligand, or NPY, a nonselective ligand, administered directly into the LHA on the stimulation of food and water intake and c-Fos expression. Both rPP and NPY significantly increased food and water intake when they were administered into the LHA, although NPY was a more potent stimulator of food intake. Furthermore, both NPY and rPP significantly stimulated c-Fos expression in the LHA. However, whereas rPP stimulated c-Fos expression in orexin neurons, NPY did not. Neither rPP nor NPY stimulated c-Fos in melanin-concentrating hormone neurons, but both activated neurons of an unknown phenotype in the LHA. These results suggest that a functional Y4 receptor is expressed on orexin neurons and that these neurons are activated in response to a ligand with high affinity for the Y4 receptor (rPP). Although these data suggest a role for central Y4 receptors, the endogenous ligand for this receptor has yet to be clearly established.

In the adult rat, arcuate-neuropeptide Y/agouti-related protein neurons have efferent projections throughout the hypothalamus and provide a potent orexigenic stimulus. At birth neuropeptide Y fibers are also present throughout the hypothalamus; however, the source of these fibers has been unknown. The present studies determined the postnatal ontogeny of arcuate-neuropeptide Y fibers into the paraventricular nucleus and dorsomedial hypothalamic nucleus, as well as the ontogeny of neuropeptide Y1 receptor expression within these areas. Agouti-related protein messenger RNA and protein expression was present exclusively in cell bodies in the arcuate throughout postnatal development, starting at P2, and was colocalized in the vast majority of arcuate-neuropeptide Y neurons. This exclusive colocalization of agouti-related protein with arcuate-neuropeptide Y neurons makes it an excellent marker for these neurons and their projections. Even though single-label neuropeptide Y fibers were abundant in the dorsomedial hypothalamic nucleus and paraventricular nucleus as early as P2, arcuate-neuropeptide Y/agouti-related protein fibers did not significantly innervate these areas until P5-6 and P10-11, respectively. In contrast, a portion of the neuropeptide Y fibers within the paraventricular nucleus as early as P2 originated from the brainstem, as indicated by their colocalization with dopamine beta hydroxylase. It remains to be determined if local sources of neuropeptide Y-expressing cells within the dorsomedial hypothalamic nucleus and paraventricular nucleus also contribute to the neuropeptide Y-immunoreactive fibers within these regions prior to the development of arcuate-neuropeptide Y/agouti-related protein projections. In addition to the dramatic change in arcuate-neuropeptide Y/agouti-related protein projections, there is also a striking change in Y1 protein expression in the hypothalamus during the first two postnatal weeks. Taken together these data suggest that the early postnatal period, during which there is a dynamic change in the hypothalamic neuropeptide Y system, may constitute a critical period in the development of this important feeding circuit., (Copyright 2003 IBRO)

Lactation is a physiological model for studying how the hypothalamus integrates peripheral signals, such as sensory signals (suckling stimulus) and those denoting energy balance (leptin), to alter hypothalamic function regulating food intake/energy balance and reproduction. The characteristics of food intake/energy balance during lactation are extreme hyperphagia, coupled with negative energy balance. The arcuate nucleus Neuropeptide Y (ARH-NPY) system is activated by: (1) brainstem projections specifically activated by the suckling stimulus, and (2) the decrease in leptin in response to the metabolic drain of milk production. NPY neurons from the ARH make direct contact with GnRH neurons and with CRH neurons in the PVH. NPY neurons also make contact with orexin and MCH neurons in the LHA, which, in turn, make contacts with GnRH neurons. Thus, the ARH-NPY system provides a neuroanatomical framework by which to integrate changes in food intake/energy with the regulation of cyclic reproductive function.

The mechanism by which lactating rats increase fluid consumption to meet the demands of milk production is unknown. Because ANG II is the most potent dipsogenic stimulus known, this study examined whether angiotensinergic signaling plays a role in enhanced drinking in lactating rats. ANG II administered intracerebroventricularly caused a significantly greater dipsogenic response in lactating rats than in control rats, suggesting that dipsogenic responsivity to ANG II is enhanced in the brains of lactating rats. The angiotensin type 1 (AT1) ANG II receptor subtype antagonist SKF-108566, also given intracerebroventricularly, caused a significant reduction in water consumption in lactating rats, whereas it did not significantly affect water intake in control rats. In contrast, stimulation of drinking by the muscarinic agonist carbachol, also administered intracerebroventricularly, did not differ between lactating and control rats. Inhibition of drinking by the muscarinic antagonist atropine also did not differ significantly between lactating and control rats. These results suggest that the increased drinking in lactating rats involves an increased responsivity to ANG II in neurons that mediate dipsogenesis, as well as an enhancement in the amount of angiotensinergic input to these ANG II-responsive neurons.

During development there is novel expression of NPY mRNA in the dorsomedial hypothalamic nucleus (DMH) and perifornical region (PFR), in addition to the arcuate nucleus (ARH). Furthermore, NPY mRNA levels peak in all regions on postnatal d 16 (P16) and decrease to adult levels by P30. The purpose of the present study was to determine whether NPY and agouti-related protein (AGRP) mRNA expression in the different hypothalamic regions on P11 and P16 are similarly affected by fasting. An examination of the full rostral to caudal extent of the hypothalamus revealed two additional regions displaying novel NPY mRNA expression, the parvocellular division of the paraventricular nucleus (PVH) and lateral hypothalamus (LH). Maternal deprivation for 36 h, used to bring about a fast, similarly increased (23-29%) NPY and AGRP mRNA expression in the ARH on P11 and P16. In contrast, NPY expression in the DMH and PFR were significantly decreased (19-30% and 48-53%, respectively), whereas NPY mRNA levels in the PVH and LH were not altered by this treatment. The increase in NPY and AGRP mRNA expression in the ARH in response to maternal deprivation suggests that these neuronal populations respond to signals of energy balance. In contrast, NPY expression in the DMH, PFR, PVH, and LH is differentially regulated by maternal deprivation or other factors associated with maternal separation.

It is well known that maternal smoking during pregnancy can lead to low birth weight and low body fat in human newborns. The purpose of this study was to determine whether chronic maternal nicotine treatment alters levels of known regulators of energy balance in the newborn offspring. Pregnant rhesus monkeys were treated with nicotine tartrate (1.5 mg/kg x d) starting on d 26 of pregnancy and maintained through d 160 of gestation. Nicotine exposure had no significant effect on absolute birth weights of the neonatal monkeys, although there was a 10% reduction in birth weights with nicotine exposure when they were normalized to maternal weight. Postnatal d 1 plasma leptin levels were significantly reduced by about 50% in the nicotine treatment group compared with saline controls, suggesting that the infant monkeys exposed to nicotine may also have lower body fat levels. In situ hybridization studies demonstrated that chronic nicotine exposure resulted in a significant decrease in arcuate NPY mRNA expression in the neonatal monkeys. In addition, there was a 2-fold increase in POMC mRNA in the arcuate nucleus in the nicotine-exposed group. These data suggest that nicotine exposure during pregnancy may increase energy expenditure in the developing fetus through actions on hypothalamic systems, resulting in lower birth weights and body fat levels.

Neuropeptide Y (NPY) displays diverse modes of action in the CNS including the modulation of feeding behavior, gonadotropin releasing hormone release, and stress responses. Many of the above physiological actions have been at least partially attributed to actions of NPY on the NPY Y5 receptor subtype. We utilized an antibody directed against the NPY Y5 receptor to characterize the distribution of this receptor in the rat brain. Using Western blot analysis, this antibody recognized a single major band at approximately 57 kD. To further verify the specificity of the antibody, animals were treated for 5 days with antisense oligonucleotides for the Y5 receptor. The antisense treatment significantly reduced food intake and body weight. Furthermore, the Y5 antibody detected a significant decrease in Y5 receptor protein. Y5-like immunoreactivity (-ir) was observed throughout the hypothalamus, thalamus, hippocampus and cortex. Double-label immunofluorescence demonstrated that Y5-ir was colocalized with the following neuronal phenotypes in the hypothalamus, gonadotropin-releasing hormone, neurophysins, corticotropin-releasing hormone, and gamma-amino butyric acid. In addition, functional interactions were demonstrated by the presence of close appositions of NPY fibers with Y5-ir expressing cells. The wide distribution of the Y5 receptor-ir, as well as the colocalization within specific neuronal populations, agrees with the distribution of the Y5 receptor mRNA and the known physiological roles of the NPY/Y5 system. The role of the NPY/Y5 receptor system as a mediator between signals of peripheral energy availability and reproductive neuroendocrine function is discussed., (Copyright 2001 S. Karger AG, Basel)

In rodents, stimulation of melanocortin-3 and -4 receptor subtypes (MC3-R and MC4-R) causes a reduction in food intake, whereas antagonism of MC3-R and MC4-R increases food intake. This report describes the effects of the stable alphaMSH analog, NDP-MSH ([Nle(4), D-Phe(7)]alphaMSH), and the endogenous alphaMSH receptor antagonist, agouti-related protein, on feeding behavior in adult male rhesus macaques. Infusion of NDP-MSH into the lateral cerebral ventricle dose dependently suppressed intake of a normally scheduled meal without affecting nonfeeding behaviors. Conversely, infusion of agouti-related protein stimulated food intake during the scheduled afternoon meal. In addition to these physiological experiments, the effect of fasting on hypothalamic POMC gene expression was assessed by in situ hybridization. Missing a single meal or fasting for 48 h caused a similar reduction in POMC gene expression in the arcuate nucleus. These results demonstrate that in the primate, central melanocortin receptors can acutely regulate food intake and suggest that the central melanocortinergic system is a physiological regulator of energy balance in primate species.

In lactating rats, ANG II receptor binding in the arcuate nucleus (ARH) and median eminence is decreased. To further evaluate brain angiotensinergic activity during lactation, we assessed angiotensinogen (AON) mRNA by in situ hybridization in forebrains of day 10 or 11 postpartum lactating and diestrous rats. AON mRNA was abundantly expressed in the ARH, preoptic, suprachiasmatic, supraoptic, paraventricular, and dorsomedial hypothalamic nuclei, and other regions, similar to that reported in male rat brains. AON mRNA levels were decreased 27% in the midcaudal ARH of lactating rats but did not differ between lactating or diestrous rats in any of the other brain areas examined. Immunofluorescence for AON and glial fibrillary acidic protein or tyrosine hydroxylase confirmed that the AON immunoreactivity in the ARH was limited to astrocytes. Confocal microscopy revealed close appositions of AON-positive astrocytes to dopaminergic neurons in the ARH. The decrease in AON mRNA in the midcaudal ARH during lactation coupled with decreased ARH ANG II receptor binding suggests that lactating rats are less subject to ANG II-mediated inhibition of prolactin secretion.

During lactation, hypothalamic levels of neuropeptide Y (NPY) and agouti related protein (AGRP) mRNA are increased, while pro-opiomelanocortin (POMC) mRNA is decreased. Serum leptin levels are also decreased during lactation. These changes may underlie the large increases of both food and water intake that occur in concert with milk production. However, additional hypothalamic substances, such as the novel peptide, orexin, may be involved. In addition, in the presence of chronically suppressed levels of serum leptin, there may be a change in leptin receptor expression in the hypothalamus. The objectives of the present study were to determine if orexin and leptin receptor mRNA levels were changed during lactation. Rats were studied on dioestrus of the oestrous cycle or on day 10 postpartum (the lactating animals were suckling eight pups). Orexin mRNA levels in the lateral hypothalamus did not differ between dioestrus and lactation. There was a significant increase in leptin receptor mRNA levels in the supraoptic nucleus during lactation compared to dioestrus. Furthermore, leptin receptor protein, as determined by immunocytochemistry, was colocalized in virtually all vasopressin and oxytocin cells in the supraoptic nucleus. Lactating animals exhibited a decrease in leptin receptor mRNA in the ventromedial hypothalamic nucleus whereas no change was apparent in other hypothalamic areas compared to the dioestrus animals. These results demonstrate that changes in orexin do not appear to contribute to the increase in food intake during lactation. It is likely that the increases in NPY and ARGP, coupled with the decrease in POMC, are primarily responsible for sustaining the chronic hyperphagia of lactation. The changes observed in leptin receptor expression in the hypothalamus, along with the suppression of serum leptin levels, also suggest that the leptin signalling system may play a significant role in the regulation of food and water intake during lactation.

Neuropeptide Y (NPY) is a potent orexigenic peptide. In the normal adult rat, hypothalamic NPY mRNA expression is limited to the arcuate nucleus (ARH). The purpose of this study was to characterize the developmental expression of NPY mRNA in the hypothalamus of the rat. In contrast to the normal adult rat, NPY mRNA was observed in the ARH, the dorsomedial hypothalamic nucleus (DMH) and the perifornical region (PFR) during development. NPY mRNA expression in all three regions increased progressively from postnatal days 0-4 (P0-4) to reach maximum levels at P16 and subsequently decreased to near adult levels by P30. The unique expression of NPY mRNA in the PFR and DMH may be important in establishing the proper management of energy homeostasis and body weight in the adult animal.

Neuropeptide Y displays diverse modes of action in the CNS including the modulation of cortical/limbic function. Some of these physiological actions have been at least partially attributed to actions of neuropeptide Y on the Y5 receptor subtype. We utilized an antibody raised against the Y5 receptor to characterize the distribution of this receptor subtype in the rat cortical/limbic system and brainstem. Y5-like immunoreactivity was located primarily in neuronal cell bodies and proximal dendritic processes throughout the brain. In the cortex, Y5 immunoreactivity was limited to a subpopulation of small gamma-aminobutyric-acid interneurons (approximately 15 microm diameter) scattered throughout all cortical levels. Double label immunofluorescence was also used to demonstrate that all of the Y5 immunoreactive neurons in the cortex displayed intense corticotropin releasing hormone immunoreactivity. The most intense Y5 immunoreactive staining in the hippocampus was located in the pyramidal cell layer of the small CA2 subregion and the fasciola cinerea, with lower levels of staining in the hilar region of the dentate gyrus and CA3 subregion of the pyramidal cell layer. Nearly all of the Y5 immunoreactive neurons in the hilar region of the hippocampus displayed gamma-aminobutyric-acid immunoreactivity. In the brainstem, Y5 immunoreactivity was most intense in the Edinger-Westphal nucleus, locus coeruleus and the mesencephalic trigeminal nucleus. The present study provides neuroanatomical evidence for the possible sites of action of the neuropeptide Y/Y5 receptor system in the control of cortical/limbic function. The presence of Y5 immunoreactivity on cell bodies and proximal dendritic processes in specific regions of the hippocampus suggests that this receptor functions to modulate postsynaptic activity. These data also suggest that the neuropeptide Y/Y5 system may play a role in the modulation of a specific population of GABAergic neurons in the cortex, namely those that contain corticotropin-releasing hormone. The location of the Y5 receptor immunoreactivity fits with the known physiological actions of neuropeptide Y and this receptor.

During lactation there are many dramatic alterations in the hypothalamic-pituitary (HP) axis, as well as an increased demand for food and water. The renin-angiotensin system (RAS) is one of the major mediators of the HP axis. This study examined the receptors for ANG II in the rat brain during lactation and diestrus. Compared with diestrus, lactating rats had significant decreases in ANG II receptor binding in several forebrain regions, most notably in the arcuate nucleus/median eminence, dorsomedial hypothalamic nucleus (DMH), and lateral hypothalamic area (LHA). In contrast, there was an increase in ANG II receptor binding in the preoptic area during lactation. These significant changes in ANG II binding in the brain during lactation support the hypothesis that changes in the RAS may contribute to the dramatic changes in the HP axis during lactation. In addition, the significant reduction in ANG II binding in the DMH and LHA may be indicative of a role in the regulation of food intake, a function only recently associated with the RAS.

Functional angiotensin IV (Ang IV) receptors (denoted AT4) are localized to the outer stripe of the medulla in the rat kidney, and may play a critical role in salt homeostasis. The purpose of this study was to determine if AT4 receptor binding in the kidney is differently regulated in the salt-sensitive spontaneously hypertensive (SH) rat compared to Wistar Kyoto (WKY) controls. AT4 receptor binding was determined using in vitro receptor autoradiography. AT4 receptor binding in the outer stripe of the medulla was similar in WKY and SH rats maintained on a 1% salt diet. A high salt diet (8%) resulted in a statistically significant increase (28%) in AT4 receptor binding in kidneys from WKY rats. However, there was no change in AT4 receptor binding in the kidneys of SH rats fed the same diet. The present data indicate that AT4 binding sites are regulated by salt intake. In addition, regulation of this receptor may be impaired in the kidneys of SH rats, explaining in part the salt-sensitivity of this strain.

It has long been known that angiotensin II (Ang II) can affect reproductive tissues such as the uterus. However, the existence of a local renin-angiotensin system (RAS) in female as well as male reproductive tissues is a relatively recent observation. Of great interest is the discovery that all components of the RAS are present in the ovary, that the ovary secretes components of the RAS into the bloodstream, and that the ovary itself is responsive to Ang II. Recent studies suggest that the primary role of Ang II in the ovary is to cause atresia in non-ovulatory follicles; however, there is also compelling data to suggest that Ang II facilitates ovulation. Male reproductive structures also contain all of the components of the RAS, gonadotropins regulate the activity of these components, and these tissues have Ang II receptors. Of great interest is the expression of testis-specific angiotensin-converting enzyme (ACE), which is located on germ cells. Recent studies using gene knock-out techniques indicate that testis ACE plays an important role in male fertility. However, the overall significance of the RAS for normal reproductive function remains questionable. There is now a body of evidence implicating the RAS in pathophysiologies associated with reproductive function, which gives rise to the possibility that drugs acting on the RAS might ameliorate some of these disorders. Considerable work remains to determine the role of Ang II in reproductive functions.

The lactating rat has been shown to lack a behavioral response and immediate early gene expression (cFos) in the hippocampus (Hipc) following intravenous or intracerebroventricular administration of an N-methyl-d-aspartate (NMDA) receptor agonist. The purpose of this study is to determine whether neurons in the Hipc have an intact postsynaptic NMDA receptor system. The presence of NMDA receptor protein was determined by Western blot analysis for the NR1, NR2A, and NR2B subunits. The presence of functional NMDA receptors in the Hipc was determined by behavioral responses and the expression of cFos immunoreactivity (-ir) in response to microinjection of an NMDA receptor agonist into the hilus of the dentate gyrus. No difference in NR1 and NR2A subunit protein in the Hipc was detected between the lactating and nonlactating rats. However, there was a 26% decrease in NR2B subunit protein in this region in the lactating rat. Lactating rats receiving NMA injections displayed hyperactive behavior, similar to that observed in the nonlactating animals receiving the same treatment. The lactating rat and the nonlactating rat also displayed equivalent bilateral cFos-ir in the dentate gyrus (DG), CA1 and CA3 regions of the Hipc in response to unilateral NMA injections into the Hipc. These data indicate that the lactating rat has an intact postsynaptic NMDA receptor system. Thus, Hipc refractoriness to peripheral and third ventricular injections of an NMDA receptor agonist may reflect inhibition of presynaptic input and glutamate release., (Copyright 1998 Elsevier Science B.V.)

Many techniques have been utilized to discern the localization of angiotensin II (Ang II) receptors to specific cellular components (glia, neuronal cell bodies and nerve terminals) in the brain. In the present study, we used lesioning techniques to localize Ang II receptors to cellular components in the rat forebrain. In the first experiment, axons ascending to the hypothalamus and forebrain from neurons in the brainstem were destroyed by unilaterally cutting the medial forebrain bundle (MFB). In the second experiment, a single injection of the neurotoxin, ibotenic acid, was injected unilaterally into the ventral portion of the bed nucleus of the stria terminalis (BSTV) to destroy neuronal cell bodies, thus determining if Ang II receptors are present on neuronal cell bodies. In both experiments, the animals were sacrificed after two weeks recovery and the brains processed for in vitro receptor autoradiography using 125I-sar1,ile8 Ang II (125I-SI Ang II). Unilateral knife-cut lesions of the MFB caused a significant reduction in 125I-SI Ang II binding in the BSTV (30+/-6%) and the piriform cortex (PC; 26+/-4%) ipsilateral to the knife cut. Unilateral injection of the neurotoxin into the BSTV failed to alter 125I-SI Ang II binding in this nucleus. These experiments suggest that at least a subpopulation of Ang II receptors in the BSTV and PC are located on terminals of neurons that have their cell bodies in the brainstem and their axons in the MFB., (Copyright 1998 Elsevier Science B.V.)

The natriuretic peptide receptor (NPR) family consists of three receptor subtypes: two transmembrane forms that contain a guanylyl cyclase intracellular domain (NPR-A and NPR-B), and one truncated form (NPR-C). Because of the lack of specific agonists and antagonists for each receptor subtype and to the difficulty to detect the presence of small quantities of NPR-B by ligand binding studies, polyclonal antibodies against a peptide whose sequence was chosen from a region of the extracellular domain of rat NPR-B that is not homologous to sequences in NPR-A and NPR-C were developed. Western blotting with affinity-purified anti-NPR-B (413-426)-Tyr revealed a polypeptide of approximately 120 kD on COS-1 cell membranes transfected with rat NPR-B cDNA. The antibody recognized a second polypeptide, approximately 5 to 10 kD smaller, which probably represents the unglycosylated receptor. Anti-NPR-B (413-426)-Tyr did not show crossreactivity to any other NPR. Western blotting analysis with anti-NPR-B (413-426)-Tyr also identified a protein of appropriate size in renal vascular membranes. These results were supported by immunohistochemistry findings that demonstrated staining for NPR-B on papillary and medullary capillaries, glomeruli, and renal arteries. This study concludes that NPR-B is present in the rat kidney, although it was only detected in vascular structures.

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to approximately 5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.

2'-Fluoro-5-methyl-beta-L-arabinofuranosyluracil (L-FMAU) is the first L-nucleoside analog with low cytotoxicity discovered to have potent antiviral activities against both hepatitis B virus and Epstein-Barr virus but not human immunodeficiency virus. This spectrum of activity is different from those of the other L-nucleoside analogs examined. L-FMAU enters cells through equilibrative-sensitive and -insensitive nucleoside transport as well as through nonfacilitated passive diffusion. L-FMAU is phosphorylated stepwise in cells to its mono-, di-, and triphosphate forms. In the present study the enzymes responsible for the first step of L-FMAU phosphorylation were identified. This is the first thymidine analog shown to be a substrate not only for cytosolic thymidine kinase and mitochondrial deoxypyrimidine kinase but also for deoxycytidine kinase. This finding suggests that the antiviral activity of L-FMAU will not be limited by the loss or alteration of any of these deoxynucleoside kinases.

The acyclic cytosine nucleoside analog cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], which has both (+)- and (-)-enantiomers, was evaluated for its anti-hepatitis B virus (HBV) activity in 2.2.15 cells and was found to have potent activity against HBV DNA synthesis. The R-(-)-enantiomer was found to be the more active of the cytallene enantiomers, with a 50% inhibition concentration against HBV synthesis (HBIC50) of 0.08 microM. Its antiviral activity could be reversed by deoxycytidine (dC) and less efficiently by cytidine. Upon removal of the R-(-)-enantiomer from culture medium, the synthesis of HBV DNA could reinitiate, which suggested that the antiviral action is reversible. The R-(-)-enantiomer was also found to be more cytotoxic than the S-(+)-enantiomer. The degree of cytotoxicity varied among the cell lines, with a 50% inhibition of cell growth at greater than 10 microM. The R-(-)-enantiomer had no effect on HBV RNA synthesis and mitochondrial DNA synthesis at a concentration of 10 times or more than the HBIC50. The two enantiomers cannot be deaminated by dC deaminase, and they can be phosphorylated by cytoplasmic dC kinase. The R-(-)-enantiomer of cytallene is the first acyclic cytosine analog with potent inhibitory activity against HBV similar to those of other L-(-)-ddC analogs.

We compared the abundance and sensitivity of atrial natriuretic peptides (ANP) receptors in the brains of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats and examined the effect of blood pressure on the abundance of brain ANP receptors in several other experimental rat models. Brain slices from SHR generated more guanosine 3',5'-cyclic monophosphate in response to ANP than brain slices from WKY rats. No differences were found in brain particulate guanylate cyclase activity in both strains of rats. In rat brain homogenates, we observed that ANP bound in a specific and saturable fashion to samples from WKY rats, but not in samples from SHR. In vitro receptor autoradiography revealed that ANP binding was reduced in the subfornical organ, the choroid plexus, and the paraventricular nucleus of SHR compared with WKY rat brains. Correction of hypertension in SHR or induction of hypertension in other strains did not affect ANP binding in any of these brain regions. Altogether, our data suggest that the increased sensitivity of SHR brains to the action of ANP may be a consequence of factors other than the abundance of receptors and that it is not secondary to the elevation of blood pressure.

The effects of angiotensin II (Ang II) as a semen extender were studied. In the first experiment, individual ejaculates from 10 bulls were split and extended in egg yolk citrate in the absence or presence of varying concentrations of Ang II (10[-5]-10[-10] M) to a final concentration of 35 x 10(6) sperm per mL. The percentage of intact acrosomes and percentage motility were determined in all treatments for all bulls at 0 h (immediately post thaw) and after incubation for 4 h at 37 degrees C. Extension of the semen with Ang II did not affect spermatozoal viability at either time studied. In the second experiment, mixed breed virgin heifers were induced into oestrus with intramuscular injections of prostaglandin F2alpha on Days 0 and 3. Animals that stood to be mounted were paired for bilateral intracornual insemination using a 0.5-mL French straw on each side approximately 8 h later. One of the paired heifers received semen containing Ang II (10[-5] M) while the other received control semen. A 1-mL aspirate of vaginal mucus was collected at hourly intervals for 8 h after insemination. Concentration of spermatozoa was determined by haemocytometry. There was a significant reduction in cumulative semen loss into the vagina of heifers inseminated with Ang II extended semen (14.4%) compared with heifers inseminated with control semen (19.7%). This suggests that Ang II, when added to extended semen, may reduce retrograde sperm loss following insemination without affecting sperm viability.

Beta-L-(-)-dioxolane cytidine [(-)-OddC] is the first nucleoside analogue with the unnatural L configuration shown to have anticancer activity. The transport and metabolism of this unique compound were studied in human prostate carcinoma DU-145 cells. (-)-OddC was translocated rapidly into the cells by both equilibrative-sensitive and -insensitive nucleoside transport systems. Accumulation of (-)-OddCMP, (-)-OddCDP, and (-)-OddCTP occurred in a time- and concentration-dependent manner, with (-)-OddCDP being the major metabolite. Elimination of (-)-OddCTP was biphasic, with an initial t1/2 of 3.5 h and a second phase t1/2 of > 20 h. The incorporation of (-)-OddCTP into DNA was concentration dependent, and toxicity was directly correlated with the amount of (-)-OddCMP present in the DNA. Treatment with (-)-OddC led to the degradation of DNA into large fragments at high concentrations, but internucleosomal laddering was not observed. The rapid membrane permeation of (-)-OddC and prolonged retention of its metabolites may contribute to the potent activity of this compound against DU-145 xenografts.

Tyr(O)CNP is an analogue of C-type natriuretic peptide (CNP) with a tyrosine residue added to the NH2 terminus to allow its iodination. In the present study, the suitability of iodinated Tyr(O)CNP as a ligand was tested, and its potency was compared with that of other natural rat natriuretic peptides or structural analogues by radioligand binding experiments. Binding studies were performed on membranes of COS-1 cells transfected with expression plasmids for either rat natriuretic peptide receptor (NPR)-A, rat NPR-B, or bovine NPR-C. 125I-ANP(99-126) was used as a ligand to assess the binding characteristics of NPR-A and -C, and 125I-Tyr(O)CNP was used to study NPR-B. Binding associated to membranes of nontransfected COS cells was always < 3% of the total binding observed in membranes from cells transfected with receptor expression plasmids. Receptor densities in transfected cells ranged from 500 to 2500 fmol/mg of protein. High performance liquid chromatography and ionspray mass spectrometry analyses revealed that the reagents used in the course of iodination (lactoperoxidase, chloramine T, or N-chloromorpholine altered the structure of Tyr(O)CNP, most likely by changing the thiol of the Met17 residue into a sulfoxide. To further evaluate the usefulness of forms of iodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability iodinated forms of Tyr(O)CNP as radioligands, we performed iodination of the peptide with cold iodine (Na-127I-). After purification by high performance liquid chromatography, three different modified peptides (i.e. Tyr(O)Met(O)17CNP, 127I-Tyr(O)Met(O)17CNP, and 127I2-Tyr(O)Met(O)17CNP) were recovered, and they were compared with CNP-22, Tyr(O)CNP, ANP(99-126), BNP-32, and des[Gin18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) NH2 (c-ANP) for their ability to bind to transfected receptors. The binding affinity of Tyr(O)CNP for NPR-A and -B receptors is similar to that of CNP. However, oxidation of the Met17 residue into methionine sulfoxide reduces the affinity of the compound for NPR-B by > 10-fold, whereas the addition of one or two iodines did not further reduce its affinity. Similar results were obtained on evaluation of the ability of the oxidized form of monoiodinated Tyr(O)CNP on the cGMP responses in cells transfected with NPR-B. In conclusion, the suitability of iodinated forms of Tyr(O)CNP as radioligands for binding studies on rat NPR-B is not optimal, and the results of studies using such compounds for the detection, identification, and quantification of this receptor should be interpreted with caution.

Pregnant women are advised against using angiotensin-converting enzyme (ACE) inhibitors due to reports of adverse effects on human fetuses. This study examined ACE binding and angiotensin II (Ang II) receptor binding in fetuses of rats treated with the ACE inhibitor fosinopril (16 mg/kg/day fosinopril, p.o. in 4 divided doses, from gestational day (gd) 13 to gd 18). Binding of the potent radiolabeled ACE inhibitor 125I-351A to ACE in the lung and aorta of gd 19 fetuses of fosinopril-treated dams was reduced by 56 and 44%, respectively, compared to fetuses from vehicle-treated dams, indicating that fosinopril or its active metabolite, fosinoprilat, crosses the placental barrier and inhibits fetal ACE. Fetal Ang II receptor binding of 125I-Sar1,Ile8 Ang II was not altered in most of the tissues examined, although reductions in binding in the adrenal of fetuses of fosinopril-treated dams approached statistical significance.

Naturally occurring nucleosides and all anticancer nucleoside analogue drugs are in the beta-D configuration. L-(-)-dioxolane-cytidine [(-)-OddC] is the first L-nucleoside analogue ever shown to have anticancer activity. This compound was converted within cells to its mono-, di-, and triphosphate metabolites and was incorporated into DNA. As with cytosine arabinoside, conversion to the monophosphate was catalyzed by cellular deoxycytidine kinase, which was essential for cytotoxicity. However, unlike cytosine arabinoside, (-)-OddC was not susceptible to degradation by deoxycytidine deaminase. Because (-)-OddC inhibited the growth of hepatocellular and prostate tumors that are generally difficult to treat, it is a promising candidate for additional testing. Our results indicate that there is a great deal of variability in the chiral specificities of cellular enzymes and demonstrate how these differences can be exploited in the design of better anti-viral and anticancer drugs.

Natriuretic peptides have been demonstrated to induce a variety of effects when administered into the brain. Most studies to date have tested the effects of 'atrial' natriuretic peptide (ANP), but C-type natriuretic peptide (CNP) has recently been suggested to be the predominant form of natriuretic peptides within the brain. We therefore have compared the amplitudes of the cyclic guanosine monophosphate (cGMP) responses induced by either ANP or CNP in slices form different rat brain regions. Whereas both peptides induced the generation of cGMP, CNP-evoked responses were never greater than those obtained with ANP, regardless of the brain region used or the age of the animal. In diencephalon, ANP even induced a significantly higher cGMP response than CNP. To test which cells were targets to the actions of the peptides, brain slices were incubated with fluorocitrate (a drug that selectively blocks the metabolism of glial cells). Fluorocitrate totally blocked the ANP-evoked cGMP responses in brain slices. In contrast, fluorocitrate reduced only partially the responses evoked by sodium nitroprusside (a drug that stimulates soluble guanylate cyclase, which is contained predominantly in neurons). Likewise, the cGMP response induced by CNP was only partially affected by fluorocitrate. These results indicate that: (1) CNP is not more potent than ANP in terms of its ability to generate cGMP in rat brains; (2) brain cells generating cGMP upon exposure to ANP are predominantly glial; and (3) CNP-responsive cells are partly glial, but belong at least in part to a different compartment than ANP-responsive cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of angiotensin II (AII) receptor binding often necessitates freezing of the tissue of interest prior to assay of radioligand binding. This study evaluated the effects of freezing of various rat tissues at different rates on 125I-sarcosine1, isoleucine8 angiotensin II (125I-SI AII) binding to AII receptor subtypes. Slow freezing in a -20 degrees C compartment significantly reduced 125I-SI AII binding to AT1 receptors in the adrenal (51%), epididymis (34%), and liver (22%). Binding of 12tI-SI AII to the AT1 receptor in the brain was not significantly reduced. In the adrenal, both the Bmax and affinity of AT1 receptors were decreased by freezing. But in the epididymis, only the affinity of AT1 receptors was decreased. Binding of 125I-SI AII to AT2 receptors in the adrenal, epididymis, and brain was also not significantly reduced by freezing. Further evaluation of the mechanism of the reduction in 125I-SI AII binding to AT1 receptors in the adrenal indicated that both the receptor density and affinity for 125I-SI AII were decreased by freezing. Rapid freezing in a dry-ice bath caused even greater reductions in 125I-SI AII binding to AT1 receptors in the adrenal. Snap freezing in liquid nitrogen decreased 125I-SI AII binding in adrenals to a similar extent as did slow freezing. These results suggest that studies of AII receptors subtypes that involve freezing of the tissues underestimate the density and affinity of the AT1 receptor subtype.

Adrenal angiotensin II (AII) receptors have been pharmacologically and structurally divided into two main subtypes, AT(1) and AT(2). Radioligand receptor binding assays with(125)I-sarcosine(1), isoleucine(8) angiotensin II ((125)I-SI AII) in the presence of losartan, an AT(1) selective ligand, and PD123177 an AT(2) selective ligand, indicated that the AT(1) subtype was predominant in membrane homogenates of the rat adrenal cortex (AT(1) Bmax=649 ± 62 fmol/mg protein; AT(2) Bmax=237 ± 29 fmol/mg protein). In membrane homogenates of the adrenal medulla, the AT(2) subtype was predominant (AT(1) Bmax=55 ± 5 fmol/mg protein; AT(2) Bmax=109 ± 29 fmol/mg protein). Overall 58% of the(125)I-SI AII binding in the rat adrenal was to the AT(1) subtypes, and 42% was to the AT(2) subtypes. The outer cortex contained 59% of the AH receptor binding sites in the adrenal, while the medulla accounted for the remaining 41%. The affinity of the AT(1) binding sites in membrane homogenates of the cortex and medulla (K( D )=672 ± 123 pM and 573 ± 85 pM, respectively) was not significantly different. The affinity for(125)I-SH AII of AT(2) binding sites in membrane homogenates was higher than that of AT, binding sites. The affinity for(125)I-SI All of AT(2) binding sites in membrane homogenates of the outer cortex (K( D )=265 ± 35 pM) was significantly less than that in the medulla (K( D )=133 ± 11 pM).In vitro receptor autoradiography also demonstrated that the AT(2) subtype in frozen sections of the cortex had a lower affinity (K( D )=1512 ± 191 pM) than that in the medulla (K( D )=867 ± 72 pM). The heterogeneous affinity of adrenal AT(2) binding sites may indicate existence of multiple AT(2) receptor subtypes in the rat adrenal.

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.

By virtue of the more than 1000-fold selectivity of losartan (DuP 753) for the AT1 angiotensin II (AII) receptor subtype compared with the AT2 subtype, [3H]losartan may be a useful radioligand for studies of the AT1 receptor subtype. Comparison of Bmax values in the liver obtained from saturation isotherms using [3H]losartan (Bmax = 194 pmol/g tissue) and [125I]sarcosine1,isoleucine8 angiotensin II (Bmax = 20 pmol/g tissue) indicated that the AII receptor concentration was approximately 10% that of the [3H]losartan binding sites. In addition, AII at concentrations as high as 10 microM displaced less than one-third of specific [3H]losartan binding in the liver and less than 80% in the whole adrenal. The presence of non-AII displaceable [3H]losartan binding in the liver did not appear to result from metabolism of the radioligand since HPLC analysis of free and bound 3H revealed that greater than 90% of the 3H eluted at the same time as the parent [3H]losartan. This suggests that [3H]losartan binds with high affinity to a site(s) other than angiotensin II receptors in the rat liver.

Our laboratory has reported previously that a unique binding site specific for the hexapeptide angiotensin (A)II(3-8), now referred to as AIV, is present in a number of tissues including bovine adrenal gland, rabbit and guinea pig heart and guinea pig kidney, liver, lung, uterus and brain. The present results extend previous findings in the guinea pig brain and identify binding sites for AIV in the neocortex, paleocortex, hippocampus, medial habenula, superior and inferior colliculi, caudate putamen, thalamus, dorsal tegmentum, central gray, red nucleus, inferior olivary, oculomotor and hypoglossal nuclei and cerebellum. Binding of [125I]AIV in selected regions was shown to be of high affinity (Kd = 0.60-1.47 nM), saturable (maximal number of binding sites = 181-449 fmol/mg of protein) and specific. This binding site was shown to be distinct from the AT1 and AT2 sites with Ki values > 10(-4) M for DuP 753, CGP42112A and PD123177. Changes at the N-terminal of the peptide, either by removal of the valine or by extension of the peptide, resulted in a large decrease in binding affinity. In contrast, C-terminal extensions resulted in little change in affinity for the binding site. Guanosine 5'-0-(3-thiotriphosphate) was shown to have no effect on binding, suggesting that the guinea pig brain binding site is not G-protein-linked. Potential functions associated with this newly discovered A binding site are discussed.

A growing body of evidence suggests that angiotensin may have a functional role in growth and development, in addition to its classical role in the maintenance of body water homeostasis. Components of the renin-angiotensin system have been identified in the rat fetus. Because of the association between the renin-angiotensin system and hypertension, we quantified angiotensin receptor binding sites in the brains of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats during perinatal development. Using in vitro receptor autoradiography we identified specific 125I-Sar1,Ile8 AII binding in several areas of the brains of perinatal rats of both strains and observed significant differences in the concentration of binding sites, at different ages in several brain nuclei. With the knowledge that components of the renin-angiotensin system appear early in development and are known to have an association with cellular growth, it is possible that an irregularity in this system occurring during neurogenesis could contribute to developmental abnormalities, as well as subsequent hypertension.

Brain angiotensin II (AII) receptors play a role in the regulation of luteinizing hormone release. This action is thought to involve luteinizing hormone-releasing hormone containing neurons in the preoptic-anterior hypothalamic (POAH) area of the brain. Previous studies have demonstrated a discrete locus of AII receptor binding sites and responsiveness within the POAH to microinjections of AII in the adult female rat, corresponding to the ventral portion of the bed nucleus of the stria terminalis (BSTV). To further characterize the age- and sex-dependent AII receptor binding in the BSTV and other brain regions, in vitro receptor autoradiography using 125I-sarcosine1, isoleucine8 AII (125I-SI AII) was performed on 2-, 4- and 10-week-old female and male rat brains. Rats of both sexes displayed an age-dependent increase in 125I-SI AII binding in the BSTV, as well as in the suprachiasmatic nucleus (SCh). There was no detectable difference in binding within the BSTV between the female and male of any age group. Prepubertal ovariectomy did not impair the expression of 125I-SI AII binding in the BSTV or the SCh of adult female rats. The developmental expression of AII receptors in the BSTV and SCh may therefore play a role in sexual maturation and regulation of sexual function in the rat.

The angiotensin II receptor subtype-specific antagonists Dup 753 (AT1) and PD 123177 (AT2) were used to characterize the angiotensin II receptor subtypes present in 18 day gestation fetal Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rat brain using in vitro receptor autoradiography. The AT2 subtype was predominant in the brain of both rat strains, even in areas that display predominantly the AT1 subtype in the adult rat brain.

Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1,isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1,isoleucine8 angiotensin II binding was strongly inhibited by 30 mM beta-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AII alpha) 125I-sarcosine1,isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AII beta) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AII alpha) receptor subtype.

In vitro receptor autoradiography, using the radiolabeled angiotensin II (Ang II) antagonist 125I-sar1,ile8 Ang II (125I-SI Ang II; 250 pM) in the absence or presence of 1 microM Ang II, was used to identify Ang II receptor binding sites in the preoptic-anterior hypothalamic (POAH) brain region of cycling female rats. A nucleus within this region, lateral to the organum vasculosum of the lamina terminalis and ventral to the anterior commissure, displayed a discrete locus of 125I-SI Ang II binding sites (385 fmol/g tissue). This nucleus, which corresponds to the area of the POAH from which Ang II is most effective at eliciting luteinizing hormone release, has been identified as the ventral portion of the bed nucleus of the stria terminalis (BSTV) by the rat brain atlas of Paxinos and Watson. The selective nonpeptidic Ang II alpha receptor antagonist Dup 753 completely inhibited the binding of 125I-SI Ang II to the BSTV and other hypothalamic nuclei, suggesting that these receptors are of the Ang II alpha subtype.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AII alpha subtype) or PD123177 (designated AII beta subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AII alpha and AII beta subtypes.

Rats were tested for dipsogenic and pressor responses to intracerebroventricularly (icv) administered Ang II and for dipsogenic responses to icv administered carbachol in the absence and presence of pertussis toxin, also administered icv. Pertussis toxin did not inhibit the pressor or dipsogenic responses to Ang II, but did inhibit the dipsogenic responses to carbachol. This suggests that the pressor and dipsogenic responses to Ang II in the brain are not mediated by a pertussis toxin-sensitive G protein, but that the muscarinic cholinergic dipsogenic response is mediated by a pertussis toxin-sensitive G protein.

The locus coeruleus (LC) is a putative site of action for angiotensin II in the brain. Immunocytochemical studies have identified angiotensin II-like immunoreactive material in nerve terminals innervating the LC, and the LC contains one of the highest densities of angiotensin II receptor binding sites in the rat brain. Recent studies using selective neurotoxins suggest that the binding sites for angiotensin II in the LC are present on noradrenergic perikarya. Angiotensin II receptors are now known to exist as two subtypes that are distinguishable both pharmacologically and biochemically. Radioligand binding studies using agonists and antagonists selective for these angiotensin II receptor subtypes indicate that the rat LC contains a mixture of the two known angiotensin II receptor subtypes, but that the PD123177-sensitive AII beta receptor subtype is predominant. Comparisons of spontaneously hypertensive rats with normotensive rats indicates that angiotensin II and its receptors in the LC are elevated in the hypertensive rat strain. Studies of the biochemical and physiological actions of angiotensin II in the LC have not yet established an agreed-upon function for angiotensin II in this nucleus.

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1,Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 greater than 10(-4) M). DuP 753 sensitive binding sites (designated AII alpha subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AII beta) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.

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.