Ontogenic expression of natriuretic peptide mRNAs in postnatal rat brain: implications for development?

The central natriuretic peptide system is composed of at least three structurally homologous and uniquely distributed peptides and receptors which are thought to be involved in the central regulation of cardiovascular and autonomic function and more recently been shown to affect cellular growth and proliferation, processes pertinent to mammalian development. As such, following our initial mapping of preproatrial natriuretic peptide (ppANP) mRNA in adult brain [M.C. Ryan, A.L. Gundlach, Anatomical localization of preproatrial natriuretic peptide mRNA in the rat brain by in situ hybridization histochemistry: in olfactory regions, J. Comp. Neurol., 356 (1995) 168-182], it was of interest to determine the ontogenic expression of natriuretic peptide mRNAs in the developing rat brain. Using in situ hybridization histochemistry of specific [35S]- or [33P]-labeled oligonucleotides, ppANP and preproC-type natriuretic peptide (ppCNP) mRNAs were detected in the developing rat brain from postnatal day 4 to day 60 (adult). PpANP mRNA was observed in many hindbrain, but only some forebrain, regions at postnatal day 4. Regional differences in the temporal expression of ppANP mRNA were apparent with ppANP mRNA detected in the medial preoptic area, mammillary nuclei and medial habenular nucleus at postnatal day 4 and in other areas including the arcuate and dorsomedial hypothalamic nuclei and in olfactory and limbic regions at postnatal day 10. A number of regions also exhibited transient expression of ppANP mRNA such as the bed nucleus of the stria terminalis and the medial cerebellar nucleus. In contrast, ppCNP mRNA was detected at relatively high levels in several regions on postnatal day 4 including olfactory nuclei, the hippocampus and particularly the pontine nucleus. The level of expression appeared to increase markedly in most regions including forebrain olfactory and hippocampal areas and in brainstem regions including the pontine nucleus, the parvocellular and lateral reticular and spinal trigeminal nuclei by postnatal days 10 and 13, but decreased from this peak to equivalent to adult levels by postnatal day 28. The differential and transient expression of the natriuretic peptides during postnatal development, together with previous reports of the ontogenic regulation of natriuretic peptide receptor expression and binding patterns, further suggests their involvement in developmental processes in the rat CNS and provides information relevant to the likely functional development of natriuretic peptide-utilizing pathways.     

Localization of C-type natriuretic peptide mRNA in rat hypothalamus

Central or peripheral administration of C-type natriuretic peptide (CNP) affects numerous neuroendocrine systems, including the hypothalamo-pituitary-gonadal, hypothalamo-pituitary-adrenocortical and hypothalamo-neurohypophysial axes. The present report characterizes the distribution of CNP mRNA in hypothalamus, providing the first definition of CNP-containing neuroendocrine circuits. In situ hybridization histochemical analysis revealed high expression of CNP mRNA in the anteroventral periventricular nucleus (AVPv) and in hypothalamic arcuate nucleus (ARC). Hybridization signals of significantly lower intensity were seen in the medial, median and periventricular preoptic area, the supraoptic, dorsomedial, ventral premammillary and lateral mammillary nuclei and in the posterior hypothalamic area. A few scattered CNP mRNA containing cells were visualized in the medial parvocellular paraventricular nucleus, posterior magnocellular paraventricular nucleus and lateral hypothalamic area. In the AVPv and ARC the pattern of CNP mRNA distribution paralleled that of ANP mRNA. The results indicate a distribution of CNP mRNA associated with key neuroendocrine systems, and underscores the potential importance of this novel natriuretic peptide in neuroendocrine regulation.     

Atrial natriuretic peptide and bradykinin interaction on norepinephrine uptake in rat adrenal medulla

Interactions between atrial natriuretic peptide (ANP) and bradykinin (BDK) on norepinephrine (NE) uptake were demonstrated in the adrenal medulla of the rat. One hundred nM ANP and 10 nM BDK increased total NE uptake. Subthreshold concentration of ANP (1nM) or BDK (1nM) reverted the effects of thre shold concentrations of both peptides (10 nM BDK and 100 nM ANP respectively). Effective concentrations of ANP and BDK acting simultaneously did not induce additive effects on total NE uptake. Threshold concentrations of both peptides increased neuronal NE uptake only. These results suggest interactions between ANP and BDK at the neuronal uptake level. They confirm that ANP and BDK take part in the regulation of the sympathetic activity in the adrenal medulla of the rat.     

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.     

Cocaine- and amphetamine-regulated transcript peptide immunohistochemical localization in the rat brain

The clinical features of Crohn's disease manifest during adolescence are varied as in adults. The potential complication of growth impairment and concomitant delay in pubertal development is unique to this population. Cytokines released from the inflamed bowel and chronic nutritional insufficiency are the major factors in the pathophysiology of growth inhibition. Hence reduction of intestinal inflammation and consistent provision of adequate nutrition are of paramount importance in management. Drug treatment mirrors that of adults; few specifically paediatric clinical trials have been conducted. Enteral nutrition is an important therapeutic alternative for young patients. There is evidence that it constitutes both a primary therapy of inflammation and a means of providing the calories needed for growth. In the setting of extensive disease, dependency on corticosteroids should be minimized through judicious administration of immunosuppressive drugs. For an adolescent with localized stenotic disease, optimal management includes a timely referral for intestinal resection as a means of providing an asymptomatic interval during which growth and pubertal development can normalize.     

Urocortin, a newly identified corticotropin-releasing factor-related mammalian peptide, stimulates atrial natriuretic peptide and brain natriuretic peptide secretions from neonatal rat cardiomyocytes

Vanadate stimulates adipocyte 2-deoxyglucose transport and GLUT-4 translocation to the membrane through an insulin receptor-independent but wortmannin-inhibitable pathway. Vanadate stimulates PI 3-kinase in anti-IRS-1 immunoprecipitates and the binding between IRS-1 and the p85alpha subunit of PI 3-kinase. In insulin-resistant adipocytes from old rats vanadate fully stimulates IRS-1-associated PI 3-kinase, but partially activates glucose uptake. We conclude that: (a) vanadate stimulates 2-deoxyglucose uptake using a pathway that converges with that of insulin at the level of PI 3-kinase; and (b) adipocytes from old rats are defective in the insulin pathway at steps located both upstream and downstream of PI 3-kinase.     

Effect of adrenal steroids on preproneurokinin-A gene expression in discrete regions of the rat brain

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.     

Streptozotocin-induced diabetes has differential effects on atrial natriuretic peptide synthesis in the rat atrium and ventricle: a study by solution-hybridization-RNase protection assay

In rats with streptozotocin (STZ)-diabetes for 2 or 4 weeks, the atrial natriuretic peptide (ANP) concentrations in the atria decreased whilst that of ANP in the plasma and ventricles increased. ANP concentrations in the hypothalamus and in the brainstem did not change in either 2- or 4-week diabetic rats. Atrial ANP content was partly restored by insulin replacement in 4-week diabetic rats. Plasma ANP concentrations and ventricular ANP contents were reversed to normal by insulin treatment in both 2- and 4-week diabetic rats. Solution-hybridization-RNase protection assay showed a significant increase in the preproANP mRNA expression in the ventricles but not in the atria. These results indicated that the STZ-diabetes increased the synthesis of ANP in the ventricles and consequently its release from the ventricles. The synthesis of ANP in the atria did not change as judged from the preproANP mRNA expression but the release of ANP from the atria might also be increased for ANP content decreased in the atria. The reason for the difference in the response of atrial and ventricular preproANP concentrations to STZ-diabetes is not known.     

Plasma brain natriuretic peptide in assessment of acute dyspnoea

Recognition of heart failure (HF) may be difficult in patients presenting with acute dyspnoea, particularly in the presence of chronic airways obstruction. Since increased secretion of the cardiac hormones atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) occurs early in the course of HF, we have assessed the value of measuring these hormones in plasma in the diagnosis of suspected HF in 52 elderly patients presenting with acute dyspnoea, and compared values with left-ventricular ejection fraction (LVEF), a standard measure of left-ventricular function, by radionuclide angiography. Patients were enrolled prospectively. On the basis of clinical findings, conventional tests, and response to specific treatment, 20 of the 52 patients were classified as having primary lung disorder (PLD), 12 as HF alone, and 20 as HF with underlying PLD (HF/PLD). Compared with findings in PLD patients, LVEF was significantly depressed in HF and HF/PLD patients (p < 0.001), whereas both plasma ANP and BNP were significantly increased (p < 0.001). Admission plasma BNP concentration more accurately reflected the final diagnosis of HF (93% sensitivity and 90% specificity when BNP > or = 22 pmol/L) than LVEF or plasma ANP concentration. When all patients were considered together, there were strong negative correlations between LVEF and log BNP (r = -0.7, p < 0.001) and log ANP (r = -0.59, p < 0.001). Our finding that plasma BNP is raised in dyspnoeic patients with HF but not in acutely breathless patients with PLD, suggests that rapid BNP assays may assist in the diagnosis of patients with acute dyspnoea.     

Cyclic guanosine monophosphate responses to atrial natriuretic factor, brain natriuretic peptide, but not C-type natriuretic peptide, and the characterization of their receptors in rat medullary thick ascending limb

The effects of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on renal medullary thick ascending limb (mTAL) have not been fully understood. The aim of this study is to examine the second-messenger responses of rat mTAL to ANF, BNP, and CNP. Characterizations of the ANF, BNP, and CNP receptors in mTAL were also performed by radioligand studies. Results showed that ANF and BNP were both capable of eliciting cyclic guanosine monophosphate (cGMP) responses in mTAL. Conversely, no cGMP response was observed upon stimulation by CNP in mTAL. The presence of ANF receptors was demonstrated by radioligand studies. One receptor site was found, and the Kd and maximum binding capacity were 4.0 +/- 0.45 nmol/L and 277.8 +/- 47.7 fmol/mg protein, respectively. BNP receptors were also found in mTAL, and ANF and BNP were sharing the same receptor. On the contrary, no CNP receptor could be shown by radioligand studies. These results suggest that guanylyl cyclase-coupled receptors (atrial natriuretic peptide receptor-A [ANPR-A]) specific for ANF and BNP are present in rat mTAL, while those for CNP (ANPR-B) are absent. ANF and BNP but not CNP act on mTAL to control water excretion.     

Differential expression and autoradiographic localization of atrial natriuretic peptide receptor in spontaneously hypertensive and normotensive rat testes: diminution of testosterone in hypertension

Previous studies have shown that the diuretic hormone atrial natriuretic peptide (ANP) also regulates the steroidogenic responsiveness in isolated Leydig cells from mouse and rat testes. In the present study, we examined the distribution of specific receptors for ANP and C-type natriuretic peptide (CNP) in the testicular compartments of 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). We used an in vitro autoradiographic procedure on slide-mounted frozen testicular sections to localize the receptors of the natriuretic peptide hormone family using 125I-ANP and 125I-CNP as radioligands. A high level of specific 125I-ANP binding sites was localized largely in the Leydig cells of the interstitial compartment; other testicular cells were not significantly labeled. On the other hand, no significant difference was observed in 125I-CNP binding sites in the testicular cells of SHR and WKY. Semiquantitative analysis of the binding sites indicated that the density of 125I-ANP receptor binding in Leydig cells of WKY testis was ninefold higher than in those of SHR testis. A moderate level of 125I-ANP binding was also observed in seminiferous tubules, particularly in the spermatids of both SHR and WKY. 125I-ANP binding in WKY spermatids was approximately 2.5-fold higher than in SHR spermatids. Northern blot analysis showed that mRNA specific for guanylyl cyclase type A (Npra) was expressed at approximately twofold higher levels in WKY than in SHR testis. ANP (1 x 10(-8) mol/L) stimulated fourfold to fivefold increased levels of testosterone production in isolated Leydig cells from normotensive WKY compared with those from SHR. These findings support a new physiological role of ANP in Leydig cells, in which a functional relationship seems to exist between testicular ANP receptor expression and testosterone production and the state of hypertension in SHR.     

Expression and distribution of brain natriuretic peptide in human right atria

OBJECTIVES: We investigated expression of brain natriuretic peptide (BNP) as well as atrial natriuretic peptide (ANP) and their genes in human right atria. Their relations with atrial pressure were also examined. BACKGROUND: The BNP plays a roll in electrolyte-fluid homeostasis such as ANP. The tissue level is reported to be elevated in the failing ventricles. However, expression and transmural distribution of BNP in the atria remain unclear. METHODS: Expression of ANP and BNP was immunohistochemically investigated in the right atrial (RA) specimens from 21 patients who had undergone cardiac surgery. The mRNA of specimens were quantitatively measured by Northern blot analysis and also evaluated by in situ hybridization. In addition, plasma levels of ANP and BNP were measured in the patients. RESULTS: The BNP immunoreactivity was diffusely seen in RA tissue of patients with mean RA pressure (mRAP) of 5 mm Hg or more, but it was noted only in the subendocardial half of the atria of those with mRAP less than 5 mm Hg. There was a significant correlation between the incidence of BNP-positive myocytes and mRAP (r = 0.850, p < 0.0001). Conversely, ANP-positive myocytes were found diffusely in all cases. In Northern blot analysis, the mRNAs levels of ANP and BNP in the atrial tissue were positively correlated with the mRAP (ANP, p = 0.775, p < 0.005 and BNP, p = 0.771, p < 0.005). In situ hybridization confirmed these findings. The mRNA levels were significantly correlated to each other (r = 0.845, p < 0.0002). Plasma ANP and BNP levels were elevated in the patients compared with that in controls; however, none were significantly correlated with the mRAP. CONCLUSIONS: Expression of BNP and BNP mRNA is augmented in the atria with increased pressure, and distributed predominantly in the subendocardial side. The level of BNP mRNA was well correlated with that of ANP mRNA. Thus, these two genes might be commonly regulated in response to atrial pressure.     

Atrial natriuretic peptide metabolism during pregnancy in the rat

OBJECTIVE: Our purpose was to determine whether plasma clearance rates and production rates of atrial natriuretic peptide 99-126 are altered during pregnancy in the rat. STUDY DESIGN: Twelve virgin and 12 late-pregnant chronically instrumented, conscious, unrestrained Sprague-Dawley rats were studied. Mean arterial pressure, heart rate, and plasma atrial natriuretic peptide levels were measured before and during a 40-minute continuous infusion of atrial natriuretic peptide (10 ng/kg/min). RESULTS: Control mean arterial pressure was 106 +/- 5 mm Hg in virgin rats versus 97 +/- 4 mm Hg in pregnant rats. Atrial natriuretic peptide infusion did not significantly affect mean arterial pressure in either group of animals but decreased heart rate in virgin rats. Basal plasma atrial natriuretic peptide levels were significantly higher in virgin than in pregnant rats (107 +/- 10 vs 78 +/- 7 pg/ml, respectively, p < 0.05). Atrial natriuretic peptide infusion significantly increased plasma levels in both groups to similar (183 +/- 19 and 154 +/- 14 pg/ml, virgin vs pregnant rats). Calculated plasma clearance rates were similar in virgin and pregnant rats (166 +/- 27 vs 155 +/- 17 ml/kg/min). Estimated production rates of atrial natriuretic peptide were higher in virgin then in pregnant rats (15.1 +/- 1.4 vs 11.4 +/- 1.1 ng/kg/min, p < 0.05). CONCLUSIONS: Plasma atrial natriuretic peptide levels are lower in chronically instrumented near-term pregnant rats compared with levels in virgin rats. This is not related to differences in plasma atrial natriuretic peptide clearance rates but rather to a decrease in production rates in late pregnancy.     

Cellular orientation of atrial natriuretic peptide in the human brain

Many peptide hormones and neurotransmitters have been detected in human neuronal tissue. The localisation of atrial natriuretic peptide (ANP) in the human brain was considered to be both interesting and relevant to the understanding of neurochemistry and brain water-electrolyte homeostasis. This vasoactive peptide hormone has been localised in rat and frog neuronal tissue. In the present study, we report the immunohistochemical localisation of ANP in autopsy samples of human brain tissue employing the avidin-biotin-peroxidase complex technique, using an antibody against a 28 amino acid fragment of human ANP. The most intense staining of immunoreactive ANP was detected in the neurones of preoptic, supraoptic and paraventricular nuclei of the hypothalamus, epithelial cells of the choroid plexus and ventricular ependymal lining cells. Immunoreactive neurones were also observed in the median eminence, lamina terminalis, infundibular and ventromedial nuclei of the hypothalamus, and in neurones of the brain stem, thalamic neurones and some neurones of the caudate nucleus. The network of ANP cells in numerous hypothalamic centres may regulate the salt and water balance in the body through a hypothalamic neuro-endocrine control system. ANP in the brain may also modulate cerebral fluid homeostasis by autocrine and paracrine mechanisms.     

Different secretion patterns of adrenomedullin, brain natriuretic peptide, and atrial natriuretic peptide during exercise in hypertensive and normotensive subjects

The purpose of this study was to investigate the effect of exercise on plasma concentrations of adrenomedullin, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) in patients with essential hypertension (n = 15) and in normotensive controls (n = 10). Exercise consisted of two fixed workloads, 40 and 80 watts of work load using a supine bicycle ergometer. Plasma levels of all three peptides at rest were significantly higher in hypertensives than in controls. Plasma concentrations of ANP increased with exercise in both groups and had greater increments in hypertensive patients than in normotensives. Plasma concentrations of BNP increased only in patients with hypertension and the levels of increase correlated with basal plasma BNP levels (r = 0.94, p < 0.001) and with left ventricular mass (r = 0.62, p < 0.01) determined by echocardiography. In contrast, plasma adrenomedullin did not change with exercise in either group. These results suggest that secretion patterns of these peptides are regulated by different mechanisms and that the amount and kind of peptides mobilized by exercise may depend on the underlying diseases or pathophysiologic condition.     

Median eminence-afferent vasoactive intestinal peptide (VIP) neurons in the hypothalamus: localization by simultaneous tract tracing and immunocytochemistry

Retrograde tract tracing and immunocytochemistry were used to investigate the CNS source of the VIP that is present in high concentrations in the hypophysial portal blood and has been shown to have a stimulatory effect on pituitary prolactin secretion. Fluoro-gold (FG), which enters the CNS through areas devoid of the blood-brain barrier, such as median eminence, was injected peripherally. Brain sections from FG-treated animals were immunostained for VIP. A small population of VIP-containing cell bodies in the parvocellular and periventricular parts of the paraventricular nucleus (PVN) was also labeled with FG. Vasoactive intestinal peptide-immunoreactive perikarya not labeled with FG were also observed in the PVN, as well as FG-labeled cells that did not contain VIP. The results suggest that some VIP-producing neurons in the PVN project to the median eminence and are, therefore, functionally related to pituitary regulation; the function of other VIP neurons in the PVN is unknown.     

Low density lipoprotein receptors in rat adipose cells: subcellular localization and regulation by insulin

The distribution of LDL receptors within subcellular compartments of isolated rat adipose cells and the effects of insulin on their expression have been assessed. By immunoblotting with specific anti-rat LDL receptor antibodies, LDL receptors were 2.3- and 4.5-fold enriched in endoplasmic reticulum-rich high-density microsomes (HDM) and Golgi complex-rich low-density microsomes (LDM), respectively, compared to plasma membranes (PM). This distribution was similar in cultured cells in which total receptors were increased 2.5-fold compared to freshly isolated cells. After correction for enzyme recoveries, LDL receptors were distributed approximately 4% in HDM, approximately 73% in LDM, and approximately 23% in PM. Insulin decreased total LDL receptors in adipose cells approximately 44%, with a 48% and 49% decrease in HDM and LDM, respectively, without any changes in PM. In contrast, insulin caused an increase of glucose transporters in PM while also decreasing glucose transporters in LDM. When adipose cells were depleted of potassium to inhibit receptor-mediated endocytosis, insulin again caused a decrease of LDL receptors in LDM but now increased LDL receptors in PM. Insulin increased the rate of LDL receptor synthesis approximately 24%, but decreased their half life approximately 40%. Thus, in isolated adipose cells the majority of LDL receptors appear to be located in an intracellular compartment that co-sediments with the Golgi complex rather than located in the PM. The LDL receptors localized in intracellular compartments seem to be functionally regulated as insulin acutely diminishes the number of receptors by apparently accelerating their rate of degradation through, as yet, incompletely determined mechanisms.