Immunoreactive Atrial Natriuretic Factor Research Articles

Influence of doxazosin on biosynthesis of S100A6 and atrial natriuretic factor peptides in the heart of spontaneously hypertensive rats.

Hypertension frequently results in severe complications in cardiovascular system and histopathological changes in the heart. To better understand the cellular processes and signaling pathways responsible for the proper functioning of the heart, we decided to check whether doxazosin affects the density of structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats. The aim of this study is to find differences in the density of the structures containing S100A6 and atrial natriuretic factor in the heart of spontaneously hypertensive rats treated with doxazosin compared to untreated animals. Fragments of heart were collected from five spontaneously hypertensive rats and five spontaneously hypertensive rats receiving doxazosin for six weeks (dose 0.1 mg per 1 kg of body weight). On the paraffin sections S100A6 and atrial natriuretic factor peptides were localized in the heart using immunohistochemistry. Positive immunohistochemical reaction for S100A6 was observed in atrial and ventricular cardiomyocytes and in the coronary vasculature. In the heart of hypertensive rats treated with doxazosin the S100A6 immunoreactivity was significantly lower compared to untreated animals. Immunodetection of atrial natriuretic factor in the heart of rats confirmed presence of peptide in atrial myocardium. Delicate atrial natriuretic factor-immunoreactivity was observed also in few ventricular cardiomyocytes. The atrial natriuretic factor-immunosignal was significantly weaker in hearts of hypertensive rats receiving doxazosin compared to spontaneously hypertensive rats untreated. Since we found that doxazosin reduces the levels of S100A6 and atrial natriuretic factor peptides in the heart of spontaneously hypertensive rats, it can be assumed that cardiovascular disorders that occur in hypertension may be associated with disturbances of cellular processes and signaling pathways.

Atrial natriuretic factor: localization in the adrenal gland of the lizard Podarcis sicula and effects on pituitary–adrenal axis activity

The occurrence of atrial natriuretic factor (ANF) immunoreactivity was investigated in the adrenal gland of the lizard Podarcis sicula by avidin-biotinylated peroxidase complex (ABC) immunocytochemical technique: ANF immunoreactivity was present in the chromaffin tissue, and was absent in the steroidogenic tissue. The role of ANF in the modulation of the pituitary–adrenal axis activity was investigated in vivo by intraperitoneal administration of ANF. The effects were evaluated by examination of the morphological and morphometrical features of the tissues, as well as the plasma levels of adrenocorticotropic hormone (ACTH), corticosterone, aldosterone, norepinephrine, and epinephrine. ANF (28 μg/100 g body wt) did not affect ACTH plasma levels, that remained almost unchanged; in contrast, corticosterone plasma levels increased from 6.45 ± 0.070 ng/ml in carrier-injected lizards to 9.69 ± 0.080 ng/ml 24 h after the injection; aldosterone levels decreased from 2.19 ± 0.010 ng/ml in carrier-injected specimens to 0.58 ± 0.003 ng/ml 24 h after the experimental treatment. In the chromaffin tissue, an increase in the number of epinephrine cells and a decrease in the number of norepinephrine cells were observed, decreasing the numeric norepinephrine/epinephrine cell ratio, from 1.4/1 of control specimens to 0.3/1 24 h after ANF administration. Moreover, norepinephrine plasma levels decreased from 998 ± 4.600 pg/ml in carrier-injected specimens to 321 ± 2.230 pg/ml 24 h after ANF administration; epinephrine plasma levels were elevated from 614 ± 3.410 pg/ml in carrier-injected specimens to 1672 ± 10.800 pg/ml 24 h after the experimental treatment. The presence of ANF in the adrenal gland suggests that, also in reptiles as in other vertebrates, this peptide, locally released from the chromaffin cells, may modulate the activity of the adrenal gland, probably in a paracrine manner. The effects of ANF on the adrenal gland suggest that this peptide may affect reptilian salt and fluid homeostasis.

Capsaicin-sensitive neural pathway mediates atrial natriuretic factor (ANF) release in response to physiological stimuli

Increases in intravascular volume are detected by mechanoreceptors situated at the junctions of the great veins with the atria. We had previously shown that localized distension of the superior vena caval/right atrial junction, simulating increased cardiac preload, elicits release of ANF remotely from the atrial appendage. We proposed that ANF secretion is stimulated via intrinsic neural pathways running from the venoatrial junctions to the appendage. We developed a technique whereby non-adrenergic, non-cholinergic sensory nerves could be selectively destroyed in the heart of adult rats by instilling capsaicin into the pericardial space. Four days later, the animals were killed, and isolated perfused atria were prepared with small balloons positioned so that the superior vena caval/right atrial junction could be discretely stretched. Immunoreactive ANF secretion into the perfusate was measured. Although distension of the venoatrial junction increased ANF secretion from the control atria, there was no such response in the denervated atria. We conclude (A) that local application of capsaicin to the heart of adult rats induces selective functional neural deficits and (B) that information regarding distension of the junction of the great veins and the atria is normally transmitted across the atrium via these nerves to stimulate ANF secretion from peptide stores located in the atrial appendage. We propose that these pathways are crucial to ensure appropriate ANF secretion in response to an increase in circulating blood volume.

Immunocytochemical localization of atrial natriuretic factor (ANF) in rat female reproductive tract: evidence for a potential hormonal regulation

In the present studies atrial natriuretic factor (ANF) was characterized immunocytochemically in the reproductive tract of immature female rats, and changes of ANF levels in response to different hormonal conditions were demonstrated. Administration of pregnant mare serum gonadotropin (PMSG) to immature animals has shown to be a useful method to synchronize growth, differentiation and atresia of ovarian follicles. ANF immunoreactivity was investigated in rat uterus and oviduct during follicular growth and estrogenic dominance (48 h after PMSG treatment) and during follicular atresia and progesterone dominance (96 h after PMSG treatment). Our immunocytochemical results showed that in rat uterus ANF was localized in endometrial mucosal and glandular epithelium and smooth muscle cells of the myometrium. In the oviduct ANF immunoreactivity was observed in mucosal cells and muscle layers. Immunocytochemical staining patterns and Western blot analysis revealed that ANF levels in rat uterus and oviduct are modulated by the hormonal status. ANF immunoreactivity was elevated during estrogenic dominance (48 h after PMSG) in uterus and oviduct. However, during progesterone dominance (96 h after PMSG) elevation of ANF immunoreactivity was observed in the uterus only. These results raise the possibility that ANF expression in rat oviduct is positively controlled by estrogen and negatively by progesterone. ANF staining in uterus during progesterone phase provides evidence that both estrogen and progesterone regulate ANF levels in uterus. The observed staining patterns indicate that ANF may have intracellular functions as well as a role in priming the extracellular environment. Accordingly, the possibility that ANF might be an important regulatory molecule for autocrine / paracrine communication within the female reproductive tract should be considered.

Sulfonylurea receptor ligands modulate stretch-induced ANF secretion in rat atrial myocyte culture.

Stretch-induced atrial natriuretic factor (ANF) secretion was studied in cultures of neonate atrial appendage myocytes. Stretch, applied for 40 min by hypotonic swelling, increased the mean area of 44 individually imaged myocytes by 4.8-8.8% (P < 0.0001) at 6 min and by 2.3-6.2% (P < 0.05) at 35 min. Stretch increased immunoreactive ANF release by 42% (P < 0.05) from a baseline of 315 pg/ml. The ATP-sensitive K(+) (K(ATP))-channel blocker tolbutamide (100 micromol/l) increased the stretch-stimulated release to 84% (P < 0.01) over baseline, whereas lower concentrations (1, 10, and 30 micromol/l) had no stimulatory effect. The K(ATP)-channel opener diazoxide (0.1, 1, 10, 30, and 100 micromol/l) inhibited stretch- plus tolbutamide-stimulated ANF release in a concentration-dependent manner, with IC(50) = 2.2 micromol/l, although 100 micromol/l diazoxide did not reduce the increase in mean cell area. The stretch-stimulated K(ATP) current, monitored in 82 whole cell recordings with sulfonylurea receptor (SUR) ligands, was inversely correlated with the stretch-induced ANF release (r(2) = 0.79, P < 0. 0001). In the absence of stretch, the K(ATP) current had no relationship with baseline ANF release, and baseline ANF release was not affected by the K(ATP)-channel modulators. The results show that SUR ligands that open K(ATP) channels inhibit stretch-induced ANF release in atrial myocytes, in correlation with the stretch-activated K(ATP) current. The subcellular site of action of the SUR ligands-plasmalemma or intracellular organelles-remains to be determined.

Water Immersion Increases Urinary Excretion of Aquaporin-2 in Healthy Humans

Many previous studies have shown that aquaporin-2 (AQP2), the vasopressin-regulated water channel, is excreted in the urine and that the excretion increases in response to vasopressin. Moreover, recently a close correlation between AQP2 excretion in urine and kidney AQP2 expression has been demonstrated, showing that urinary excretion of AQP2 is a reliable indicator for AQP-2 function. As head-out water immersion causes an expansion in the central vascular volume equal to that induced by 2 liters of saline, without modifying plasma composition, we used immersion in water to evaluate if the response to acute expansion of the central vascular volume could involve vasporessin (AVP) and AQP2. In healthy subjects, concentrations of plasma atrial natriuretic factor (ANF) and AVP, and urinary AQP2 were measured during a 2-hour immersion period. In all subjects, immersion caused a prompt and marked increase in immunoreactive ANF (23.0 ± 2.12 pg/ml at second hour vs. 2.17 ± 0.42 pg/ml at baseline) and in urinary excretion of AQP2 (23.9 ± 2.69 pmol/mg creatinine at second hour vs. 4.42 ± 0.14 pmol/mg creatinine at baseline), while a significant decrease was found in plasma AVP. Recovery was associated with a prompt return to pre-study levels. These findings demonstrate that heat-out water immersion stimulates urinary excretion of AQP2 in absence of an increase in plasma AVP.

Atrial natriuretic factor and brain natriuretic peptide gene expression in the spontaneous hypertensive rat during postnatal development

The increase in natriuretic peptides (NP), atrial natriuretic factor (ANF), and brain natriuretic peptide (BNP) production and release by cardiocytes that occurs in hypertension has been considered to be a compensatory mechanism against ventricular overload. Studies on NP production in the spontaneously hypertensive rat (SHR), an experimental model of human hypertension, have produced controversial results and were carried out when hypertension was already established (> 17 weeks). At this time, age-related physiologic and molecular changes in cardiac muscle are difficult to separate from those related to hypertension, ie, increased ANF production and plasma levels. In addition, most of the studies used male rats because the rate of increase in arterial blood pressure—as well as the level to which it rises—is greater in males than in females. Studies of a similar nature using female SHR are not available. The aim of this work was to determine 1) whether ANF and BNP production and secretion increase with the development of hypertension in genetically hypertensive rats; 2) whether a sexual dimorphism in ANF and BNP production and secretion is present in the genetically hypertensive rat during the development of hypertension; and 3) whether the demand for ANF and BNP is the same from each chamber of the heart under these experimental conditions. Age-matched male and female SHR, Wistar-Kyoto (WKY), and Sprague Dawley (SD) rats at 2, 4, and 8 weeks of age were used. The normotensive SD were included to provide a wider basis for baseline findings, as WKY rats are not always a suitable control for SHR due to genetic variations. Natriuretic peptide plasma levels and tissue content were measured by radioimmunoassay. ANF, BNP, as well as α- and β-myosin heavy chain (MHC) mRNA were estimated by Northern blot analysis. Blood pressure (BP) of more than 150 mm Hg was found only in 8-week-old male SHR. Plasma immunoreactive (ir)ANF and irBNP increased significantly at puberty (8 weeks) in both male and female SHR. The earliest molecular change encountered during the development of hypertension was a significant increase in BNP mRNA in the right and left atria from both male and female 8-week-old SHR. In the ventricles from both male and female SHR, there was no increase in the ratio of left ventricular wet weight/body weight, no increase in ventricular ANF mRNA transcripts, and no myosin heavy chain isoform switch (a protein marker of hypertrophy). irBNP ventricular concentration, however, increased significantly in both male and female SHR, but only in female SHR was there a concomitant increase in BNP mRNA. These results suggest that 1) ANF and BNP production are not coordinated in all cardiac compartments during the development of hypertension; 2) upregulation of BNP in the atria from male and female SHR is the earliest event detected at 8 weeks; 3) the prehypertensive stage, in the genetically hypertensive female rats, is associated with an increase in ventricular irBNP concentration and BNP mRNA; 4) there is a dissociation between BP and plasma levels of NP; and 5) as well, there is a dissociation between NP gene expression and MHC isoform switch. The regulation of NP is not coordinated in either gender during the development of hypertension. The activation of the BNP gene in female SHR suggests that BNP might play an important role at the onset of hypertension.

Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development.

Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat alpha-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II-III tadpoles and in successive larval stages. Soon after hatching, stages II-III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI-XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone.

Atrial natriuretic factor (ANF) and ANF receptor C gene expression and localization in the respiratory system: effects induced by hypoxia and hemodynamic overload.

Atrial natriuretic factor (ANF) and ANF receptor C (ANF.RC) expression have been investigated in healthy and cardiomyopathic hamsters (CMPH) with widespread necrosis of the diaphragm and myocardium leading to respiratory and heart failure. ANF- and ANF.RC-producing cells were localized in different structures of the respiratory system, and the regulation of their expression by the individual and/or combined action of hypoxia and hemodynamic overload was analyzed. The study was performed in 20-, 90-, and 150-day-old animals using immunohistochemistry, in situ hybridization, Northern blot, and RIA analyses. ANF was shown to be expressed in the tracheo-bronchial epithelium and muscle and, to a lesser extent, in the alveolar wall and muscular media of the pulmonary arteries and extraparenchymal pulmonary veins in both healthy hamsters and CMPH. In 150-day-old CMPH, hypoxia (PaO2 < 50 mm Hg) induced a 10-fold increase in ANF messenger RNA accumulation and a 6-fold increase in the immunoreactive ANF (IR-ANF) concentration in lungs, as quantitated by RIA. As plasma IR-ANF concentrations were elevated in all CMPH age groups, it was most likely produced by the myocardium. ANF.RC messenger RNA was homogeneously distributed throughout the entire respiratory system and was increased 2-fold in hypoxic 150-day-old CMPH only. These results suggest that ANF originating in the respiratory system exerts only paracrine effects on different structures of the respiratory system in addition to the action of circulating ANF. Hemodynamic overload (left ventricular end-diastolic pressure, 17.20 +/- 3.80 mm Hg) might contribute to enhanced ANF gene expression only in extraparenchymal pulmonary vein walls of 150-day-old CMPH. We also propose that ANF.RC overexpression might be a protective mechanism operated via either ANF clearance or inhibition of adenylate cyclase activity to counteract exaggerated smooth muscle relaxation.

Renal effects of TAPP, a highly selective μ‐opioid agonist

1. The effect of i.v. administration of TAPP, a highly selective and exclusively peripherally-acting mu-opioid receptor agonist, on urine output, urinary sodium, potassium and cyclic GMP, and on plasma immunoreactive atrial natriuretic factor (IR-ANF) levels was studied in conscious normally hydrated female rats (200-250 g). 2. TAPP treatment produced a significant dose-dependent increase of urine output and urinary sodium, potassium and cyclic GMP excretion during the first hour. The highest TAPP dose used (2.5 mg kg-1. body weight) elicited a 10 fold elevation of urine output from 0.23 +/- 0.06 ml h-1 to 2.5 +/- 0.3 ml h-1 (n = 18) accompanied by augmented sodium [from 17.0 +/- 4.7 mu Eq h-1 to 79 +/- 12.7 mu Eq h-1, n = 18 (P < 0.001)], potassium [from 9.5 +/- 2.5 mu Eq h-1 to 39.4 +/- 6.6 mu Eq h-1, n = 18 (P < 0.005)], and cyclic GMP excretion [from 191 +/- 21 pmol h-1 to 1340 +/- 322 pmol h-1, n = 18 (P < 0.001)]. Plasma IR-ANF rose from 22 +/- 4 pg ml-1 to 508 +/- 22 pg ml-1 (n = 18) (P < 0.001) 5 min after administration of TAPP (2500 micrograms kg-1). 3. TAPP lowered systemic blood pressure, also in a dose-related manner, 1-5 min after injection. This decrease in blood pressure was transient and did not last more than 10 min. 4. Pretreatment with the opioid antagonist naloxone (0.8 mg per rat) abolished the diuretic, natriuretic and kaliuretic effect of TAPP (250 micrograms kg-1); urine output dropped from 1.16 +/- 0.15 ml h-1, n = 12, to the control value of 0.15 +/- 0.06 ml h-1, n = 12 (P < 0.001), sodium excretion fell from 57.5 +/- 11 mu Eq h-1, to 21.3 +/- 8.5 mu Eq h-1, n = 12 (P < 0.001), and potassium excretion decreased from 45.4 +/- 9.7 mu Eq h-1, n = 12, to 16.1 +/- 7.0 mu Eq h-1, (P < 0.001). 5. Pretreatment with anti-ANF serum (0.4 ml) abolished the diuretic effect of TAPP: urine output diminished significantly from 1.93 +/- 0.28 to 0.88 +/- 0.29 ml h-1 (P < 0.01) (n = 6). The TAPP-induced diuretic action, increased sodium/potassium excretion and elevated urinary cyclic GMP levels were also reversed by anti-ANF antibodies. 6. Since TAPP is totally unable to cross the blood-brain barrier, the ensemble of these observations led to the conclusion that the diuretic, natriuretic, kaliuretic and hypotensive effects produced by this mu-opioid agonist through interaction with peripheral mu-opioid receptors occur via ANF release.

Volemie Status Influences the Response of Plasma Atrial Natriuretic Factor to Positive Airway Pressure

STUDY OBJECTIVE; To evaluate interactive effects of volemic status and positive end-expiratory pressure (PEEP) on the plasma levels of atrial natriuretic factor (ANF) in assist-controlled mechanical ventilation (MV). Three successive protocols applied in randomized order to each participant. Clinical investigation laboratory. Twenty-one young, healthy adults. The three protocols were as follows: (1) MV+PEEP, normovolemia; (2) MV+PEEP, hypervolemia; and (3) spontaneous breathing (SB), hypervolemia. In protocols 1 and 2, a preliminary period of SB lasting 2 h was followed by MV alone (0.5 h), MV+20 cm H2O PEEP (1 h), and a recovery period of SB (1.5 h). Hypervolemia was induced by the continuous i.v. infusion of 3 L of 0.9% NaCl in 5 h (protocols 2 and 3). Heart rate, BP, and the plasma levels of immunoreactive ANF and catecholamines were measured serially. During hypervolemia, ANF significantly decreased when PEEP was added to MV (protocol 2: from 31.1 +/- 2.7 to 20.7 +/- 1.5 fmol/mL; p < 0.01). This did not occur in normovolemia (protocol 1: from 20.0 +/- to 16.7 +/- 1.2 fmol/mL; p = NS). The different effects of MV+PEEP in normovolemia and hypervolemia were not related to differences in circulating catecholamine levels. These results demonstrate for the first time (to our knowledge) that volemic status modulates the response of plasma ANF to PEEP in humans. The role of ANF in the water and salt retention induced by MV with PEEP might be limited to hypervolemic conditions.

Stretch-induced release of atrial natriuretic factor from the heart of rainbow trout (Oncorhynchus mykiss)

We investigated the effect of both cardiac filling and after-load pressures on the release of atrial natriuretic factor (ANF) from rainbow trout hearts (Oncorhynchus mykiss). Our experimental approach was to perfuse hearts in situ and collect perfusate ejected from the heart in 1-min fractions and assay for immunoreactive ANF-(99-126) (human and canine). Increases in cardiac filling pressure produced repeatable and sustained increases in cardiac output. At the same time, there was an immediate and sustained increase in ANF secretion. ANF secretion increased sixfold from the control level of 30.68 ± 4.76 pg∙min−1∙g−1wet heart mass for a fourfold increase in cardiac output from 15.24 ± 0.73 mL∙min−1∙kg−1body mass. ANF secretion was unaffected by increases in after-load pressure. Measurement of immunoreactive ANF content in trout myocardial tissues revealed ANF in both the atrium and ventricle, but 92% of the total ANF store was contained within the atrium. This suggests that in rainbow trout, the atrium serves as the primary cardiac tissue for ANF release. These data are consistent with the mechanism of ANF release for mammalian hearts, and provide additional evidence that ANF secretion in vertebrates is mediated by atrial stretch and not ventricular stretch.

Alpha 1-adrenergic stimulation of isolated rat atria results in discoordinate increases in natriuretic peptide secretion and gene expression and enhances Egr-1 and c-Myc expression.

We studied the effects of alpha1-adrenergic stimulation on atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) secretion and gene expression in isolated right atria. The early-response genes Egr-1 and c-myc were also studied as potential markers of transcriptional activation after alpha1-adrenergic stimulation. Isolated right atria from rats were stimulated for up to 8 h by the alpha1-adrenergic agonist phenylephrine (PE). PE at 10, 50, or 100 microM stimulated the secretion of immunoreactive (ir) ANF, beginning at 0.5 h and peaking after 1.5 h, IrANF secretion remained significantly elevated for 8 h with 100 microM PE, reached control levels after 5 h with 10 microM PE, and after 6 h microM PE with 50 microM PE, PE at 50 or 100 microM stimulated irBNP secretion after 15 min, which peaked at 1 h, and thereafter remained above control levels. Calculation of irANF/irBNP ratios revealed that their stimulated secretion was not coregulated. PE caused significant changes in steady state transcript levels for the genes studied. After 6 h, 50 microM PE caused a 49% increase in ANF messenger RNA (mRNA) levels. BNP mRNA levels were increased by 135% after 6 h and by 77% after 8 h. Egr-1 mRNA levels were increased by 81% after 4 h, 167 after 6 h, and 40% after 8 h of treatment, mRNA levels of c-myc were increased by 49% after 4 h and 53% after 6 h. PE-induced increases in secretion and gene expression were inhibited by the alpha1-adrenergic receptor antagonist prozosin (10 microM). We conclude that both ANF and BNP secretion from atria can be stimulated by PE, and that their secretion is not coregulated. The kinetics of enhanced natriuretic peptide gene expression and secretion did not change in parallel, suggesting that these processes are not acutely coordinated. The enhanced expression of Egr-1 and c-myc suggests that they may be involved in the modulation of atrial gene expression in response to alpha1-adrenergic stimulation. The results presented suggest that compensatory adrenergic activation such as those seen in several clinical entities may be one of the factors that provide long-term enhanced natriuretic peptide production, thus contributing to the maintenance of cardiovascular homeostasis.

Glucocorticoids inhibit D1B, but not D2, receptor-mediated effects on hypothalamic atrial natriuretic factor neurons.

Recent evidence suggests that ANF neurons of the hypothalamus are dopamine sensitive, and the catecholamine may exert a direct stimulatory or inhibitory effect on the neurons mediated through D1 or D2 receptors, respectively, in a manner related to the differential dopamine binding sensitivity of the two receptor subtypes. Employing well characterized ANF RIA and colorimetric Northern blot analysis with synthetic oligonucleotide probes complementary to pro-ANF messenger RNA (mRNA), we report here the effect of dexamethasone (DM), a potent synthetic glucocorticoid, on DA-stimulated ANF neurons in long term primary cultures of neonatal rat hypothalamic cells. Although DM alone did not affect basal secretion of immunoreactive ANF, it approximately halved immunoreactive ANF secretion induced by D1 agonist, SKF38393 (P < 0.01). The effect of DM was both time dependent and dose related, with an EC50 of 0.1 nM; it was blocked by 100 nM RU38486 (P < 0.05), a glucocorticoid receptor antagonist, but not by 100 nM RU28318, a mineralocorticoid receptor antagonist. In addition, the effect of DM was mimicked by corticosterone (EC50, 10 nM), but not deoxycorticosterone. The increased expression of pro-ANF mRNA signal induced by the D1 agonist in culture was suppressed by DM in a similar manner. In contrast, DM did not modulate ANF production and secretion induced by D2 agonist, quinpirole. Furthermore, reverse transcription-polymerase chain reaction demonstrated that D1B, but not D2, receptor mRNA expression was selectively suppressed by glucocorticoids. Thus, we conclude that in monolayer cultures of rat hypothalamic neurons, glucocorticoids differentially modulate dopamine receptor-induced responsiveness of ANF neurons by down-regulating D1B, but not D2, receptor-mediated changes. Hence, in severe stress, high levels of circulating glucocorticoids may negate the D1B-induced stimulatory response but allow dopamine to suppress the function of hypothalamic ANF neurons through D2 receptor activation.

Glucocorticoids inhibit D1B, but not D2, receptor-mediated effects on hypothalamic atrial natriuretic factor neurons

Recent evidence suggests that ANF neurons of the hypothalamus are dopamine sensitive, and the catecholamine may exert a direct stimulatory or inhibitory effect on the neurons mediated through D1 or D2 receptors, respectively, in a manner related to the differential dopamine binding sensitivity of the two receptor subtypes. Employing well characterized ANF RIA and colorimetric Northern blot analysis with synthetic oligonucleotide probes complementary to pro-ANF messenger RNA (mRNA), we report here the effect of dexamethasone (DM), a potent synthetic glucocorticoid, on DA-stimulated ANF neurons in long term primary cultures of neonatal rat hypothalamic cells. Although DM alone did not affect basal secretion of immunoreactive ANF, it approximately halved immunoreactive ANF secretion induced by D1 agonist, SKF38393 (P < 0.01). The effect of DM was both time dependent and dose related, with an EC50 of 0.1 nM; it was blocked by 100 nM RU38486 (P < 0.05), a glucocorticoid receptor antagonist, but not by 100 nM RU28318, a mineralocorticoid receptor antagonist. In addition, the effect of DM was mimicked by corticosterone (EC50, 10 nM), but not deoxycorticosterone. The increased expression of pro-ANF mRNA signal induced by the D1 agonist in culture was suppressed by DM in a similar manner. In contrast, DM did not modulate ANF production and secretion induced by D2 agonist, quinpirole. Furthermore, reverse transcription-polymerase chain reaction demonstrated that D1B, but not D2, receptor mRNA expression was selectively suppressed by glucocorticoids. Thus, we conclude that in monolayer cultures of rat hypothalamic neurons, glucocorticoids differentially modulate dopamine receptor-induced responsiveness of ANF neurons by down-regulating D1B, but not D2, receptor-mediated changes. Hence, in severe stress, high levels of circulating glucocorticoids may negate the D1B-induced stimulatory response but allow dopamine to suppress the function of hypothalamic ANF neurons through D2 receptor activation.

Attenuation of renal effects of atrial natriuretic factor during rat pregnancy.

The influence of pregnancy on renal responses to atrial natriuretic factor (ANF) was determined in urethane-anesthetized Sprague-Dawley rats. Infusions of ANF caused a significantly greater increase in urinary excretion of fluid, sodium, and potassium in virgin than in pregnant (13-15 days and 21 days) rats. Guanosine 3',5'-cyclic monophosphate (cGMP) excretion, mean arterial pressure, plasma immunoreactive ANF, and glomerular filtration rate (GFR) following ANF infusions were not different in virgin and gravid rats, although increments in GFR over basal were greater in virgin than in gravid animals. Renal responses to ANF normalized during postpartum and were attenuated by progesterone treatment of virgin rats. Natriuretic effects of infusions of ANF plus ANF-(4-23) (a ligand for clearance receptors) or of ANF plus thiorphan (an endopeptidase inhibitor) in virgin and pregnant rats did not differ; ANF-(4--23) and thiorphan alone caused greater natriuresis in pregnant than in virgin rats. Effects of ANF on cGMP production by collecting duct cells isolated from virgin and pregnant rats did not differ. We concluded that the attenuation in the renal effects of ANF during pregnancy might be mediated by progesterone by an increase in the intrarenal metabolism of ANF and might reflect physiological adjustment to facilitate fluid/electrolyte expansion.

D1 receptors mediate dopamine stimulation of immunoreactive atrial natriuretic factor (ANF) release and pro-ANF messenger ribonucleic acid expression of rat hypothalamic neurons in culture.

Atrial natriuretic factor (ANF) and its smaller congeners are produced and released from rat brains to regulate the cardiovascular system, drinking behavior, and neurohormone release at the central level. In the hypothalamus, the adenylyl cyclase-cAMP system plays an important role in modulating the function of ANF neurons in the paraventricular nuclei and the periventricular regions that receive rich dopaminergic inputs. We report here a novel observation of a dopamine (DA) D1 agonist, SKF-38393, modulating immunoreactive (ir) ANF and pro-ANF messenger RNA (mRNA) expression in rat hypothalamic neurons. In long term primary cultures of neonatal rat hypothalamic cells, treatment with SKF-38393 increased irANF secretion in a time-related and dose-dependent manner, with an ED50 of approximately 10(-7) M and a concentration producing maximum effect of 10(-5) M. This stimulating effect of SKF-38393 was mimicked by 10(-5) M DA, a physiological ligand for D1 receptors. Furthermore, both of the stimulatory effects were abolished by SCH-23390, a D1 antagonist. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures, as examined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D1 receptor was colocalized in approximately 90% of the irANF-positive cells in the cultures. In addition, cholera toxin, an irreversible activator of adenylyl cyclases, markedly increased irANF secretion and cAMP production in a dose-dependent manner. This effect mimicked that of D1 agonist-stimulated ANF release, in that the latter also concurrently enhanced cAMP production in the hypothalamic cultures. Furthermore, the stimulatory effect of D1 agonist on irANF release was markedly suppressed by rp-cAMPS, a cAMP antagonist. We, thus, conclude that the release and gene expression of ANF in rat hypothalamic neurons are directly stimulated by DA acting through its D1 receptors on ANF neurons; this effect may operate at the genomic level and is mediated at least in part through activation of the cAMP-dependent kinase-A pathway.

D1 receptors mediate dopamine stimulation of immunoreactive atrial natriuretic factor (ANF) release and pro-ANF messenger ribonucleic acid expression of rat hypothalamic neurons in culture

Atrial natriuretic factor (ANF) and its smaller congeners are produced and released from rat brains to regulate the cardiovascular system, drinking behavior, and neurohormone release at the central level. In the hypothalamus, the adenylyl cyclase-cAMP system plays an important role in modulating the function of ANF neurons in the paraventricular nuclei and the periventricular regions that receive rich dopaminergic inputs. We report here a novel observation of a dopamine (DA) D1 agonist, SKF-38393, modulating immunoreactive (ir) ANF and pro-ANF messenger RNA (mRNA) expression in rat hypothalamic neurons. In long term primary cultures of neonatal rat hypothalamic cells, treatment with SKF-38393 increased irANF secretion in a time-related and dose-dependent manner, with an ED50 of approximately 10(-7) M and a concentration producing maximum effect of 10(-5) M. This stimulating effect of SKF-38393 was mimicked by 10(-5) M DA, a physiological ligand for D1 receptors. Furthermore, both of the stimulatory effects were abolished by SCH-23390, a D1 antagonist. These immunoassay findings were accompanied by corresponding changes in the abundance of pro-ANF mRNA in the cultures, as examined by colorimetric Northern blot analysis. By combining the techniques of in situ hybridization and immunocytochemistry, the mRNA of D1 receptor was colocalized in approximately 90% of the irANF-positive cells in the cultures. In addition, cholera toxin, an irreversible activator of adenylyl cyclases, markedly increased irANF secretion and cAMP production in a dose-dependent manner. This effect mimicked that of D1 agonist-stimulated ANF release, in that the latter also concurrently enhanced cAMP production in the hypothalamic cultures. Furthermore, the stimulatory effect of D1 agonist on irANF release was markedly suppressed by rp-cAMPS, a cAMP antagonist. We, thus, conclude that the release and gene expression of ANF in rat hypothalamic neurons are directly stimulated by DA acting through its D1 receptors on ANF neurons; this effect may operate at the genomic level and is mediated at least in part through activation of the cAMP-dependent kinase-A pathway.

901-29 Effects of Positive EndExpiratory Pressure Ventilation on Right Atrial Size and Atrial Natriuretic Factor Release in Humans

Atrial natriuretic factor (ANF) release is augmented by atrial distention whereas positive end-expiratory pressure (PEEP) ventilation has the opposite effect. We tested the hypothesis that PEEP-induced decrease in ANF secretion is mediated by a decrease in right atrial (RA) distention. Accordingly, eight hemodynamically stable mechanically ventilated patients (age: 22-49) in sinus rythm and without kidney or cardiovascular disease were studied. A central venous catheter (3Fr) and a fluid-filled probe were inserted into the RA and esophagus, respectively, in order to measure transmural RA pressure (tP). A radial artery catheter was placed to monitor blood pressure (BP) and withdraw blood samples for the measurement of plasma immunoreactive ANF (irANF). Blood pressure, diuresis, tP, endsystolic RA area at the level of the foramen ovale (acquired using transesophageal echocardiography at end-expiration) and irANF were measured at the end of 3 consecutive 30 min periods of: (1) mechanical ventilation at zero end-expiratory pressure (ZEEP1); (2) application of PEEP + 15 cm of H 2 O; and (3) after withdrawal of the PEEP (ZEEP2). Variations of irANF and RA area were directly correlated (r = 0.82, P &lt; 0.001)). Results are expressed as mean ± SEM; *: p &lt; 0.05 versus ZEEP1 values. ZEEP 1 PEEP + 15 ZEEP 2 irANF(pg/ml) 14.9 ± 1.9 10.7 ± 1.1 * 13.8 ± 2 RA(cm 2 ) 14.4 ± 1.1 11.2 ± 1.6 * 14.2 ± 1.2 tP(mmHg) 4.2 ± 0.6 1.2 ± 0.2 * 2.7 ± 0.5 BP(mmHg) 137.9 ± 4.4 114.6 ± 7.4 * 134.2 ± 4.0 Diuresis(ml/min) 1.08 ± 0.19 0.60 ± 0.16 * 1.02 ± 0.23 As expected, PEEP resulted in a decrease in both RA area and tP as well as ANF secretion. These data support the hypothesis that PEEP-induced decreases in circulating ANF are related to decreased RA distention, which may contribute to the antidiuresis noted in patients ventilated with positive end-expiratory pressure.

Mechanisms of release of atrial natriuretic factor in vivo.

The aim of the study was to define the relationship between the concentration of immunoreactive atrial natriuretic factor (ANF) in the plasma and atrial stretch, atrial pressure and atrial wall stress during changes in blood volume within the physiological range. Advantage was taken of the potentiation of the release of ANF, in response to blood volume expansion, in the anaesthetized rabbit, by section of the carotid sinus, aortic depressor and vagus nerves. Mean arterial pressure and right and left atrial pressures were measured. Right and left atrial dimensions were measured by sonomicrometry. Blood volume was expanded by 20% and then decreased at 1% of the blood volume per minute for 40 min, before and after section of the nerves. Plasma ANF did not change significantly in response to either the increase or decrease in blood volume in the presence of intact nerves. After the nerves were sectioned, blood volume expansion significantly increased IR-ANF (34.6 +/- 12 to 260.6 +/- 89.8 pg/ml). The relationship between ANF and the changes in left atrial dimensions (extension ratio) was exponential. A significant linear correlation was found between ANF and left atrial pressure and left atrial wall stress. Over a range of atrial pressures from 1-10 cm H2O the atrial wall was extremely distensible, but there were only minor changes in ANF. At higher atrial pressures there was little further extension of the atrial wall but there were much larger increases in plasma ANF. In response to changes in blood volume within the physiological range cardiovascular reflexes normal maintain atrial dynamic function within a narrow range and there is little stimulus for the release of ANF. In conditions in which the cardiovascular reflexes are unable to maintain atrial function constant then there will be changes in the release of ANF and there will be an exponential relationship between atrial wall stretch and plasma ANF.