Dopaminergic mediation of the diuretic and natriuretic action of centrally administered rat atrial natriuretic factor (99-126)

Intracerebroventricular administration of either rat atrial natriuretic factor (99-126) or dopamine to conscious male hydrated rats resulted in an increase in urinaryvolume and sodium excretion. This activity was prevented, in both cases, by nonselective dopamine antagonist haloperidol (2.5 or 1.25 mg/kg sc, 18 and 2 hr before intracerebroventricular administration of atrial natriuretic factor). Our findings suggest that atrial natriuretic factor exerts its centrally mediated effects on sodium and water metabolism, at least in part, via a dopaminergic mechanism.     

Natriuretic and diuretic action of centrally administered rat atrial natriuretic peptide (99-126): possible involvement of aldosterone and the sympathoadrenal system

Intracerebroventricular (ICV) administration of rat atrial natriuretic peptide (99-126) (rANP) to conscious male hydrated rats resulted in a dose-related increase in urinary volume and sodium excretion over a 6-h period of urine collection. A diminished mineralocorticoid effect on the kidneys may explain the natriuretic phenomenon. This hypothesis was tested by ICV rANP injection (1.25 microgram/5 microL) in conscious hydrated rats pretreated beforehand with d-aldosterone (20 micrograms/kg, ip). Although the absolute amount of sodium excreted was reduced, aldosterone did not affect rANP-induced sodium output at 1 and 3 h. Rats that were sham-operated or bilaterally adrenalectomized after 4 days were pretreated with aldosterone and given an oral water load followed by ICV rANP or saline. The possible participation of the peripheral sympathetic nervous system in the central action of rANP was evaluated in rats pretreated with 6-hydroxydopamine. In sympathectomized and adrenalectomized rats natriuresis and diuresis were still evident after rANP. Our results indicate that the natriuretic effect of ICV rANP is independent of mineralocorticoids. Likewise, diuresis and natriuresis can occur in the absence of the adrenal glands and are independent from the neural tone that the adrenergic system exerts on sodium reabsorption.     

Evidence for a dopaminergic mechanism for the diuretic and natriuretic action of centrally administered atrial natriuretic factor

1. Intracerebroventricular (IVT) administration of rat atrial natriuretic factor (ANF) (99-126) to conscious male hydrated rats induces a dose-dependent increase in urine and sodium excretion. The possible involvement of brain dopaminergic system in the IVT-ANF-induced diuresis and natriuresis was evaluated. 2. Central sympathectomy (6-OHDA, 250 micrograms/5 microliters, IVT; 72 and 48 hr before IVT-ANF) inhibited both the diuretic and the natriuretic action of centrally administered ANF, suggesting that in the brain ANF requires the integrity of central noradrenergic and/or dopaminergic systems function for its actions. 3. Intracerebroventricular injection of haloperidol and intragastric administration of domperidone prevent the diuretic and natriuretic response to centrally administered ANF. 4. Our data suggest a neuromodulatory action of ANF within the brain and demonstrate an interaction of the peptide with brain dopaminergic systems.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Localization of atrial natriuretic peptide, ANP-(99-126) binding sites in the rat thymus and spleen with quantitative autoradiography

Binding sites for atrial natriuretic peptide, ANP-(99-126) were studied in lymphoid organs of the rat with quantitative autoradiography. Tissue sections were incubated in the presence of 0.13 nM 125I-ANP-(99-126) followed by autoradiography using [3H]-Ultrofilm, and the results were analyzed by computerized densitometry and comparison to 125I-standards. Specific ANP binding sites were localized in the medulla and the cortex of the rat thymus and in the white pulp of the rat spleen, with apparent binding sites concentrations of 93, 65, and 126 fmol/mg protein, respectively. The presence of ANP binding sites in areas related to the maturation and function of lymphocytes, and to the production of thymic hormones, suggests the possibility of a role of circulating ANP in the modulation of the immune response.     

Effects of centrally administered galanin (1-16) on galanin expression in the rat hypothalamus

In the rat hypothalamic magnocellular neurons, galanin coexists with vasopressin and might be involved in hydro-osmotic regulation. In the present study, we investigated the ability of galanin to also regulate the osmotically stimulated expression of galanin itself in hypothalamic magnocellular neurons. Ten minutes after galanin injection, galanin mRNA rate decreased in salt-loaded rats whereas the level of galanin immunoreactivity increased. Both effects were suppressed by the injection of a galanin antagonist together with galanin. Moreover, electron microscope studies demonstrated synaptic contacts between galanin-containing fibers and magnocellular neurons. Galanin may exert inhibitory roles in the regulation of magnocellular neurons. However, galanin and vasopressin expression displayed differences upon galanin injection. Possible mechanisms underlying these discrepancies are further discussed.     

Endopeptidase-24.11 in human plasma degrades atrial natriuretic factor (ANF) to ANF(99-105/106-126)

We previously demonstrated the presence of ANF(99-126), and ANF(99-126) cleaved between Cys105 and Phe106 (cleaved ANF), in human coronary sinus plasma. We now report that cleaved ANF is formed when synthetic ANF(99-126) is added to human plasma. When synthetic ANF(99-126) was incubated in heparinized human plasma, HPLC analysis showed two degradation products. The main product was shown by amino acid and sequence analysis to be cleaved ANF. Degradation of ANF was inhibited by EDTA and phosphoramidon. These findings are consistent with the action of endopeptidase EC 3.4.24.11, which may play an important part in the biological inactivation of ANF.     

The secretion of atrial natriuretic factor-(99-126) by cultured cardiac myocytes is regulated by glucocorticoids

Atrial natriuretic factor (ANF) is stored in mammalian atria primarily as ANF-(1-126), the precursor to the known circulating form of the hormone ANF-(99-126). When primary cultures of atrial myocytes were maintained in a complete serum-free medium, they contained and secreted an ANF-(1-126)-like peptide. The addition of dexamethasone to the culture medium, however, resulted in the secretion of a molecule with chromatographic characteristics identical to ANF-(99-126), although the intracellular storage form of ANF was unchanged. Radiosequencing and amino acid analysis confirmed that the cultures maintained in dexamethasone secreted authentic ANF-(99-126). Chronic exposure of the cells to dexamethasone also resulted in a significant increase in the quantity of immunoreactive ANF both contained and secreted by the cultures. Dexamethasone stimulated ANF processing and secretion by atrial cultures in a dose-dependent manner, with an approximate EC50 of 10 nM. This stimulation could be reversed by removing the glucocorticoid from the culture medium. ANF processing was also stimulated by the specific glucocorticoid receptor agonist RU 28362, and both DEX- and RU 28362-stimulated ANF processing was inhibited by the specific glucocorticoid receptor antagonist RU 38486. Ventricular cells, which possess few granules and release ANF in a constitutive fashion, were also capable of processing ANF in a glucocorticoid-dependent fashion. Medium freshly removed from atrial cultures did not convert ANF-(1-126) to ANF-(99-126) nor was exogenous ANF-(1-126) efficiently processed when added to the medium of actively processing cultures. These results indicate that the post-translational processing of ANF-(1-126) to ANF-(99-126) likely occurs within or in close association with the cardiac myocytes and is not dependent on the presence of large quantities of secretory granules. Furthermore, it is apparent that both the expression and the post-translational processing of ANF by cultured cardiac myocytes is specifically regulated by glucocorticoids.     

Hyperthermic effect of centrally administered natriuretic peptides in the rat.

The effects of atrial natriuretic peptide (ANP-28), brain natriuretic peptide (BNP-32) and C-type natriuretic peptide (CNP-22) on body temperature were investigated in rats. Intracerebroventricular administration of each peptide in doses of 400 or 1000 ng caused a dose-related elevation in colon temperature 30 and 60 min after injection. A 40 ng dose of ANP-28 was also hyperthermic at 60 min. An intramuscular (i.m.) injection of noraminophenazone (a cyclooxygenase inhibitor) abolished the natriuretic peptide-induced hyperthermia. The results show that natriuretic peptides may participate in thermoregulatory processes in the central nervous system, and that their hyperthermic effect may be mediated via a cyclooxygenase-involved pathway.     

Rapid natriuretic action of aldosterone in the rat.

Rapid, nongenomic actions of aldosterone have been demonstrated in a number of cell types in vitro, including renal cell lines, but there remains little direct evidence that it is able to exert rapid effects on the kidney in the whole animal. Accordingly, the aim of this study was to determine whether aldosterone induces rapid changes in the renal handling of electrolytes or acid-base balance in the anesthetized rat. With the use of a servo-controlled fluid replacement system, spontaneous urine output by anesthetized male Sprague-Dawley rats was replaced with 2.5% dextrose. After a 3-h equilibration and a 1-h control period, rats were infused with aldosterone (42 pmol/min) or vehicle for 1 h. Aldosterone infusion induced a rapid (within 15 min) increase in sodium excretion that peaked at 0.24 +/- 0.08 compared with 0.04 +/- 0.01 micromol x min(-1) 100 x body weight(-1) (P = 0.041) in the vehicle-infused rats. This natriuresis was not associated with changes in glomerular filtration rate; urine flow rate; potassium, chloride, or bicarbonate excretion; or urine pH. The mechanisms involved are unclear, but because we have previously shown that aldosterone stimulates a rapid (4 min) increase in cAMP generation in the rat inner medullary collecting duct (IMCD) (Sheader EA, Wargent ET, Ashton N, and Balment RJ. J Endocrinol 175: 343-347, 2002), they could involve cAMP-mediated activation of the cystic fibrosis transmembrane conductance regulator chloride channel, which drives sodium secretion in the IMCD.     

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.     

Atrial natriuretic peptide, ANP(99-126), receptors in rat thymocytes and spleen cells

A single class of saturable, specific binding sites for the circulating form of atrial natriuretic peptides, ANP(99-126), was identified in rat thymus and spleen and in isolated thymocytes and spleen cells using quantitative autoradiographic techniques. In the thymus, the relative potency of ANP analogs to inhibit [125I] ANP(99-126) binding was ANP(99-126) = ANP(103-126) greater than ANP(111-126) greater than ANP(103-125). ANP(103-123) could not displace [125I]ANP(99-126) binding. Addition of ANP(99-126) stimulated the formation of cyclic GMP in isolated thymocytes and spleen cells in a dose-dependent manner. Our results indicate that immune cells have specific ANP receptors which could be coupled to guanylate cyclase activation and may play a role in the regulation of the immune response.     

Two-dimensional 1H-NMR investigation of the water conformation of the atrial natriuretic factor (ANF 101-126)

The complete sequential assignment of the ¹H-nmr resonance frequencies of the active fragment of the rat atrial natriuretic factor (ANF 101–126) has been performed. Two-dimensional nmr techniques have been employed, including phase-sensitive nuclear Overhauser spectroscopy (NOESY), relayed coherence transfer spectroscopy (RELAY), and J-correlated spectroscopy (COSY). Experiments were performed both in D₂0 and H₂0 solutions at different pH values. With few exceptions, resonance frequencies were practically pH independent. NOESY spectra were recorded using both 300- and 500-ms mixing times, and no long-range connectivities were observed, leading to the conclusion that ANF 101–126 has no defined secondary nor tertiary structure in water in the pH range used (2.73–5.21).     

Urodilatin (CDD/ANP-95-126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (alpha-hANP/CDD-99-126)

Atrial natriuretic peptide (CDD/ANP-99-126) is rapidly inactivated by a membrane preparations from dog kidney cortex. Inactivation occurs by cleavage of the ring structure in the position between Cys-105 and Phe-106. A unique proteolytic product separated by HPLC on reverse-phase column appears as a single peak which elutes prior the intact peptide. In contrast, CDD/ANP-95-126 (urodilatin) which is released from the kidney is not destroyed by proteolysis using an identical membrane preparation.     

Effect of rat cardionatrin I (rat ANF 99-126) on the response of toad skin to angiotensin II

The atrial natriuretic peptide cardionatrin I (cardionatrin I is ANF 99-126) was used in studies directed to assess its effects on osmotic water permeability (Posm) and short-circuit current (SCC) in isolated toad skin. Results showed that ANF 99-126 (10(-7) M) added to the dermal side of the skin had no effect on basal Posm or SCC. However, ANF 99-126 (3.3 x 10(-8) M) was able to produce a 50% reversible inhibition of the maximal Posm response to angiotensin II (AII) (3.2 x 10(-8) M). These effects were seen when the skins were preincubated with ANF 99-126 for 10 min or less before the addition of AII. Longer preincubation appeared to inactivate ANF 99-126 through proteolysis. ANF 99-126(10(-7) M) failed to inhibit the SCC response to AII (10(-5) M) in toad skin. These results are compatible with a modulatory function for ANF on several systems including those involved in the regulation of extracellular fluid volume.     

Effect of atrial natriuretic peptide on adrenal renin and aldosterone

The effect of atrial natriuretic peptide (ANP) on adrenal renin and aldosterone was investigated in anesthetized rats. Under pentobarbital anesthesia 40 mg/kg), intravenous infusion of ANP (0.25 micrograms/kg/min) for 45 min failed to alter the adrenal renin, adrenal aldosterone, and plasma aldosterone (PA). In this condition, intraperitoneal injection of ACTH (10 micrograms/kg) significantly increased the adrenal renin (from 2.4 +/- 0.1 to 5.0 +/- 0.08 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 13.6 +/- 1.3 to 22.7 +/- 2.3 ng/mg protein, P less than 0.01) and PA (from 59.8 +/- 5.8 to 75.5 +/- 7.4 ng/dl, P less than 0.05), respectively. Under ACTH stimulation, ANP infusion induced significant decreases in adrenal renin (from 5.0 +/- 0.08 to 2.8 +/- 0.2 ng/mg protein/h, P less than 0.05), adrenal aldosterone (from 22.7 +/- 2.3 to 16.2 +/- 1.8 ng/mg protein, P less than 0.05) and PA (from 75.5 +/- 7.4 to 61.6 +/- 4.9 ng/dl). These results suggest a possible role for adrenal renin in the mechanism underlying the inhibitory effect of ANP on aldosterone production in vivo.     

Renal effects of administered atrial natriuretic peptide in the conscious, aging rat

Studies were performed in conscious, chronically catheterized male Sprague-Dawley rats to investigate the effect of administered atrial natriuretic peptide (ANP) on blood pressure, renal hemodynamics and urinary electrolyte excretion. Studies were performed on young adult (3-4 month old) rats and on aging rats (18-24 months of age). Low dose ANP (80 ng/kg/min for 60 min) had no effects on renal hemodynamics in either young or old rats and produced only a slight blood pressure reduction in young animals. No effect on urinary electrolyte excretion was evident in young rats whereas in the old animals, low dose ANP produced large rises in the rate of sodium excretion, fractional excretion of sodium and urine flow rate. A four fold higher dose of ANP evoked a moderate natriuretic and a marked antihypertensive response in young rats. Time control studies indicated that time alone had no influence on urinary sodium excretion rate, the fractional excretion of sodium or urine flow rate. These studies indicate a much enhanced sensitivity to the natriuretic effects of administered ANP by the kidneys of old rats.     

Sites of action of angiotensin II, atrial natriuretic factor and guanabenz, on aldosterone biosynthesis.

It is well known that atrial natriuretic factor (ANF) inhibits aldosterone biosynthesis. Recent studies showed that amiloride can also inhibit adrenal steroidogenesis. Since the antihypertensive agent, guanabenz, is structurally related to amiloride, we have examined its action on aldosterone biosynthesis. The aim of this work was to localize the sites of action of angiotensin II (AII) and of ANF on steroidogenesis and to compare the effects of guanabenz to ANF. Trilostane, an inhibitor of 3 beta-hydroxysteroid dehydrogenase was used to separately study the early and late pathways of aldosterone biosynthesis. The different steps of steroidogenesis are stimulated by AII. ANF inhibits the formation of pregnenolone, the steps between progesterone and deoxycorticosterone, deoxycorticosterone and corticosterone and finally, corticosterone and aldosterone with ED50 of 114 +/- 17, 199 +/- 90, 14 +/- 3 and 92 +/- 34 pM of ANF, respectively, and around 70% of inhibition. These steps are also inhibited by guanabenz with ED50 of 66 +/- 17 microM for the formation of pregnenolone, 1.6 +/- 1.3, 3.3 +/- 1.7 and 29 +/- 4 microM for the last 3 steps. The percentage of inhibition by guanabenz was at least 80% for all the steps except for progesterone to deoxycorticosterone which is less than 35%. These results indicate that the major site of action of both AII and ANF could be at the level of intracellular signal transduction for the activation of mitochondrial steroidogenic enzymes or for the transport of steroids to mitochondria. We also showed that guanabenz mimics the inhibitory effects of ANF. This study with guanabenz suggests that it might be a prototype for a new family of antihypertensive agents.     

Dissociation and enrichment of rat atrial myocytes containing atrial natriuretic factor (ANF)

Methodologies developed for the dissociation and subsequent enrichment of muscle and nonmuscle cells from atrial myocardium were used to evaluate the contribution of these cell populations to the natriuretic, diuretic and vasoactive properties of crude atrial tissue extracts. Suspensions of single cells, which contained approximately 34% myocytes, were prepared from atrial tissue blocks with a collagenase-trypsin digestion followed by gentle mechanical disruption. Differential centrifugation and unit gravity sedimentation techniques were employed to enrich the 'muscle' and 'nonmuscle' cell suspensions to a purity of approximately 91 and 95%, respectively. Cell extracts were bioassayed for natriuretic activity in saline-expanded, pentobarbital-anesthetized, female rats. Extracts obtained from 'initial' and 'muscle' cell suspensions significantly enhanced sodium and chloride excretion as well as urine flow while extracts from 'nonmuscle' cell suspensions had no effect on renal function. Sodium excretion was dose-dependent and increased linearly with increasing numbers of extracted and infused myocytes. This simple two-step centrifugation and sedimentation protocol can be utilized to obtain enriched atrial myocyte populations for subsequent physiologic and biochemical studies.