Influence of isoproterenol on plasma immunoreactive atrial natriuretic peptide and plasma vasopressin in the anesthetized rabbit

Changes in levels of plasma immunoreactive atrial natriuretic peptide (IR-ANP) were measured in response to administration of isoproterenol in the anesthetized, vagotomized rabbit. A dose-dependent increase in plasma IR-ANP was seen in response to 10 min isoproterenol infusions between 0.1 and 10.0 g/kg/min. The time course of these responses showed the maximum levels of IR-ANP to be attained 10 min after the cessation of infusion. In rabbits in which plasma vasopressin (AVP) levels were also measured, the maximum levels of AVP were attained during the infusion period. There was no correlation between levels of AVP and IR-ANP suggesting that AVP released into the plasma did not affect directly the release of IR-ANP. The changes in IR-ANP in response to isoproterenol were significantly reduced in rabbits which had been administered the beta-1-adrenoceptor blocking agent, atenolol. In six rabbits in which the vagi remained intact, the increases in IR-ANP were reduced and became significant only with 10 g/kg/min isoproterenol infusion. The results demonstrate that isoproterenol infusion increases the level of plasma IR-ANP in the anesthetized rabbit and suggest that this is through an effect on the heart rather than on peripheral vessels.     

A sensitive enzyme immunoassay for atrial natriuretic polypeptide

A sensitive sandwich enzyme immunoassay for mammalian alpha-atrial natriuretic polypeptide (alpha-ANP) has been developed using Fab' fragments of affinity purified rabbit anti-alpha-hANP[6-28] polyclonal antibodies (R3) conjugated to horseradish peroxidase. Immunoplates were coated with (F(ab')2) fragments of anti-alpha-hANP monoclonal antibodies (HA53). Without using a pre-concentration step the detection limit of alpha-hANP in plasma was 5 pg/ml. The assay was sufficient accuracy and reproducibility for general use of, i.e., the coefficients of within-assay and between-assay variation were 6.4-10.7% and 9.7-15.3%, respectively. Using the assay, the basal plasma alpha-hANP level of healthy persons was 15.3 +/- 8.3 pg/ml (mean +/- SD, n = 40).     

Atrial natriuretic polypeptide: topographical distribution in the rat brain by radioimmunoassay and immunohistochemistry

The widespread distribution of neurons containing alpha-atrial natriuretic polypeptide-like immunoreactivity in the rat brain was demonstrated using radioimmunoassay and immunohistochemistry in conjunction with specific antisera. The highest concentrations of alpha-atrial natriuretic polypeptide-like immunoreactivity were in the hypothalamus and septum, with low but still appreciable concentrations in the mesencephalon, cerebral cortex, olfactory bulb and thalamus by radioimmunoassay. Immunohistochemical studies clearly showed that the perikarya of immunoreactive neurons are most prevalent in the ventral part of the lateral septal nucleus, periventricular preoptic nucleus, bed nucleus of the stria terminalis, periventricular and dorsal parts of the paraventricular hypothalamic nucleus, ventromedial nucleus, dorsomedial nucleus, arcuate nucleus, median mamillary nucleus, supramamillary nucleus, zona incerta, medial habenular nucleus and the periaqueductal grey matter. Scattered neurons were seen in the cingulate cortex, endopiriform nucleus, lateral hypothalamic area, and pretectal and dorsal thalamic areas. In addition to the areas mentioned above, high concentrations of immunoreactive varicose fibers were seen in the glomerular layer of the olfactory bulb, external layer of the median eminence, central to paramedian parts of the interpeduncular nucleus and the paraventricular hypothalamic nucleus. The globus pallidus, medial and central amygdaloid nuclei, dorsal raphe, dorsal parabrachial nucleus, locus coeruleus, vagal dorsal motor nucleus, solitary nucleus and some circumventricular organs, including the subfornical organ and organum vasculosum laminae terminalis, contained considerable numbers of immunoreactive varicose fibers. In dehydrated rats and homozygous Brattleboro rats, the pattern of alpha-atrial natriuretic polypeptide-immunoreactive neurons and varicose fibers was qualitatively similar to that seen in normal conditioned rats. This study gives an atlas of the distribution of the alpha-atrial natriuretic polypeptide-containing neuronal system in the rat brain and provides the groundwork for studying the influence of this new peptide on various brain functions.     

Antidipsogenic action of alpha-human atrial natriuretic polypeptide administered intracerebroventricularly in rats

Intracerebroventricular (i.c.v.) administration of alpha-human atrial natriuretic polypeptide (alpha-hANP) in a dose of 5 micrograms did not change water intake in normal rats, while 0.1 micrograms of angiotensin II (AII) and 0.5 micrograms of carbachol caused a marked increase in water intake for 30 min after i.c.v. injections. The water intake induced by 0.1 micrograms of AII was significantly suppressed by the simultaneous administration of 2 and 5 micrograms of alpha-hANP. However, alpha-hANP did not affect the water intake caused by 0.5 micrograms of carbachol. In 24-h water-deprived rats, alpha-hANP in doses of 2 and 5 micrograms pronouncedly inhibited the water intake. alpha-hANP did not change the food intake in 24-h fasted rats nor the locomotor activity in normal rats. These findings suggest that alpha-hANP in the central nervous system may play an important role in controlling drinking behavior, interacting with AII.     

Inhibition of neuronal Cl-stimulated Mg-ATPase by alpha-human atrial natriuretic polypeptide

Effects of alpha-human atrial natriuretic polypeptide (alpha-hANP) on neuronal membrane-bound adenosine triphosphatases (ATPases) (Na, K-ATPase, Cl-stimulated Mg-ATPase and anion-insensitive Mg-ATPase) were examined using rat brain microsomes. This peptide inhibited Cl-stimulated Mg-ATPase in a dose-dependent manner. The inhibition was non-competitive with respect to ATP, and was not affected by Cl-, the most potent stimulator of this enzyme. The activities of Na,K-ATPase and anion-insensitive Mg-ATPase were not changed by alpha-hANP at all.     

Lack of effect of alpha-human atrial natriuretic polypeptide on volume reabsorption and p-aminohippuric acid secretion in the rabbit proximal straight tubule

To assess a possible direct tubular action of alpha-human atrial natriuretic peptide (alpha-hANP), we studied the effects of alpha-hANP on volume reabsorption (JV), transepithelial voltage (VT), and p-aminohippuric acid (PAH) secretion in the rabbit proximal straight tubules (PST) by in vitro microperfusion technique. In superficial PST (SFPST), addition of alpha-hANP at various concentrations (10(-7), 10(-9), 10(-11) M) to the bath solution did not alter JV significantly. Bath alpha-hANP (10(-7) M) did not change VT in SFPST, either. In juxtamedullary PST (JMPST), addition of alpha-hANP (10(-7) M) to the bath solution changed neither VT nor JV significantly. The alpha-hANP (10(-7) M) in the luminal fluid caused no changes in either VT or JV in SFPST. Furthermore, alpha-hANP (10(-7) M) in the bath did not change the rate of PAH secretion in SFPST. Thus, we could not obtain any evidence for direct tubular action of alpha-hANP in rabbit PST. Accordingly, alpha-hANP may increase solute excretion without directly affecting solutes transport in PST of the rabbit kidney.     

A new radioimmunoassay for human alpha atrial natriuretic peptide and its physiological validation

A new sensitive, specific radioimmunoassay for human alpha atrial natriuretic peptide (hANP) and a novel extraction method for hANP have been developed. Antiserum to hANP (Keq = 1.923 x 10(11) l/mol) showed no cross-reactivity with related analogues. Displacement of 50% bound 125I-hANP occurred at 4.7 +/- 0.1 fmol/tube (n = 15). The limit of detection of plasma hANP after extraction of 2 ml plasma with Florisil was 1.2 pmol hANP/litre plasma. The recovery of synthetic hANP from plasma over the range 6.5-162.5 pmol/l was 71.2 +/- 1.9%. Inter- and intra-assay coefficients of variation were 13.1% and 10.1% respectively. Extracted plasma was stored at -80 degrees C without loss in immunoreactivity. The radioimmunoassay was physiologically validated in man by measuring plasma hANP following central volume expansion - (a) plasma hANP rose from 2.5 +/- 0.5 to 4.1 +/- 0.6 pmol/l in 9 normal volunteers after postural change from seated to lying (b) infusion of normal saline (2 litre/2 hour) increased hANP from 1.6 to 4.3 pmol/l (n = 2). Infusion of hANP (2 pmol/kg/min) increased plasma hANP to 19.9 +/- 3.4 pmol/l (n = 6).     

The inotropic effect of atrial natriuretic factor in the anesthetized rabbit

This study was designed to investigate whether atrial natriuretic factor (ANF) administered over the physiological, pathological and pharmacological range has a negative inotropic action on the heart. Anesthetized rabbits were infused with increasing doses of ANF (0.05, 0.25 and 0.5g kg^–1min^–1), while measuring hemodynamic variables including the maximum rate of change of left ventricular pressure (dP/dt _max) as an index of inotropic state. Plasma levels of immunoreactive ANF (iANF) were measured to relate the hemodynamic changes to actual plasma levels of the peptide. Administration of ANF was associated with decreases in blood pressure, left ventricular pressure and dP/dt _max so that after 0.5 g kg^–1 min^–1 infusion, these variables had decreased by 21±2 mmHg, 21±5.3 mmHg and 925±175 mmHg/s, respectively (P<0.01). There were no significant changes in right atrial pressure, left ventricular end-diastolic pressure or heart rate. Since dP/dt _max can be influenced by changing hemodynamic variables and baroreflex changes, a second group of rabbits was studied in which afterload and heart rate were held artificially constant. Again, in this group of rabbits, infusions of ANF led to decreasing inotropic state, so that at the highest infusion rate, a 14% decrease in dP/dt _max was observed (P<0.05). By comparison, hydralazine, a drug which causes active vasodilatation but no direct inotropic action, significantly (P<0.01) decreased blood pressure, left ventricular pressure and dP/dt _max when infused at a rate of 10 g kg^–1 min^–1. However, in animals in which afterload was controlled, hydralazine did not affect any of the variables measured. The results indicate that ANF does have a negative inotropic action in the anesthetized rabbit.     

Identification and distribution of atrial natriuretic polypeptide in ventricular myocardium of humans with myocardial infarction

The expression of atrial natriuretic polypeptide (ANP) in the ventricles of human hearts with myocardial infarction (MI) was studied immunohistochemically. Immunoreactive myocytes were identified in the ventricular tissues of all of 16 hearts with old MI (both with and without heart failure) and in all five hearts with subacute MI, but not in any of the eight hearts without MI nor in the five with acute MI. In the nonfailing hearts with MI, ANP positive myocytes surrounded the areas of infarction, and were also seen in the subendocardium of the infarcted segment. In the failing hearts with MI, ANP expression was noted in the whole ventricular subendocardial region, in addition to the border of infarcts. The sites of ANP expression corresponded well to those of marked stress attributable to tissue shrinkage or fibrosis due to MI, haemodynamic overload, or both. It thus appears that ANP expression is augmented in human hearts with MI regardless of the presence or absence of heart failure, and it is suggested that regional mechanical stress on the ventricular myocardium, as well as haemodynamic overload, may be very closely associated with ventricular ANP expression.     

Lack of direct action of α-human atrial natriuretic polypeptide on the in vitro perfused segments of Henle's loop isolated from rabbit kidney

We examined direct effects of -human atrial natriuretic polypeptide (-hANP) on water and NaCl transport across the segments of Henle's loop by the in vitro microperfusion technique. In the medullary and the cortical thick ascending limb, 10^–6 M -hANP did not affect the transmural voltage (V _t) and the lumen to bath³⁶Cl flux whether it was added to the perfusate or to the bath. In the thin ascending limb, 10^–6 M -hANP did not affect the lumen to bath³⁶Cl flux. The peptide may not affect permselectivity of the thin ascending limb, since it did not influence the diffusion potential generated in the presence of a NaCl gradient. In the descending limb, 10^–6 M -hANP did not affect osmotic water permeability, whether it was added to the perfusate or to the bathing fluid. From these observations, we conclude that -hANP does not show direct effects on water and NaCl transport in the segments of Henle's loop at least under our experimental conditions. Therefore, natriuresis by -hANP may be caused either by an action on nephron segments other than Henle's loop or by an action on renal vasculatures.     

Possible involvement of central atrial natriuretic polypeptide in regulation of hypothalamo-pituitary-adrenal axis in conscious rats

The effects of intracerebroventricular (i.c.v.) administration of angiotensin II (AII) and atrial natriuretic polypeptide (ANP) on the plasma corticosterone level were studied in conscious, unrestrained rats. Although i.c.v. injection of ANP had no apparent effect on the basal plasma corticosterone level, it attenuated the plasma corticosterone increase induced by centrally injected AII dose-dependently. These results suggest that ANP in the brain is involved in the regulation of the hypothalamo-pituitary-adrenal axis.     

Changes in plasma atrial natriuretic peptide during exercise in healthy volunteers

Graded exercise was performed in three healthy volunteers. Plasma levels of immunoreactive atrial natriuretic peptide (iANP) were determined at different workloads. Unchanged or slightly decreased plasma levels of iANP were observed during light exercise, whereas at medium to high workloads a considerable increase in plasma levels of iANP was found. Factors responsible for the increase in plasma levels of iANP might include elevated right atrial pressure and increased plasma levels of epinephrine and norepinephrine.     

Hyperbaric diuresis is associated with decreased antidiuretic hormone and increased atrial natriuretic polypeptide in humans.

When men are exposed to a hyperbaric environment, urine flow increases. In order to elucidate the mechanism of this hyperbaric diuresis, a dry saturation dive experiment was carried out. Five male subjects were exposed to a 16-21 ATA (atmospheric pressure absolute) helium-oxygen (He-O2) environment for 4 days. Five blood samples were obtained in the early morning (0600-0630 h): once at predive 1 ATA air, 3 times at 16-21 ATA He-O2, and once at postdive 1 ATA air. Eight-hour timed urine samples, 0600-1400 h, 1400-2200 h, and 2200-0600 h (night urine), were collected throughout the experimental period. Urine flow markedly increased by the exposure to hyperbaria in the presence of constant creatinine clearance. The increase was mostly attributable to the urine flow during 2200-0600 h. The secretion of antidiuretic hormone (ADH) was suppressed at daytime and night during the exposure. On the other hand, the secretion of atrial natriuretic polypeptide (ANP) increased solely at night during hyperbaria and correlated with the increases of both the nocturnal urine flow and the nocturnal urinary excretion of sodium. These results suggest that both suppressed ADH secretion and stimulated ANP secretion cause hyperbaric diuresis.     

Human atrial natriuretic factor (ANF). Characterisation of a monoclonal antibody panel and its use in radioimmunoassay

The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1-28) is described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (KD = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma.     

Effect of atropine on plasma atrial natriuretic peptide concentration in healthy subjects

This work shows that an intravenous infusion of atropine increases the concentration of atrial natriuretic peptide (ANP) in the plasma of healthy subjects. The effect is discussed in reference to the heart rate and the role of the autonomic nervous system in the control of ANP secretion by atrial cardiocytes.     

Concomitant increase in plasma atrial natriuretic peptide and cyclic GMP during volume loading

Summary To investigate the effects of fluid expansion on endogenous atrial natriuretic peptide (ANP) and cyclic 3,5-guanosine monophosphate (cGMP), four male volunteers were studied before, during and after intravasal volume loading. Volume expansion was performed by intravenous infusion of 2,000 ml isotonic saline solution within 30 min. Mean plasma ANP levels increased 2.5-fold from 31.2 pg/ml to 81.7 pg/ml 40 min after the start of infusion. Plasma cGMP levels paralleled the rise in ANP, shwoing a mean cGMP increment from 2.7 pmol/ml to a maximum of 8.2 pmol/ml. Both ANP and cGMP levels were back to basal levels 120 min after termination of the infusion. Stimulation of endogenous ANP release by volume loading suggests that ANP is involved in the regulation of fluid homeostasis in man. The parallel rise in plasma cGMP levels supports the idea that cGMP is a mediator for the effects of ANP.Abbreviations ANP atrial natriuretic peptid - cGMP cyclic 3,5-guanosine monophosphate - PRA plasma renin activity     

The distribution of atrial natriuretic polypeptide (ANP)-containing cells in the adult rat heart

Summary The present study was performed to clarify the distribution of ANP-containing cells in the adult rat heart by immunostaining for ANP using antiserum against -human ANP. ANP-immunoreactive cells were generally present in the atrial walls except for the sinoatrial node. In the ventricular walls, they were distributed in the impulse conducting system, particularly the left bundle branch, Purkinje fibers on the left side of the interventricular septum, and those in the false tendons in the left ventricle, while they were sporadically seen in the atrioventricular node and bundle of His. The immunoreactive cells contained specific granules that were positive for ANP. These findings demonstrate that ANP-containing cells are present in the atrial and ventricular walls.