[Contribution of radioimmunologic methods to the diagnosis of endocrine diseases].
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Endocrine System Diseases↗
Biomedical subjects
J Gutkowska
Publications and source records attributed to J Gutkowska.
Radioreceptor assay for atrial natriuretic factor.
Interest in accurate measurement of atrial natriuretic factor (ANF) in biological fluids and various tissues has been stimulated by recent data indicating the possible role of ANF in the homeostasis of salt and water. The presence of high-affinity binding sites for ANF in rat glomeruli has allowed us to develop a rapid, sensitive, and simple radioreceptor assay (RRA). A saturable high-affinity binding site on the membranes of rat glomeruli has been characterized by a dissociation constant of 33 pM and binding capacity of 396 fmol/mg protein. Rat plasma extracts or atrial homogenates or standards were incubated with radioiodinated ANF and a preparation of rat glomerular membranes. The receptor-bound and free radioactivity were separated by filtration on Whatman GF/C paper after 1 h incubation at room temperature. The sensitivity of the RRA was 2.08 fmol. The effective concentration of standard ANF that displaced 50% of labeled receptor-bound ANF (EC50) was 43.3 +/- 2.6 fmol/ml (n = 7). Both intra- and interassay coefficients of variation were smaller than 11%. This RRA assay has been compared with radioimmunoassay (RIA). High correlations for 19 plasma extracts and 34 atrial homogenates (r = 0.973 and r = 0.954, respectively) tested by RRA and RIA were obtained. This good correlation between the two methods suggests that the immunoreactive material found in rat plasma and atrial homogenates also displays biological activity.
Animals↗
Clonidine stimulates atrial natriuretic factor (ANF) release in water-deprived rats.
To determine the effect of clonidine, an alpha 2-adrenergic agonist, on atrial natriuretic factor (ANF) release during water deprivation, plasma immunoreactive ANF (IR-ANF) arginine vasopressin, diuresis and natriuresis were measured in rats which had been deprived of water for 24 and 48 hr after intravenous (IV) administration of 50 micrograms clonidine. In normally-hydrated rats clonidine produced a marked elevation of plasma IR-ANF from 40.5 +/- 4.6 pg/ml to 1064 +/- 22 pg/ml (mean +/- SEM) and sodium excretion from 73.3 +/- 6.8 microEq to 723.4 +/- 62.3 microEq. Clonidine evoked an increase in plasma IR-ANF from 16.6 +/- 5.9 pg/ml to 229.5 +/- 60 pg/ml (mean +/- SEM) after 24 hr water deprivation and from 13.6 +/- 7.4 pg/ml to 104.8 +/- 21 pg/ml (mean +/- SEM) after 48 hr water deprivation. Clonidine did not induce any significant changes in vasopressin levels. During 24 hr and 48 hr water deprivation vasopressin rose from 3.1 +/- 0.3 pg/ml to 7.3 +/- 1.3 pg/ml and 8.4 +/- 0.6 pg/ml (mean +/- SEM), respectively. In normally-hydrated rats clonidine produced a marked diuresis and natriuresis. These effects and urinary cGMP excretion were significantly inhibited by anti-ANF antibodies. Clonidine caused a significant increase in urine output in 24 hr water-deprived rats but the response was markedly lower than that seen in normally-hydrated rats. In conclusion, clonidine stimulates ANF release both in normally-hydrated and water-deprived rats. The diuretic effect of clonidine appears to be related to ANF release but not to inhibition of vasopressin.
Animals↗
Atrial natriuretic factor increases after a protein meal in man.
1. The renal function changes induced by dietary protein are thought to result from the activity of hormonal factors that remain as yet undefined. Since a meat meal and high dose atrial natriuretic factor (ANF) infusions have similar effects on glomerular filtration rate, natriuresis and kaliuresis, we decided to investigate the possibility that a protein meal could stimulate ANF activity. 2. We studied 10 normal volunteers who had a fixed protein and sodium intake for 7 days before the experiments. The subjects received a meat meal (1-1.5 g of protein/kg) and, on a separate occasion, a carbohydrate meal that had a similar caloric, sodium and potassium content. Diuresis was stimulated with water ingestion, and urine collections were obtained before the meals (baseline) and after the meals for a period of 3 h. Blood samples were obtained 30 min and 5 min before the meals and every hour for 3 h in the period after the meal. 3. The protein meal, but not the carbohydrate meal, was associated with parallel increments in plasma immunoreactive ANF (i-ANF), natriuresis, kaliuresis and glomerular filtration rate (estimated from creatinine clearances) which reached peak values 2-3 h after the meal. The mean increment of plasma i-ANF after the protein meal represented a twofold increase over baseline levels. 4. We conclude that ANF may participate in the physiological response to an oral protein load.
Atrial Natriuretic Factor↗
Atrial natriuretic factor in experimental and human hypertension.
Plasma levels of immunoreactive atrial natriuretic factor (IR-ANF) were evaluated by radioimmunoassay in several models of experimental hypertension and in human hypertension. Plasma levels of IR-ANF are consistently increased in all models of experimental hypertension studied so far. This is accompanied by a decrease of IR-ANF levels in the left atrium at certain times. Plasma levels of IR-ANF are not increased in human essential hypertension, except in the severe form (diastolic blood pressure above 110 mmHg). Peripheral levels of IR-ANF in renovascular hypertension do not differ from normal but are increased above normal in aortic blood.
Animals↗
The atrial natriuretic factor in hypertension.
Plasma immunoreactive atrial natriuretic factor (IR-ANF) concentration measured by radioimmunoassay after extraction on Sep-Pak cartridges was studied in 64 control normotensive subjects, 25 patients with labile essential hypertension, 67 patients with mild essential hypertension (diastolic pressure between 90 and 105 mm Hg and no left ventricular hypertrophy) and 9 patients with moderate to severe essential hypertension (diastolic pressures between 105 and 120 mm Hg). An additional group of 16 patients under medication but without effective control of their blood pressure and with diastolic pressure above 110 mm Hg also was studied. Results show that plasma IR-ANF concentrations are within normal range in patients with labile, mild, and moderate hypertension. In view of the reported increased right and left atrial pressures and distension in patients with mild and moderate hypertension, these findings strongly suggest a state of hyporesponsiveness of the atria to release ANF.
Animals↗
Amlodipine in elderly hypertensive patients: pharmacokinetics and pharmacodynamics.
Elderly (65-73 years) and young (28-34) hypertensive patients received amlodipine by i.v. infusion (2.5, 5.0, or 10.0 mg). Patients were then started on oral amlodipine 2.5 mg daily for 2 weeks, at the end of which amlodipine disposition and effect were evaluated over one 24-h dose interval. Patients were treated subsequently with amlodipine in an escalating dose protocol (maximum 10.0 mg once daily) for 12 weeks to control blood pressure. After i.v. amlodipine, clearance tended to be decreased in elderly as compared with young patients with resulting prolongation in elimination half-life (64 +/- 20 vs. 48 +/- 8 h; mean +/- SD). Maximum decrease in systolic blood pressure (SBP) after i.v. doses tended to be greater in the elderly (-34 +/- 15 vs. -23 +/- 15 mm Hg) and maximum decrease in diastolic blood pressure (DBP) was similar in the two groups (-21 +/- 10 vs. -18 +/- 7 mm Hg). SBP was significantly decreased after 14 weeks' therapy in the elderly at doses ranging from 2.5 to 10.0 mg per day (171 +/- 17 to 149 +/- 22 mm Hg; p less than 0.01). DPB was decreased both at 2 and 14 weeks' therapy in the elderly (baseline 100 +/- 7, 2 weeks 93 +/- 5, 14 weeks 90 +/- 5 mm Hg; p less than 0.01 vs. baseline).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Distribution of atrial natriuretic factor-like immunoreactivity in the brain of the frog Rana ridibunda.
The localization of atrial natriuretic factor (ANF)-like immunoreactivity in the central nervous system of the frog Rana ridibunda was examined by the indirect immunofluorescence technique, using an antiserum against synthetic ANF (Arg101-Tyr126). Immunoreactive cell bodies were principally found in the dorsal and medial pallium, the medial septal nucleus, the ventrolateral and anteroventral areas of the thalamus, the lateral forebrain bundle, the posterolateral thalamic nuclei, the preoptic nucleus, the dorsal infundibular nucleus, and the anteroventral tegmentum nucleus of the mesencephalon. Numerous cell bodies and a very dense fiber bundle were visualized in the interpeduncular nucleus. All the areas mentioned above contained a high density of immunoreactive fibers. In addition, the amygdala, the infundibular nucleus, the median eminence, and most of the areas of the mesencephalon contained a moderate number of ANF-positive nerve processes. In the frog pituitary, fibers and nerve terminals were found in the peripheral zone of the neural lobe. The intermediate and anterior lobes of the frog pituitary were totally devoid of ANF immunoreactivity. These results indicate that ANF-like material is widely distributed in the frog brain and that ANF may be involved in various brain functions including neuroendocrine regulations.
Animals↗
Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary.
The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.
Adrenal Cortex↗
Vasorelaxation and vascular binding sites for atrial natriuretic peptide in pregnant rats.
To investigate the role of atrial natriuretic peptide (ANP) in pregnancy, we measured, in cyclic and pregnant female rats (9- and 21-days pregnant), the vascular responsiveness to ANP using helical strips of the thoracic aorta, the binding characteristics of 125I-labeled ANP in a membrane preparation of the mesenteric vascular bed, and the plasma level and the atrial content of immunoreactive ANP (IR-ANP). On aorta strips, concentration-response (C-R) curves to phenylephrine (PE) were measured and were slightly displaced to the right in the aorta of both groups of pregnant rats in comparison with the cyclic rats. There was a potentiation of the relaxant response of ANP on the PE-precontracted aortic strips of 9-day pregnant rats but it was not statistically modified in tissues of 21-day pregnant rats in comparison with strips from cyclic rats. The number of binding sites (Bmax) for ANP in the mesenteric vascular bed was similar in cyclic rats and the two groups of pregnant rats. The dissociation constant (KD) of ANP was lower in 9-day pregnant rats than in cyclic and 21-day pregnant ones. Plasma IR-ANP was not different in 9-day pregnant rats and cyclic rats but was markedly decreased at the end of gestation. Atrial content of IR-ANP increased at the end of gestation, but not in midpregnancy in comparison with cyclic rats. These results indicate that despite the reported important increase in blood volume during gestation the secretion of ANP is not increased and suggest that the ANP-volume relationship is reset during pregnancy in the rat.
Animals↗
Atrial distension of isolated rabbit hearts and release of atrial natriuretic factor.
Interventions that increase atrial pressures in humans or laboratory animals release atrial natriuretic factor (ANF) into the circulation. We studied the relation between distension of the right or left atrium and release of ANF in retrograde-perfused isolated rabbit hearts. A fluid-filled balloon within the right or left atrium was inflated to a mean pressure of 5, 10, 15, or 20 mmHg, and ANF in the cardiac effluent was measured by radioimmunoassay. The slope of the regression line relating ANF release to atrial distending pressure was steeper for the left than right atrium (P less than 0.001), indicating that, at comparable increases in mean pressures, the left atrium releases more ANF than does the right atrium. Left atrial tissue concentration of ANF was greater than right atrial (1.58 +/- 0.15 vs. 1.05 +/- 0.09 micrograms ANF/mg protein, P less than 0.01). In contrast to previous studies showing right atrial dominance in rats, the left atria of isolated, perfused rabbit hearts contain more ANF and release more in response to atrial distension.
Animals↗
Glomerular ANF receptor regulation during changes in sodium and water metabolism.
The concentration of atrial natriuretic factor (ANF) in atria and plasma was investigated in relation to the regulation of renal glomerular ANF receptors in the rat during changes in water and sodium intake. A decrease in plasma immunoreactive ANF (IR-ANF) was observed after 4 days of water deprivation or after 1 wk on a low-sodium diet, whereas animals offered 1% NaCl in their drinking water had elevated plasma ANF values. Atrial IR-ANF was lower in water-deprived and higher in sodium-restricted rats than in their respective controls. A low-sodium intake or water deprivation increased the density of glomerular ANF receptors, whereas the inverse occurred with a high-salt consumption. It is concluded that an inverse correlation exists between ANF plasma concentration and renal glomerular ANF receptor sites.
Animals↗
Hemodynamic and renal responses to volume expansion in dogs with cardiac denervation.
Hemodynamic responses, renal function, and plasma levels of immunoreactive atrial natriuretic factor (irANF) were examined following volume expansion (VE) in normal (N) conscious dogs and in conscious dogs with cardiac denervation (CD). Base-line urine flow was consistently greater (P less than 0.05) in dogs with CD (0.54 +/- 0.06 ml/min) than in N (0.29 +/- 0.03 ml/min) dogs but sodium excretion did not differ between N (2.80 +/- 0.58 mu eq.min-1.kg body wt-1) and CD (3.53 +/- 0.75 mu eq.min-1.kg-1) groups. With VE (18 ml/kg of 3% dextran in saline), mean arterial pressure (MAP) increased (P less than 0.01) by 16 +/- 3 from 103 +/- 4 mmHg in N dogs but did not change from pre-VE base line (103 +/- 2 mmHg) in dogs with CD. At 10 min after VE, urine flow increased more (P less than 0.01) in N dogs (1.39 +/- 0.24 ml/min) than in dogs with CD (0.26 +/- 0.09 ml/min). At that time, increases in sodium excretion were also greater (P less than 0.01) in N (9.13 +/- 1.96 mu eq.min-1.kg-1) dogs than in dogs with CD (1.06 +/- 0.68 mu eq.min-1.kg-1). With VE, increases in irANF plasma levels were not different in N dogs (40 +/- 12 from 34 +/- 5 pg/ml) and in dogs with CD (27 +/- 3 from 45 +/- 7 pg/ml). In dogs with CD, when MAP was increased by aortic constriction to mimic responses observed in N dogs, renal responses were similar to those of N dogs.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The atrial natriuretic factor in hypertension. State of the art lecture.
Studies were conducted to assess the effects of bolus injections and infusions of human atrial natriuretic factor (ANF) in control subjects and patients with mild essential hypertension, and to measure plasma immunoreactive ANF (irANF) concentration in a large group of patients with essential hypertension. The results are compared with those obtained by other groups on the measurements of plasma irANF in hypertensive patients. It appears that plasma irANF concentrations are not increased in patients with mild essential hypertension despite the evidence of increased preload and of atrial distention as reported by others. This suggests a hyporesponsiveness of the atria to release ANF.
Atrial Natriuretic Factor↗
Atrial natriuretic factor immunoreactivity in human fetal lung tissue and perfusates.
Immunoreactive atrial natriuretic factor (ANF) was detected in human fetal homogenates and perfusates using a sensitive and specific radioimmunoassay for the 28 amino acid (C-terminal) fragment. Three peaks of ANF immunoreactive material were found in the lung homogenates. With high performance liquid chromatography, the elution characteristics of the first immunoreactive peak were the same as those of circulating human ANF. The other two peaks have not been characterized, although one had a position similar to the 126 amino acid rat prohormone (Asn 1-Ile 110-Tyr 126). The time course of release of immunoreactive ANF by perfused human fetal lungs was also studied. It is suggested that ANF may play a role in early pulmonary function.
Atrial Natriuretic Factor↗
Atrial natriuretic peptide in rats with experimental cirrhosis of the liver without ascites.
Plasma levels of atrial natriuretic peptide (ANP) have been measured in eight sodium-retaining cirrhotic nonascitic rats and eight control animals before and after extracellular volume expansion by isotonic saline infusion (30 ml/kg BW, 20 min). In addition, disappearance of [125I]ANP was studied in six control and six cirrhotic rats. The effect of infusing synthetic rat ANP-(1-28) (1 microgram) on mean arterial pressure and renal function has been also studied. In basal conditions, cirrhotic rats showed higher ANP plasma levels than control animals (71.1 +/- 16.6 vs. 43.9 +/- 5.1 pg/ml; P less than 0.05). After extracellular volume expansion, ANP increased in both control and cirrhotic rats; the increase in cirrhotic was higher than that in control rats (88 +/- 27% vs. 33 +/- 8%; P less than 0.05). Disappearance of iodinated ANP from plasma was identical in control and cirrhotic rats. ANP infusion induced a larger decrease in mean arterial pressure in control (21 +/- 5%) than in cirrhotic rats (9 +/- 2.5%). ANP induced comparable increases in glomerular filtration rate and renal plasma flow in both groups of animals. However, diuretic and natriuretic effects were markedly impaired in cirrhotic animals. Thus, urinary flow increased by 91 +/- 18 microliters/min in control animals, but only by 37 +/- 7 microliters/min in cirrhotic animals. Fractional sodium excretion increased to 1.7 +/- 0.44% in controls and to 0.54 +/- 0.12% in cirrhotic rats (P less than 0.05). It is concluded that the defect in renal handling of sodium in cirrhotic rats is not due to a lack of ANP synthesis or release. In addition, these animals show an impaired renal response to ANP.
Animals↗
Is clonidine-induced diuresis mediated by atrial natriuretic factor?
The effect of intracerebroventricular (ICV) administration of clonidine on urine output, urinary sodium excretion, urinary cGMP, and plasma immunoreactive atrial natriuretic factor (IR-ANF) was studied in conscious, normally hydrated rats. Clonidine treatment evoked a significant dose-dependent increase in urine output. A 20-fold elevation was noted after the highest clonidine dose (2 micrograms/rat). The observed diuresis was accompanied by enhanced sodium excretion, which with the highest dose (2 micrograms) of clonidine increased from 1.6 +/- 0.36 to 39.4 +/- 10.5 meq/liter (P less than 0.001). Plasma IR-ANF rose from 30.7 +/- 8.8 to 113.3 +/- 32.3 pg/ml plasma 5 min after the 0.5 micrograms clonidine dose (P less than 0.05), and urinary cGMP excretion was augmented from 8.49 +/- 4.29 to 27.7 +/- 5.0 pmol/min 1 h after 0.5 micrograms clonidine (P less than 0.05). Pretreatment with peripherally administered anti-ANF serum abolished the diuretic effect of intracerebroventricularly administered clonidine; urine output decreased from 1.49 +/- 0.41 to 0.42 +/- 0.21 ml/h. The urinary cGMP level after anti-ANF serum treatment fell from 25.0 +/- 7.56 to 7.1 +/- 3.5 pmol/min (P less than 0.05). Peripheral pretreatment with the alpha 2-antagonist yohimbine or the opioid antagonist naloxone partially abolished clonidine's diuretic impact: urine output dropped from 1.91 +/- 0.55 to 0.42 +/- 0.18 and 0.46 +/- 0.18 ml/h (P less than 0.05), respectively. At the same time, plasma IR-ANF decreased from 113.3 +/- 32.2 to 30.3 +/- 11.4 after yohimbine and to 24.6 +/- 12.1 pg/ml after naloxone treatment (P less than 0.05). These data suggest that ANF may be involved in the mechanism of diuresis of centrally applied clonidine, which appears to enhance ANF release through its central stimulation of opiate and alpha 2-adrenergic receptors.
Animals↗
ANF in experimental congestive heart failure.
The plasma and cardiac levels of immunoreactive (IR) atrial natriuretic factor (ANF) were measured during the entire lifespan of cardiomyopathic hamsters, which eventually develop spontaneous congestive heart failure, and were correlated with immunohistochemical, ultrastructural, and immunocytochemical changes in the secretory apparatus of atrial and ventricular cardiocytes. Plasma IR-ANF rose in the early stages of the disease, reached a maximum in moderate heart failure, and declined thereafter but remained above control values. The peptide decreased constantly in the atria during the evolution of the disease but increased markedly in the ventricles. Its highest levels were found in the inner half of the left ventricle. In atrial cardiocytes, the size and complexity of the Golgi complex increased with the progression of the disease, whereas the number, size, and IR-ANF content (as assessed by the immunogold technique) of secretory granules decreased constantly. In ventricular cardiocytes, the size of the Golgi complex increased, and typical secretory granules were present in approximately 20% of these cells, regardless of their localization in the myocardium. The results suggest that stimulation of ANF secretion in atrial cardiocytes leads to a dissociation between synthesis and release, the latter being maximal according to ultrastructural and immunocytochemical criteria. In ventricular cardiocytes, the same stimulation culminates in increased synthesis and the possibility of release via two pathways: one constitutive, the other regulated. Thus, the elevated plasma levels of IR-ANF in congestive heart failure may be derived from secretion by both atrial and ventricular cardiocytes.
Animals↗