Specific binding of atrial natriuretic factor to renal glomeruli in Doca- and Doca-salt-treated rats correlation with atrial and plasma levels

Since volume expansion and high blood pressure (BP) are known stimuli of atrial natriuretic factor (ANF) release, and since this peptide may be involved in mineralocorticoid escape, we investigated the effects of chronic deoxycorticosterone (DOCA) and DOCA-NaC1 treatment on renal glomerular ANF receptor density and affinity in relation to atrial and plasma ANF levels. An increase in plasma immunoreactive ANF (IR-ANF) was observed both after two and four weeks of treatment. IR-ANF concentrations were elevated in the left atrium only in four-week DOCA treated rats. Administration of the mineralocorticoid alone resulted in a decreased density of glomerular ANF receptors in both time periods investigated. DOCA-NaC1-treated animals presented an increased receptor density during the pre-hypertensive stage (2 weeks) and a reduced density in the later hypertensive period (4 weeks). Receptor affinity in both groups was identical to that in the controls after 2 weeks and was augmented after 4 weeks of treatment. Our data suggest that the down-regulation of renal glomerular ANF receptors during chronic DOCA-NaC1 administration may play a role in the maintenance of high BP in this model of volume-expanded hypertension.     

Comparative biological activities of ANF (Arg 101-Tyr 126) and the synthetic form of circulating ANF (Ser 99-Tyr 126)

The biological activities of ANF (Arg 101-Tyr 126) and of the circulating form, ANF (Ser 99-Tyr 126), were compared in the following assays: precontracted rabbit aortic strip and chick rectum, rat natriuresis, inhibition of aldosterone secretion and receptor affinity in bovine and rat adrenal zona glomerulosa cells, and receptor affinity in rabbit aorta and rat mesenteric artery cells. The results demonstrate that both peptides share the same biological activities. It is concluded that the addition of two amino acids to the N-terminal of ANF (Arg 101-Tyr 126) does not modify its biological characteristics, validating thus previous research employing this peptide.     

Partial characterization and solubilization of receptors for atrial natriuretic factor in rat glomeruli

Specific receptors for atrial natriuretic factor (ANF) have been identified and solubilized in glomeruli from rat kidney. Radioiodinated synthetic ANF (Arg 101-Tyr 126) bound to a single class of high affinity (Kd 27 +/- 24 pM) sites with a density of 390 +/- 230 fmole/mg protein. The binding was time- and temperature-dependent, saturable and reversible. The ANF-receptor complex was not affected by angiotensin II, ACTH or vasopressin. Solubilization with 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]- 1-propane sulfonate (CHAPS) slightly increased the affinity for ANF (Kd 5.0 +/- 3.3 pM) without affecting the density (250 +/- 110 fmole/mg protein). Similar results were found with 1% Triton X-100. ANF-related peptides interact generally in the same way with non-solubilized and solubilized receptors, indicating a fully preserved specificity of the receptors.     

Identification of a biologically active circulating form of rat atrial natriuretic factor

An atrial natriuretic peptide has been isolated from plasma of morphine treated rats by means of glass beads extraction, immunoaffinity chromatography, and reverse phase HPLC. 1.3 micrograms of immunoreactive material was obtained. The biological activity of this material was found comparable to that of ANF (Arg 101 - Tyr 126) on the inhibition of basal aldosterone secretion by rat adrenal zona glomerulosa cells and the displacement curve of iodinated ANF from ANF receptors in a mesenteric artery preparation. Gas phase amino acid sequencing indicated that it is related to ANF (Ser 99 - Tyr 126). These results suggest that the maturation of ANF may require a tryptic-like cleavage after a single Arg residue.     

Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa

We have recently shown that synthetic rat atrial natriuretic factor (ANF) directly inhibits mineralocorticoid and glucocorticoid secretion in cultured bovine adrenal cells with a potency of 100 pM. [125I]iodo-ANF was used in the present study to characterize potential receptor sites in bovine zona glomerulosa membranes. ANF binds to a class of high affinity binding sites with a pK of 10.2 and a density of 1.3 pmol/mg protein. Detailed competition curves with ANF document a class of high affinity sites with a pK of 10.2 and also a second class of lower affinity sites with a pK of 8.5. Nonspecific binding amounts to less than 10% of [125I]iodo-ANF binding at concentrations less than 100 pM. High affinity binding of [125I]iodo-ANF is reversible with a half-time of association of 15 minutes at 25 pM and a half-time of dissociation of 140 minutes. Monovalent cations Na, Li and K equipotently enhance [125I]iodo-ANF specific binding. Divalent cations Mg, Ca and Mn also increase [125I]iodo-ANF specific binding, with Mn being the most active cation. No effect of guanine nucleotide could be detected on ANF binding. The binding of [125I]iodo-ANF is very specific and is not inhibited by 1 microM angiotensin II, ACTH, VIP, somatostatin, Leu-enkephalin, dynorphin or by the N-terminal of POMC. The N-terminal fragment ANF-(1-16) is also completely inactive. Reduction of the disulfide bridge of ANF inactivates the peptide. This enabled the development of a highly specific radio-receptor assay for ANF with a minimum detectable dose of 2 femtomoles. The results document the specific receptor involved in the potent inhibitory effect of ANF on adrenal steroidogenesis and indicate that bovine adrenal zonal glomerulosa provide a highly sensitive system for studying the recently discovered atrial natriuretic factor.     

The blood pressure decrease-induced sympathetic discharge following atrial natriuretic factor administration may offset its natriuretic action

Conscious SHR and WKY rats were infused during 7 days with ANF (Arg 101-Tyr 126), 100 ng/hr/rat, by means of miniosmotic pumps and their basal blood pressure (BP), changes in sodium excretion and urinary catecholamines compared with those at the last day of the infusion. The SHR initial BP of 181 +/- 3 mmHg gradually declined to 137 +/- 5 mmHg. No significant change in blood pressure was observed in the ANF-infused WKY group. However, WKY rats exhibited an increased sodium excretion and urinary dopamine/norepinephrine ratio when compared to sham-infused rats. No such differences were observed in SHR. It is suggested that an ANF-induced withdrawal of the renal sympathetic tone permits the manifestation of its natriuretic action in WKY rats. When, however, a BP decrease predominates, as in SHR, this decrease results in a reflex sympathetic discharge with a renal sympathetic activity over-riding the ANF induced natriuresis seen in WKY rats. Secondary sympathetic responses to the ANF-induced BP decrease have to be thus taken into account when a dissociation between the hypotensive and natriuretic action of ANF is observed in vivo.     

Isolation and NH2-terminal sequence of a highly conserved human and porcine pituitary protein belonging to a new superfamily: Immunocytochemical localization in pars distalis and pars nervosa of the pituitary and in the supraoptic nucleus of the hypothalamus

The isolation and purification of a 21,000-Da (pI 4.9) novel protein from porcine anterior pituitary and whole human pituitary is described. Comparison of the NH₂-terminal sequence of the first 77 and 81 residues of the human and porcine homologs shows only one conservative substitution at residue 12, namely an Ala for a Thr between these two species. Such high sequence homology is also reflected in their amino acid composition. A computer data-bank search using a mutation data matrix and comparison with 338,327 segments of proteins revealed that this substance should be classified as belonging to a new protein superfamily. Immunocytochemical staining, using an antibody produced against a synthetic fragment, revealed the presence of immunostainable material in the anterior and posterior lobe of the pituitary and in the supraoptic nucleus of the hypothalamus. No staining was observed in the intermediate lobe of the pituitary. Furthermore, purified neurointermediate lobe secretory granule preparations were also shown to contain this novel polypeptide.     

Changes in membrane phospholipid distribution during platelet activation

(1) Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A₂ from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. (2) Incubation with phospholipase A₂ alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxydizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. (3) Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A₂ and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. (4) The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. (5) It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.     

Atrial natriuretic factor in the vena cava and sinus node

We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node.