Gaétan Thibault

Effect of native and synthetic atrial natriuretic factor on cyclic GMP.

Mammalian atrial cardiocyte granules contain a potent natriuretic and diuretic peptide. Since cGMP appears to be involved in the modulation of cholinergic and toxin-induced sodium transport, we examined the effect of atrial natriuretic factor (ANF) on this nucleotide. Atrial but not ventricular extracts elicited approximately a 28-fold increase of urinary cGMP excretion parallel to the natriuresis and diuresis. The atrial extracts also elevated cGMP levels in kidney slices and primary cultures of renal tubular cells. The effect of ANF on cGMP appeared to be specific since antibodies which were capable of inhibiting the ANF-induced diuresis also suppressed cGMP excretion. Furthermore, during the course of ANF purification, the ANF-induced increase of cGMP production by kidney cells paralleled the heightened specific natriuretic activity of the atrial factor. A synthetic peptide (8-33)-ANF similarly increased urinary plasma and kidney tubular cGMP levels. The exact mechanism of action of ANF on cGMP remains to be elucidated, but indirect inhibition of cGMP phosphodiesterase appears to participate in its effect.

Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats.

Endothelin-1 (ET-1) is a powerful vasoconstrictor peptide produced in the endothelium of blood vessels that may play an important role in the control of local blood flow and could be involved in the pathogenesis of hypertension. We investigated immunoreactive ET-1 (ir-ET-1) levels in acid extracts from blood vessels of deoxycorticosterone acetate (DOCA)-salt and spontaneously hypertensive rats. We found that segments of thoracic aorta and the mesenteric vascular bed contain significantly more ir-ET-1 (11.84 +/- 0.84 and 17.30 +/- 1.89 fmol, respectively) than uninephrectomized control rats (1.78 +/- 0.20 and 9.19 +/- 0.63 fmol, respectively; p < 0.001). High performance liquid chromatography showed that ir-ET-1 of blood vessels of DOCA-salt hypertensive rats eluted in the same position as synthetic ET-1. Significantly increased ir-ET-1 was localized by immunohistochemistry in endothelial cells of aorta and large and small mesenteric arteries of DOCA-salt hypertensive rats. In contrast to the latter, in spontaneously hypertensive rats, vascular content of ir-ET-1 was similar to that of blood vessels of Wistar-Kyoto control rats, at both 6 and 16 weeks of age. High levels of vascular ET-1 may explain the downregulation of vascular endothelin receptors previously described in DOCA-salt hypertensive rats. Furthermore, this suggests that ET-1 may be involved in the maintenance of high blood pressure in mineralocorticoid hypertension.

Radioautographic localization of125I-atrial natriuretic fator (ANF) in rat tissues

SummaryRats were injected either with synthetic125I-Arg 101-Tyr 126 atrial natriuretic factor (ANF) or with125I-ANF together with an excess of cold Arg 101-Tyr 126 ANF. Binding sites in various tissues were accepted depending on two criteria: displacement of radioactivity by cold ANF and absence of localization of silver grains on putative target cells in the presence of cold ANF. Binding sites were localized on zona glomerulosa cells and on adrenergic and noradrenergic cells of adrenal medulla, on hepatocytes, on the base of mature epithelial cells of villi in the small intestine, on smooth muscle cells of the muscularis layer of the colon and on the base of epithelial cells of the ciliary bodies. In addition, binding sites were localized in the vasculature of kidney, adrenal cortex, lung and liver. Binding sites were particularly numerous on renal glomerular endothelial cells. These results indicate that ANF may have important hemodynamic effects in kidney, lung, liver and adrenal cortex, may regulate water and ion transport in small intestine and ciliary bodies and may have metabolic effects in the liver. The presence of binding sites on the zona glomerulosa is in agreement with the important inhibitory effect of the peptide on aldosterone secretion.

Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands

SummaryAntibodies produced in the mouse by repeated intraperitoneal injections of partly purified atrial natriuretic factor (low molecular weight peptide (LMWP) and high molecular weight peptide (HMWP)) have been used to localize these factors by immunohistochemistry (immunofluorescence and immunoperoxidase method) and by immunocytochemistry (protein A-gold technique) in the heart of rats and of a variety of animal species including man and in the rat salivary glands. Immunofluorescence and the immunoperoxidase method gave identical results: in the rat, atrial cardiocytes gave a positive reaction at both nuclear poles while ventricular cardiocytes were consistently negative. The cardiocytes of the right atrial appendage were more intensely reactive than those localized in the left appendage. A decreasing gradient of intensity was observed from the subpericardial to the subendocardial cardiocytes. The cardiocytes of the interatrial septum were only lightly granulated. Sodium deficiency and thirst (deprivation of drinking water for 5 days) produced, as already shown at the ultrastructural level, a marked increase in the reactivity of all cardiocytes from both atria with the same gradient of intensity as in control animals. Cross-reactivity of intragranular peptides with the rat antibodies allowed visualization of specific granules in a variety of animal species (mouse, guinea pig, rabbit, rat, dog) and in human atrial appendages. No reaction could be elicited in the frog atrium and ventricle although, in this species, specific granules have been shown to be present by electron microscopy in all cardiac chambers. With the protein A-gold technique, at the ultrastructural level, single labeling (use of one antibody on one face of a fine section) or double labeling (use of two antibodies on the two faces of a fine section) showed that the two peptides are localized simultaneously in all three types (A, B and D) of specific granules. In the rat salivary glands, immunofluorescence and the immunoperoxidase method showed reactivity exclusively in the acinar cells. The reaction was most intense in the acinar cells of the parotid gland. In the sublingual gland, only the serous cells, sometimes forming abortive “demi-lunes”, were reactive. In the submaxillary gland, the reaction was weaker and distributed seemingly haphazardly in the gland. The most constantly reactive cells were localized near the capsule while many cells did not contain visible reaction product.

Resistance Artery Mechanics, Structure, and Extracellular Components in Spontaneously Hypertensive Rats Effects of Angiotensin Receptor Antagonism and Converting Enzyme Inhibition

BACKGROUND Altered vascular mechanics resulting from changes in collagen and integrins may influence resistance artery structure and function and, therefore, peripheral resistance and blood pressure in spontaneously hypertensive rats (SHR). METHODS AND RESULTS Effects of age, angiotensin-converting enzyme inhibition (fosinopril, 10 to 30 mg/kg per day), and AT(1)-receptor antagonism (irbesartan, 50 mg/kg per day) on vascular structure, mechanics, and composition were assessed in SHR. Systolic blood pressure was elevated in young SHR (130+/-2 mm Hg) compared with Wistar-Kyoto (WKY) rats (106+/-2 mm Hg). In adult SHR, the rise in systolic blood pressure (44+/-3 mm Hg) was blunted by fosinopril (18+/-1 mm Hg) and irbesartan (9+/-3 mm Hg). Lumen diameter of mesenteric resistance arteries was smaller and media/lumen ratio was greater in young and adult SHR versus WKY rats. Growth index was 24% in untreated adult SHR versus WKY rats; these values were -35% for fosinopril-treated and -29% for irbesartan-treated SHR versus untreated SHR. Isobaric wall stiffness was normal despite increased stiffness of wall components in adult SHR vessels. Irbesartan partially prevented stiffening of wall components in SHR. The collagen/elastin ratio was greater in adult SHR vessels (6.5+/-1.3) than in WKY (3.2+/-0.4) vessels. Expression of alpha(v)beta(3) and alpha(5)beta(1) integrins was increased in SHR aged 20 versus 6 weeks. Expression of alpha(5)beta(1) integrins was lower in young SHR, and alpha(v)beta(3) integrins were overexpressed in adult SHR versus WKY rats. Irbesartan and fosinopril attenuated differences in the collagen/elastin ratio and integrin expression. CONCLUSIONS Wall components of mesenteric resistance arteries stiffen with age in SHR. Interrupting the renin-angiotensin system has normalizing effects on integrin expression and composition, stiffness, and growth of the arterial wall.

Direct radioimmunoassay of atrial natriuretic factor

A direct radioimmunoassay of atrial natriuretic factor (ANF) has been developed. The method uses a synthetic 26 amino-acid fragment (8-33 ANF) of the native peptide. Antibodies have been prepared in rabbits immunized with the peptide coupled to thyroglobulin. The radiolabelled tracer prepared by iodination according to the Chloramine-T method has been purified by HPLC followed by affinity chromatography on Sepharose-4B anti-ANF. Dextran-coated charcoal has been used for separation of free from antibody bound radioactivity. Higher ANF content has been found in the right rat atrium than in the left. These results have been confirmed by bioassay.

Relationship of specific granules to the natriuretic and diuretic activity of rat atria.

The isolation of several fractions from rat atrial homogenates, by the use of differential and sucrose gradient centrifugation, indicates that the diuretic and natriuretic activity is restricted to the fractions rich in specific granules. Our preliminary results suggest that the active substance is a small peptide which is probably different from the natriuretic substance(s) already known.

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.

Endothelin-1 and Angiotensin II Receptors in Cells From Rat Hypertrophied Heart: Receptor Regulation and Intracellular Ca2+ Modulation

This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca2+]i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca2+]i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: > 90% of ET receptors in cardiomyocytes are of the ETA subtype, whereas fibroblasts had a nearly equal proportion of the ETA and ETB subtypes. ET-1 receptor densities, affinities, and ET-1-induced [Ca2+]i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca2+]i responses induced by Ang II at concentrations > 10(-8) mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT1) on fibroblasts was significantly reduced (42,970 +/- 3330 versus 73,870 +/- 7940 sites per cell for control cells, P < .01), but AT1 receptor affinity was unchanged after volume overload. The maximum increase in [Ca2+]i evoked by 10(-6) to 10(-4) mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ETA subtype and fibroblasts possessing both ETA and ETB receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT1 receptors on fibroblasts, whereas total binding and [Ca2+]i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.

Atrial natriuretic factor is a circulating hormone

The radioimmunoassay of atrial natriuretic factor (ANF) has been applied for determination of immunoreactive ANF (IR-ANF) in rat plasma. Immunoreactive ANF has been extracted from rat plasma by immunoaffinity column on Sepharose-4B anti-ANF or by Vycor glass. The mean concentrations of IR-ANF in ether anesthetized rats were found to be 1.61 +/- 0.14 ng/ml in female and 1.25 +/- 0.21 ng/ml in male rats when extracted on Sepharose-4B anti-ANF, and 1.21 +/- 0.10 ng/ml in females and 1.02 +/- 0.11 ng/ml in males when extracted by Vycor glass. A close linear correlation has been observed between the plasma IR-ANF concentrations in aorta and jugular vein. The described results indicate that atrial cardiocytes secrete atrial natriuretic factor into plasma. The heart is, therefore, an endocrine organ.