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.     

Characterization of insulin receptors in the bovine adrenal cortex and medulla.

In order to identify insulin receptors in the bovine adrenal cortex and medulla, we have studied 125I-porcine insulin binding to the membrane preparations from the bovine adrenal cortex and medulla. 125I-porcine insulin bound not only to the bovine adrenal cortex but to the medulla in time-, temperature-, and pH-dependent manners. The maximum levels of 125I-porcine insulin binding in the two tissues were observed at 4 degrees C for 24 h of incubation, and its optimum pH ranged from 7.6 to 8.0. Under these conditions, at tracer concentration of porcine insulin (200 pg/ml), 10.4% and 6.6% of 125I-porcine insulin added to each reaction tube bound specifically to 10(5) x g-pellet fractions (microsomal membrane) from the cortical tissue (0.3 mg of protein) and from the medullary tissue (2 mg of protein), respectively. 125I-porcine insulin binding was observed predominantly in the microsomal membrane from the bovine adrenal cortex, and in a 15,000 x g- pellet fraction (synaptosomal membrane) from the bovine adrenal medulla. Scatchard analysis of binding data yielded curvilinear plots in each tissue. Analysis of curvilinear plots based on two sites model revealed similar affinity constant between the cortex and medulla. Receptor concentration of the cortex was several times higher than that of the medulla. In the two bovine adrenal tissues, human proinsulin and insulin-like growth factor I (IGF-I) had about 1/100 potency compared to porcine insulin in displacing 125I-porcine insulin binding. Porcine glucagon added with concentration up to 10(-6) M did not inhibit 125I-porcine insulin binding to both the cortex and the medulla.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.     

Purification and primary structure of pro-aldosterone secretion inhibitory factor from bovine adrenal chromaffin cells

This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.     

Quantitative in vitro autoradiographic characterization of [125I]angiotensin III binding sites in rat adrenal gland

A single class of high-affinity binding sites for [125I]angiotensin III and [125I]angiotensin II were found in rat adrenal medulla and zona glomerulosa by quantitative autoradiography. In the medulla, Kd were 1.46 and 1.16 nM, and Bmax 1700 and 1700 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. In the zona glomerulosa, Kd were 0.86 and 0.90 nM, and Bmax 790 and 560 fmol/mg protein, for [125I]angiotensin II and [125I]angiotensin III, respectively. Unlabeled angiotensin III and angiotensin II displaced [125I]angiotensin III with similar potency in both adrenal zona glomerulosa and medulla. Our findings suggest that angiotensin III and angiotensin II might share the same binding sites in adrenal gland and support the hypothesis of a role for angiotensin III in the adrenal medulla and zona glomerulosa.     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

Purification and sequence determination of bovine atrial natriuretic factor

We report the purification and the sequence determination of bovine atrial natriuretic factor (ANF) in acid extracts of bovine atrial appendages. The monitoring of the activity along the purification steps was performed with a radio-receptor assay using bovine adrenal cortex membranes sites and 125I ANF. Bovine ANF was separated by carboxymethyl agarose gel chromatography from catecholamines and major protein contaminants. It behaved as a 3 K dalton peptide on Sephadex G-50. The active fractions were then subjected to high performance liquid chromatography (HPLC) using a sulfopropyl cation exchange column. Subsequent purification steps by reverse phase mode on Ultrapore RPSC, Vydac TP 218 and uBondapak using acetonitrile gradients led to the obtention of a pure fraction which amino acid sequence was identical to that for human ANF. This confirms the high degree of homology of ANF structure among mammalian species and advocates the use of the radio-receptor assay based on bovine adrenal receptor for measuring human ANF.     

Photoaffinity labeling of atrial natriuretic factor receptor in bovine and rat adrenal cortical membranes

Radioiodinated synthetic atrial natriuretic factor (ANF) bound to a single class of high affinity binding sites in the plasma membrane from bovine adrenal cortex with a KD of 7.4 X 10(-10) M. The binding affinities of related peptides showed close parallelism to their potencies in natriuretic and vasorelaxant activities. Incubation of adrenal membranes with radioiodinated 4-azidobenzoyl ANF or a similar derivative of its analogue followed by photolysis resulted in specific radiolabeling of a protein band in SDS gel electrophoresis with an apparent Mr of 124,000 in bovine or Mr of 126,000 in rat, which was abolished by inclusion of unmodified ANF in the incubation. Prevention of the labeling was dependent on the concentration of ANF and was not observed with atriopeptin I or with unrelated peptides, angiotensin II, ACTH or [Arg8] vasopressin. These results indicate specific covalent labeling of ANF-receptor or its subunit by the photoaffinity ligands.     

Amiloride potentiates atrial natriuretic factor inhibitory action by increasing receptor binding in bovine adrenal zona glomerulosa

The interaction of atrial natriuretic factor (ANF) with the diuretic amiloride was studied in bovine adrenal zona glomerulosa. Amiloride enhances 2 to 3-fold high affinity binding of [125I] ANF to zona glomerulosa membrane receptor with an ED50 of 10 microM. This effect is due to a recruitement of high affinity receptor sites and to an increase of their affinity from a Kd of 23 to 8 pM. This enhancing effect is almost equipotently elicited by guanabenz, while clonidine is 20-fold less potent and arginine is inactive. ATP reduces by 30 to 50% [125I] ANF binding with an IC50 of 50 microM. Amiloride and ATP opposite effects on [125I] ANF binding are mutually competitive. Low concentrations of amiloride (less than 100 microM) potentiate the inhibitory effect of ANF in hormone-stimulated steroid secretion with a 3-fold decrease in ANF IC50 at 10 microM amiloride. Higher concentrations of amiloride (greater than 100 microM) directly inhibit aldosterone secretion with an IC50 of 500 microM and a maximum of 80 to 100% reversal of stimulation by various secretagogues. These results indicate that amiloride synergistically potentiates ANF inhibitory action by altering ANF receptor binding properties. They also suggest a role for sodium transport and for phosphorylation-dephosphorylation mechanisms in the mode of action of ANF.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Identification of ω-Conotoxin Binding Sites on Adrenal Medullary Membranes: Possibility of Multiple Calcium Channels in Chromaffin Cells

Binding of 125I-omega-conotoxin GVIA and [3H]nitrendipine to membranes from bovine adrenal medulla was investigated to test for the presence of N- and L-type Ca2+ channels in adrenal chromaffin cells. Saturable, high-affinity binding sites for 125I-omega-conotoxin and [3H]nitrendipine were detected in a membrane fraction from adrenal medulla. [3H]Nitrendipine binding sites were found to have a KD of 500 +/- 170 pM and a Bmax of 26 +/- 11 pmol/g of protein. 125I-omega-Conotoxin binding sites had a KD of 215 +/- 56 pM and a Bmax of 105 +/- 18 pmol/g of protein, about four times the number of sites found for [3H]nitrendipine. 125I-omega-Conotoxin binding was potently inhibited by unlabeled toxin and Ca2+ but was unaffected by dihydropyridines, verapamil, and diltiazem. [3H]Nitrendipine binding was not affected by omega-conotoxin, whereas it was inhibited by other dihydropyridines. Bay K 8644 potentiated K+-evoked cytosolic Ca2+ transients measured by fura-2 fluorescence, and this potentiation was completely blocked by nifedipine. In contrast, omega-conotoxin had no effect on Bay K 8644-evoked Ca2+ transients. Thus, the binding sites for omega-conotoxin and for nitrendipine appear to be different. The results confirm the presence of L-type Ca2+ channels and open the possibility of N-type Ca2+ channels as the omega-conotoxin binding sites in chromaffin cell membranes.     

Characterization of gingival epithelium epidermal growth factor receptor

The binding characteristics of gingival epithelium epidermal growth factor (EGF) receptor were investigated using epithelial cell membranes from bovine gingiva. The binding of [125I]EGF was found to be time and protein concentration dependent, reversible, and specific. Unlabeled EGF competed for [125I]EGF binding with IC50 of 0.25nM and maximum displacement of 93% at 0.81nM. Scatchard analysis of the binding data inferred the presence of two binding sites, one of high affinity (Kd = 3.3 nM and Bmax = 47.3fmol/mg protein) and the other of a low affinity (Kd = 1.6 microM and Bmax = 1.9pmol/mg protein). Crosslinking of [125I]EGF to gingival membranes followed by polyacrylamide gel electrophoresis and autoradiography revealed a receptor protein of 170kDa.     

Atrial natriuretic factor (ANF) binding sites in frog kidney and adrenal.

Atrial natriuretic factor (ANF) binding sites were localized and quantified in kidney and adrenal of the frog Rana temporaria by quantitative in vitro autoradiography. [125I]-rat ANF(99-126) binding was present in kidney glomeruli and in the outer layer of interrenal tissue in the adrenal gland. ANF binding exhibited positive cooperativity with a half-maximal binding concentration (EC50) of 102 +/- 16 pM in glomeruli and 93 +/- 19 pM in interrenal tissue (n = 8). The corresponding maximal binding capacities (Bmax) were 1.33 +/- 0.16 and 1.21 +/- 0.36 fmol/mm2. [125I]-Rat ANF(99-126) binding was competitively displaced by unlabeled ANF analogues with an intact disulfide bridge showing a lower affinity than the iodinated ligand. The presence of ANF binding in glomeruli and steroidogenic interrenal cells suggests physiological functions of ANF for the osmomineral regulation in the frog by influencing glomerular filtration rate and adrenal steroid secretion.     

Bombesin-like immunoreactivity in bovine adrenal medulla

The presence of immunoreactive (ir)-bombesin in bovine adrenal medulla, isolated adrenal chromaffin cells and subcellular fractions of the adrenal medulla was demonstrated using a specific antibody to the synthetic peptide. High levels of ir-bombesin were detected in acid (HCl) extracts of the adrenal tissue (27 pmol/g) and isolated cells (0.35 pmol per 10(6) cells). Subpopulations of adrenal chromaffin cells were also obtained by centrifugation of the original cell preparation through a stepwise bovine serum albumin gradient (cell layers I, II and III). The highest concentration of ir-bombesin (0.77 pmol/10(6) cells) was found in a cell population (cell layer I) enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6). At the subcellular level, ir-bombesin was mainly concentrated in the secretory granules (0.61 pmol/mg protein) along with catecholamines (1097 nmol/mg protein), but a relatively high concentration of ir-bombesin (0.26 pmol/mg protein) was also found in the microsomal fraction. Isolation and high performance liquid chromatography (HPLC) analysis of adrenomedullary ir-bombesin revealed the presence of four molecular forms, one of them corresponding to gastrin releasing peptide (GRP), another one (major peak) eluting closely to synthetic neuromedin B and another one coeluting with GRP-(18-27). HPLC analysis of the molecular forms of ir-bombesin in the microsomes and secretory granules indicated that GRP- and neuromedin B-like materials can be generated between the two fractions.     

The Posttranslational Arginylation of Proteins in Different Regions of the Rat Brain

The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.     

Porcine brain natriuretic peptide receptor in bovine adrenal cortex

The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10(-8) M and 10(-7) M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of [125I]-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10(-10) M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).     

Characterization, distribution and plasticity of atrial natriuretic factor binding sites in brain

The [125I]iodotyrosyl derivative of atrial natriuretic factor [( 125I])ANF) apparently binds to a single class of high affinity sites in guinea pig brain membrane preparations. Ligand selectivity pattern reveals that the structural requirements of brain [125I]ANF binding sites are similar to those reported in most peripheral tissues. In vitro receptor autoradiographic studies demonstrate that the brain distribution of [125I]ANF binding sites is species dependent. In rat, high levels of binding are found in olfactory bulb, subfornical organ, area postrema, choroid plexus, and ependyma. In guinea pig, these regions are also enriched with [125I]ANF binding in addition to various thalamic nucleic, amygdala, hippocampus, and cerebellum. In monkey, high densities of sites are seen in the cerebellar cortex. This suggests that brain ANF receptor sites could mediate ANF effects related to the central integration of cardiovascular parameters, as well as other actions not associated with these systems. As in the periphery, it appears that brain [125I]ANF binding sites are associated with guanylate cyclase. Moreover, the density of [125I]ANF receptor binding sites is altered in certain brain regions in spontaneously hypertensive rats and in cardiomyopathic hamsters, demonstrating the plasticity of brain ANF receptors. Thus, ANF and ANF receptors are complementary facets of a new neurotransmitter-neuromodulator system present in mammalian brain.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Photoaffinity labeling of atrial natriuretic factor receptors of rat kidney cortex plasma membranes

Synthetic rat atrial natriuretic factor (ANF) was derivatized with the N-hydroxysuccinimide ester of [125I]iodoazidosalicylic acid to yield a radioactive photoaffinity probe. Incubation of purified plasma membranes from rat kidney cortex with this photoaffinity probe resulted in the specific labeling of a 140-kDa glycoprotein. The photoaffinity labeling of this protein was inhibited by ANF but not by reduced and alkylated ANF nor by other unrelated peptides. A 140-kDa band was also specifically labeled in liver plasma membranes but not in adipocyte plasma membranes. These observations suggest strongly that the 140-kDa glycoprotein is the ANF receptor.