A. De Léan

The amino acid sequences of frog heart atrial natriuretic-like peptide and mammalian ANF are closely related.

Despite few studies conducted in non‐mammalian species, there has been a number of reports pertaining to the occurrence of a natriuretic‐like substance in lower organisms. Thus, an immunoreactive substance reacting with antibodies directed against mammalian atrial natriuretic factor has previously been detected both in heart atria and ventricles of a chordate, the frog. This substance was isolated and purified from frog heart atria and its amino acid sequence established. The sequence, Ala‐Pro‐Arg‐Ser‐Ser‐Asp‐Cys‐Phe‐Gly‐Ser‐Arg‐Ile‐Asp‐Arg‐Ile‐Gly‐Ala‐Gln‐Ser‐Gly‐Met‐Gly‐Cys‐Gly‐Arg‐(Phe), is highly homologous to known mammalian ANF sequences. However, when aligned with the complete mammalian ANF precursor sequence at positions 121 to 151, it exhibits a single amino acid insertion at position 129 and other substitutions at positions 121, 125, 133 ,135, 144, 147 and 148. Some evidence is also presented concerning the occurrence of uncleaved frog pronatriodilatin, the precursor form of ANF. This study represents the first report pertaining to the structure of a non‐mammalian ANF and its precursor.

Phosphorylation of atrial natriuretic factor R1 receptor by serine/threonine protein kinases: evidences for receptor regulation

The 130 kDa atrial natriuretic factor receptor (ANF-R1) purified from bovine adrenal zona glomerulosa is phosphorylated in vitro by serine/threonine protein kinases such as cAMP-, cGMP-dependent and protein kinase C. This phosphorylation is independent of the presence of ANF (99–126) and there is no detectable intrinsic kinase activity associated with the ANF-R1 receptor or with its activated form. In bovine adrenal zona glomerulosa cells, TPA (phorbol ester) induces a marked inhibition of the ANF-stimulated cGMP accumulation as well as of the membrane ANF-sensitive guanylate cyclase catalytic activity without any change in the binding capacity or affinity for 125I-ANF. However, we have demonstrated a significant 32P incorporation in the ANF-R1 receptor of the TPA-treated cells. The effect of TPA on the zona glomerulosa ANF-R1 receptors was abolished by calphostin C, a specific protein kinase C inhibitor. Altered ANF actions due to blunted response of guanylate cyclase to ANF could be a consequence of the ANF receptor phosphorylation by excessive activity of protein kinase C and might be involved in the pathogenesis of hypertension.

Pituitary adenylate-cyclase activating polypeptide (PACAP) evokes long-lasting secretion and de novo biosynthesis of bovine adrenal medullary neuropeptides

Recently, the pituitary adenylate-cyclase activating polypeptide (PACAP) has emerged as a potential noncholinergic neuromodulator of adrenal medullary function. In support of this hypothesis, we documented PACAPs effects on the secretion and biosynthesis of neuropeptides by cultured bovine chromaffin cells. Data presented in this study indicate that PACAP is a potent and efficacious secretagogue of leucine-enkephalin which was coreleased with catecholamines with identical profiles. In comparison to nicotinic activation, however, rates of PACAP-induced secretion were substantially slower but persisted for several hours causing a prolonged increase in the tonic release of both transmitters and peptides. Interestingly, renewal of intracellular pools of neuropeptides was also stimulated by PACAP but not the vasoactive intestinal peptide (VIP). Indeed, the higher incorporation of [35S]-labeled amino acids into atrial and brain natriuretic peptides (ANP, BNP) provided strong evidence that PACAP directly activated de novo biosynthesis. Of particular importance was PACAPs net preferential stimulation of the biosynthesis of BNP, similar to the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously the differential regulation by protein kinase A (PK-A) and protein kinase C (PK-C) activators we have previously reported. PACAP-induced secretion and biosynthesis appeared to be mediated by the PACAP-specific type I receptors known to activate adenylate cyclase and phospholipase C. We verified that PACAP did indeed stimulate the production of cyclic AMP and inositol phosphates in our cell system. These findings suggest that the dual signaling properties of type I receptors may be important for PACAPs differential effect on the biosynthesis of natriuretic peptides. We conclude that PACAP might assume important noncholinergic trans-synaptic regulation of the adrenal medulla by releasing and modifying intragranular catecholamine and neuropeptide contents.

Atrial natriuretic factor inhibits the early pathway of steroid biosynthesis in bovine adrenal cortex

We have previously determined that atrial natriuretic factor (ANF) is a potent inhibitor of steroid secretion in cultured bovine zona glomerulosa and fasciculata cells. The present report describes a comparison of the effect produced by ANF on aldosterone, deoxycorticosterone and progesterone secretions by zona glomerulosa cells and on cortisol, corticosterone and progesterone secretions by zona fasciculata cells. The equipotent inhibitory action of ANF on the stimulated secretion of these steroids in both cell types indicates a common site of action prior to progesterone synthesis at which ANF inhibits the steroidogenic pathway.

The H295R human adrenocortical cell line contains functional atrial natriuretic peptide receptors that inhibit aldosterone biosynthesis

The inhibitory effect of atrial natriuretic peptide (ANP) on angiotensin II (AII)-stimulated aldosterone secretion has been previously studied in rat and bovine adrenal zona glomerulosa cells in primary culture. However the understanding of the mode of action of ANP at the molecular level has been hampered by limitations of those primary cell culture systems and by the lack of cell lines from human adrenal cortex. Here we demonstrate the presence of fully functional ANP receptors in the recently characterized AII-responsive adrenocortical carcinoma cell line H295R. Specific saturable binding of 125I-rANP to H295R cell membrane preparations revealed a single class of high affinity binding sites with a density of 20 fmol/mg of protein. The pharmacological profile of this ANP receptor was documented by competitive binding of 125I-rANP with naturally occurring natriuretic peptides. rANP was the most potent with a Kd of 42 pM. pBNP32 was less potent with a Kd of 174 pM. 125I-rANP binding was not competed by pCNP (NPRB-specific ligand) nor by C-ANF (NPRC-specific ligand). Photoaffinity labeling of membrane preparations with 125I-BPA-ANP revealed a single specific protein of molecular weight around 130 kDa. This protein was further identified by immunodetection with a specific antibody directed to the human ANP-specific receptor NPRA. Natriuretic peptides stimulated cGMP production by the receptor-coupled guanylate cyclase with the same specificity. Aldosterone production by AII-stimulated H295R cells was dose-dependently inhibited by rANP with an ED50 of 1.5 nM. In addition, we used this model to test two chimeric analogs of ANP and BNP. pBNP1 and pBNP3 were, respectively, 4- and 2-fold more potent than rANP in competing for 125I-rANP binding with Kd of 10 and 20 pM. pBNP1 was 24-fold more potent in inhibiting AII-stimulated aldosterone production with ED50 of 63 pM. pBNP1 is therefore the most potent natriuretic peptide analog tested. In summary, the human H295R cell line contains NPRA receptors positively coupled to the particulate guanylate cyclase and that antagonize angiotensin II stimulation of aldosterone secretion.

Comparative effect of pituitary adenylate cyclase-activating polypeptide on aldosterone secretion in normal bovine and human tumorous adrenal cells.

The purpose of this study was to investigate the mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in stimulating aldosterone production in two different models: bovine adrenal zona glomerulosa (ZG) cells in primary culture and the human adrenocortical carcinoma cell line H295R. PACAP binds to two major groups of receptors: type I, which prefers PACAP38 and PACAP27 over vasoactive intestinal peptide (VIP); and type II, which has approximately equal affinity for PACAP38, PACAP27, and VIP. The type I subclass comprises multiple splice variants that can be distinguished by their specificity to PACAP38 and PACAP27 in their activation of adenylate cyclase and phospholipase C. Type II PACAP/VIP receptors couple only to AC. In bovine ZG cells, PACAP38 and PACAP27 stimulated aldosterone production in a dose-dependent manner, whereas VIP was ineffective. In H295R cells, PACAP38, PACAP27, and VIP dose-dependently stimulated aldosterone production with roughly the same ED50. In bovin...

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines.

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R1A and ANF-R1C subtypes. A10 and NIH cells which express high density of ANF-R2 receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R1A and ANF-R1C subtypes are modulated in the same manner. In the presence of Mn2+, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg2+ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R1A, the ANF-R1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R1A subtype.

C-type natriuretic peptide in bovine chromaffin cells. The regulation of its biosynthesis and secretion.

We report here the regulation of the biosynthesis and the secretion of C‐type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and ‐C activators induced a 6‐fold increase of intracellular levels of CNP‐(1–103). The biosynthesized CNP‐(1–103) was co‐released with its mature forms, typically CNP‐(51–103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.

Purification of Radioiodinated Peptides with PRP-1 Polystyrene Cartridges and HPLC: Application to Atrial Natriuretic Factor and Vasopressin

Abstract A simple and rapid cleanup procedure is described for the purification of iodinated peptides using PRP-1 polystyrene cartridges following the radioiodination process. The method is validated using different volumes and solvent systems and compared to the standard Sep-Pak C18 procedure. In this study, the method is used to prepare 125I-labeled atrial natriuretic factor and arginine-vasopressin which are further purified by reverse phase HPLC giving maximally obtainable specific activity required for the radioimmunoassays of these peptides.