Atrial natriuretic factor prohormone peptides are present in a variety of tissues.

Utilizing two sensitive and specific radioimmunoassays which immunologically recognize 1) the 98 amino acid (a.a.) N-terminus and 2) the 28 a.a. C-terminus (i.e., a.a. 99-126) of the 126 a.a. atrial natriuretic (ANF) prohormone, various tissues including aorta, kidney, small intestine, colon, liver, spleen, lung, and testis were investigated to determine if the ANF prohormone was present in any of these tissues in addition to its previously demonstrated presence in heart and brain. Aorta with 62.3 +/- 3 ng of the N-terminus/g of tissue and 51.6 +/- 1.8 ng of the C-terminus of the ANF prohormone/g of tissue had the highest concentration of the ANF prohormone of the previously undescribed ANF prohormone-containing tissues. The next highest concentration of the ANF prohormone was in the intestine, followed by lung and spleen. Pancreas, liver and kidney had similar levels of immunologically recognized ANF prohormone (approximately 1/50 of the aorta), while the testis and cerebrum had low levels. These results suggest that a much larger variety of tissues synthesize and/or store the ANF prohormone than is presently thought.     

The circadian rhythm of the N-terminus and C-terminus of the atrial natriuretic factor prohormone

Circadian variation in the circulating concentrations of the N-terminal and C-terminal portions of the atrial natriuretic factor prohormone (pro ANF) was evaluated in 8 men, ages 41-47, who have been followed for 19 years with respect to circadian variation in physiological variables including blood pressure and clinical chemistries. The N-terminus of the ANF prohormone contains two peptides consisting of amino acids 1-30 and 31-67 while the C-terminus contains 1 peptide (amino acids 99-126) of this 126 amino acid prohormone which lower blood pressure and have natriuretic properties. To determine if either the N-terminus and/or the C-terminus of the prohormone have a circadian variation in their circulating plasma concentrations these 8 men had blood samples obtained for radiommunoassay every 3 hr during a 24-hr period. Three radiommunoassays which immunologically recognize (1) the whole N-terminus (i.e. amino acids 1-98), (2) the midportion of the N-terminus (amino acids 31-67) and (3) the C-terminus (amino acids 99-126) of the ANF prohormone were utilized. The whole N-terminus, the midportion of the N-terminus which circulates after being proteolytically cleaved from the rest of the N-terminus, and the C-terminus each had a peak circulating concentration between 0400 and 0700 which were significantly (P less than 0.001) higher than their concentrations at any other time throughout the 24-hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Increased release of the N-terminal and C-terminal portions of the prohormone of atrial natriuretic factor during immersion-induced central hypervolemia in normal humans

The role of peptides from the N terminus and C terminus of the 126 amino acid atrial natriuretic factor (ANF) prohormone in modulating renal sodium and water handling has not been defined. Since water immersion to the neck (NI) provides an acute central volume expansion identical to that produced by 2 liters of saline but without plasma compositional change, immersion to the neck was used to assess the N-terminal and C-terminal portions of the ANF prohormone response to acute central blood volume expansion in seven seated sodium-replete normal subjects. Both the C terminus, which contains amino acids 99-126 and is identical to ANF, and the whole N terminus (i.e., amino acids 1-98) increased promptly with NI and peaked after 1 hr of immersion. A Mr 3900 peptide from the midportion of the N terminus consistent with amino acids 31-67 (i.e., pro-ANF-31-67) also increased with NI and followed a pattern of increasing circulating concentration nearly identical to that of the whole N terminus of the prohormone, except that its maximal concentration was at the second hour of the 3 hr of NI. With cessation of immersion, ANF decreased to preimmersion levels within 1 hr whereas the N terminus and pro-ANF-31-67, although their circulating concentrations were decreasing, were still significantly elevated at 1 hr. These findings suggest that the increase in plasma ANF, the N terminus of the ANF prohormone, and pro-ANF-31-67 from the midportion of the N terminus, with natriuretic properties similar to ANF, contribute to the natriuretic response to NI, implying a physiologic role for these atrial peptides in modulating volume homeostasis in humans.     

Identification and characterization of three distinct atrial natriuretic factor receptors. Evidence for tissue-specific heterogeneity of receptor subtypes in vascular smooth muscle, kidney tubular epithelium, and Leydig tumor cells by ligand binding, photoaffinity labeling, and tryptic proteolysis

Three distinct atrial natriuretic factor (ANF) receptors have been identified and characterized from rat thoracic aortic cultured vascular smooth muscle (RTASM) cells, kidney tubular epithelium (MDCK), and Leydig tumor (MA-10) cells. These include 1) a disulfide-linked 140-kDa protein found in RTASM cells, which was reduced by dithiothreitol (DTT) to 70 kDa, 2) a 120-135-kDa single polypeptide protein, specific to MDCK and MA-10 cells whose Mr was not reduced by DTT, and 3) a 66-70-kDa protein prevalent in both RTASM and MDCK cells, which was not reduced by DTT. After incubation of RTASM cells with 4-azidobenzoyl 125I-ANF, labeling of the 140-kDa protein was blocked by both full-length ANF(99-126) and truncated ANF103-123. In contrast, the labeling of the 120-kDa receptor in MDCK cells was blocked only by full-length ANF(99-126). However, labeling of the 68-70-kDa receptor in both RTASM and MDCK cells was blocked by full-length ANF(99-126) and truncated ANF(103-123). Binding of 125I-ANF(99-126) to RTASM and MDCK cells was rapid, specific, and saturable with a Kd of 1.5 x 10(-10) M and binding capacity (Bmax) of 2.1 x 10(5) sites/RTASM cell and Kd 4.5 x 10(-10) M and Bmax 5 x 10(4) sites/MDCK cell, respectively. Binding of 125I-ANF(99-126) to RTASM cells was displaced with both full-length ANF(99-126) and truncated ANF(103-123), however, binding to MDCK cells was efficiently displaced only with full-length ANF. Both ANF(99-126) and ANF(103-123) stimulated cGMP in RTASM cells but only ANF(99-126) elicited cGMP in MDCK cells. Tryptic proteolysis of the high Mr single chain receptor produced only a 68-kDa fragment, whereas disulfide-linked 140-kDa receptor yielded 52-, 38-, 26-, and 14-kDa fragments. These data provide direct biochemical evidence for three distinct ANF receptors which might be linked to diverse physiological functions of ANF such as natriuresis in the kidney, vasorelaxation in vascular smooth muscle, and steroidogenic responsiveness in Leydig cells.     

The endocrine heart and natriuretic peptides: histochemistry, cell biology, and functional aspects of the renal urodilatin system

 This review focuses on some selected aspects of the endocrine heart and natriuretic peptides. The endocrine heart is composed of specific myoendocrine cells of the cardiac atria. The myoendocrine cells synthesize and secrete the natriuretic peptide hormones which exhibit natriuretic, diuretic, and vasorelaxant properties. Immunohistochemical analyses show that natriuretic peptides of the A-type and B-type are localized not only in the specific granules of these myoendocrine cells but also in many other organs including the brain, adrenal medulla, and kidney. Also, their receptors are detected in many organs showing the multiple functions of these regulatory peptides. Of the members of the natriuretic peptide family, ANP (ANP for atrial natriuretic peptide; also denominated cardiodilatin, CDD), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and the A-type, including its renal form, urodilatin, are emphasized in this review. Urodilatin is localized in the kidney, differentially processed, and secreted into the urine. The intrarenal synthesis and secretion is the basis for a paracrine system regulating water and sodium reabsorption at the level of the collecting duct. CDD/ANP-1-126, cleaved from a precursor of 126 amino acids in the heart to a 28-amino acid-containing circulating molecular form (CDD/ANP-99-126), and urodilatin (CDD/ANP-95-126) share similar biochemical features and biological functions, but urodilatin may be more involved in the regulation of body fluid volume and water–electrolyte excretion, while circulating CDD/ANP-99-126 is responsible for blood pressure regulation. The physiological and pharmacological properties of these peptides have great clinical impact, and as a consequence urodilatin is involved in drug development for the treatment of acute renal failure, cardiomyopathia, and acute asthma. Accepted: 8 July 1998     

Contribution of blood and systemic circulation to the processing of pro-(atrial natriuretic factor).

Atrial natriuretic factor-(Asn1-Tyr126)-peptide, the 13.6 kDa propeptide of atrial natriuretic factor (ANF), is stored in the secretory granules of atrial cardiocytes. ANF-(Ser99-Tyr126)-peptide, the 28-amino-acid species, is the circulating form of this hormone in the rat. As the site of maturation of the prohormone is still unknown, the present study was undertaken to understand the contribution of the circulation to the maturation process of pro-ANF. 125I-ANF-(Asn1-Tyr126)-peptide was incubated with whole rat blood, plasma or serum for different time intervals, and the products were analysed. There was minimal activation of the propeptide in either whole blood or plasma. Incubation with serum, however, resulted in the formation of an 11 kDa and a 3 kDa peptide which corresponded respectively to the N-terminal and C-terminal parts of the propeptide. These results suggest that hydrolysis of the propeptide in serum is brought about by enzymes that may be stimulated during coagulation but which may not play a major role in the activation of pro-ANF in the circulation. Plasma analysis at different time intervals after prohormone injection indicated a non-specific hydrolysis of the pro-ANF molecule. The disappearance rate curves, obtained with radiolabelled pro-ANF, suggested the presence of two components with half-lives of 2.1 +/- 0.4 min and 52.5 +/- 8.4 min respectively. A metabolic clearance rate of 1.49 +/- 0.22 ml/min and an initial distribution volume of 47.4 +/- 8 ml were calculated. These results indicate that the maturation of pro-ANF to its active circulating form takes place before it is released into the circulation.     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.     

Proatrial natriuretic factor is phosphorylated by rat cardiocytes in culture

Proatrial natriuretic factor (proANF) is phosphorylated in primary cultures of neonatal rat cardiocytes. Rittenhouse et al. (Rittenhouse, J., Moberly, L., O'Donnell, M. E., Owen, N. E., and Marcus, F. (1986) J. Biol. Chem. 261, 7607-7610) observed that cyclic AMP-dependent protein kinase phosphorylated synthetic peptides related to atrial natriuretic factor (ANF) and that phosphorylated ANF peptides were more effective in stimulating Na/K/Cl cotransport in smooth muscle cells than nonphosphorylated forms. In our studies, rat cardiocytes in culture were incubated with [32P]orthophosphoric acid, and ANF-related peptides in cell extracts and culture media were isolated using antisera to ANF. Both atrial and ventricular cardiocytes contained and secreted phosphorylated proANF, a 126-amino acid precursor of ANF. Phosphorylated and nonphosphorylated isoforms of proANF were resolved by isoelectric focusing; approximately 35% of the proANF secreted by cardiocytes was phosphorylated. proANF is phosphorylated on a serine residue localized to a 42-amino acid tryptic fragment (proANF residues 26-67). We conclude that proANF is phosphorylated by rat cardiocytes but not within the portion of the molecule destined to become ANF (proANF residues 99-126). Phosphorylation may have a role in the cellular mechanisms of proANF storage and secretion or in the modulation of potential biological activities of the circulating amino-terminal portion of proANF.     

The responses of atrial natriuretic factor concentrations to acute volume changes in conscious rats

A rapid and sensitive radioimmunoassay has been developed for measurements of atrial natriuretic factor (ANF) in rat plasma. The antiserum, raised to rat ANF (99-126), cross-reacts with rat ANF (103-123), ANF (103-125), ANF (103-126) but not with smaller fragments, human ANF (99-126), angiotensin II, bradykinin or vasopressin. The plasma ANF concentration is 181 +/- 24 pg/ml (N = 24) in the unstressed conscious rats (Charles River CD, male). The ANF immunoreactivity in the plasma extracts was verified by HPLC analysis, which displayed one major immunoreactive peak of ANF corresponding to rat ANF (99-126) and some smaller fragments. Intravenous injection of saline elevated circulating ANF, whereas acute volume depletion by hemorrhage, water deprivation and furosemide diuresis greatly lowered plasma ANF. The prompt response of plasma ANF levels to acute changes in volume status is consistent with the proposed role of ANF as a volume-regulatory hormone.     

Temporal (Circadian) and Functional Relationship Between Atrial Natriuretic Peptides and Blood Pressure

Long-acting natriuretic peptide, vessel dilator, and atrial natriuretic factor consisting of amino acids (a.a.) 1 to 30, 31 to 67, and 99 to 126 of the 126-a.a. atrial natriuretic factor (ANF) prohormone, respectively, circulate in humans and have potent vasodilatory properties. To determine if these atrial natriuretic peptides are directly related to blood pressure in clinically healthy normotensive humans, we obtained 24-h profiles of vessel dilator, long-acting natriuretic peptide, ANF, and blood pressure in 10 men in 1988 and 11 men in 1993 (seven men were studied twice) to compare circulating concentrations of atrial natriuretic peptides with naturally occurring changes in blood pressure. Overall, vessel dilator, long-acting natriuretic peptide, and ANF each had significant (p > 0.001) circadian rhythms, with peak concentrations late during sleep (at 04:00 h) being nearly twice their concentrations in the afternoon and evening. This high-amplitude circadian change allowed for the refinement of normal limits for ANF peptides by computing 3-hourly tolerance intervals (chronodesms) against which to compare time-specified single samples for normality. Systolic, diastolic, and mean arterial blood pressure also had significant circadian rhythms (p > 0.001) with peaks and troughs that were exactly opposite those of the ANF peptides. In addition to this inverse temporal relationship, there was a significant inverse correlation between absolute values for blood pressure and each ANF peptide (p > 0.001), implying a functional relationship. These data suggest that in addition to other well-established neurochemical factors, the ANF peptides (vessel dilator, long-acting natriuretic peptide, and ANF) are important for the maintenance of blood pressure and modulation of its circadian rhythm.     

Disappearance of atrial natriuretic factor from circulation in the rat

The rate of disappearance of radioiodinated forms of 3 different atrial natriuretic factors (ANF (Ser 99-Tyr 126), ANF (Arg 101-Tyr 126), ANF (Ser 103-Tyr 126)) from circulation in the rat was studied. Before proceeding to study the half-life of these peptides, the biological activity of their cold iodinated forms was examined. Upon incorporation of iodine into the ANF molecule, there was a 2 to 5-fold loss in their binding affinities to mesenteric arteries and adrenal capsules as compared to their respective uniodinated forms. A similar loss in their potency to inhibit basal aldosterone release from adrenal zona glomerulosa cells was observed. The rate of disappearance of the radioiodinated peptides from plasma was very fast; the half-life of ANF (Ser 99-Tyr 126) was 16.8 +/- 0.9 sec. Similar values were also obtained for ANF (Arg 101-Tyr 126) and ANF (Ser 103-Tyr 126). The in vivo disappearance of ANF from plasma is probably due to the binding to receptors in the cells since in vitro incubation of ANF (Ser 99-Tyr 126) with rat plasma caused only a slight loss in its immunoreactivity in the first 5 minutes. Hepatectomy and nephrectomy did not cause any major prolongation of the disappearance rate suggesting that these two organs may not be the primary sites involved in the removal of this peptide from circulation.     

Specific binding sites for prohormone atrial natriuretic peptides 1-30, 31-67 and 99-126

Two peptides with vasodilatory properties consisting of amino acids 1-30 and 31-67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1-30), (31-67), and (99-126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1-30), (31-67), and (99-126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1-30), (31-67), and ANF were 2.57, 59.91 and 40 fmols/10(6) cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1-30), (31-67), and (99-126) (ANF). Each peptide bound to DDT1 MF-2 membranes between 10(-8) to 10(-11) M but could only bind to the other peptides' receptors at concentrations of 10(-6) and 10(-7)M. These results suggest that proANF(1-30) and proANF(31-67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Atrial natriuretic factor-like peptide and its prohormone within single cell organisms.

The present investigation was designed to determine if the atrial natriuretic peptide hormonal system is present within single cell organisms. Paramecium multimicronucleatum were examined with 3 sensitive and specific radioimmunoassays which recognize the N-terminus [amino acids 1-98; proANF(1-98)], the midportion of the N-terminus [amino acids 31-67; proANF(31-67)] and C-terminus (amino acids 99-126; ANF) of the 126 amino acid atrial natriuretic factor (ANF) prohormone. ProANF(1-98), proANF(31-67), and ANF-like peptides were all present within these unicellular organisms at concentrations of 460 +/- 19 pg/ml, 420 +/- 15 pg/ml, and 14.5 +/- 2 pg/ml, respectively. These concentrations are similar to their respective concentrations in the plasma of the rat (Rattus norvegicus). These results suggest that even single cell organisms contain the atrial natriuretic peptide-like hormonal system.     

Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues

The 98 amino acid (a. a.) N-terminus of the 126 a. a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a. a. 1–30 (proANF 1–30), a. a. 31–67 (proANF 31–67) and a. a. 79–98 (proANF 79–98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a. a. 99–126, C-terminus of prohormone). ProANF 1–30 and proANF 31–67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1–30, 31–67, and 79–98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2) — cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1–30, proANF 31–67, proANF 79–98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 M concentrations, with no further increase in activity above their 1 M concentrations. Half-maximal (EC₅₀) enhancement of particulate guanylate cyclase occurred at 0.15 ± 0.01, 0.3 ± 0.02, 0.5 ± 0.03, and 0.9 ± 0.03 nM for proANF 1–30, proANF 31–67, proANF 79–98 and ANF, respectively. ProANFs 1–30, 31–67, 79–98, and 99–126 (i.e., ANF) each increased cyclic GMP but not cyclic AMP levels in tissue slices of liver, lung, small intestine, heart, and testes. None of these peptides enhanced either adenylate cyclase or the soluble 100,000 G form of guanylate cyclase. The ability of these N-terminal peptides to enhance particulate guanylate cyclase activity in a wide variety of tissues suggests that they may have effects in a much wider variety of tissues than presently thought.     

Circadian Rhythm of Prohormone Atrial Natriuretic Peptides 1-30, 31-67 and 99-126 in Man

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminal end of the prohormone of the atrial natriuretic factor (pro ANF), vasodilate aortas in vitro, lower blood pressure in vivo, and have natriuretic properties similar to the atrial natriuretic factor (ANF, amino acids 99-126 of the prohormone). It has been recently discovered that pro ANF 1-30 and pro ANF 31-67 as well as ANF circulate in man. To determine if these three peptide hormones have a circadian variation in their circulating plasma concentrations, eight housestaff volunteers were studied on a day when they were in the hospital for 24 hr. These 5 men and 3 women, ages 25 to 39 had blood samples taken at 0800, 1200, 1600, 2000, 0000, 0400 and 0800 on the following day. One-half of these house officers were up all night while the other half went to sleep from midnight to 0800 and had their 0400 plasma samples drawn while in a supine position. The peak level for all three peptide hormones was at 0400 for both supine and upright subjects. It was concluded that there are circadian rhythms in normal, active people of these three peptide hormones, whose peak levels are at 0400 irrespective of posture.     

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.     

Studies of the penetration of the blood brain barrier by atrial natriuretic factor

The atrial natriuretic factors (ANF) have been detected in various areas of the brain. To determine whether circulating blood borne ANF could contribute to the ANF content in the central nervous systems we examined the ability of ANF-99-126 or ANF-102-126 to penetrate the blood brain barrier. Carotid artery injections of [3H] inulin with [125I] ANF in anesthetized rabbits resulted in a comparably minimal brain uptake index (BUI) for each labeled substance as measured in cerebral cortex extracts. Injection of [3H] HOH and [125I] ANF resulted in a mean BUI in cortex of 4.9 +/- .6 (SEM)% for ANF relative to triated water; this low uptake was not significantly saturable. The BUI ratio for ANF/HOH in olfactory bulb was somewhat higher though still low, at 7.0 +/- 9%, possibly reflecting the high density of ANF receptors in this structure. Infusion of [125I] ANF into the carotid artery of anesthetized rabbits resulted in little radioactivity being detected in the cerebrospinal fluid. Infusion of unlabeled ANF, which raised plasma levels as high as 26.3 ng/ml, resulted in little change in CSF levels. Our results demonstrate that the uptake of ANF into the brain is minimal and supports the idea that local synthesis of ANF predominantly accounts for the brain pool of this peptide.