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.     

Food Intake and Body Positional Change Alter the Circadian Rhythm of Atrial Natriuretic Peptides Excretion into Human Urine

The 98 amino acid (a.a.) N-terminus of the 126 a.a. atrial natriuretic factor prohormone contains two natriuretic and vasodilatory peptides consisting of a.a. 1–30 (proANF 1–30) and a.a. 31–67 (proANF 31–67). The N-terminus and C-terminus (a.a. 99–126, i.e., ANF–also a vasodilatory peptide) circulate normally in humans with a circadian peak at 04:00 h in plasma. To determine if the N-terminus and C-terminus of the ANF prohormone are present in urine and possibly have a circadian variation in urine, six healthy volunteers had urine samples hourly while awake and every 3 h during sleep for five consecutive days obtained for radioimmunoassay. The sleep-awake pattern was varied so that after 2 days of normal sleep (supine)-awake (upright) positions, these volunteers were supine from 15:00 h on the third day until 10:00 h of the fourth day. They were then upright until 19:00 h that day when they became supine again until 02:30 h, and then were upright until 10:00 h of day 5. Three radioimmunoassays that immunologically recognize (a) the whole N-terminus (i.e., amino acids 1–98), (b) the midportion of the N-terminus (amino acids 31–67), and (c) the C-terminus of the ANF prohormone were utilized. ProANF 1–98, proANF 31–67, and the ANF radioimmunoassays each detected their respective peptides in urine. A circadian peak for each of these peptides was detected at 04:00 to 05:00 h whether the person was supine or upright during the night, which were significantly (p < 0.001) higher than their concentrations in the afternoon of the previous days. Assuming a supine position during the day caused a significant (p < 0.01) two- to threefold increase in these peptides in the urine. Food intake also increased the concentrations of proANF 1–98, proANF 31–67, and ANF in urine (p < 0.001). Fluid intake when abstaining from food throughout the day lowered the concentration of these peptides in the urine. It was concluded that there is a circadian rhythm in both the N-terminus and C-terminus of the ANF prohormone excretion into urine with a peak at 04:00 h irrespective of posture, but that both posture and food and fluid intake throughout the day significantly influence the excretion of these peptides into the urine, with supine posture and food increasing their concentrations in the urine while fluid intake decreases their concentrations in the urine.     

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.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

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.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Consistent Changes in the Circadian Rhythms of Blood Pressure and Atrial Natriuretic Peptide in Congestive Heart Failure

We demonstrated in previous works that the circadian rhythms of blood pressure (BP) and atrial natriuretic peptide (ANP) are antiphasic in normal subjects and in essential hypertension. The aim of the present study was to assess the circadian rhythms of BP and ANP in 20 patients with stable congestive heart failure (CHF), divided into two groups of 10 according to their New York Heart Association functional class. A matched control group of 10 normal volunteers was also studied. Noninvasive BP monitoring at 15-min intervals was performed for 24 h. Peripheral blood samples were also obtained at 4-h intervals starting from 08:OO h. The mean (±SEM) circadian mesors of ANP plasma levels were 13.4 ± 1.7 pmol/L in the control group, 28.6 ± 2.4 pmol/L in the group of 10 patients in class 11, and 81.5 ± 12 pmol/L in the group of 10 patients in class 111-IV. In normal subjects, plasma ANP concentration was highest at 04:OO h (21.5 ± 2.7 pmol/L) and lowest at 16:OO h (8.8 ± 2.4 pmol/L; p < 0.01). Both groups of patients with CHF showed no significant circadian change in the plasma levels of ANP and also a significantly blunted circadian rhythm of BP. Cosinor analysis confirmed the loss of the circadian rhythms of ANP and BP in CHF patients. Our findings support the existence of a causal relationship between the circadian rhythms of ANP and BP.     

Atrial natriuretic prohormone peptides 1-30, 31-67, and 79-98 vasodilate the aorta

Human prohormone atrial natriuretic peptides 1-30, 31-67, and 79-98 caused vasodilation of porcine aortas which began in 30 seconds and was maximal at 10 minutes. These three peptides were found to be equally potent to atrial natriuretic factor in their vasodilatory activity which was found with or without endothelium present. This vasodilation was associated with a 4 to 5 fold increase in cyclic GMP in the aorta secondary to activation of particulate guanylate cyclase [E.C. 4.6.12]. These data demonstrate that three N-terminal peptide segments of the atrial natriuretic factor prohormone cause vasodilation.     

Elevation by Atrial Natriuretic Factors of Cyclic GMP Levels in Astroglia-Rich Cultures from Murine Brain

Atrial natriuretic factors, peptide hormones originally found in the heart, slowly but strongly elevate the level of cyclic GMP in primary astrocyte-rich cultures derived from brains of newborn rats or mice but not in neuron-rich cultures prepared from embryonic rat brain. In the absence of a phosphodiesterase inhibitor, a plateau level of cyclic GMP is obtained within 10 min. In the presence of the inhibitor 3-isobutyl-1-methylxanthine, the concentration of cyclic GMP continues to rise, even after 30 min. The elevation of the level of cyclic GMP in response to atrial natriuretic factor is much more pronounced in the rat cultures than the mouse cultures. Even at peptide concentrations of 1 microM, plateaus of the concentration-response curves are not yet reached. The potencies of the active peptides vary over a range of approximately 1.5 orders of magnitude, with atriopeptins II and III and auriculin A being the most potent ones. These results suggest (a) that atrial natriuretic factors may regulate functions of glial cells, most likely of astrocytes, in brain and (b) that such cultures may be useful tools in defining such astroglial functions.     

Atrial Natriuretic Factors Stimulate Accumulation and Efflux of Cyclic GMP in C6–2B Rat Glioma and PC12 Rat Pheochromocytoma Cell Cultures

Atrial natriuretic factors (ANFs) were tested for their effects on cyclic GMP production in two neurally derived cell lines, the C6-2B rat glioma cells and the PC12 rat pheochromocytoma cells. These cell lines were selected because both are known to possess high amounts of the particulate form of guanylate cyclase, a proposed target of ANF in peripheral organs. Previous studies from our laboratory have shown that ANF selectively activates particulate, but not soluble, guanylate cyclase in homogenates of a variety of rat tissues and that one class of ANF receptor appears to be the same glycoprotein as particulate guanylate cyclase. In the present study we found that four analogs of ANF stimulate cyclic GMP accumulation in both C6-2B and PC12 cells with the rank order of potency being atriopeptin III = atriopeptin II greater than human atrial natriuretic polypeptide greater than atriopeptin I. Atriopeptin II (100 nM) for 20 min elevated cyclic GMP content in C6-2B cells fourfold and in PC12 cells 12-fold. Atriopeptin II (100 nM) for 20 min also stimulated the efflux of cyclic GMP from both C6-2B cells (47-fold) and PC12 cells (12-fold). Accumulation of cyclic GMP in both cells and media was enhanced by preincubation with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (250 microM). After 20 min of exposure to atriopeptin II, cyclic GMP amounts in the media were equal to or greater than the amounts in the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor (ANF) binding sites in brain and related structures

Visualization of [¹²⁵I]ANF binding sites in rat brain by an autoradiographic technique demonstrated that these sites are highly localized in areas such as the olfactory bulb, subfornical organ, area postrema and nucleus tractus solitarius. This distribution suggests that certain cardiovascular effects of ANF could be centrally mediated and that the existence of brain ANF-related peptides should be considered. Finally, moderate densities of [¹²⁵I]ANF binding sites are found in the rat and guinea pig eye while low densities are seen in pituitary and pineal gland.     

血浆B型脑钠肽对心力衰竭和呼吸困难的诊断价值

目的:探讨血浆脑钠肽(BNP)水平在心力衰竭(CHF)T和呼吸困难诊断中的应用。方法:采用免疫荧光快速测试法测定56例已确诊心衰患者、40例心源性呼吸困难患者、29例肺源性呼吸困难患者和30例健康人血浆BNP的含量。结果:心衰组患者血浆BNP水平明显高于对照组,差异显著(P<0.01);心源性呼吸困难组患者的BNP值水平(1032.2±879.8 pg/ml)与肺源性呼吸困难组患者的BNP值水平(67.1±43.6 pg/ml)比较有显著性差异(P<0.01)。结论:检测BNP水平可为临床诊断CHF及心源性与肺源性呼吸困难的鉴别诊断提供重要依据。     

血浆B型脑钠肽对心力衰竭和呼吸困难的诊断价值

目的：探讨血浆脑钠肽（BNP）水平在心力衰竭（CHF）T和呼吸困难诊断中的应用。方法：采用免疫荧光快速测试法测定56例已确诊心衰患者、40例心源性呼吸困难患者、29例肺源性呼吸困难患者和30例健康人血浆BNP的含量。结果：心衰组患者血浆BNP水平明显高于对照组,差异显著（P〈0．01）;心源性呼吸困难组患者的BNP值水平（1032．2±879．8pg／ml）与肺源性呼吸困难组患者的BNP值水平（67．1±43．6pg／ml）比较有显著性差异（P〈0．01）。结论：检测BNP水平可为临床诊断CHF及心源性与肺源性呼吸困难的鉴别诊断提供重要依据。     

Absence of Seasonal Variation in the Phase of the Endogenous Circadian Rhythm in Humans

Humans may be subject to seasonal variations, as evidenced by the existence of seasonal affective disorder (SAD) and midwinter insomnia. However, some recent studies have shown that the seasonal variation in the phase of the circadian rhythm is relatively weak in healthy humans. In the present study, evidence is found that there is no seasonal variation in the phase of the endogenous circadian rhythm at all. Body temperature, cortisol excretion, and subjective alertness of six subjects recorded under constant routine conditions showed no systematic seasonal variation in circadian phases. This finding indicates that secondary zeitgebers blocked or counterbalanced the seasonal variation in the entrainment effect of the natural photo-period. The human being may live in an environment in which the photoperiod has lost its status of primary zeitgeber. (Chronobiology International, 15(6), 623-632, 1998)     

Decrease of Atrial Natriuretic Peptide Content in Rat Superior Cervical Sympathetic Ganglion After Denervation and Axotomy

We found atrial natriuretic peptide (ANP), known as a humoral factor in regulating body fluid volume and blood pressure, in considerable quantities in rat superior cervical sympathetic ganglion (SCG) by radioimmunoassay after separation with reverse-phase HPLC. Although the ANP content of the immature rat 1 week after birth was low, it doubled at 2 weeks and then increased gradually, until it reached the adult level. Denervation caused a rapid decrease in the ANP content to half of the intact SCG level after 3 h, which then fell to 10% of the control value on day 2 after operation. The time course of ANP content reduction after denervation was similar but rather faster than that of activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, an observation suggesting that ANP may partly contribute to cholinergic synaptic transmission. On the other hand, axotomy produced a rather slower decrease in the ANP content than did denervation. Enucleation and sialoadenectomy also caused a considerable reduction of the ANP content. Thus, part of the ANP found in the ganglion is apparently transported from sympathetically innervated extraganglionic organs via retrograde axoplasmic flow.     

Atrial Natriuretic Hormones Raise the Level of Cyclic GMP in Neural Cell Lines

Atriopeptin III and related atrial natriuretic peptide hormones strongly elevate the level of cyclic GMP in three neural tumor cell lines. At peptide concentrations of 1 microM clear-cut plateaus of the dose-response curves are not yet reached. Atriopeptin III increases the intracellular concentration of cyclic GMP to a maximum in the course of 30-40 min. The effect of atriopeptin III on the cellular cyclic GMP level is independent of the concentration of extracellular Ca2+ and is not affected by the Ca2+ ionophore A23187. These results suggest (1) that atrial natriuretic hormones may play an important role in the nervous system, and (2) that cultured neural cells may be useful tools in the elucidation of the mechanisms of action of these hormones.     

Atrial natriuretic peptide in heart and specific binding in organs from fetal and newborn rats

To assess the possibility that atrial natriuretic peptide plays a role in salt and water balance during early mammalian development, we examined hearts from fetal and neonatal rates for the presence of this peptide and presumed target tissues for their ability to bind the hormone. Immunohistochemistry was used to localize and radioimmunoassay to quantify this peptide in heart. Immunoreactive atrial natriuretic peptide was visualized in the fetal heart on day 17.5 post-conception. It was distributed throughout the atrial appendages and free wall and, in ventricle, in the trabeculae carnae and chordae tendineae. The concentrations of immunoreactive atrial natriuretic peptide in atria of rats on day 19.5 post-conception were one-tenth of those in the adult. Levels of this peptide in fetal ventricle were low and virtually absent from the adult tissue. Specific binding of radiolabelled atrial natriuretic peptide measured by whole organ counting occurred in several organs from 19.5-day fetal and neonatal rats. A number of these tissues, including the kidney, ileum, adrenal, lung and liver, are targets for and/or bind the peptide in adult rats. Specific binding in these tissues was localized using autoradiography at anatomical sites similar to those in adult organs. Specific binding was also seen in fetal but not neonatal skin. In the kidney, binding was associated with immature as well as mature glomeruli. These findings support the proposition that atrial natriuretic peptide may function in the perinatal rat as it does in the adult and, in addition, may play a unique role during fetal life.     

Cyclic GMP Regulates Nicotine-Induced Secretion from Cultured Bovine Adrenal Chromaffin Cells: Effects of 8–Bromo–Cyclic GMP, Atrial Natriuretic Peptide, and Nitroprusside (Nitric Oxide)

Methacholine, atrial natriuretic peptide (ANP), nitroprusside (nitric oxide), angiotensin II, and bradykinin raised cyclic GMP (cGMP) levels in cultured bovine adrenal chromaffin cells. The role of cGMP in secretion from chromaffin cells was examined using 8-bromo-cGMP. This analogue had no effect on basal secretion or secretion due to angiotensin II, bradykinin, or a high K+ level but potentiated secretion due to low doses of nicotine. At supramaximal doses of nicotine, 8-bromo-cGMP inhibited secretion. These effects of 8-bromo-cGMP were not due to changes in the nicotine-induced rise in cytosolic calcium concentration. A potentiation of secretion due to low doses of nicotine was also found following simultaneous addition of ANP or nitroprusside, a result suggesting that ANP and nitric oxide (endothelium-derived relaxing factor) could be important regulators of secretion from adrenal chromaffin cells.     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

Effect of Atrial Natriuretic Peptide on Sodium-Glucose Cotransport in the Rat Small Intestine

GonzÁLez Bosc, L. V., P. A. Elustondo, M. C. Ortiz and N. A. Vidal. Effect of atrial natriuretic peptide on sodium-glucose cotransport in the rat small intestine. Peptides 18(10) 1491–1495, 1997.—Atrial natriuretic peptide (ANP) decreases sodium absorption in small intestine of rats in vitro under sodium concentration-gradient conditions (SCG) and this effect may be mediated by the inhibition of the sodium/glucose cotransporter (SGLT). In order to assess this hypothesis, the effects of ANP, phloridzine (Phlz) and methylene blue (MB), added alone or together, using a voltage clamp technique in Ussing’s chamber with SCG were studied. ANP and Phlz significantly decreased potential difference and short circuit current. Effects of Phlz and ANP were not additive. The addition of MB alone did not affect ion transport, whereas it abolished ANP effects. These data suggest that ANP blocks the SGLT through mechanisms mediated by cGMP and/or NO.