Ser-Leu-Arg-Arg-atriopeptin III: the major circulating form of atrial peptide.

Vasopressin induces a concentration-dependent increase in atriopeptin immunoreactivity in plasma. Rat plasma, rat atrial extract, and synthetic atriopeptin III (APIII) produced parallel displacement curves of iodine-125-labeled APIII binding to specific antiserum. Fractionation of plasma atriopeptin immunoreactivity by reverse-phase high-performance liquid chromatography showed that the major portion consists of two species of low molecular weight peptides in a ratio of 10 to 1. Both peaks exhibited potent vasorelaxant activity, suggesting the presence of the carboxyl terminal Phe-Arg sequence of atriopeptin in each species. Sequence determination of the purified peptides indicated that the major peptide is Ser-Leu-Arg-Arg-APIII and the minor peptide APIII. It appears that the former is the major species of atrial peptide in the rat circulation and that it is the product of selective cleavage of the high molecular weight precursor.

Atriopeptins: correlation between renal vasodilation and natriuresis.

The effect of atrial peptides on renal function was studied in intact anesthetized dogs. A quantitative comparison of bolus intra-arterial injections demonstrated a rank order potency as renal vasodilators and natriuretic/diuretic agents as follows: ser-leu-arg-arg-atriopeptiin III (SLRR-APIII) greater than high molecular weight artrial peptide greater than or equal to atriopeptin (AP)III = APII much greater than API (essentially inactive). A sustained infusion of APIII was employed in order to study the temporal and quantitative correlation of the renal functional changes induced by the atrial peptide. Both intra-arterial and intravenous administration of the peptide produced concentration-dependent increases in renal blood flow, urine volume, sodium excretion, and osmotic clearance. Infusion of APIII into the renal artery did not alter systemic blood pressure or heart rate. Intravenous infusions of APIII required 10 times higher doses to induce the changes in renal vascular resistance and electrolyte excretion, and a fall in blood pressure and tachycardia resulted. The natriuretic-diuretic effect of the atriopeptins appears to be closely associated with renal vasodilation, exhibiting a positive linear correlation between the peptide-induced changes in sodium excretion and changes in renal blood flow.

Vasodilator properties of a family of bioactive atrial peptides in isolated perfused rat kidneys.

The isolated Krebs-perfused rat kidney was used for the quantitative and qualitative evaluation of the family of peptides derived from rat atrial extracts. Renal resistance changes were measured in perfused rat kidneys continuously infused with norepinephrine. The low molecular weight peptide fraction from rat atrial extracts was purified to obtain six peptides. The 21 amino acid peptide, designated atriopeptin I, was previously demonstrated to be natriuretic and to relax intestinal but not vascular smooth muscle strips (in vitro) and to be an extremely weak renal spasmolytic in the isolated perfused rat kidney. On the other hand, the 23 amino acid peptide (which has a Phe-Arg carboxy-terminal extension on atriopeptin I), designated atriopeptin II, and atriopeptin III (the Phe-Arg-Tyr carboxy-terminal extension) were natriuretic and spasmolytic (in vitro) on both intestinal and vascular smooth muscle. Both atriopeptin II and III produced a profound concentration-dependent decrease in renal resistance in the norepinephrine-constricted rat kidney preparation. A comparative study of the six peptides isolated from atrial extracts indicates that the Phe-Arg or Phe-Arg-Tyr carboxy-terminal extension of the basic 21 amino acid sequence is essential for the renal vasorelaxant activity. A purified high molecular weight peptide (designated atriopeptigen) is impotent relative to the low molecular weight atriopeptin II and III as a renal spasmolytic in isolated perfused rat kidneys and reduces renal resistance only after in vitro proteolytic cleavage. Thus, the low molecular weight peptides atriopeptin II and III appear to be the active species that mediate the renal vasodilation produced by atrial extracts.

Atriopeptin-immunoreactive neurons in the brain: presence in cardiovascular regulatory areas.

Antisera to atriopeptin III and to a cyanogen bromide fragment of the precursor molecule atriopeptigen were prepared and used to examine the distribution of atriopeptin-like immunoreactive material in the heart and brain of the rat. Granules of this material were seen in myocytes throughout the right and left atria and were densest in the perinuclear region. The distribution of atriopeptin-like immunoreactive material in the heart is consistent with previous reports of atrial secretory granules. In the brain neurons containing the material were observed in the hypothalamus and the pontine tegmentum. Atriopeptin in the brain may serve as a neurotransmitter in neural systems controlling blood volume and composition, the same physiological functions regulated by blood-borne atriopeptin.

Atriopeptins: bioactive peptides derived from mammalian cardiac atria.

Mammalian atria possess bioactive peptides that are natriuretic-diuretic and potent relaxants of vascular and nonvascular smooth muscle. Characterization of the biological activity of rat atrial extracts indicates two major peaks, having apparent molecular weight of 20,000-30,000 (atriopeptigen) and less than 10,000 (atriopeptins). The amino acid sequence of atriopeptins I, II and III have been determined, and it has been found that their structures are only slightly different. Atriopeptin I (twenty-one amino acid residues); ser-ser-cys-phe-gly-gly-arg-ile-asp-arg-ile-gly-ala-gln-ser-gly-leu-gly- cys- asn-ser) relaxes intestinal but not vascular smooth muscle strips, and is natriuretic. Atriopeptins II and III (23 and 24 residues; the 21-sequence of I with the addition of phe-arg or phe-arg-tyr at the C-terminus, respectively) relax intestinal and vascular smooth muscle strips and are potent natriuretics. Since atriopeptigen and the atriopeptins exhibit similar biological effects the possibility of a precursor-product relationship was tested. Mild proteolytic digestion (1IU/ml trypsin) of atriopeptigen activates this peptide and reduces its apparent molecular weight. Examination of whether the atria of Krebs perfused isolated hearts released the bioactive atrial peptides revealed the presence in the cardiac effluent of a trypsin-labile substance that was natriuretic-diuretic and a smooth muscle relaxant. To determine which form of the atrial peptide (e.g. atriopeptigen or atriopeptin) is released by the atria the cardiac effluents were concentrated and partially purified. The cardiac effluent contained a substance(s) similar to atriopeptin, but did not appear to possess the less-active high molecular weight peptide, atriopeptigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Atriopeptin release from the isolated perfused rabbit heart.

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (less than 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.

The sequence of an atriopeptigen: a precursor of the bioactive atrial peptides.

The high molecular weight fraction ( atriopeptigen -APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides ( Thibault , et al. (1984) FEBS Letters 167, 352-356, and Kangwa , et al., Biochem. Biophys. Res. Commun 119, 933-940) provide the complete primary structure of an 111 residue APG.

An atrial peptide is a potent renal vasodilator substance.

Renal intra-arterial administration of rat atrial extracts elicits a concentration dependent renal vasodilation (independent of prostaglandin or dopamine release) in anesthetized rats. The atrial extracts do not alter skeletal musculature (hindlimb) vascular resistance or systemic arterial blood pressure. The high molecular weight peptide fraction of atrial extracts obtained by gel filtration, reduces renal resistance intra-arterially only after proteolytic activation (in vitro) or following intravenous (i.e., systemic in vivo activation) administration. The low molecular weight peptide fraction of the atrial extract which is active intra-arterially as a renal vasodilator has been further purified to yield two major peptides. The 21 amino acid peptide, designated atriopeptin I, was previously demonstrated to be natriuretic and to relax intestinal but not vascular smooth muscle strips. This peptide exerted little or no intra-arterial effect on renal resistance. The 23 amino acid peptide (the phenylalanine-arginine C terminal extension of atriopeptin I), designated atriopeptin II, was natriuretic and spasmolytic (in vitro) on both intestinal and vascular strips and was a potent renal vasodilator in vivo. Thus, the renal vasodilator activity present in cardiac atrial extracts appears to derive from a proteolytic process which selectively generates the 23 amino acid peptide, atriopeptin II. Further cleavage with the loss of phenylalanine-arginine C-terminal, as occurs with atriopeptin I, markedly suppresses the renal vasodilation.

Atriopeptins: a family of potent biologically active peptides derived from mammalian atria.

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des- ser1 -, des- ser1 -ser2-, and des- ser21 - atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.

Kallikrein activation of a high molecular weight atrial peptide.

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.

Proteolytic activation of a bioactive cardiac peptide by in vitro trypsin cleavage.

Mammalian cardiac atria possess several unidentified biologically active peptides. Fractionation of rat atrial extracts by gel filtration chromatography revealed two major fractions [apparent molecular weights of 20,000-30,000 (peak I) and less than 10,000 (peak II)], both of which were potent natriuretic agents (eliciting a 25-fold increase in sodium excretion) and smooth muscle relaxants. Vigorous treatment with trypsin (100 units/ml at 37 degrees C for 15 min) of both fractions abolished all biological activity. Further purification of the lower molecular weight fraction (peak II) by ion-exchange chromatography indicated two subfractions that possessed potent natriuretic activity and that preferentially relaxed either intestinal (designated peak IIA) or vascular (peak IIB) smooth muscle assay tissues. The similarity of the biological effect of the high (peak I) and low (peak II) molecular weight peptides led us to test the possibility of precursor-product relationship. Mild proteolytic treatment of the high molecular weight peptide with trypsin (1 unit/ml at room temperature) markedly enhanced the smooth muscle relaxant activity. Subsequent analysis of the trypsin (1 unit/ml)-treated high molecular weight peptide (peak I) by gel filtration and ion-exchange chromatography revealed that the peptide now resembled the low molecular weight peptides (peaks IIA and IIB) present in the original atrial extract. These data suggest that the cardiac atria contain a relatively inactive (smooth muscle relaxant) high molecular weight peptide and suggest that biologically active low molecular weight peptides can subsequently be generated by proteolytic cleavage.

Purification and sequence analysis of bioactive atrial peptides (atriopeptins).

Mammalian cardiac atria have several biologically active peptides that exert profound effects on sodium excretion, urine volume, and smooth muscle tone. In the present study two such peptides of low molecular weight were purified and separated from each other on the basis of differences in charge, hydrophobicity, and biological profile. The first peptide, designated atriopeptin I, exhibits natriuretic and diuretic activity and selectivity relaxes intestinal smooth muscle but not vascular smooth muscle strips. The second peptide, atriopeptin II, is a potent natriuretic and diuretic that relaxes both intestinal and vascular strips. Sequence analysis of atriopeptin I indicates that it is composed of 21 amino acids, of which serine and glycine residues predominate. The amino terminal sequence of atriopeptin II up to residue 21 is the same as that of atriopeptin I, with the addition of the Phe-Arg extension at the carboxyl terminus. Both peptides appear to be derived from a common high molecular weight precursor (designated atriopeptigen); their biological selectivity and potency may be determined by the site of carboxyl terminal cleavage.

Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria.

Mammalian atrial extracts possess natriuretic and diuretic activity. In experiments reported here it was found that atrial, but not ventricular, extract also causes relaxation of isolated vascular and nonvascular smooth muscle preparations. The smooth muscle relaxant activity of atrial extract was heat-stable and concentration-dependent and could be destroyed with protease. Rabbit aortic and chick rectum strips were used for the detection of atrial biological activity. The atrial activity was separated by column chromatography into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. The atrial substance that copurified with the smooth muscle relaxant activity in both peaks caused natriuresis when injected into conscious rats. It appears that atria possess at least two peptides that elicit smooth muscle relaxation and natriuresis, suggesting an endogenous system of fluid volume regulation.

Characterization of increased plasma dopamine-beta-hydroxylase activity in rats with experimental diabetes.

Plasma dopamine-beta-hydroxylase (pDBH) activity is markedly elevated in chronic experimental streptozotocin (STZ) diabetes in the rat. Several possible explanations, all potentially relevant to the pathophysiology of diabetes, could explain this observation. The objective of this paper was to further delineate the behavior of pDBH in diabetes and examine several possible mechanisms fot the increase. Plasma DBH increases within 1 day of STZ administration, is fivefold elevated within 1 wk, and slowly reaches ninefold control values after several months. Similar changes result from alloxan-induced diabetes. The increase in pDBH activity correlates well with the severity of diabetes as assessed by plasma glucose levels. Reversal of the diabetic state with insulin administration or islet cell transplantation results in the decrease of pDBH activity toward normal values. Plasma DBH is not increased in hyperglycemic obese (ob/ob) mice, suggesting a primary dependence of pDBH elevation on reduced levels of insulin and not hyperglycemia per se. Guanethidine-sympathectomized and sympathectomized/adrenal demedullated animals, with 60% and 25% of control levels of pDBH, respectively, show the same percentage increase in pDBH activity with induction of diabetes, thus, the increase in pDBH does not result from a selective activation or dysfunction of the sympathetic nervous system or adrenal medulla in diabetic animals. No evidence is found for the alteration of the kinetic parameters, molecular size, or charge of pDBH in diabetes. Several mechanisms for the increase are considered.

The structure of rat proalbumin.

The structure of rat proalbumin, a liver precursor to rat serum albumin, has been determined to consist of the hexapeptide Arg-Gly-Val-Phe-Arg-Arg attached to the NH2 terminus of the polypeptide chain of rat serum albumin. Edman degradation of a proalbumin preparation for 14 rounds gave the major sequence Arg-Gly-Val-Phe-Arg-Arg-Glu-Ala-His-Lys-Ser-Glu-Ile-Ala. A comparison of cyanogen bromide fragments suggests that these two proteins differ only in this respect. On treatment with cyanogen bromide, these proteins gave three classes of peptides with molecular weights of 30,000, 10,000, and smaller than or equal to 5,000. A combination of gel filtration, electrofocusing, and ion exchange established that these peptides were indistinguishable, with exception of those of 10,000 molecular weight. By amino acid and sequence analyses this fraction from rat serum albumin was found to be the NH2-terminal fragment. Radiochemical amino acid and sequence analyses show that the NH2-terminal hexapeptide is the major fragment released from proalbumin by limited tryptic hydrolysis. The protein that remains cannot be distinguished from rat serum albumin.