Isomorphous replacement of cystine with selenocystine in endothelin: oxidative refolding, biological and conformational properties of [Sec3,Sec11,Nle7]-endothelin-1.

Air re-oxidation of fully reduced human endothelin-1 under optimized conditions yields the natural isomer with parallel disulfide bridges and the non-natural isomer with crossed disulfide bridges at a ratio of 3:1. In view of the recently determined highly reducing redox potential of selenocysteine (-381 mV) in peptides, the half-cystine residues Cys3 and Cys11 of the natural isomer of endothelin-1 were replaced by selenocysteine. Taking advantage of the high stability of the diselenide group toward reducing agents for disulfides a regioselective disulfide bridging of the second cysteine pair allowed for straightforward preparation of the [Sec3,Sec11, Nle7]-endothelin-1. NMR structural analysis showed conformational preferences of this endothelin analog that were identical to those of the natural hormone. Similarly, the bioactivity data confirmed that replacement of cysteine residues with selenocysteine was without detectable effect on receptor recognition and signal transduction. Both findings strongly support that the exchange of sulfur against selenium produces a fully isomorphous molecule as recently observed for similar exchanges at the level of methionine residues in proteins. Moreover, oxidative refolding of the fully reduced [Sec3,Sec11,Nle7]-endothelin-1 fulfilled the expectation that the redox potential of the selenocysteines would dictate quantitative formation of the natural isomer. These results suggest that the selenocysteine approach, besides offering an interesting chemical tool for induction of correct oxidative folding of multiple cysteine-containing peptides, should even allow for the preparation of non-natural isomers and thus for studying conformational preferences of folding intermediates in peptides and proteins.

Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) from rat aorta possess specific receptors for a novel potent vasorelaxant peptide, adrenomedullin (AM). To elucidate its receptor coupling to guanine nucleotide-binding stimulatory protein and the structural requirement of the AM molecule to its vascular receptors, we have studied the effects of guanine nucleotides on [125I]human (h) AM binding and adenylate cyclase activity in cultured rat VSMC, and the effects of various synthetic hAM analogs on [125I]hAM binding and the cAMP response. Guanosine 5'-O-(3-thiotriphosphate) dose dependently inhibited [125I]hAM binding to rat VSMC membranes. hAM stimulated adenylate cyclase activity, and its effect was additive with GTP. hAM-induced cAMP formation was abrogated by pretreatment with cholera toxin, but not by that with pertussis toxin. Intact hAM-(1-52)-NH2 and N-terminal truncated derivatives [hAM-(13-52)-NH2, hAM-(16-52)-NH2] almost equally inhibited [125I]hAM binding and stimulated cAMP formation, whereas removal of C-terminal Tyr52 residue [hAM-(1-51)-NH2] remarkably decreased receptor-binding activity and the cAMP response. The effects of hAM-(1-52)-OH, hAM-(1-51)-OH, and a linear hAM analog ([carbamoylmethyl-Cys16,21]hAM-NH2) were far less potent on receptor binding and the cAMP response than that of hAM-(1-52)-NH2. The C-terminal fragment [hAM-(33-52)-NH2] and the N-terminal fragment [hAM-(1-10)-OH] had neither receptor-binding nor adenylate cyclase activity. hAM-(22-52)-NH2 had no agonistic effect, but showed an antagonistic effect on the hAM-induced cAMP response. These data suggest that vascular AM receptors are functionally coupled to adenylate cyclase via guanine nucleotide-binding stimulatory protein. Studies of the structure-activity relationship of hAM revealed that the cyclic structure formed by the disulfide bridge and amidation of the C-terminal residue of the AM molecule are critical for receptor binding and subsequent cAMP generation and suggest that the C-terminal fragment hAM-(22-52)-NH2 may be an antagonist for vascular AM receptors.

A novel natriuretic peptide isolated from eel cardiac ventricles.

A new natriuretic peptide, which exhibits the entire spectrum of actions known to be characteristic of atrial and brain natriuretic peptides (ANP and BNP), was isolated from eel cardiac ventricles and has been named ventricular natriuretic peptide (VNP). The primary structure of eel VNP is characterized by its uniquely long C-terminal 'tail' that extends from the second half-cystine. Thus, eel VNP appears to be a novel natriuretic peptide of a type not found in mammals. With respect to natriuretic (rat) and vasodepressor (rat and eel) activities, eel VNP is much more potent than human ANP in eels and almost equipotent in rats. Strong tachyphylaxis is observed for the vasodepressor effect in both rats and eels, whereas it is not observed for the natriuretic effect in rats.

Binding of synthetic beta-human atrial natriuretic peptide to cultured rat vascular smooth muscle cells.

We have studied the effects of synthetic beta-human atrial natriuretic peptide (beta-hANP), an antiparallel dimer of alpha-hANP, on receptor binding and cGMP generation in cultured rat vascular smooth muscle cells and compared the effects with those of alpha-hANP. Characteristics of temperature-dependent binding and degradation of 125I-beta-hANP were similar to those of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for beta-hANP with a maximal binding capacity one-half that of alpha-hANP. Parallel and antiparallel dimers were equipotent in inhibiting the binding and stimulating intracellular cGMP formation, of which the maximal effect was about one-half that of alpha-hANP. Reverse-phase high performance liquid chromatography revealed that most of beta-hANP added to cells was converted to a small molecular mass component corresponding to alpha-hANP after incubation. These data suggest that the less potent effect of beta-hANP in receptor binding and cGMP generation may be partly accounted for by the possible conversion of beta-hANP to alpha-hANP at the site of target cells.

A new natriuretic peptide isolated from cardiac atria of trout, Oncorhynchus mykiss.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells.

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [125I]rAM revealed the presence of a single class of high-affinity (Kd 1.3 x 10(-8) M) binding sites for rAM in VSMC. The apparent Ki of rat calcitonin gene-related peptide (rCGRP) was 3 x 10(-7) M. Affinity labeling of VSMC membranes with [125I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP, rAM stimulated cAMP formation with an approximate EC50 of 10(-8) M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranolol, indomethacin, or quinacrine, but inhibited by a CGRP receptor antagonist, human CGRP[8-37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.

Synthesis and specific pressor activity of [1-aspartic acid,5-valine,9-serine]angiotensin I ("fowl angiotensin I").

[Asp1, Val5, Ser9]angiotensin I was synthesized by Merrifield's solid-phase procedure. The dansylated derivative of this angiotensin was cochromatographed on the TLC with the dansylated angiotensin decapeptide isolated from white leghorn fowl. Either angiotensin showed identical behavior. The per mole pressor activity of the synthetic decapeptide (in rats anesthetized with pentobarbital and treated with pentolinium) as compared to mammalian angiotensins, namely, [Ile5]angiotensin I, [Val5]angiotensin I, [Ile5]angiotensin II, and [Val5]angiotensin II, was 157, 181, 114, and 85%, respectively.

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

A Ca(2+)-sensitive electrode was used to measure the Ca2+ concentration of the medium containing the heavy fraction of the fragmented sarcoplasmic reticulum (SR) prepared from guinea-pig psoas muscle. Among K(+)-channel blockers tested, 4-aminopyridine (4-AP), tetraethylammonium (TEA) and charybdotoxin elicited Ca2+ release from the SR, but apamin and glibenclamide did not. These results suggest that a reduction of SR K+ conductance leads to Ca2+ release from the SR.

Eel ventricular natriuretic peptide: isolation of a low molecular size form and characterization of plasma form by homologous radioimmunoassay.

Ventricular natriuretic peptide (VNP) with 25 amino acid residues was isolated from the low molecular weight fraction of acid extracts of eel cardiac ventricles. No other short forms of VNP were recovered from the fraction. This peptide was named eel VNP(1-25) because it was a C-terminally truncated form of the previously isolated eel VNP(1-36). As observed before with eel VNP(1-36), eel VNP(1-25) had a much higher (146-fold) vasodepressor activity than human atrial natriuretic peptide (ANP) in eels, but was a third to a half as active in rats with respect to vasodepressor and natriuretic activities. Eel VNP(1-25) was generally less potent than eel VNP(1-36) for vasodepressor and natriuretic effects. A specific radioimmunoassay (RIA) has been developed for the measurement of eel VNP. The antiserum, raised against eel VNP(1-36), was highly specific and did not exhibit significant cross-reactivity with eel ANP and C-type natriuretic peptide, even though their amino acid sequences have more than 60% homology with that of eel VNP. The sensitivity of assay was 0.5 fmol/tube for eel VNP(1-36) with more than 99% confidence. Such high sensitivity permitted direct assaying of VNP with only a few microliters of plasma. In fresh water eels, the concentration of VNP in the cardiac ventricle was higher than those in the atrium or brain and that of ANP in the ventricle. Thus, VNP seems to be a ventricular hormone. Although ANP is a major circulating hormone in mammals, the plasma concentration of VNP was threefold higher than that of ANP.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel angiotensin I isolated from an elasmobranch fish.

It is believed that the renin-angiotensin system evolved initially in primitive bony fishes and is absent from elasmobranchs. We have isolated angiotensin I from the incubates of plasma and kidney extracts of an elasmobranch fish, Triakis scyllia, using eel vasopressor activity as an assay system. Its sequence was determined to be H-Asn-Arg-Pro-Tyr-Ile-His-Pro-Phe-Gln-Leu-OH. Dogfish angiotensin I is teleost-like because of an asparagine residue at position 1 but it is mammalian-like because of an isoleucine residue at position 5. The unique and most important substitution in dogfish angiotensin I is a proline residue at position 3 which may cause significant changes in its tertiary structure. A glutamine residue at position 9 is also unique among all angiotensin Is sequenced to date. Dogfish angiotensin I is more potent than rat angiotensin I in its vasopressor activity in the dogfish but the relationship is reversed in the rat. Thus angiotensin receptors as well as the hormone molecules appear to have evolved during vertebrate phylogeny. Our findings establish the elasmobranch renin-angiotensin system and support the hypothesis that the renin-angiotensin system is a phylogenetically old hormonal system which plays important roles in cardiovascular and fluid homeostasis.

A C type natriuretic peptide is a vasodilator in vivo and in vitro in the common dogfish.

The effects of an elasmbranch cardiac C-type natriuretic peptide (dogfish CNP-22) on arterial blood pressure were investigated in vivo in chronically cannulated dogfish Scyliorhinus canicula and in vitro by a myographic technique using the distal part of the first branchial artery. In-vivo dogfish CNP-22 caused a dose-dependent reduction in mean arterial blood pressure which was much more potent than that of alpha-human ANP. In-vitro dogfish CNP-22 also caused a dose-dependent relaxation which was independent of the endothelium. These results are in marked contrast to those obtained in similar studies on other vertebrate species in which CNP exhibited only mild hypotensive effects compared to both atrial and brain natriuretic peptides. This study indicates the importance of using homologous peptides in determining the physiological role of natriuretic peptides in non-mammalian vertebrates.

Centrally induced vasopressor and sympathetic responses to a novel endogenous peptide, adrenomedullin, in anesthetized rats.

Possible central actions of adrenomedullin were explored and compared with the peripheral effects by injecting it into the lateral ventricle, cisterna magna, and femoral vein in urethane-anesthetized rats. Adrenomedullin, 1.0 to 3.0 nmol/kg, injected intravenously (i.v.), caused a transient vasodepression of about 10 to 30 mm Hg, dose dependently, which lasted for < 15 min. On the other hand, intracerebroventricular (ICV) and intracisternal (IC) injections of adrenomedullin elicited sustained elevations of arterial pressure of gradual onset, dose dependently; the arterial pressure started to rise at about 3 min after the injection, and gained peak response after > 20 min. The pressor response lasted for > 2 h. Heart rate was not significantly influenced by these doses of adrenomedullin. The abdominal sympathetic outflow was markedly increased in relation to the blood pressure elevation. The time-course of the responses was quite similar with both ICV and IC injections. Hypotensive effects of i.v. injected adrenomedullin was partially attenuated, and the centrally induced vasopressor responses were abolished by the pretreatment with human calcitonin gene-related peptide (hCGRP)-receptor antagonist, hCGRP(8-37). These findings indicate that the receptors for adrenomedullin exist in the brain, and that the receptor site may be anatomically far from the surface of the brain and the ventricular system because the onset of the pressor response was delayed. Or, CGRP and adrenomedullin may share the same receptors, particularly in the brain.

Structure-activity relationships of alpha-human atrial natriuretic peptide.

The spasmolytic activity of synthetic alpha-human atrial natriuretic peptide (alpha-hANP) and its related peptides was determined in vitro using the chick rectum and the rat aorta. Natriuretic activity was also measured in the anesthetized rat, alpha-hANP-(7-28), with the NH2-terminal hexapeptide truncated, had greater spasmolytic and natriuretic effect than did alpha-hANP-(1-28). These responses were reduced by truncation of the COOH-terminal residues. alpha-hANP-(7-23), the cyclic structure of alpha-hANP-(1-28), exhibited weak aortic relaxation and natriuretic activities. However, alpha-hANP-(7-23) produced a greater relaxation than did alpha-hANP-(1-28) in the chick rectum. Elimination of Gly at position 9 reduced the spasmolytic and natriuretic activity. Substitution of amino acid residues at position 8, 12 and 13 changed these activities. Analogues containing the ethylene linkage instead of the disulphide bond had weak biological activity. These results indicate that the size of the 17-amino acid ring and the COOH-terminal residues of alpha-hANP are important for the expression of spasmolytic and natriuretic activity.

Receptor binding and vasoconstrictor activity of big endothelin.

We studied the effects of synthetic porcine big endothelin (ET), a putative 39-residue intermediate deduced from cDNA sequence analysis, on receptor binding activity in cultured rat vascular smooth muscle cells as well as its vasoconstrictive activity in rat pulmonary artery. Big ET was about two orders of magnitude less active than ET in the receptor binding and vasoconstriction assays. Our data are consistent with the contention that big ET is an intermediate which can be converted into fully bioactive 21-residue mature ET.

Effects of homologous atrial, brain, and C-type natriuretic peptides on isolated heart and blood vessels of bullfrog.

The cardiovascular effects of homologous natriuretic peptides (fNPs) were examined in the bullfrog, Rana catesbeiana. Synthetic bullfrog atrial natriuretic (fANP), brain natriuretic (fBNP) and C-type natriuretic peptides (fCNP I and fCNP II), were tested in vitro and compared under the same experimental conditions. All frog NPs produced a significant, and concentration-dependent, reduction in tension (relaxant effect) in the isolated dorsal aorta. Frog CNP II exhibited similar vasorelaxation profile as that of fANP, while fBNP and fCNP I had lower activity than fANP. Frog NPs inhibited norepinephrine induced contraction and fCNP II was most potent. In isolated preparations of atrium and ventricle, fCNP I and II produced a significant, and concentration-dependent, reduction in tension, but neither fANP nor fBNP produced any significant effects. Frog CNP II is most potent among fNPs in relation to reduce the cardiac output. Chronotropic responses of the heart to administrations of NPs were insignificant. The present results for the first time showed that fNPs play roles in the control of cardiovascular system, both blood vessels and heart, in the bullfrog.

Amino acid sequence of sardine calcitonin and its hypocalcemic activity in rats.

A novel calcitonin (CT) was isolated from the spotlined sardine, Sardinops melanostictus. The primary structure of sardine CT was determined as follows: H-Cys-Ser-Asn-Leu-Ser-Thr- Cys-Ala-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Ser-Tyr-Pro-Arg- Thr- Asn-Val-Gly-Ala-Gly-Thr-Pro-NH2. This amino acid sequence was different from that of salmon CT in 4 amino acid residues at positions 8th, 21th, 27th and 29th. As judged by the international method of CT bioassay, hypocalcemic activity of sardine CT was calculated as 4156 IU/mg. When compared for durability of CTs, it was found that sardine CT was significantly more potent than that of salmon CT. This is the first report of CT from a marine species of teleost.

Blood pressure regulation by angiotensin in the spontaneously hypertensive rats.

Plasma renin activity (PRA) was subnormal or normal in the main strain of spontaneously hypertensive rats (SHR). PRA increased greatly in the stroke-prone substrain of SHR (SHRSP) at 20-30 weeks of age. Captopril (SQ 14,225) is an orally active angiotensin-converting enzyme inhibitor. The drug acutely decreased blood pressure moderately in SHR, and markedly in SHRSP. Participation of the renin-angiotensin system in the pathogenesis of hypertension in SHR may be limited. Etiology of hypertension in connection with renal excretory function and the central and peripheral nervous system is discussed.

Central and peripheral cardiovascular actions of adrenomedullin 5, a novel member of the calcitonin gene-related peptide family, in mammals.

Adrenomedullin 5 (AM5) is a new member of the calcitonin gene-related peptide (CGRP) family identified in teleost fish. Although its presence was suggested in the genome database of mammals, molecular identity and biological function of AM5 have not been examined yet. In this study, we cloned a cDNA encoding AM5 in the pig and examined its cardiovascular and renal effects. Putative mature AM5 was localized in the middle of prohormone and had potential signals for intermolecular ring formation and C-terminal amidation. The AM5 gene was expressed most abundantly in the spleen and thymus. Several AM5 genes were newly identified in the database of mammals, which revealed that the AM5 gene exists in primates, carnivores, and undulates but could not be identified in rodents. In primates, nucleotide deletion occurred in the mature AM5 sequence in anthropoids (human and chimp) during transition from the rhesus monkey. Synthetic mature AM5 injected intravenously into rats induced dose-dependent decreases in arterial pressure at 0.1-1 nmol/kg without apparent changes in heart rate. The decrease was maximal in 1 min and AM5 was approximately half as potent as AM. AM5 did not cause significant changes in urine flow and urine Na+ concentration at any dose. In contrast to the peripheral vasodepressor action, AM5 injected into the cerebral ventricle dose-dependently increased arterial pressure and heart rate at 0.1-1 nmol. The increase reached maximum more quickly after AM5 (5 min) than AM (15-20 min). AM5 added to the culture cells expressing calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of the receptor activity-modifying proteins (RAMPs), the combination of which forms major receptors for the CGRP family, did not induce appreciable increases in cAMP production in any combination, although AM increased it at 10(-)(10)-10(-)(9) M when added to the CLR and RAMP2/3 combination. These data indicate that AM5 seems to act on as yet unknown receptor(s) for AM5, other than CLR/CTR+RAMP, to exert central and peripheral cardiovascular actions in mammals.

Chronic osmotic stimuli increase salusin-beta-like immunoreactivity in the rat hypothalamo-neurohypophyseal system: possible involvement of salusin-beta on [Ca2+]i increase and neurohypophyseal hormone release from the axon terminals.

Salusin-alpha and -beta were recently discovered as bioactive endogenous peptides. In the present study, we investigated the effects of chronic osmotic stimuli on salusin-beta-like immunoreactivity (LI) in the rat hypothalamo-neurohypophyseal system. We examined the effects of salusin-beta on synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) and neurohypophyseal hormone release from both freshly dissociated SONs and neurohypophyses in rats. Immunohistochemical studies revealed that salusin-beta-LI neurones and fibres were markedly increased in the SON and the magnocellular division of the paraventricular nucleus after chronic osmotic stimuli resulting from salt loading for 5 days and dehydration for 3 days. Salusin-beta-LI fibres and varicosities in the internal zone of the median eminence and the neurohypophysis were also increased after osmotic stimuli. Whole-cell patch-clamp recordings from rat SON slice preparations showed that salusin-beta did not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. In vitro hormone release studies showed that salusin-beta evoked both arginine vasopressin (AVP) and oxytocin release from the neurohypophysis, but not the SON. In our hands, in the neurohypophysis, a significant release of AVP and oxytocin was observed only at concentrations from 100 nm and above of salusin-beta. Low concentrations below 100 nm were ineffective both on AVP and oxytocin release. We also measured intracellular calcium ([Ca(2+)](i)) increase induced by salusin-beta on freshly-isolated single nerve terminals from the neurohypophysis devoid of pars intermedia. Furthermore, this salusin-beta-induced [Ca(2+)](i) increase was blocked in the presence of high voltage activated Ca(2+)channel blockers. Our results suggest that salusin-beta may be involved in the regulation of body fluid balance by stimulating neurohypophyseal hormone release from nerve endings by an autocrine/paracrine mechanism.

Chemical structure of angiotensin in the bullfrog Rana catesbeiana.

Crude bullfrog angiotensin was prepared by modified Boucher's procedures. The kidney extract was incubated in vitro with homologous plasma. Crude angiotension, 15 micrograms equivalent to [Asp1, Ile5]angiotensin II in rat vasopressor assay, was purified further by three steps by SEP-PAK C18 cartridge, SP-Sephadex column chromatography, and high-performance liquid chromatography on Finepak SIL C18 column. Amino acid analysis was done on 64 ng of the purified material after acid hydrolysis. The fluorescent peptide mapping technique was used to confirm the amino acid sequence. It is proposed that bullfrog angiotensin is [Asp1, Val5, Asn9]angiotensin I.