Actions of angiotensin peptides on the rabbit pulmonary artery

The presence of the angiotensin AT1A-like receptor subtype in the pulmonary artery and AT1B-like receptor subtype in the pulmonary trunk of the rabbit has been reported in two earlier studies. The present study further investigated these receptor subtypes using five other angiotensins (namely angiotensin II, angiotensin III, angiotensin IV, angiotensin-(1-7) and angiotensin-(4-8)). The direct action of the angiotensins on the rabbit pulmonary arterial and trunk sections and the ability of each angiotensin to further contract or relax preconstricted sections of the pulmonary artery and trunk were studied using the organ bath set-up. The effects of angiotensin III on the 3H overflow from re-uptaken [3H]noradrenaline in the electrically-contracted rabbit pulmonary arterial and trunk sections were also studied. The contractile response of the arterial and trunk section had the following rank order potency: angiotensin II > angiotensin III > angiotensin IV. The contractile response to these angiotensins was greatly reduced or absent in the pulmonary trunk. Angiotensin II further contracted the preconstricted arterial and trunk sections. In contrast, angiotensin III further contracted the preconstricted arterial section but relaxed the preconstricted trunk section. Angiotensin IV similarly relaxed the preconstricted trunk section but had minimum effect on the preconstricted arterial section. Angiotensin-(1-7) and angiotensin-(4-8) had no effect on both sections. The actions of the three angiotensins were inhibited by losartan, an AT1-selective antagonist. Indomethacin, a cyclo-oxygenase inhibitor, inhibited the relaxation caused by angiotensin III and angiotensin IV in the trunk section. The effects of angiotensin III on the electrically preconstricted sections of the pulmonary trunk and artery were not accompanied by any significant changes in 3H overflow. The differential responses produced by angiotensin II and its immediate metabolites via two positionally located and functionally opposing receptor subtypes suggest that the pulmonary trunk and artery is not a passive conduit but an important regulator of blood flow from the heart to the lung.     

Angiotensin III and IV activation of the brain AT1 receptor subtype in cardiovascular function

The present investigation determined that native angiotensins II and III (ANG II and III) were equipotent as pressor agents when ICV infused in alert rats, whereas native angiotensin IV (ANG IV) was less potent. An analogue of each of these angiotensins was prepared with a hydroxyethylamine (HEA) amide bond replacement at the N-terminus, yielding additional resistance to degradation. These three angiotensin analogues, HEA-ANG II, HEA-ANG III, and HEA-ANG IV, were equivalent with respect to maximum elevation in pressor responses when ICV infused; and each evidenced significantly extended durations of effect compared with their respective native angiotensin. Comparing analogues, HEA-ANG II had a significantly longer effect compared with HEA-ANG III, and HEA-ANG IV, whereas the latter were equivalent. Pretreatment with the AT₁ receptor subtype antagonist, Losartan (DuP753), blocked subsequent pressor responses to each of these analogues, suggesting that these responses were mediated by the AT₁ receptor subtype. Pretreatment with the specific AT₄ receptor subtype antagonist, Divalinal (HED 1291), failed to influence pressor responses induced by the subsequent infusion of these analogues. These results suggest an important role for Ang III, and perhaps ANG IV, in brain angiotensin pressor responses mediated by the AT₁ receptor subtype.     

Laser Raman spectroscopic analysis of angiotensin peptides' conformation

In this study we examined the conformation and side chain environments of angiotensins I, II, III, and [Sar¹-Ile⁵-Ala⁸]angiotensin II using laser Raman spectroscopy. The positions of the amide I bands for all four peptides were found between 1664 and 1673 cm^?1. D₂O exchange studies confirmed the positions of the amide I and amide III bands. The positions of the amide I bands for all the angiotensins were found at approximately 1665 cm^?1 and the amide III bands were all located between 1265 and 1278 cm^?1. From the positions and intensities of the amide I and III bands we concluded that all peptides share the same overall conformation consisting of β-turn structure. Spectral analysis indicated that although the spectra for all the peptides were qualitatively identical there was evidence that the angiotensin conformations were more flexible in the aqueous phase than the solid phase. Examination of the $\text{850}\text{830}$ cm^?1 tyrosine doublet suggested that the tyrosine residue in the peptides is exposed to the solvent environment and becomes more exposed as the peptide length is decreased. Therefore, there are some localized conformational differences among the angiotensins. The conformational data yielded by this study leads us to conclude that the various biological properties ascribed to the angiotensins are not due to different conformations of the peptides. The biological differences could perhaps be attributed to localized interactions of the individual amino acid residues with themselves and with the hormone receptors.     

Evaluation of angiotensins II and III as vasoconstrictors in the hepatic and hindlimb vasculatures of dogs

Previous work has demonstrated that intravenously administered angiotensin II is more potent than angiotensin III as a systemic vasopressor agent. We tested the hypothesis that this difference in potency is caused at least partially by angiotensin II being more potent than angiotensin III as a vasoconstrictor in the hindlimb and hepatic vasculatures. The effects of angiotensins II and III on hindlimb and hepatic blood flow were evaluated in 14 dogs anesthetized with pentobarbital. Blood flows were measured electromagnetically. Graded doses of angiotensins II and III were administered as bolus injections directly into the arterial supply of the hindlimb and liver. On the basis of duration and graphic integration of the flow responses, but not on the basis of absolute changes in amplitude, angiotensin II was significantly more potent than angiotensin III as a vasoconstrictor in the hindlimb vasculature. In the hepatic circulation the flow changes produced by angiotensin II and angiotensin III were not significantly different on the basis of duration, graphic integration, or amplitude. We conclude that (i) differential vasoconstrictor responses of the hindlimb, but not the hepatic circulation, to angiotensins II and III contribute to the difference in systemic vasopressor potency between these two peptides, and (ii) because flow responses are an integral event with duration and constantly varying amplitude, evaluation of vasoconstrictor potency based only upon amplitude of the flow changes can be misleading.     

Enzymatic formation of angiotensins II and III in the hindlimb circulation of dogs

Experiments were performed in 14 anesthetized dogs to (1) to determine if the reductions in hindlimb blood flow produced by [des-Asp1] angiotensin I were due to its local enzymatic (kininase II) conversion to angiotensin III and (2) to quantitate the extent of conversion of angiotensin I to angiotensin II and of [des-Asp1] angiotensin I to angiotensin III in the hindlimb circulation. Graded doses of these peptides were administered as bolus injections directly into the left external iliac artery while measuring flow in this artery electromagnetically. Dose-response relationships were determined before and during the inhibition of kininase II activity with captopril or antagonism of angiotensin receptor sites with [Ile7] angiotensin III. Captopril inhibited the vasoconstrictor responses to angiotensin I and [des-Asp1] angiotensin I, but did not affect the responses to angiotensins II or III, or norepinephrine. [Ile7] angiotensin III inhibited the vasoconstrictor responses to all four angiotensin peptides but did not alter the responses to norepinephrine. These findings indicate that the hindlimb vasoconstrictor responses to [des-Asp1] angiotensin I were due to the local formation of angiotensin III. The extent of conversion of [des-Asp1] angiotensin I to angiotensin III that occurred in one transit through the hindlimb arterial circulation was estimated to be 36.7%, which was not different from the estimated 36.4% conversion of angiotensin I to angiotensin II. We conclude that angiotensin I and [des-Asp1] angiotensin I are converted to their respective vasoactive forms (angiotensins II and III) to a similar extent in the hindlimb circulation via the action of kininase II.     

Granuloma macrophages in murine schistosomiasis mansoni generate components of the angiotensin system

The angiotensin cascade was recently detected in liver granulomas of murine Schistosomiasis mansoni, suggesting an immunoregulatory role for angiotensins in inflammation. In this study, isolated liver granulomas were fractionated into macrophage or lymphocyte-eosinophil-rich populations to determine the cellular origin of these hormones. Immunoreactive angiotensins I, II, and III (AI, AII, and AIII) were detected in granuloma macrophage homogenates by radioimmunoassay and chromatography. No angiotensins were associated with the lymphocyte-eosinophil fraction. Isolated granuloma macrophages, but not the lymphocyte-eosinophil fraction, retained appreciable angiotensins when cultured in vitro and spontaneously released these peptides into the culture medium. Similarly, culture of these cells in the presence of exogenous angiotensinogen or AI resulted in additional AI and/or AII/III appearing in the medium. These data support the contention that granuloma macrophages generate angiotensins from both endogenous and exogenous substrates.     

The involvement of angiotensins in the control of prostatic epithelial cell proliferation in the rat.

The effects of captopril (the inhibitor of the angiotensin-converting enzyme) and of angiotensins II and IV (3-8 fragment of angiotensin II) on cell proliferation of the prostatic epithelium was investigated in the rat. The incorporation of bromodeoxyuridine into cell nuclei was used as an index of cell proliferation. It was found that the treatment with captopril resulted in the suppression of prostatic epithelial cell proliferation. The antiproliferative effect of captopril was reversed (at least partially) by a simultaneous treatment with either angiotensin II or angiotensin IV. The effects of angiotensins were not blocked by the administration of losartan--AT1 angiotensin receptor blocker. These findings suggest the involvement of angiotensins in the control of prostatic growth, acting via the receptors different from the AT1-subtype (presumably via AT4 receptors).     

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.     

Chain length effects on helix-hairpin distribution in short peptides with Aib-DAla and Aib-Aib segments

The Aib-D Ala dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and Boc-Aib-Xxx-NHMe (4, 8), where Xxx=DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7), and Boc-Aib-Aib-NHMe (8) helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3 OH. The β-turn conformations (type-I'/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc-Aib-D Ala-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 3(10)-helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc-Val-Aib-DAla-Leu-NHMe (3) adopts a novel α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of an NOE between Val (1) NH?HNCH3 (5) in CD3OH suggests, that the solid state conformation is maintained in methanol solutions.     

Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Dipeptidyl-aminopeptidase III was isolated from cells of the cellular slime moldDictyostelium discoideum in the culmination stage of development. The enzyme was purified 18-fold by precipitation with ammonium sulfate and gel filtration chromatography and was shown to have a molecular weight of 158,000 and a sharp pH optimum at pH 10.2 and to be inhibited by sulfhydryl reagents. The enzyme acted upon the artificial substratearginyl-arginyl-β-naphthylamide, producing arginyl-arginine andβ-naphthylamine but notarginyl-β-naphthylamide. Activity towardarginyl-arginyl-β-naphthylamide was strongly inhibited by physiological concentrations of angiotensin III and, to a lesser extent, by angiotensins I and II and other angiotensin-related peptides but not by enkephalin peptides. Several dipeptides known to inhibit mammalian dipeptidyl-aminopeptidase III also inhibited theDictyostelium enzyme. Incubation of the enzyme preparation with angiotensins resulted in their conversion into a complex mixture of products. Thus dipeptidyl-aminopeptidase III fromDictyostelium closely resembles the mammalian enzyme in many of its characteristics.     

Effects of angiotensins on cellular hypertrophy and c-fos expression in cultured arterial smooth muscle cells.

An increase in cell size and protein content was observed when quiescent arterial smooth muscle cells in culture were incubated with either angiotensin II or III. These effects were inhibited by the specific angiotensin type-1 receptor antagonist losartan (DuP753) but not by CGP42112A. In parallel, a transient and dose-dependent induction of c-fos was demonstrated not only with angiotensins II and III but also with angiotensin I. Both angiotensins II and III exerted their maximal effect at 1 microM, while angiotensin I needed a tenfold-higher concentration to exert an identical effect. As for hypertrophy, losartan also inhibits angiotensin-induced c-fos expression, suggesting that this gene may be involved into the hypertrophic process. Angiotensin-I-mediated c-fos induction is partially inhibited by the angiotensin-converting enzyme inhibitors captopril and trandolaprilate; given that an angiotensin-converting enzyme activity was detected in these smooth muscle cell cultures, these results suggest that angiotensin-I-induced c-fos expression is mediated in part via angiotensin-I conversion to angiotensin II, but also by other unidentified pathway(s). Angiotensin I could essentially induce smooth muscle cell hypertrophy by indirect mechanisms, while angiotensins II and III act directly on smooth muscle cells.     

Steroids as potential modulators of angiotensin receptors in bovine adrenal glomerulosa and kidney

To test the hypothesis that there is feedback inhibition of adrenal angiotensin receptors by substances released in response to the peptides, we measured binding of labeled angiotensins in the presence of various steroids. Approximately half of the 70 steroids tested inhibited binding of labeled angiotensin II and III to intact and broken cells from bovine adrenal glomerulosa and kidney, but the concentrations required for inhibition were relatively high. The most potent inhibitors were 3 alpha, 5 beta tetrahydroaldosterone and tetrahydrodeoxycorticosterone (ID50 = 8 x 10-5 M). Kinetic analysis showed that inhibition was mostly competitive. among steroids whose reduced congeners were tested, potency increased in the sequence: parent steroid less than 5 alpha dihydroderivative less than 5 beta dihydro derivative less than 3 alpha, 5 beta tetrahydro-derivative. Tetrahydrodeoxycorticosterone inhibited aldosteronogenesis by intact cells at concentrations that inhibited angiotensin binding. Steroids differentially inhibited binding of labeled angiotensins in II and III, and discriminated between receptors in adrenal glomerulosa and kidney. The results provide additional evidence for heterogeneity of angiotensin receptors, and lead to the prediction that any normal or pathological inhibition of angiotensin receptors by steroids will be mediated by reduced derivatives.     

Angiotensin III as well as angiotensin II regulates water flow through aquaporins in a clam worm

Angiotensin III has been reported to exist in various animals and tissues. The physiological role, however, is still unclear except that brain angiotensin III is a central regulator of vasopressin release. In this study, angiotensin III as well as angiotensin II enhanced an increase in body weight of clam worms of Perinereis sp. under a hypo-osmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo- and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms.     

Direct evidence for local generation and release of angiotensin II in human vascular tissue

A direct measurement of both angiotensins I and II immunoreactive substances was made in the perfusate from isolated human umbilical vein perfused with Krebs-Ringer solution which was free of any component of the renin-angiotensin system. The identity of the immunoreactive peptides was confirmed as angiotensin I and angiotensin II by high-performance liquid chromatography in reference to standard compounds. The rate of release of angiotensins was 41.9 +/- 7.4 and 63.4 +/- 12.0 pg for angiotensins I and II, respectively, during the first perfusion period of 30 min, and it remained stable at least for 3 hours. Angiotensin-converting enzyme inhibitor captopril, added to the perfusion medium (10(-9) to 5 x 10(-6) M), suppressed immunoreactive angiotensin II release in a dose-dependent fashion; the maximal percent inhibition of angiotensin II release evoked by captopril (5 x 10(-6) M) was approximately 56%. These results taken together with the previous observations of presence of essential components of the renin-angiotensin system in vascular tissue provide direct evidence for local generation and subsequent release of angiotensin II in vascular beds of human beings.     

High-performance liquid chromatographic separation of renin-angiotensin system peptides and most of their metabolic fragments

We describe here a gradient HPLC procedure for the separation, and quantification by UV absorption of renin tri- and tetradecapeptide substrates, angiotensins I, II, III, IV and V, angiotensin-derived peptides, and peptidase inhibitors including amastatin, bestatin, pepstatin, lisinopril, a renin peptide inhibitor, Z-Pro-prolinal, N-[1-(R,S)-carboxy-2-phenylethyl]-L-Ala-L-Ala-L-Phe-p-aminobenzoate, and phosphoramidon. Most peptides and peptidase inhibitors were baseline-resolved within 32 min. The overall intra- and inter-assay precisions ranged from 0.8 to 5.9 (n=6) and 2 to 13% (n=6), respectively. There was a linear relationship (correlation coefficients> or =0.9660) between peak height and peptide amount injected. In conclusion, the present method when combined with a peptidase-inhibitor paradigm can lead to the identification of renin-angiotensin system metabolizing enzymes, and when combined with radioimmunoassay can enhance the specificity of angiotensin measurement.     

Capillaries of the adrenal cortex possess aminopeptidase A and angiotensin-converting-enzyme activities.

The enzymes required to convert the prohormone angiotensin I into angiotensins II and III, secretagogues of aldosterone, are enriched in association with capillary endothelium isolated from rat adrenal cortex. Thus the secretion of aldosterone may be controlled, in part, by processing of peptides occurring within the adrenal gland itself.     

Angiotensin III: A Potent Inhibitor of Enkephalin-Degrading Enzymes and an Analgesic Agent

Various angiotensins, bradykinins, and related peptides were examined for their inhibitory activity against several enkephalin-degrading enzymes, including an aminopeptidase and a dipeptidyl aminopeptidase, purified from a membrane-bound fraction of monkey brain, and an endopeptidase, purified from the rabbit kidney membrane fraction. Angiotensin derivatives having a basic or neutral amino acid at the N-terminus showed strong inhibition of the aminopeptidase. Dipeptidyl aminopeptidase was inhibited by angiotensins II and III and their derivatives, whereas the endopeptidase was inhibited by angiotensin I and its derivatives. The most potent inhibitor of aminopeptidase and dipeptidyl aminopeptidase was angiotensin III, which completely inhibited the degradation of enkephalin by enzymes in monkey brain or human CSF. The Ki values for angiotensin III against aminopeptidase, dipeptidyl aminopeptidase, endopeptidase, and angiotensin-converting enzyme, which degraded enkephalin, were 0.66 X 10(-6), 1.03 X 10(-6), 2.3 X 10(-4), and 1.65 X 10(-6) M, respectively. Angiotensin III potentiated the analgesic activity of Met-enkephalin after intracerebroventricular coadministration to mice in the hot plate test. Angiotensin III itself also displayed analgesic activity in that test. These actions were blocked by the specific opiate antagonist naloxone.     

Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Central ventilatory and cardiovascular actions of angiotensin peptides in trout

In the brains of teleosts, angiotensin II (ANG II), one of the main effector peptides of the renin-angiotensin system, is implicated in various physiological functions notably body fluid and electrolyte homeostasis and cardiovascular regulation, but nothing is known regarding the potential action of ANG II and other angiotensin derivatives on ventilation. Consequently, the goal of the present study was to determine possible ventilatory and cardiovascular effects of intracerebroventricular injection of picomole doses (5-100 pmol) of trout [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, ANG IV, and ANG 1-7 into the third ventricle of unanesthetized trout. The central actions of these peptides were also compared with their ventilatory and cardiovascular actions when injected peripherally. Finally, we examined the presence of [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, and ANG IV in the brain and plasma using radioimmunoassay coupled with high-performance liquid chromatography. After intracerebroventricular injection, [Asn(1)]-ANG II and [Asp(1)]-ANG II two ANG IIs, elevated the total ventilation through a selective stimulatory action on the ventilation amplitude. However, the hyperventilatory effect of [Asn(1)]-ANG II was threefold higher than the effect of [Asp(1)]-ANG II at the 50-pmol dose. ANG III, ANG IV, and ANG 1-7 were without effect. In addition, ANG IIs and ANG III increased dorsal aortic blood pressure (P(DA)) and heart rate (HR). After intra-arterial injections, none of the ANG II peptides affected the ventilation but [Asn(1)]-ANG II, [Asp(1)]-ANG II, and ANG III elevated P(DA) (50 pmol: +80%, +58% and +48%, respectively) without significant decrease in HR. In brain tissue, comparable amounts of [Asn(1)]-ANG II and [Asp(1)]-ANG II were detected (ca. 40 fmol/mg brain tissue), but ANG III was not detected, and the amount of ANG IV was about eightfold lower than the content of the ANG IIs. In plasma, ANG IIs were also the major angiotensins (ca. 110 fmol/ml plasma), while significant but lower amounts of ANG III and ANG IV were present in plasma. In conclusion, our study suggests that the two ANG II isoforms produced within the brain may act as a neurotransmitter and/or neuromodulator to regulate the cardioventilatory functions in trout. In the periphery, two ANG IIs and their COOH-terminal peptides may act as a circulating hormone preferentially involved in cardiovascular regulations.     

Distinct localization of renin and angiotensins in separate subcellular fractions of the rat adrenal cortex.

The subcellular localization of renin and immunoreactive angiotensins I and II was studied in rat adrenal cortical tissues. The identity of the immunoreactive angiotensins was confirmed as angiotensin I and angiotensin II by radioimmunoassay and high-performance liquid chromatography, respectively, with reference to standard compounds. By differential centrifugation of tissue homogenate in 0.25 M sucrose/30 mM Tris-HCl/l mM EDTA, pH 7.4, specific immunoreactive renin was found to be localized principally (60%) in the mitochondrial fraction (P2), whereas about 40% of both angiotensins I and II was contained in the soluble fraction; only 18-20% of both peptides was contained in the P2 fraction. On Percoll density gradient centrifugation of P2, renin was fractionated mostly in a denser band whereas angiotensins I and II were contained in a lighter density area closely corresponding to mitochondrial and lysosomal marker enzymes. These results suggest that renin and angiotensins in the cells of the rat adrenal gland reside in different subcellular compartments and argue against intracellular formation of angiotensins by renin in renin granules.