Adrenal, kidney, and heart angiotensins in female murine Ren-2 transfected hypertensive rats.

We analyzed by high-performance liquid chromatography and radioimmunoassay angiotensin I (Ang I), Ang II, Ang-(1-7), and metabolites in the adrenal, kidney and heart of normotensive female Sprague-Dawley (SD) and transgenic hypertensive [TGR(mRen-2)27] rats carrying the murine Ren-2d renin gene. The monogenetic model of hypertensive rats had significant increases in adrenal Ang II; whereas in the kidney Ang II was unchanged, but Ang I and Ang-(1-7) were significantly lower. Cardiac Ang I, Ang II, and Ang-(2-10) were significantly reduced in transgenic rats, while Ang-(2-7) was increased. In SD and transgenic rats kidney and adrenal angiotensins increased primarily during estrus or proestrus. In female transgenic rats the increased adrenal Ang II and the sustained renal Ang II may contribute to the established phase of hypertension.

Measurement of immunoreactive angiotensin peptides in rat tissues: some pitfalls in angiotensin II analysis.

Angiotensin II, the major effector peptide of the renin-angiotensin system, is an endocrine and paracrine regulator of tissue function. To determine its physiological role, it is important to quantify angiotensin II and related fragment peptides in tissues and plasma as a first step toward understanding angiotensin II metabolism within tissues. A fully characterized, sensitive, and reproducible immunochemical assay has been developed for quantitating angiotensin II immunoreactivity in tissues and plasma. We identified two methodological events of critical importance, incompletely addressed in previously reported studies. First, the nonspecific interference resulting from Sep-Pak processing was found to be due to hydrophobic impurities in the octade-casilane absorbent which were eliminated by washing the Sep-Pak with tetrahydrofuran and hexane before use. Second, a significant discrepancy was observed in the recoveries of angiotensin II and 125I-angiotensin II added to tissue extracts following high-pressure liquid chromatography. Angiotensin II immunoreactivity extracted from decapitated rat adrenal gland, brain, and kidney (target organs for angiotensin II), ovary and uterus (potential target organs for angiotensin II), and plasma has been characterized. The predominant component of the angiotensin II immunoreactivity was the biologically active octapeptide angiotensin II. However, in the brain, the ratio of angiotensin II to C-terminal angiotensin II immunoreactive fragments was lower than observed in other tissues studied. Other angiotensin II C-terminal immunoreactive peptide fragments-the biologically active heptapeptide and the biologically inactive angiotensin(3-8) and angiotensin(4-8)--were also detected in variable quantities in the various tissues.

Serum bactericidal testing with the Autobac system.

Current methodology for the serum bactericidal test requires a minimum of 48 h. A procedure was devised for performing this test with the Autobac system (General Diagnostics, Div. Organon Inc., Raleigh, N.C.) in a shortened time span. All titers obtained with the Autobac were compared against results obtained with a standardized tube dilution procedure. The Autobac low-thymidine eugonic broth performed comparably to the tube dilution diluent, a 1:1 ratio of pooled human serum and cation-supplemented Mueller-Hinton broth (99.2% correlation between bactericidal endpoints). Over 300 tests were conducted by using stock reference bacterial strains, clinical isolates, pooled human serum seeded with antimicrobial agents, and serum from patients on antimicrobial therapy. With the Autobac procedure, serum inhibitory titers can be reported in 3 to 4 h (93.4% correlation with the tube dilution procedure). Serum bactericidal titers can be obtained in 24 h without the necessity of subculturing (95.6% correlation). With the exception of staphylococci tested against penicillin, serum bactericidal titers can be obtained in 3 to 4 h (88.4% correlation). The Autobac procedure can provide the clinical laboratory with a rapid, reliable method for performing the serum bactericidal test.

Characterization of receptors for angiotensin-induced drinking and blood pressure responses in conscious rats using angiotensin analogs extended at the N-terminal.

Angiotensin II analogs with N-terminal extensions were synthesized to examine their effects on the brain and vascular angiotensin II (Ang II) receptors of the rat. Ang II, Crinia-Ang II, Thr.Ala.Gly-Ang II and Val. Ser.Leu.Thr.Ala.Gly-Ang II were all found to elicit drinking and raise blood pressure when given into the cerebrospinal fluid (CSF), and elevate blood pressure when given intravenously. When given intracerebroventricularly, the order of potency of the peptides in eliciting blood pressure and drinking responses was: Ang II (100%) = Crinia-Ang II (100%) greater than Thr.Ala.Gly-Ang II (10% blood pressure, 15% drinking) greater than Val.Ser.Leu.Thr.Ala.Gly-Ang II (5%). The order of pressor potency did not change when the Ang II analogs were given intravenously, but compared with the central effects, there was a marked difference in the relative potencies of the peptides. The potencies were: Ang II (100%) greater than Crinia-Ang II (80%) greater than Thr.Ala.Gly-Ang II (60%) greater than Val.Ser.Leu.Thr.Ala.Gly-Ang II (20%). Blood pressure and drinking responses produced by all of these peptides were markedly attenuated by the Ang II receptor antagonist, [Sar1,Thr8] Ang II. These findings indicate a difference in the Ang II receptors present in the brain and the periphery. However, no differences were noted between the central Ang II receptors mediating the pressor responses and the central Ang II receptors stimulating drinking behavior.

Studies on angiotensin II and analogs: impact of substitution in position 8 on conformation and activity.

Affinity, residual agonist activity, and inhibitor properties of a series of angiotensin II analogs modified at the COOH-terminal position (X8-substituted peptides) have been probed for structure/conformation-biological activity relationships. The results emphasize (i) the large impact that subtle conformational variations caused by structural alterations in the position 8 side chain have on biological properties, (ii) the implication of the COOH-terminal carboxyl group in both affinity and intrinsic activity, and (iii) the influence that the bulkiness of the side chain in position 8 of antagonists has on the local conformation at the COOH terminus and thus on the inhibitory properties. In the hormone, the phenylalanine-8 ring is required for its steric influence and aromaticity to ensure a fully active conformation at the COOH terminus. Especially, correct orientation of the position 8 carboxyl group relative to the phenyl group of the phenylalanine residue may be necessary for agonistic activation of the angiotensin receptor complex. Replacement of the aromatic ring on the COOH-terminal residue by a nonaromatic group leads to incorrect orientation of the carboxyl group and causes the appearance of antagonist properties. Although the steric effects of the side chain can be modulated by specific interaction of its chemical groups (if any) with the peptide backbone, we found a good correlation between the size of the side chain-e.g., the steric parameter V gamma (the van der Waals volume consisting of the C alpha, C beta, and C gamma atoms), the conformational properties in the backbone (3J HC alpha-NH), and the binding capacities in all compounds tested.

Cerebrospinal fluid angiotensin II immunoreactivity is not derived from the plasma.

To elucidate whether the presence of angiotensin II immunoreactivity (ANG II-ir) in the cerebrospinal fluid (CSF) of the dog is in part due to passage of the peptide across the CSF-blood-brain barrier, [Ile5] angiotensin II (ANG II) was infused intravenously for 7 days in conscious, trained dogs at a rate of 10 micrograms/kg/day. Mean arterial pressure (MAP) and heart rate were monitored each day, and samples of arterial blood and CSF (with a catheter secured into the cisterna magna) were drawn at regular intervals for determination of catecholamine levels, ANG II-ir, and electrolyte levels. Within 2 days after ANG II infusion, MAP stabilized at 35 +/- 1 mm Hg (mean +/- SE, p less than 0.001) above control values. The hypertension was associated with bradycardia, suppressed plasma renin activity, and a fall in both plasma and CSF Na+ concentrations. These changes coincided with a considerable and sustained decrease in the levels of plasma and CSF norepinephrine. On the other hand, levels of epinephrine and K+ in the two compartments remained unchanged. Although concentration of ANG II-ir in plasma was augmented markedly (368% above control values, p less than 0.001), ANG II-ir in the CSF remained within the low values measured in the control period.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and immunological properties of dog brain isorenin.

A neutral protease with angiotensin I-forming activity which could readily be separated from acid proteases and plasma and renal renin was obtained from extracts of dog brain. This enzyme has an apparent mol wt of 40,000 by Sephadex chromatography. On chromatofocusing, it displays isoelectric points of 7.92, 7.73, and 7.42, and thus, it is a basic protein, in contrast to either renal or plasma renin which are acidic proteins. This brain enzyme does not react with antibodies specific for dog kidney renin. Since the brain enzyme forms angiotensin I from renin substrate at neutral pH, yet can be separated from and has isoelectric points different from renal renin, it is an isoenzyme of the kidney counterpart. The majority of the renin-like activity of dog brain is due to this isoenzyme.

Restricted dietary sodium intake alters peripheral but not central angiotensin II receptors.

Male Sprague-Dawley rats were maintained on either a normal or low-sodium diet for 5 weeks to examine whether dietary sodium restriction alters angiotensin II (Ang II) receptors. The receptor sites in the hypothalamus-thalamus-septum (H-T-S) region of the brain, the adrenal glands and bladder visceral smooth muscle were measured by saturation isotherm binding assays using 125I-Ang II. Compared to control rats, the low-sodium diet group showed a smaller weight gain, reduced water intake, elevated hematocrit, and decreased urinary sodium concentration. In addition, sodium-depleted rats had a 10-fold elevation in plasma renin activity. However, neither binding affinity of 125I-Ang II to the brain H-T-S region nor its density was significantly different between the two groups. In contrast, both the 125I-Ang II binding density and dissociation constant in the adrenal gland were significantly elevated, while the binding density of 125-I-Ang II in the bladder smooth muscle was significantly decreased in the sodium-restricted group. These results suggest that dietary sodium depletion does not alter Ang II receptors in the rat brain areas wherein Ang II exerts the majority of its central actions.

Evidence for the existence of a family of biologically active angiotensin I-like peptides in the dog central nervous system.

A family of angiotensin I-like peptides has been derived from endogenous precursors present in dog cerebrospinal fluid after incubation with species homologous renin. These peptides are immunologically and pharmacologically similar to [Ile5]angiotensin I, and have molecular weights ranging between 1300 and 2200 daltons. The presence of precursors in the cerebrospinal fluid able to generate various biologically active angiotensin I-like peptides dissimilar to plasma angiotensin I supports the concept of a local angiotensin I-forming system in the brain.

Alterations in plasma and cerebrospinal fluid norepinephrine and angiotensin II during the development of renal hypertension in conscious dogs.

The levels of norepinephrine (NE), epinephrine (E), and angiotensin II immunoreactivity (AIIir) in plasma and in cerebrospinal fluid (CSF) were measured in eight conscious dogs before and during a 28-day period in the development of two-kidney, one clip (2K1C) hypertension produced by a two-step procedure. The early phase (less than 7 days) of hypertension following partial constriction of the renal artery was accompanied by tachycardia and increases in concentrations of NE and AIIir in both plasma and CSF; E did not change. One week later blood pressure remained elevated (107 +/- 2 after vs 88 +/- 2 mm Hg before clipping, p less than 0.05), but other variables returned to control values. Occlusion of the partially constricted renal artery caused severe hypertension that was initially associated with a transient decrease in levels of NE in both plasma and CSF and a sustained rise in plasma and CSF concentrations of AIIir that persisted for as long as 2 weeks after the second operation. None of these effects was seen in nine sham-operated dogs. Since activation of the renal pressor system is associated with time-related changes in the concentrations of NE and AII in both plasma and CSF, these observations indicate early involvement of both sympathetic and renin-angiotensin systems in the pathogenesis of renovascular hypertension.

Effects of chronic sodium depletion on canine brain renin and cathepsin D activities.

The activities of brain renin and cathepsin D were measured in brain regions of 10 dogs on a normal sodium intake (65 mEq Na+/day) and 10 other dogs placed on a low sodium diet (less than 4 mEq Na+/day) for 21 days and given a diuretic. The purpose of this study was twofold: to assess the effect of sodium depletion on brain renin activity; and to assess in the same regions alterations in brain renin and cathepsin D activities. Sodium depletion caused a ninefold increase in plasma renin activity, hemoconcentration, and hyponatremia. In the presence of marked hyperreninemia, the average cerebral renin activity was reduced significantly; the most pronounced changes occurred in the upper and lower brain-stem regions. Cerebrospinal fluid renin was increased by 30%, but this change was not significant in sodium-depleted dogs. There were no significant alterations in cathepsin D activity whether assessed in total or regional brain areas. These observations support the view that there is an inverse relationship between plasma and brain renin activity in chronically sodium-depleted dogs. Additionally, evidence is provided that brain renin activity is modified independently from cathepsin D activity.

Brain renin: localization in rat brain synaptosomal fractions.

The distribution of brain renin activity was determined in subcellular fractions of rat brain prepared by discontinuous density gradient centrifugation. The highest amounts of brain renin activity occurred in both the light and heavy synaptosomal fractions, while the activity of choline acetyltransferase was elevated only in the light synaptosomal fraction. These results indicate an intraneuronal localization of brain renin.

Responses of juxtaglomerular cell suspensions to various stimuli.

Cell suspensions were prepared from rat renal cortical tissue by dispersion with 0.1% collagenase. Unit gravity sedimentation in a 1%-4% Ficoll gradient resulted in a single-cell suspension enriched in juxtaglomerular (JG) cells. Both the cellular renin activity and the amount of renin released into the supernatant increased with time when the suspensions were incubated for 1 hour at 37 degrees C in tissue culture medium. These cells responded to epinephrine and norepinephrine by increasing both synthesis and release of renin. The response was blocked by timolol but not by phenoxybenzamine. Cell suspensions prepared in the same manner but using 0.25% trypsin as the dispersing enzyme neither synthesized nor released renin into the tissue culture medium when similarly incubated. Trypsin-dispersed cells did not respond to catecholamine stimulation. Renin synthesis and release in collagenase-dispersed JG cells were unaltered by changes in Na, K, or Ca ion concentrations. Angiotensin II inhibited release, while saline extracts of clipped kidney from renal hypertensive rats stimulated renin release by these cells.

Synthesis of [alpha-methyltyrosine-4]angiotensin II: studies of its conformation, pressor activity, and mode of enzymatic degradation.

Modifications in angiotensin II and its antagonistic peptides that should have increased in vivo half-lives but not reduced biological activity were studied by determining the effect of alpha-methylation of the tyrosine in position 4. [alpha-Methyltyrosine-4]angiotensin II, synthesized by the solid-phase procedure, showed 92.6 +/- 5.3% pressor activity of angiotensin II. Incubation with alpha-chymotrypsin for 1 hr indicated absence of degradation although, under the same conditions, angiotensin II was completely degraded to two components. Comparison of the 1H NMR spectra in aqueous solution and the circular dichroism spectra in trifluoroethanol of angiotensin II and [alpha-methyltyrosine-4]angiotensin II suggested that alpha methylation of the tyrosine residue in angiotensin II does not lead to major changes in the overall solution conformation. These results are in contrast to those obtained with N-methylation in position 4, which drastically reduced the biological activity and produced remarkable changes in the peptide backbone and a severe limitation in rotational freedom of the side chains in tyrosine. Thus, it may be possible to synthesize potent angiotensin II analogs that have greater resistance to enzymatic degradation by alpha-methylation in position 4 (or 5) and simultaneous suitable modification at the NH2 and COOH termini.

Amino acid side chain conformation in angiotensin II and analogs: correlated results of circular dichroism and 1H nuclear magnetic resonance.

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life in vivo of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding N-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of N-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (i) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (ii) N-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-N-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-N-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (i) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (ii) greater impact of N-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.

Role of renin-angiotensin system in chronic renal hypertensive rats.

The role of renin-angiotensin system has been examined in the maintenance of hypertension in acute and chronic two-kidney (36 weeks) and chronic one-kidney (12 weeks) Goldblatt hypertensive rats using three inhibitors of this system. The inhibitors used were URI-73A, a synthetic analog of lysophosphatidylethanolamine, which inhibits renin both in vivo and in vitro, SQ14,225, a potent converting enzyme inhibitor, and [Sar1, Thr8] angiotensin II, an angiotensin II antagonist. When the inhibitors were administered in acute (high renin) hypertensive rats, they all lowered blood pressure significantly. However, in the chronic (low renin) hypertensive phase, both renin and converting enzyme inhibitors lowered blood pressure, whereas, Sar1, Thr8 failed to lower blood pressure. The renin inhibitor lowered plasma renin activity (PRA), and SQ14,225 and [Sar1, Thr8] Ang II increased PRA. Further studies on water and electrolyte balance with one-kidney model hypertensive and uninephrectomized control rats showed no change in plasma volume. However, there was increased 24-hour urinary output and increased sodium excretion. This study indicates that in chronic renal hypertensive rats, blood pressure reduction is possible by either renin on converting enzyme inhibitor, but not by angiotensin antagonists. Since volume did not change either during the development or reversal of hypertension, volume did not appear to play a major role in the maintenance of hypertension.