New angiotensin I isolated from a reptile, Alligator mississippiensis.

Angiotensin I (ANG I) of the American alligator Alligator mississippiensis was isolated from incubates of homologous plasma and kidney extract, and its amino acid sequence was determined as H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ala-Leu-OH. The presence of strongly hydrophobic alanine at position 9 is unusual among ANGs I sequenced to date. Since alanine can be converted to serine by a one-point mutation of the triplet nucleotides, the phylogenetic proximity of the alligator to birds, whose ANG I has serine at position 9, is confirmed by the chemical evolution of the hormone structure. The alligator [Asp1,Val5,Ala9] ANG I increased arterial blood pressure of conscious alligators, but it was no more potent than avian [Asp1,Val5,Ser9] ANG I. The alligator ANG I was also equipotent to the homologous ANG I in the quail and rat. It is concluded that substitutions at position 9 have little influence on the vasopressor activity of ANG I.

Effects of converting enzyme inhibition and alpha receptor blockade on the angiotensin pressor response in the American alligator.

This study examines the effects of two converting enzyme inhibitors (captopril and enalaprilat) and two alpha-adrenergic receptor antagonists (phentolamine and phenoxybenzamine) on the pressor response produced by exogenous angiotensin I ([Asp1, Val5, Ser9] ANG I, fowl) and [Val5] angiotensin II (ANG II) in the American alligator (Alligator mississippiensis). Bolus administration of ANG I at 0.1, 0.5, and 1.0 micrograms/kg; ANG II at 0.05, 0.1, and 0.5 micrograms/kg; or norepinephrine (NE) at 2 micrograms/kg elicited dose-dependent increases in arterial blood pressure. Captopril (0.5 mg/kg/hr) and enalaprilat (300 micrograms/kg/hr) significantly reduced the response to ANG I, but not ANG II or NE. Both phenoxybenzamine (0.25 mg/kg/min) and phentolamine (1 mg/kg/hr) effectively blocked the NE pressor response (84 and 88%, respectively) and attenuated (42-80%) the pressor effects of ANG I and ANG II. These results support previous work suggesting the alligator may possess a renin-angiotensin system with characteristics similar to those found in mammals and other vertebrates. In addition, the pressor response to exogenously administered ANG I and ANG II was attenuated by alpha adrenergic receptor blockade and thus may be due, in part, to secondary catecholamine release.

The pressor response to exogenous angiotensin I and its blockade by angiotensin II analogues in the American alligator.

We examined the pressor response to exogenous, nonnative angiotensin I (ANG I; bullfrog, turtle, and fowl) in the conscious American alligator, Alligator mississippiensis. In addition, the inhibitory effects of three ANG II analogues ([Sar1, Ala8], [Sar1, Thr8], and [Sar1, Ile8]ANG II) on the pressor responses to angiotensin I (fowl ANG I, [Asp1, Val5, Ser9]) were also examined. Intravenous administration of bullfrog, turtle, and fowl ANG I at 0.1, 0.5, and 1.0 micrograms/kg produced dose-dependent increases in arterial blood pressure. [Val5]ANG II at 0.05, 0.1, and 0.5 micrograms/kg, or NE at 2 micrograms/kg also produced dose-dependent increases in blood pressure. [Sar1, Ile8]ANG II and [Sar1, Ala8]ANG II (10 micrograms/kg/min) both attenuated the pressor response to fowl ANG I whereas [Sar1, Thr8]ANG II (10 micrograms/kg/min) produced no significant blockade. These data demonstrate: (1) All three exogenous ANG I molecules exert potent vasopressor responses in the alligator, (2) [Sar1, Ile8]ANG II is the most effective ANG antagonist, and (3) the alligator appears to possess a renin-angiotensin system similar to that found in other vertebrates.

Salt intake and plasma renin activity in the freshwater turtle.

In mammals, a well-documented inverse relationship exists between oral salt intake and plasma renin activity (PRA). We carried out this study to determine if a similar relationship exists in the freshwater turtle Pseudemys scripta. Three groups of turtles (N = 8 for each group) were fed different amounts of salt dissolved in distilled water (2 cc/kg). The high salt group received 580 mg (10 mEq) NaCl/kg twice per week, the medium salt group received 290 mg (5 mEq) NaCl/kg twice per week, and the low salt (control) group received 2 cc/kg distilled water twice per week with no salt. The animals were weighed weekly and blood was collected by cardiac puncture after 3 and 6 weeks of salt loading and again 3 weeks after termination of the salt loading. PRA and plasma electrolytes (PNa, PK, PCl) were determined. The low salt (control) group showed no changes in any of the measured variables throughout the 9-week study. At 3 weeks the following mean values were obtained for the control group: PNa, 119.5 +/- 7.2 mEq/liter; PK, 5.0 +/- 1.0 mEq/liter; PCl, 74.1 +/- 2.6 mEq/liter; PRA, 1.0 +/- 0.2 ng/ml/hr. In the medium salt group, no significant changes were seen until 3 weeks after termination of the salt loading when PNa and PRA were elevated to 137.9 +/- 3.6 mEq/liter and 1.9 +/- 0.2 ng/ml/hr, respectively (P less than 0.05 compared to control). In the high salt group, PNa (136.5 +/- 6.5 mEq/liter) was elevated after 3 weeks of salt loading. At 6 weeks, PNa (136.8 +/- 4.9 mEq/liter) remained elevated in the high salt group and PCl (86.3 +/- 1.3 mEq/liter) and PRA (1.7 +/- 0.2 ng/ml/hr) also increased significantly (P less than 0.05 compared to control in each case). Three weeks after termination of salt loading, PNa (136.8 +/- 3.3 mEq/liter). PCl (88.0 +/- 1.6 mEq/liter), and PRA (1.9 +/- 0.2 ng/ml/hr) remained elevated (P less than 0.05 in each case) in the high salt group while PK dropped to 3.5 +/- 1.6 mEq/liter (P less than 0.05). The results show that after 6 weeks of salt loading sufficient to raise PNa in the turtle, PRA was significantly elevated.

The effects of adrenocorticotropin hormone and angiotensin II on adrenal corticosteroid secretions in the freshwater turtle Pseudemys scripta.

This study examined the effects of angiotensin II (ANG II) and mammalian adrenocorticotropin hormone (ACTH) on adrenal corticosteroid secretions in the freshwater turtle, Pseudemys scripta. Synthetic turtle ANG II ([Asp1, Val5] ANG II) was infused at rates of 1, 10, and 100 ng/kg/min in conscious turtles while monitoring blood pressure (BP). One 60-min saline (0.6%) infusion preceded the ANG II infusions; two followed. Blood samples were collected at 30- and 60-min intervals and the plasma was frozen at -20 degrees until assay. Mammalian ACTH was infused at doses of 0.1 and 1.0 IU/min; the procedures were followed as delineated above. The plasma was assayed for corticosterone, cortisol, and aldosterone utilizing radioimmunoassay techniques. Infusions of exogenous, native ANG II at subpressor and pressor rates elicited dose-dependent increases in BP, which rose from a control mean of 22.6 +/- 5.8 mm Hg to a maximum mean value of 38.2 +/- 11.0 mm Hg (P less than 0.05 compared to control), and plasma corticosterone concentrations, which rose from a control mean of 6.6 +/- 2.8 ng/ml to a maximum mean value of 27.2 +/- 2.6 ng/ml (P less than 0.05 compared to control). Furthermore, both BP and corticosterone levels returned toward control levels during the final saline recovery period, suggesting that neither physical stress suffered by the animal nor blood volume changes due to infusions and blood sampling affected these parameters. ACTH failed to alter either BP or corticosterone. Neither ANG II nor ACTH had any effect on plasma cortisol or aldosterone concentrations--which fell below the minimal detection levels for these assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of plasma renin activity in the freshwater turtle.

Components of the renin angiotensin system have been identified in many nonmammalian vertebrates. However, in many of these animals, including reptiles, the physiological functions and importance of the system remain unclear. To aid in the study of the system in a reptile we modified a commercially available radioimmunoassay (RIA) kit containing antibody against human angiotensin I (ANG I) for use in the freshwater turtle, Pseudemys scripta. Cross-reactivity between anti-human ANG I antibodies (Rainen Angiotensin I RIA Kit, New England Nuclear) and turtle ANG I was demonstrated. Cross-reactivity with the antibody in two other human ANG I assay kits (Travenol-Genentech and Biotecx) was very limited. Blood for assay was collected from conscious turtles in EDTA, centrifuged, and the plasma frozen at -20 degrees. Turtle ANG I was generated by incubation at 0.5 ml plasma at pH 5.5 for 2 hr at 30 degrees with addition of dimercaprol and 8-hydroxyquinoline. Angiotensin generation increased with temperature and with generation time. The recovery of turtle ANG I added to turtle plasma prior to incubation was 92-97%. The assay procedure was used to measure plasma renin activity (ng/ml/hr incubation) from unstimulated turtles.

Blockade of the pressor response to angiotensins I and II in the bullfrog, Rana catesbeiana.

We examined the effects of three angiotensin II (AII) analogs, [Sar1,Ala8] AII, [Sar1,Ile8] AII, and [Sar1,Thr8] AII, phenoxybenzamine and captopril on the pressor response to angiotensins I (AI) and II (AII) and norepinephrine (NE) in the bullfrog, Rana catesbeiana. Injection of AI and AII at 0.25, 0.5, and 1.0 micrograms/kg, or NE at 3 micrograms/kg elicited dose-dependent rises in blood pressure. [Sar1,Ile8] AII (10 micrograms/kg/min) significantly blocked the pressor effects of all AI and AII doses. [Sar1,Ala8] AII blocked only the highest dose, and [Sar1,Thr8] AII produced no blockade. Captopril (0.1 mg/kg bolus + 0.5 mg/kg/hr) significantly reduced the response to AI, but not AII or NE. Phenoxybenzamine (5-10 mg bolus + 1 mg/kg/hr) blocked NE, and partially inhibited (36-49%) the pressor effects of AI and AII. These results demonstrate that (1) [Sar1,Ile8] AII is a potent angiotensin antagonist in the bullfrog, while [Sar1,Ala8] AII is partially effective and [Sar1,Thr8] AII is largely ineffectual; (2) captopril is an effective converting enzyme inhibitor; and (3) a portion of the angiotensin response can be inhibited by alpha-receptor blockade and is apparently due to catecholamine release.

Renal responses to diuretics in the turtle.

We administered the diuretics furosemide and ethacrynic acid to conscious freshwater turtles to assess changes in renal function and plasma renin activity (PRA) in an animal which lacks a loop of Henle. Furosemide (2 and 5 mg/kg) produced no changes in blood pressure, hematocrit, plasma electrolytes, glomerular filtration rate (GFR), or PRA. Furosemide doubled urine volume while sodium excretion increased 20-fold and chloride and potassium excretion increased 12-fold (P less than 0.05 in each case). Net potassium secretion was observed. Ethacrynic acid (2 and 5 mg/kg) also produced no changes in blood pressure, hematocrit, plasma electrolytes, or PRA. At the lower dose GFR increased by 40% and urine volume nearly doubled (P Less than 0.05 in each case). Sodium, chloride, and potassium excretion increased roughly 10-fold (P less than 0.05 in each case). At the higher dose, GFR increased by 80% and urine volume more than doubled (P Less than 0.05 in each case). Sodium excretion rose 40-fold, chloride excretion rose 25-fold, and potassium excretion rose 10-fold (P less than 0.05 in each case). At both doses net potassium secretion occurred. The results demonstrate that both drugs inhibit tubular reabsorption in the turtle, acting primarily on distal segments of the nephron. The failure of either drug to alter PRA suggests that the turtle lacks a tubular mechanism for altering renin release.

Angiotensin and norepinephrine effects on isolated vascular strips from a reptile.

In order to better understand the vascular effects of angiotensin II (AII) in lower vertebrates, the contractile responses of aortic strips from the freshwater turtle Pseudemys scripta elegans were studied. Circumferential strips from the left aortic arch were suspended in a tissue bath in 25 degrees C reptilian Ringer's solution at pH 7.5. Both [Asn1,Val5] AII (10(-9)-10(-5) M) and norepinephrine (NE) (10(-8)-10(-4) M) produced dose-dependent contractions. The threshold dosage for AII was between 10(-9) and 10(-8) M and for NE between 10(-8) and 10(-7) M. Pretreatment with [Sar1,Ile8] AII (10(-6) M) significantly attenuated the response to [Asn1,Val5] AII at 10(-5) M and totally blocked the response at lower AII concentration (P less than 0.01 in each case). The response to AII was unaffected by phentolamine (10(-6) M). Phentolamine abolished the response to NE at concentrations of 10(-6) M or less (P less than 0.01) and attenuated the response by 48% at 10(-5) M (P less than 0.05) and 43% at 10(-4) M (P less than 0.05) NE. The response to NE was unaffected by [Sar1,Ile8] AII. The results demonstrate that [Asn1,Val5] AII has a direct contractile effect on turtle vasculature and that two functionally distinct vascular receptor populations for AII and NE are present in the turtle. Since phentolamine did not affect the responses to AII, it also appears that angiotensin-evoked norepinephrine release from the sympathetic nerve terminals in the vessel is not involved in the angiotensin-induced contractions in this preparation.

Systemic and renal effects of angiotensin II in the freshwater turtle Pseudemys scripta elegans.

This study examined the renal and systemic effects of angiotensin II (ANG II) in the freshwater turtle, Pseudemys scripta elegans. We infused [Asn1, Val5]ANG II at doses of 1, 10, 100, and 500 ng X kg-1 X min-1 in conscious turtles while monitoring blood pressure, glomerular filtration rate (GFR), urine volume, and plasma and urine electrolytes. Saline (0.6%) was infused for two 1-h control periods at 1 ml X kg-1 X h-1 followed by a 1-h infusion of saline with ANG II added. Saline alone was then infused for either two or three 1-h recovery periods. Control values before ANG II infusion averaged 21 mmHg for blood pressure, 1.8 ml X h-1 for urine volume, and 8.0 ml X kg-1 X h-1 for GFR. At 1 (n = 7) and 10 ng X kg-1 X min-1 (n = 6) ANG II produced no significant changes in blood pressure or renal function. Angiotensin infusion at the higher rates of 100 (n = 6) and 500 ng X kg-1 X min-1 (n = 11) significantly elevated blood pressure during ANG II infusion (29 and 44 mmHg, respectively), while electrolyte excretion remained unchanged. Despite the increased blood pressure, GFR and urine volume tended to decrease. At all infusion rates, plasma electrolytes remained unchanged. The results suggest that ANG II is a potent constrictor of preglomerular vessels in the kidney of the turtle.

Arterial baroreceptor reflex control of heart rate in two species of turtle.

Attempts were made to stimulate an arterial baroreceptor reflex in anesthetized and conscious pond turtles. In turtles anesthetized with either alpha-chloralose or pentobarbital, occlusion of the ascending or descending aortas produced no reflex heart rate (HR) changes. In pentobarbital-anesthetized turtles, direct electrical or mechanical stimulation of potential baroreceptor sites along the central aortic arches and carotid arteries produced no significant changes in either HR or blood pressure (BP). Occlusion of the common carotid arteries also produced no HR or BP changes. Intravenously administered nitroglycerin lowered BP but caused no reflex tachycardia in anesthetized turtles. Phenylephrine and angiotensin elevated BP in the anesthetized turtle but caused no reflex bradycardia. In conscious turtles phenylephrine increased BP and nitroglycerin decreased BP. Neither response produced a statistically significant HR change, although HR tended to decrease transiently with phenylephrine and increase with nitroglycerin. These HR changes were abolished by atropine. Rapid intra-arterial infusion of 6% dextran transiently raised BP but caused no reflex bradycardia. These experiments suggest that cardiovascular regulation in the turtle is accomplished without a major contribution from arterial baroreceptor reflexes.

Sodium and angiotensin in the pathogenesis of experimental renovascular hypertension.

The effects of simultaneous angiotensin blockade and sodium depletion on the development of one-kidney renovascular hypertension were studied in rats. In sodium-replete rats, systolic blood pressure (SBP) increased from 102 +/- 2 to 153 +/- 11 mmHg by the 12th day after unilateral nephrectomy and subsequent partial occlusion of the renal artery with a 0.22-mm silver clip. When changes in body fluid volume were minimized by sodium restriction in a second group of rats, the increase in SBP from 98 +/- 4 to 149 +/- 7 mmHg after clipping was not different from that in sodium-replete animals. Inhibition of the angiotensin-converting enzyme with SQ 14,225 during sodium restriction prevented the SBP from increasing above 101 +/- 3 mmHg by the 12th day after nephrectomy and clipping. Once SQ 14,225 administration was discontinued, SBP rose significantly to 148 +/- 5 mmHg within 5 days. Because previous studies have shown that neither sodium depletion nor angiotensin blockade alone prevented the development of one-kidney renovascular hypertension, it is concluded that the increase in blood pressure resulting from renal artery constriction and contralateral nephrectomy was prevented only by suppression of both the renin-angiotensin system and body fluid volume.

Hypertension produced by sodium depletion and unilateral nephrectomy: a new experimental model.

Unilateral nephrectomy of sodium-restricted male Sprague-Dawley rats produced a sustained elevation in systolic blood pressure (SBP) that was reversed by sodium repletion. A chronic intraperitoneal infuson of SQ14,225 prevented the development of hypertension in sodium-deplete unilaterally nephrectomized rats. Sodium depletion of two-kidney rats increased SBP to a lesser extent, while unilateral nephrectomy of sodium replete animals had no effect. These results provide evidence for a new model of experimental hypertension in the rat and emphasize the importance of a renal component, as demonstrated by unilateral nephrectomy, in the maintenance of normal pressure-volume relationships.

Circadian changes in plasma renin activity and plasma aldosterone concentration in two-kidney hypertension rats.

Circadian changes in plasma renin activity (PRA) and plasma aldosterone concentration (PAC) in normal and hypertensive rats were determined by measurements at 8 a.m., 4 p.m. and 12 midnight (MN). For the normals, PRA and PAC were highest at 4 p.m. Animals made hypertensive by constriction one renal artery with the other kidney intact were studied after 4, 5, 7 and 10 weeks; the clear-cut circadian rhythm for PRA in normals had disappeared but for PAC the circadian rhythm was present in the 4-, 5- and 10-week groups. Both PRA and PAC were elevated in all four hypertensive groups compared with the normal controls and there was a highly significant correlation between PRA and PAC. The 4 p.m. peak value for PAC was much higher in relation to the 8 a.m. and 12 MN values values in the hypertensive animals than in the normals. Sodium balance studies failed to demonstrate any appreciable differences among the groups. When the hypertensive animals were divided into two groups on the basis of the level of hypertension, the rats with moderate hypertension showed an average elevation in PRA which was significant in only the 4- and 7-week groups whereas PRA was elevated in all four groups with severe hypertension. Thus, the present data help to define the activity of the renin-angiotensin-aldosterone system in two-kidney, one clip hypertension in the rat.

Changes in cardiac output during the development of renal hypertension in sodium-depleted dogs.

1. Chronic renovascular hypertension developed in uninephrectomized, sodium-depleted dogs in association with a decrease in cardiac output. 2. With sodium and volume repletion of these animals, cardiac output returned to normal but the high level of arterial pressure was unchanged; consequently, the peripheral arterioles dilated. 3. These observations provide evidence against the theory of whole-body autoregulation.