Gregory A. Stephens

Blockade of angiotensin pressor activity in the freshwater turtle

Abstract In order to study the pharmacological characteristics of the renin-angiotensin system in the turtle, Pseudemys scripta elegans , angiotensin I (AI) and angiotensin II (AII) were administered intravenously to conscious turtles along with angiotensin antagonists, a converting enzyme inhibitor, and adrenergic blockers. Both AI and AII produced dose-dependent vasopressor responses. Infusion of the AII analogs [Sar 1 , Ala 8 ] AII or [Sar 1 , Ile 8 ] AII at 10 μg/kg/min significantly reduced the pressor responses to both AI and AII while the pressor action of norepinephrine (3 μg/kg) was unaffected. Neither AII analog exhibited a sustained pressor action at this infusion rate. The converting enzyme inhibitor SQ 20881 (1 mg/kg bolus + 0.5 mg/kg/hr) did not reduce the pressor effect of AII but significantly reduced the response to AI. The α-adrenergic antagonists phentolamine (0.5 mg bolus + 1 mg/kg/hr) and phenoxybenzamine (0.25 mg/kg/min) almost abolished the pressor response to norepinephrine and significantly decreased the vasopressor action of AI and AII. The results suggest that an angiotensin-converting enzyme-like mechanism may exist in the turtle and that the pressor response to angiotensin in the turtle may be due largely to catecholamines.

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.

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 Ringers 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.

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)

Renal responses to diuretics in the turtle

SummaryWe 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<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<0.05 in each case). Sodium, chloride, and potassium excretion increased roughly 10-fold (P<0.05 in each case). At the higher dose, GFR increased by 80% and urine volume more than doubled (P<0.05 in each case). Sodium excretion rose 40-fold, chloride excretion rose 25-fold, and potassium excretion rose 10-fold (P<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 alterig renin release.

Response of plasma renin activity to hypotension and angiotensin converting enzyme inhibitor in the turtle

SummaryWe examined the physiological role of the renin-angiotensin system in response to hemorrhagic and pharmacologic hypotension in the freshwater turtle,Pseudemys scripta elegans. Blood pressure (BP), heart rate (HR), plasma renin activity (PRA), and plasma electrolytes were determined for 1 h prior to and 5 h after hemorrhage or vasodilator administration. With hemorrhage (23% blood volume) BP significantly decreased from 26.7 mm Hg to 17.4 mm Hg (P<0.01) within the first hour after hemorrhage and remained below control levels (P<0.05) until reinfusion of the shed blood at 5 h. HR increased within one hour (21 to 31 beats/min,P<0.01) and then slowly fell towards control levels. PRA also increased initially (2.5 to 6.5 ng/ml·h at 1 h,P<0.05). Infusion of the converting enzyme inhibitor SQ 20881 (0.5 mg/kg·h) during hemorrhage prolonged the hypotension, further elevated PRA and blunted the HR increase. The vasodilators diazoxide and papaverine produced the same degree of hypotension as hemorrhage. Diazoxide (4.5 mg/kg) decreased BP from 24.9 to 14.5 mm Hg (P<0.01) and increased PRA with no change in HR. Papaverine (10 mg/kg) produced a similar fall in BP (25.4 to 12.3 mm Hg,P<0.01) with no PRA change and a transient fall in HR. The data suggests that the renin angiotensin system plays a role in the maintenance of BP in the turtle in response to acute hypotension, but this role may be considerably less than observed in other animals, including mammals.

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.

Effects of converting enzyme inhibition and α 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.

Characterization of lipoproteins from the turtle, Trachemys scripta elegans, in fasted and fed states.

The lipid and apolipoprotein composition of VLDL, IDL, LDL, HDL(2) and HDL(3) were examined in the turtle, Trachemys scripta elegans, in fasted and fed states. The lipid composition of turtle lipoproteins was very similar to their human counterparts. The major apolipoprotein found in LDL, IDL and VLDL, which has a molecular weight of approximately 550 kD, is a homologue of apoB100. The major apolipoprotein found in both HDL(2) and HDL(3), has a molecular weight of 28-kD and is homologous to human apoA-I. HDL(3) also contains a 6.5 kD protein that is homologous to apoA-II, while HDL(2) has two low molecular weight proteins of 6 kD and 7 kD which are also found on the triglyceride rich lipoproteins (TRL). The 7 kD protein is homologous to apoC-III, while the 6 kD protein has a similar size and distribution as apoC-II or apoC-I. In addition, HDL(2) also possesses a protein of 15.8 kD that has no obvious mammalian homologue. In both size and apolipoprotein composition, turtle HDL(2) resembles human HDL(2b) while turtle HDL(3) resembles human HDL(3). In the fasted state, turtles contained very little TRL. When fed a high fat diet, the amount of IDL and LDL sized particles increased significantly.

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.