Blockade of angiotensin pressor activity in the freshwater turtle

GENERAL

AND

COMPARATIVE

45, 364-371

ENDOCRINOLOGY

Blockade of Angiotensin

(1981)

Pressor Activity in the Freshwater Turtle

GREGORY A .STEPHENS Physiology

Section,

School

of Life

and Health

Sciences,

University

of Delaware,

Newark,

Delaware

19711

Accepted March 5, 1981 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 k1 and AI1 produced dosedependent vasopressor responses. Infusion of the AI1 analogs [Sari, Ala”] AI1 or [ Sar’, Iles] AI1 at 10 Fg/kg/min significantly reduced the pressor responses to both AI and AI1 while the pressor action of norepinephrine (3 pg/kg) was unaffected. Neither AI1 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 AI1 but significantly reduced the response to AI. The a-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.

The renin-angiotensin system has been found in all vertebrates phylogenetically above the elasmobranchs (Taylor, 1977; Nishimura, 1980). In these animals, which include teleosts (Mizogami et al., 1968; Chester Jones et al., 1969; Sokabe et al., 1970; Nishimura and Sawyer, 1976), amphibians (Johnston et al., 1967; Nakajima et al., 197 1; Nolly and Fasciolo, 197 1; Fruchter et al., 1980), reptiles (Capelli et al., 1970; Nothstine et al., 1971), and birds (Taylor et al., 1970; Chan and Holmes, 197 1), infusion of synthetic angiotensin, kidney extract, or kidney extract incubated with homologous plasma produced a vasopressor response. Synthetic angiotensins were also vasopressor in the dogfish shark (Opdyke and Holcombe, 1976), even though elasmobranchs do not possess juxtaglomerular cells (Capreol and Sutherland, 1968; Nishimura et al., 1970) and are not thought to produce renin. In reptiles, juxtaglomerular cells have

been found in several species (Kaley and Donshik, 1965; Sokabe et al., 1969) and infusion of synthetic angiotensin II or kidney extract with or without prior incubation with plasma produces a pressor response (Capelli et al., 1970; Nothstine et al., 197 1; Stephens et al., 1975). Although this pressor response to synthetic angiotensin suggests a reptilian renin- angiotensin system chemically similar to mammals, the biochemical character of repitilian angiotensin and angiotensin receptors is uncertain, as are the physiological functions of the system in the Reptilia. In the present study we sought to characterize systematically the pressor responses to synthetic angiotensin I (AI) and angiotensin II (AII) in the turtle, Pseudemys scripta elegans, and to determine the effect upon these responses of two competitive angiotensin antagonists, an angiotensin-converting enzyme inhibitor, and two a-adrenergic receptor antagonists. 364

0016-6480/81/l 10364-08$01.00/O Copyright @ 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

ANGIOTENSIN

BLOCKADE

METHODS Commercially supplied red-eared turtles, Pseudemys scripla elegans, weighing 780 to 1135 g were used in the study. The animals were housed in tanks with access to water and a dry basking area and were fed liver and fish weekly. Prior to experimental use, each animal was anesthetized with pentobarbital and cooling, and a polyethylene catheter (PE 90) with a silicone rubber tip was surgically implanted in an axillary artery. The artery was exposed by opening a small hole (1 .&cm diameter) in the carapace with a trephine. A second polyethylene (PE 60) and silicone rubber catheter was inserted into the left subclavian vein through a hole in the plastron. Both holes were sealed with dental acrylic cement and the animals were allowed to recover for at least 3 days. Synthetic human AI (Asp, Arg, Val, Tyr, Ile, His, Pro, Phe, His, Leu) (Vega Biochemicals), [Va15] AI1 amide (CIBA), and norepinephrine (Levophed, Winthrop) were the pressor agents used in the study. The other drugs used were [Sat-‘, Ilea] AI1 (Vega Biochemicals), [Sari, Alas] AI1 (Norwich-Eaton Pharmaceuticals) , SQ 2088 1 (Squibb), phentolamine mesylate (Regitine, CIBA), and phenoxybenzamine-HCl (Smith, Kline & French). All the drugs were diluted with 0.6% NaCl for injection. All experiments were carried out in conscious turtles placed in small plastic tanks (17 x 28 cm) containing approximately 3 cm of water. Blood pressure was continuously recorded from the axillary artery catheter with a Statham P23 ID pressure transducer and a Grass Model 7D recorder. The animal’s ECG and the cardiotachometer putput of heart rate were recorded from electrodes placed on the carapace. All drugs were administered as bolus injections through the subclavian vein catheter in a volume of 0.2 ml or less and flushed in with 0.3 ml reptilian saline (0.6% NaCl). Saline vehicle alone produced no blood pressure or heart rate changes. A total of 18 animals were used in the study with each animal being used up to four times with different blocking agents. To insure complete recovery between procedures, experiments were conducted only every second or third day and after phenoxybenzamine administration the turtles were not reused. During the experiments a slow saline infusion (0.5 ml/hr) was maintained continuously. AI and AI1 were injected into unanesthetized turtles at doses of 0.1, 0.5, and 1 .O p,g/kg and norepinephrine was injected at a dose of 3 Fg/kg. The order of injections was randomized and no evidence of tachyphylaxis was observed. After these control responses were obtained, the saline infusion (0.5 ml/hr) was continued with the addition of either an AI1 analog [Sar’, Alas] AI1 ( 10 p,g/kg/min) or [Sar’, Ile8] AI1 ( 10 kg/kg/ min), or the angiotensin converting enzyme inhibitor SQ 2088 1 ( 1 mg/kg bolus plus 0.5 mg/kg/hr), or an a-adrenergic receptor blocker, phentolamine (0.5 mg/

IN

365

TURTLES

kg bolus plus 1 mg/kg/hr) or phenoxybenzamine (0.25 mglkgimin). Beginning 30 min after the start of inhibitor infusion the angiotensin and norepinephrine injections were repeated. The differences in the peak responses at each dose prior to and during inhibitor infusion were compared with a paired t test.

RESULTS Angiotensin

and Norepinephrine

The control mean blood pressure for all conscious turtles prior to administration of any drugs averaged 22.6 -c- 1 .O mm Hg. Both AI and AI1 injected intravenously produced dose-dependent increases in arterial blood pressure. With AI the pressor responses averaged 5.0 + 0.6, 9.6 t 0.8, and 13.8 + 0.7 mm Hg at doses of 0.1, 0.5, and 1 .O pg/kg, respectively. With AI1 the same dosages produced pressor responses averaging 6.9 f 0.7, 12.8 ZL 0.7, and 17.9 + 1.2mm Hg, respectively. The pressure rose more rapidly with AII, achieving a peak 2.8 + 0.2 min after injection compared to 3.6 f 0.2 min after AI injection. Norepinephrine (3 pg/kg) was also pressor in the turtles, increasing blood pressure by an average of 20.0 + 0.8 mm Hg for all experiments. The duration of the pressor action was also dose dependent and averaged 20 to 30 min for the angiotensins and norepinephrine. The responses of heart rate to these agonists were quite variable between animals, and when averaged within experimental groups, heart rate was not significantly altered in any case. Infusion of [Sar l, Ala81 AI1 at 10 pg/ kg/min produced no change in the resting blood pressure of conscious turtles (18.8 + 0.9 mm Hg prior to infusion, 17.9 ~fr 0.8 mm Hg during infusion). In two turtles a transient rise in blood pressure of 4 mm Hg was observed with the start of the analog infusion, but the elevation lasted less than 5 min. This analog significantly reduced the pressor responses to AI1 at all three doses (P < 0.05-0.01) and to AI at the two highest

366

GREGORY

INCREASE IN

MEAN

ARTERIAL

A. STEPHENS

24-

24-

22. 20-

22. INCREASE

IS-

IN MEAN

16.

16-

ARTERIAL

,6-

BLOOD

14-

PRESSURE

12IO-

mm Hg

ANGIOTENSIN

I

ANGIOTENSIN

II

2os

8-

61

I

*.*l

I

l *

6;/II XI

4 2 rl 01

05

l * _______-401

d/MO

FIG. 1. Increase in mean blood pressure in conscious turtles to control injections of AI and AI1 (closed circles, solid line) and during infusion of 10 kg/kg/min [Sar’, Alas] AI1 (closed triangles, dashed line). Each point is the mean of six trials. ':P < 0.05, **P < 0.01 compared to control values.

doses (P < 0.05-0.01, Fig. 1). The blood pressure response to norepinephrine (3 p,g/kg) was unaffected (Table 1). The angiotensin antagonist [Sar’, Ile8] AI1 infused at 10 pg/kg/min also caused no change in resting blood pressure (24.4 f 0.9 mm Hg prior to infusion, 24.9 L 0.8 during infusion). [Sax-‘, Ile8] AI1 was a slightly more effective angiotensin antagonist in the turtle than [Sari, Alas] AII, significantly reducing the pressor responses to AI and AI1 at all doses (P < 0.01 in all cases, Fig. 2). The response to norepinephrine (3 pg/kg) was again unchanged (Table 1).

PRESSOR

RESPONSES

TO NOREPINEPHRINE

t

+/----

-------

CI -i

** &--‘-----------

05

-F--“--

05

I o/o1 DOSE

FIG. 2. Increase in mean blood pressure in conscious turtles to control injections of AI and AI1 (closed circles, solid line) and during infusion of 10 FgJkgJmin [Sar l, Ilea] AI1 (closed triangles, dashed line). Each point is the mean of six trials. +P < 0.05, *:*P < 0.01 compared to control values.

Converting

Enzyme Inhibitor

The angiotensin-converting enzyme inhibitor SQ 20881 given as an intravenous bolus of 1 mgfkg and a continuing infusion of 0.5 mg/kg/hr significantly reduced the vasopressor response to AI at all three dose levels (P < 0.05-O.Ol), while the pressor action of AI1 was unchanged ( P > 0.05, Fig. 3). Administration of the converting enzyme inhibitor was without effect on the preinjection blood pressure (23.5 ~fr 0.9 mm Hg con-

TABLE 1 (3Fg/kg) PRIOR TO AND DURING

ANTAGONIST

INFUSION

Increase in mean arterial blood pressure (mm Hg)

[Sat-‘, Ala81 AI1 ( 10 PgJkgJmin) [Sar’, Ilee] AI1 ( 10 PgJkgJmin) SQ 20881 (1 mgJkg + 0.5 mgJkgJhr) Phentolamine (0.5 mgJkg/hr + 1 mgJkgJhr) Phenoxybenzamine (0.25 mgJkgJmin) Notr~. Values are means ? SE, N= 6. ;:P < 0.05, compared to control values. 2:4:P< 0.01 compared to control values.

IO

kg/kg

Control

Antagonist

17.5 1 1.9

17.5 2 1.8

20.7 2 1.6

18.5 k 1.4

22.3 k 2.2

18.3 + 1.8*

18.8 ” 2.8

4.4 i 1.8””

20.7 I!Z 1.7

2.2 it 0.8**

ANGIOTENSIN

24

ANGIDTENSIN

I

ANGKlTENSlN

BLOCKADE

367

IN TURTLES ANGIOTENSIN

II

I

ANGIOTENSIN

II

22

INCREASE

20

INCREASE

,6

IN

ARTERIAL

‘6

ARTERIAL

BLOOD

:;

IN

MEAN

MEAN

BLOOD PRESSURE

PRESSURE

,.

mm Hg

mm

Hg

6 6

01 DOSE

FIG. 3. Increase in mean blood pressure in conscious turtles to control injections of AI and AI1 (closed circles, solid line) and during infusion of SQ 20881 (1 mg/kg + 0.5 mg/kg/hr) (closed triangles, dashed line). Each point is the mean of six trials. *P < 0.05, **P < 0.01 compared to control values.

trol, 24.7 + 1 .O mm Hg during infusion). The response to norepinephrine (3 pg/kg) was reduced by 18% (P < 0.05) during converting enzyme inhibitor infusion (Table 1). a-Adrenergic

Blocking

IO/O1

@g/kg

Drugs

Phentolamine (0.5 mg/kg bolus plus 1 mg/kg/hr infusion) alone decreased the resting blood pressure of the turtles from 22.8 f 0.7 mm Hg to 18.1 I~I 1.1 mm Hg (P < 0.05). Phentolamine reduced the response to norepinephrine (3 pg/kg) by 77% (P < 0.01, Table 1) and the responses to AI and AI1 by 39 and 50%, respectively. The decreases in the pressor responses to the angiotensins were significant (P < 0.05-0.01) except for the 0.5 @g/kg dose of AI (Fig. 4). Phenoxybenzamine (0.25 mg/kg/min) also decreased the resting blood pressure (23.6 + l.OmmHgcontrol, 18.5 + 1.1 mm Hg during infusion, P < 0.01). At this dosage phenoxybenzamine was a more effective adrenergic antagonist than phentolamine, decreasing the pressor response to norepinephrine (3 pg/kg) by 89% (P < 0.01, Table 1). The responses to AI and AI1 were also significantly reduced by 87% for both agents (P < 0.01 in all cases, Fig. 5).

DOSE

&g/kg

FIG. 4. Increase in mean blood pressure in conscious turtles to control injections of AI and AI1 (closed circles, solid line) and during infusion of phentolamine (0.5 mg/kg + 1 mg/kg/hr) (closed triangles, dashed line). Each point is the mean of six to control trials. *:P < 0.05, *4:P < 0.01 compared values.

DISCUSSION In a number of turtle species incubation of kidney extract with homologous plasma generates a vasopressor substance pharmacologically similar to mammalian angiotensin (Kaley and Donshik, 1965; Sokabe et al., 1969; Capelli et al., 1970, Nothstine et al., 197 1; Stephens, unpublished observation). It has been assumed that this pressor substance is angiotensin I since ethylenediaminetetraacetate (EDTA), which inhibits the conversion of angiotensin I to 11 24

ANGIOTENSIN

I

ANGIOTENSIN

II

22 INCREASE IN

MEAN

ARTERIAL

16

BLOOD

16 14

PRESSURE

12

mm

Hg

I

20

IO 6 6 4 2 01

05

IO/O1 DOSE

05

IO

pg/kg

FIG. 5. Increase in mean blood pressure in conscious turtles to control injections of AI and AI1 (closed circles, solid line) and during infusion of 0.25 mg/kg/min phenoxybenzamine (closed triangles, dashed line). Each point is the mean of six to control trials. *P < 0.05, j:*P < O.Ol-compared values.

368

GREGORY

A. STEPHENS

in mammals (Skeggs et al., 1956), was added to the incubation mixture. Although the structure of this turtle angiotensin-like substance is not known, reptilian angiotensin-like compounds appear to be peptides with structures similar but not identical to mammalian angiotensins (Nakajima et al., 197 1). A similar angiotensin-like compound in the snake, Elaphe climocophora, had a tyrosine at position 9 and eluted from a SE-Sephadex column at a position similar to [Aspl, Ile5] AI (Nakajima et al., 197 1). We have found in the turtle that the compound is bound by human angiotensin I antibody (Stephens and Creekmore, 1979) suggesting a structure similar to human angiotensin I. In the turtle we observed dose-related pressor responses to injections of synthetic human AI and AI1 amide. The magnitude of the pressor responses was somewhat less than that found in the eel and rat (Nishimura et al., 1978)) while the duration of the pressor action of the angiotensins and norepinephrine was longer than seen in mammals. The decreased pressor action may reflect a lesser receptor affinity for these synthetic angiotensin molecules in the turtle and the prolonged response is probably due to less efficient degradation mechanisms. In mammals both [Sari, Ala81 AI1 and [SaP, Ile8] AI1 are potent competitive inhibitors of the vasopressor action of AI1 (Tucker et al., 1972; Johnson and Davis, 1973). These analogs were also effective in the turtle, significantly reducing the vasopressor actions of both AI and AII. The inhibition does not appear to be due to a generalized decrease in vasopressor responsiveness since the pressor responses to norepinephrine were unaffected. This ability of the turtle vascular receptors to bind mammalian angiotensin and to be blocked by the same competitive analogs suggests a close similarity of vascular angiotensin receptors in

turtles and mammals. In mammals these compounds are often transiently pressor (Tucker et al., 1972; Johnson and Davis, 1973)) and in the eel [Sari, Ilea] AI1 produced a sustained pressor response at the same dose employed in the turtle (Nishimura et al., 1978). We observed no sustained pressure elevation, but only a transient, weak pressor action with [Sar’, Alas] AI1 in two of the animals. The mammalian converting enzyme inhibitor SQ 2088 1 significantly reduced the vasopressor response to AI but not AI1 in the turtle. The results suggest that AI must first be converted to AI1 in the turtle to exhibit its pressor action, a finding consistent with those in mammals (Erdos, 1977). The lengthened time until the peak of the pressor responses with AI compared to AI1 is also consistent with this hypothesis. An angiotensinconverting enzyme or enzymes may exist in the turtle. It must be remembered, however, that in mammals other proteolytic enzymes are also capable af converting AI to AII, so the mechanism in the turtle may or may not parallel that in mammals. Throughout this study the control vasopressor responses to AI were approximately 70% as great as the responses to AII. This is in contrast to the nearly equipotent responses to AI and AI1 reported in the eel (Nishimura et al., 1978) and spiny dogfish (Opdyke and Holcombe, 1976). This difference may be due to incomplete conversion of the synthetic AI to AI1 by the turtle or to a lesser receptor affinity for the [Ile5] angiotensin than the [Va15] angiotensin molecule. The less potent AI pressor response may also be the result of high angiotensinase activity in the turtle, but the prolonged duration of the responses to both AI and AI1 suggest angiotensinase activities lower than in mammals. A reduced AI to AI1 conversion might be due to the use of a foreign AI for which the converting mechanism has a lesser

ANGIOTENSIN

BLOCKADE

affinity than native turtle AI, or it may reflect an inability of the converting mechanism to rapidly convert the large bolus of AI. It is also possible that the converting mechanism may not be as efficient in turtles as in higher forms, even with endogenous turtle angiotensin, but this seems unlikely in light of the results in the eel and spiny dogfish. It has been suggested that [Val’] angiotensins may predominate in nonmammalian species (Nakajima et al., 1971), and if native turtle angiotensin has a valine at position 5, the turtle angiotensin receptor may have a lesser affinity for [Ile5] angiotensins. This does not seem to be the case in the spiny dogfish, however, where [Ile5] AI and [Va15] AI1 amide were equipotent (Opdyke and Holcombe, 1976). SQ 20881 also blunted the norepinephrine pressor response. Although the reason is unclear, a general decrease in responsiveness does not seem to be the explanation since the AI1 response was not altered. Since SQ 20881 may affect enzyme systems other than the angiotensin-converting enzyme, the decrease may be due to inhibition of an enzyme mediating the response to norepinephrine. The converting enzyme may inhibit the breakdown of bradykinin in the turtle, but control blood pressure was not altered during infusion of the converting enzyme inhibiter, so this does not appear to be a potent mechanism. These results with SQ 20881 are generally consistent with the findings of others. In the eel (Nishimura et al., 1978) SQ 20881 blocked the pressor response to an eel angiotensin-like substance (presumably AI) and to fowl AI ([Va15, Serg] AI), and in the spiny dogfish SQ 20881 blocked the pressor response to [Asp’, Ile5] AI (Opdyke and Holcombe, 1976). The finding of a mechanism for AI to AI1 conversion in the dogfish is particularly intriguing since neither jux-

IN TURTLES

369

taglomerular cells nor renal renin-like activity have been found in the elasmobranchs (Capreol and Sutherland, 1968; Nishimura et al., 1970). The angiotensin-converting enzyme activity found in the spiny dogfish may be due to another enzyme, perhaps a bradykininase, since these two enzymes are identical in mammals (Erdos, 1977). The a-adrenergic antagonists phentolamine and phenoxybenzamine blocked the pressor action of norepinephrine and partially blunted the pressor responses to AI and AII. Phenoxybenzamine completely inhibited the pressor responses to AI and AI1 at the two lower doses. These results appear similar to those in the dogfish where the pressor action of [As& Va15] AI1 was completely abolished after phentolamine administration (Opdyke and Holcombe, 1976). Phentolamine also lowered the control blood pressure in the dogfish as did both blockers in the turtle. In contrast, phentolamine and phenoxybenzamine did not alter the resting aortic pressure in the eel and reduced the pressor response to [Asnl, Va15] AI1 by approximately 35%. In mammals adrenal catecholamine release can be stimulated by angiotensins in several species (Peach, 1974) and the response to angiotensins in turtles may be due to catecholamine release, either from the adrenergic nerve endings or from adrenal tissue. In turtles discrete adrenal glands are found adhered to the medial sides of the kidneys (Gabe, 1970) and adrenergic nerve terminals are known to release catecholamines (Burnstock, 1969), but the effect of angiotensins on these structures has not been examined. Opdyke and Holcombe suggested that in the dogfish the angiotensin vasopressor action may be entirely due to catecholamine release, stimulated by a direct action of angiotensin on chromaffin tissue, or that angiotensin and norepinephrine may act through a common receptor. In the turtle

370

GREGORY

it seems unlikely that a common adrenergic-angiotensin receptor is involved since the angiotensin II analogs blocked the angiotensin response without altering the vasopressor action of norepinephrine. It appears likely, however that the pressor response to angiotensin in the turtle may be largely due to catecholamines. Both a-adrenergic blockers lowered the control blood pressure in the turtle. It is assumed that this is due to blockade of sympathetic vasoconstrictor tone to the resistance vessels. In this situation vasoconstrictor effects of AI1 may be damped by the increased capacitance of the vasular bed, particularly if the AI1 effects occur Only in limited areas of the circulation. For these reasons the mechanism of angiotensin-mediated vasoconstriction in the turtle vasculature needs further study with in vitro methods where the variables can be more adequately controlled. We have demonstrated the vasotx-essor action of synthetic rnammalian~angiotensins in the turtle and the ability of several inhibitors to blunt these responses. The general characteristics of the renin-angiotensin system including an AI to AI1 converting mechanism appear similar to those in mammals, but the physiological significance of the systern in these primitive animals is still unclear. The turtle kidney possesses no loop of Henle and no macula dense but does have glomeruli and granulated epithelial cells. Whether the system in the turtle functions primarily in blood pressure regulation as suggested by Nishimura in teleosts (Nishimura et al., 1978) or in the control of salt balance awaits further study.

ACKNOWLEDGMENTS The SQ 20881 was generously supplied by Mr. S. J. Lucania of the Squibb Institute for Medical Research and the [Sar’, Alas] AI1 was generously supplied by Dr. Keith Ellis of Norwich-Eaton Pharma-

A. STEPHENS

ceuticals. The work was supported the University of Delaware Research American Heart Association of NIH-Biomedical Research Support PHS S07RR070 16-06.

by grants from Foundation, the Delaware, and Program Grant

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