Pressor responses to alligator Angiotensin I and some analogs in the spectacled caiman (Caiman crocodilus)

General and Comparative Endocrinology 147 (2006) 150–157 www.elsevier.com/locate/ygcen

Pressor responses to alligator Angiotensin I and some analogs in the spectacled caiman (Caiman crocodilus) David G. Butler ¤ Department of Zoology, Ramsay Wright Laboratories and Department of Physiology, Medical Sciences Building, University of Toronto, Toronto, Ont., Canada M5S 3G5 Received 15 August 2005; revised 14 October 2005; accepted 16 December 2005 Available online 21 February 2006

Abstract Discovery of the chemical structure of alligator (Alligator mississipiensis) [Asp1, Val5, Ala9]-Angiotensin I (ANG I) has permitted the investigation of cardiovascular responses to this peptide and its analogs in spectacled caimans (Caiman crocodilus), close relatives of alligators. ANG I and [Asp1, Val5]- Angiotensin II (ANG II) i.v. gave dose-dependent increases in mean arterial pressure but there was no pressor response to [Val4]-ANG III (ANG III). Pressor responses to a series of doses of ANG II were compared with a range of doses of norepinephrine (NE) and epinephrine (E) which were found to be only about 1/100 as potent as ANG II on a molar basis. The replacement of d-leu10in the alligator ANG I molecule with l-leu10 almost stopped its conversion to ANG II and attenuated the pressor response. [Asp1, Val5, Ala9]-ANG I (1–9), and ANG (1–7) both failed to increase arterial blood pressure, even at the relatively high non-physiological test dose of 194 pmol kg bw¡1 i.v. Captopril blocked angiotensin converting enzyme (ACE) and prevented the pressor response to ANG I whereas the mammalian AT1 inhibitor Losartan attenuated, but did not completely block the pressor response to ANG II. These are the Wrst experiments which test the cardiovascular responses to alligator ANG I and its analogues in any crocodilian species. © 2005 Elsevier Inc. All rights reserved. Keywords: Alligator ANG I; ANG I analogs; Cardiovascular; Caimans

1. Introduction A complete mammalian-type juxtaglomerular apparatus is not found in reptiles (Sokabe and Ogawa, 1974). Reptilian nephrons are glomerular, but possess neither loops of Henle nor a macula densa (MD). Reptilian aVerent arteriolar juxtaglomerular (JG) cells release renin in response to renal vascular hypotension. Renin binds angiotensinogen and releases the decapeptide Angiotensin I (ANG I) which, in turn, is modiWed by angiotensin converting enzyme (ACE) to yield Angiotensin II (ANG II), the active pressor peptide (see Kobayashi and Takei, 1996 for review). Sildorf and Stephens (1992a,b) have reported a signiWcant positive correlation between dose and the pressor response to Fowl ANG I in American alligators (Alligator *

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mississipiensis). These pressor responses were blocked by the ACE inhibitor Captopril. Moreover, the ANG II analog, Sarile, blocked the pressor responses to Fowl ANG I in A. mississipiensis. ANG II may also increase the synthesis and release of aldosterone from reptilian adrenocortical cells and release the reptilian antiduretic peptide arginine vasotocin (Blair-West et al., 1971; Butler, 1972). Hypotension and or blood volume contraction led to an ANG II-dependent increase in thirst and sodium appetite in Iguanas (Iguana iguana) (Fitzsimons and Kaufman, 1977) and in Nile crocodiles (Balment and Loveridge, 1989). All of these known responses to ANG II are directed towards re-establishing normovolemia, electrolyte balance and a “normal” systemic arterial blood pressure in reptiles. In general, osmotic regulation including the involvement of the RAS may be diVerent and possibly more complex in the ocean-going crocodilians than in fresh water Alligatorids.

D.G. Butler / General and Comparative Endocrinology 147 (2006) 150–157

After Takei et al. (1993) discovered the amino acid sequence for crocodilian (A. mississipiensis) ANG I, it became possible to study the cardiovascular responses to this native peptide in a crocodilian, the spectacled caiman (Caiman crocodilus). Among the living Crocodilia, the genus Caiman is the nearest relative of the genus Alligator. In addition, ANG I is thought to be a fairly conserved molecule. Nevertheless, the possibility that some structural diVerences may exist between Alligator and Caiman ANG I should not be overlooked. One may now measure pressor responses, if any, to synthetic analogs of alligator ANG I to learn about molecular structure–function relationships. In addition, this study measured the cardiovascular responses to [Asp1, Val5]-ANG II and [Val4]-ANG III. Both ANG I and ANG III increased dorsal aortic blood pressure in eels (Butler and Oudit, 1995) and rats (Scheur and Perrone, 1993; Wright et al., 1985) but there was no measurable pressor response to [Val4]-ANG III in domestic ducks (Butler et al., 1998). Recently, ANG (1–7) has been shown to evoke a vasodilation in mesenteric vessels in mammals, both directly and indirectly (Carey and Siragy, 2003; Oudit et al., 2003; Paula et al., 1995; Ueda et al., 2000). It was therefore of interest to test the cardiovascular responses to ANG (1– 7) in spectacled caimans. 2. Materials and methods 2.1. Animals Fifteen male spectacled caimans (C. crocodilus) were housed in suitable holding tanks under a 12L:12D light cycle at a temperature of 28–30 °C and fed mice and rats. Their weights ranged from 7–18 kg by the time they were used for experiments. Their care was supervised by the Provincial Veterinarian and the University of Toronto Veterinarian under a license (permit) issued to DGB by the University of Toronto Animal Care Committee.

2.2. Preparation for measurements of cardiovascular responses Each caiman was selected randomly from the holding tanks. Its jaws were taped shut with duct tape with little or no impairment of breathing. The caiman was then secured, in the supine position, with duct tape to a specially designed plywood holding board. The caiman’s head was covered with a cotton towel and if noise was kept to a minimum, the caiman remained quiet, nearly motionless, and apparently unstressed throughout the entire experimental procedure. Next, the right posterior limb was extended to access the right femoral vein and artery via a 5 cm incision through the skin following induction of local anesthesia with 3–4 ml of Xylocaine s.c. Appropriate lengths of heparin-Wlled PE 20, Intramedic tubing were inserted into the femoral vein for injection of test drugs and hormones and into the femoral artery for measurements of arterial blood pressure. These catheters were tied Wrmly in place with 5–0 surgical silk. The incision was closed with 9 mm stainless steel Michel clips (Clay Adams, Sparks, MD, USA) and the area was inWltrated with Ampicillin. The caiman was allowed 30 min to rest before the measurements of cardiovascular function were started. When the experiments were completed the catheters were removed and the caiman placed in specially designed plywood box with good ventilation for approximately 10 days to allow the catheter incision to heal. If the caiman was to be tested again after a 2-day rest period, the catheters were Wlled with heparin solution, heat sealed and taped to the skin (see Experiment 2). The caiman was then placed in a dry holding box, supplied with drinking water and food ad libitum and transferred to a clean box each day. Finally, the caiman was returned to the

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communal holding tank supplied with water. Wound healing was so complete that eventually it was nearly impossible to locate the site of the original skin incision. If the caiman was used for a second series of experiments, the catheters were placed in the opposite (left) limb.

2.3. Blood pressure measurements Femoral systolic and diastolic arterial pressures were measured with a Model 1050 BP pressure transducer (UFI, Morro Bay, CA, USA) and an MP 100 system (Biopac Systems, Santa Barbara, CA, USA). The data were analyzed using Waveform Acquisition and Analysis Software (AcqKnowledge Version 3.5.3, BIOPAC Systems). The pressure transducer and the zero point level were adjusted to the height of the caiman’s heart. The system was calibrated against a static heparinized saline reservoir. Mean arterial blood pressure (MABP) was expressed as the sum of the diastolic pressure and 1/3 of the pulse pressure. The undamped frequency of the heparinized saline-Wlled system was 30 Hz and the damping ratio was 0.10.

2.4. Experimental groups 2.4.1. Measurements of the cardiovascular responses to a series of doses of [Asp1, Val5]-ANG II were made in a total of Wve caimans The experiment started with a measurement of MABP and heart rate (HR) following an i.v. injection of 0.2 ml of 0.9% NaCl solution. This was taken as the initial baseline measurement. A series of doses of ANG II (9.69, 15.2, 30.3, 77.5, 96.9, 194, 485, and 969 pmol kg bw¡1 i.v.) were each injected in 0.9% saline quickly followed by a 0.5 ml saline wash to clear any remaining peptide from the venous catheter. The MABP was allowed to return to baseline after the injection of each test dose of ANG II and prio to injection of the next test dose in the series. 2.4.2. Measurements of the cardiovascular responses to a series of doses of norepinephrine and epinephrine Each of the Wve caimans used in Experiment 1 above was held over for a two day rest period. Each experiment started with a measurement of baseline MABP and HR followed by a test of the response to a series of i.v. doses (1.6, 3.13, 6.26, 12.50, 18.0, and 31.3 nmol kg bw¡1 i.v.) of norepinephrine. After a 15 min break, a second epinephrine series was run using the same doses and protocol. 2.4.3. Measurement of cardiovascular responses to [Asp1, Val5, Ala9]-ANG I, and its analogs Six new caimans were selected from stock for these experiments. After a preliminary baseline measurement of MABP and HR 194 pmol kg bw¡1 i.v. of each of the following compounds were tested in sequence as follows: [Asp1, Val5, Ala9]- ANG I; [d-leu10]-ANG I; ANG I (1–9); [Asp1, Val5]ANG II; ANG (1–7); [Val4]-ANG III; [Asp1, Val5, Ala9]- ANG I (repeat), and [Asp1, Val5]-ANG II (repeat). As in Experiments 1 and 2 the MABP was allowed to return to baseline before each successive peptide was tested. Injections of [Asp1, Val5, Ala9]-ANG I and [Asp1, Val5]-ANG II were repeated at the end of the series to show that the caiman was still responsive to ANG I and ANG II. 2.4.4. Inhibition of the pressor response to ANG I following the injection of Captopril First, the baseline MABP was recorded. Then the peak MABP was measured following an i.v. injection of 194 pmol kg bw¡1 ANG I and again after an injection of 194 pmol kg bw¡1 ng of ANG II. Next, Captopril (46.0 mol kg bw¡1 i.v. in 1.5 ml of 0.9% saline) was injected to block angiotensin converting enzyme (ACE). After a 1 h interval, a second dose of ANG I (194 pmol kg bw¡1 i.v.) was injected to measure completeness of the ACE blockade. A Wnal injection of ANG II (194 pmol kg bw¡1 i.v.) was used to ensure that the cardiovascular system remained sensitive to the peptide during ACE blockade. The venous catheter was always washed through with 0.5 ml of 0.9% NaCl solution after an injection and MABP was subsequently allowed to return to baseline prior to the injection of each successive test peptide.

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2.4.5. Inhibition of the pressor response to [Asp1, Val5]-ANG II with the mammalian AT1 blocker Losartan Peak MABP was measured in response to a series of i.v. injections of ANG II (10, 19, 39, 78, 97, 194, 490, 960, and 2000 pmol kg bw¡1i.v.) before, and 40 min after, an i.v. injection of 21.7 mol kg bw¡1 of Losartan.

pril (N-[S-3-mercapto-2-methylpropionyl]-L-proline, (Fwt. 217.3), Squibb Canada, Montreal, P.Q., Canada) and Losartan ((2-n-butyl-4-chloro-5hydroxymethyl-1-[2-(1H-tetrazole-5-yl) biphenyl-4-yl] methyl) imidazole, (Fwt. 461.01), DuPont Merck, Wilmington, DE, USA), were generous gifts.

2.5. Peptides and drugs

2.6. Statistical methods

[Asp1, Val5]-ANG II and [Val4]-ANG III were purchased from Peninsula Laboratories (Belmont, CA) [Asp1, Val5, Ala9]-ANG I, [Asp1, Val5, Ala9, d-leu10]-ANG I, [Asp1, Val5, Ala9]-ANG I (1–9), and [Asp1, Val5]ANG (1–7) were all synthesized by the Peptide Technology Laboratory at the Banting Institute, University of Toronto. Heparin sodium (10,000 USP U ml¡1; Hepalean; Organon Teknika, Toronto, ON); Ampicillin (Penbritin-500; Ayherst Laboratories, Montreal, PQ); Xylocaine (2% lidocaine hydrochloride, Astra-Zeneca, Mississauga, ON). Epinephrine bitartrate (Fwt. 333.3), and norepinephrine bitartrate (Fwt. 337.3); Capto-

Values given in Figs. 1 and 3, and Table 1 are means § SEM. Comparison of means were made using Student’s t test. Treatment groups in Fig. 1 were compared using a repeated measures ANOVA and linear regressions were Wtted to data in Table 1. All statistical methods were applied using Statgraphics Plus, Version 5.1; Manugistics, Rockville, MD, USA.

Fig. 1. Percentage diVerences between baseline MABP (mean arterial blood pressure) and the peak MABP () versus the log10 of each dose (pmol kg bw¡1) of hormone. A range of test doses of Angiotensin II, norepinephrine, and epinephrine series was tested in each of Wve spectacled caimans. Values are means § SEM.

3. Results 3.1. Pressor response to [Asp1, Val5]- ANG II and catecholamines Fig. 1 illustrates the successive pressor responses, expressed as the diVerence between baseline and peak MABP () to a series of increasing doses of [Asp1, Val5]ANG II, norepinephrine (NE), and epinephrine (E). ANG II is t100 times as potent as the norepinephrine and epinephrine on a molar basis. A repeated- measures ANOVA showed that both the NE and E curves were signiWcantly diVerent from the ANG II curve (P < 0.05) and from each other (P < 0.05). Fig. 2 illustrates the successive pressor responses in one caiman to a series of increasing doses of [Asp1, Val5]- ANG II. Note that as the doses increase, there is a shortened time interval (min) to peak MABP and a lengthened decay time deWned as the time lapse between peak MABP and the subsequent return to baseline MABP. Numerical data for the cardiovascular responses in Wve caimans to a series of increasing doses of [Asp1, Val5]-ANG II is given in Table 1. Here, there is a positive correlation between dose of [Asp1, Val5]-ANG II and systolic pressure

Fig. 2. Pressor responses by a caiman to a range of i.v. doses of [Asp1, Val5]-ANG II. Each injection of hormone was followed immediately by a 0.5 ml of 0.9% saline to clear the injection catheter.

Table 1 Cardiovascular responses of spectacled caimans (Caiman crocodilus) to a series of i.v. doses of [Asp1, Val5]-Angiotensin II Arterial blood pressures (mmHg)

Dose ANG II (pmol kg bw¡1)

% diV

Diastolic

% diV

% diV

% diV

% diV

Peak

Baseline

9.69

Baseline Peak

56.7 § 7.8 59.0 § 8.6

10.6 § 2.2

49.0 § 6.4 51.6 § 7.3

10.0 § 1.3

53 § 7.0 55.7 § 8.0

10.6 § 1.5

5.94 § 1.6 7.40 § 1.8

21.8 § 16.1

23.8 § 2.9 19.8 § 1.7

9.9 § 4.8

3.0 § 0.9

6.4 § 2.1

Baseline Peak

55.1 § 7.5 64.0 § 7.1

17.9 § 5.4

48.1 § 6.3 55.4 § 5.7

17.2 § 5.6

51.9 § 6.8 59.8 § 6.3

17.0 § 5.4

5.42 § 0.80 6.60 § 1.4

24.2 § 2.5 20.6 § 14.6

24.8 § 2.7

2.6 § 3.3

4.2 § 0.6

9.6 § 0.7

Baseline Peak

56.4 § 7.3 65.7 § 7.2

17.7 § 3.8

48.9 § 6.4 55.8 § 5.9

15.5 § 5.1

52.7 § 6.7 61.2 § 6.4

17.3 § 3.9

5.9 § 0.90 7.5 § 1.8

24.1 § 10.9

22.8 § 2.9 24.4 § 4.4

5.1 § 6.6

4.3 § 0.8

13.5b § 1.6

Baseline Peak

56.9 § 6.7 70.8 § 6.0

27.0 § 9.7

49.5 § 5.5 59.9 § 5.0

23.0 § 8.4

53.4 § 6.1 65.6 § 5.3

25.2 § 9.1

5.84 § 1.0 8.60 § 1.8

52.9 § 30.1

24.2 § 3.3 23.4 § 3.0

2.7 § 3.1

3.9 § 0.5

24.2d § 7.2

Baseline Peak

55.8 § 6.2 75.2 § 7.8

36.3d § 10.4

48.2 § 4.9 64.3 § 7.0

34.4 § 10.9a

52.2 § 5.4 69.7 § 7.3

34.9 § 10.5d

5.34 § 0.80 8.08 § 1.6

24.2 § 3.8 48.7 § 10.1

25.0 § 2.9

5.7 § 5.4

3.7 § 0.7

25.4d § 7.4

Baseline Peak

55.0 § 6.5 78.1 § 7.8

43.7c § 8.6

47.8 § 5.1 66.2 § 6.0

40.4 § 9.7b

51.7 § 5.8 72.2 § 6.7

41.5 § 9.0c

5.34 § 0.9 9.06 § 2.0

70.0 § 17.8d

23.0 § 4.0 25.0 § 3.3

18.0 § 10.4

2.9 § 0.2

25.8c § 3.5

Baseline Peak

55.5 § 6.5 86.4 § 7.3

58.1d § 8.5

47.6 § 5.1 73.6 § 7.2

56.2 § 11.8d

51.8 § 5.7 79.7 § 6.9

56.2 §10.3c

5.06 § 1.2 8.06 § 1.7

22.8 § 4.5 32.3 § 7.2

28.4 § 13.1

3.2 § 0.7

22.0c § 2.5

Baseline Peak

56.2 § 6.2 86.8 § 7.3

55.8d § 5.7

48.1 § 4.6 74.9 § 5.6

57.2 § 7.1d

52.4 § 5.3 81.0 § 6.2

56.2 § 6.2d

5.24 § 1.0 8.12 § 2.2

23.4 § 4.3 31.6 § 5.4

36.2 § 3.7c

2.9 § 0.6

27.1c § 4.1

75.5 96.9 194 485 969

4.8 § 1.9

HR (bpm)

58.4 § 7.9

30.3

54.6 § 6.8

PP

Saline

15.2

50.6 § 6.0

MABP

0

26.6 § 2.8

64.7 §18.3 52.6 § 20.2

Values are means § SEM. Comparison are made between baseline and peak responses. Percentage (%) diVerences are  as a percentage of baseline values. n D 5 Caimans. a P < 0.05. b P < 0.025. c P < 0.005. d P < 0.001 compared with % diVerence at a dose of 9.69 pmol ANG II kg bw¡1 i.v.

D.G. Butler / General and Comparative Endocrinology 147 (2006) 150–157

Systolic

Time to peak and return to baseline (min)

153

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(22.9 + 0.043 *Dose ANG II; correlation coeYcient (r) D 0.64; P < 0.01); diastolic pressure (20.6 + 0.046 *Dose ANG II; r D 0.652; P < 0.01); and MABP (21.7 + 0.435 *Dose ANG II; r D 0.65; P < 0.01). There was a signiWcant but gradual increase in partial pressure (PP) in response to increasing doses of ANG II even though the correlation coeYcient was relatively low (32.5 + 0.0337 *Dose ANG II; r D 0.32; P < 0.05). HR increased signiWcantly with an increased dose of ANG II (1.79 + 0.039 *Dose ANG II; r D 0.65; and P < 0.01). The observation that the time (min) to reach peak MABP shortened with increasing doses of ANG II was observed in a single caiman (Fig. 2) but not in the group of Wve caimans (Table 1) where the response was masked by individual variability (3.77–0.001 *Dose ANG II; r D 10.3; P < 0.10). Nevertheless, there was a clearly expressed increase in the time (min) for the peak MABP to return to baseline (16.0 + 0.013 *Dose ANG II; r D 0.39; P < 0.05). Table 1 shows that the baseline MABP was remarkably constant over the entire range of test doses there being little evidence of tachyphylaxis. The overall cardiovascular responses to ANG II are consistent with the hypothesis that it is the cardiovascular eVector peptide of the caiman RAS. 3.2. Pressor responses to alligator [Asp1, Val5, Ala9]-ANG I and some chemical analogs Each of the test peptides was injected in the same dose of 194 pmol kg bw¡1 i.v. There was a signiWcant and comparable pressor response to ANG I and ANG II both before and after injecting the series of test peptides (Fig. 3). When d-leu10 was substituted for l-leu10 in alligator ANG I, almost all of the pressor activity was lost. There was only a slight pressor response in 3 of the 6 test caimans and no response in the remaining 3 so that the average MABP was not signiWcantly diVerent from the baseline MABP. If l-leu10 was deleted from the alligator ANG I molecule, all pressor activity was lost (Fig. 3). Phe8 was

Fig. 3.  Peak MABP (mean arterial blood pressure) following an i.v. injection of 194 pmol kg bw¡1 of each test peptide. Each peptide was tested in six caimans. Values are means § SEM. The annotation “ns” indicates that the peak MABP did not diVer from the baseline MABP.

deleted from the C-terminal end of [Asp1, Val5]-ANG II to give ANG I (1–7) and Asp1 was removed from the N-terminal to give ANG III (2–8). Neither molecule gave a measurable pressor response (Fig. 3). Average baseline MABP’s (mmHg; n D 6) for each of the test peptides listed in sequence, in Fig. 3 were as follows: ANG I (1–10) 60.9 § 1.1; ANG I (d-leu), 61.7 § 1.6; ANG (1–9), 62.9 § 2.8; ANG II, 64.5 § 1.3; ANG (1–7), 64.3 § 1.3; ANG III (2–8), 64.0 § 1.1; ANG I (1–10), 64.9 § 1.9; and ANG II, 63.0 § 1.8. These relatively constant baseline pressures showed that repeated injections of diVerent peptides did not induce a tachyphylaxis. 3.3. Pressor responses to alligator ANG I and ANG II before and after Captopril Fig. 4 illustrates the pressor response to ANG I and ANG II before and 40 min after an i.v. injection of 46.0 mol kg bw¡1 of the ACE inhibitor Captopril. Results for the Captopril experiment (Fig. 4) support the assumption that alligator [Asp1, Val5, Ala9]-ANG I is converted to [Asp1, Val5]-ANG II the eVector pressor peptide in spectacled caimans. ACE blockade with Captopril prevented this step and blocked completely the pressor response to ANG I. 3.4. Pressor responses in a caiman before and after an i.v. injection of 21.7 mol kg bw¡1 of the mammalian AT1 receptor antagonist Losartan Peak MABP following a series of i.v. injections of ANG II ranging from 10 to 2000 pmol kg bw¡1; (see Section 2, Experiment 5.) was measured before, and 40 min after, partial receptor blockade (Fig. 5).

Fig. 4. Pressor responses by a caiman to 194 pmol kg bw¡1 of ANG I and ANG II were observed before, and 40 min after, the i.v. injection of 46.0 mol kg bw¡1 of the ACE inhibitor Captopril.

D.G. Butler / General and Comparative Endocrinology 147 (2006) 150–157

Fig. 5. The peak MABP in a caiman following a range of doses of [Asp1, Val5]-ANG II given before, and 40 min after, an i.v. injection of 21.7 mol kg bw¡1 of the mammalian AT1 receptor antagonist Losartan.

4. Discussion Cardiovascular responses to synthetic Fowl ANG I have been measured in three species of reptiles: The alligator, Alligator mississipiensis, (Sildorf and Stephens, 1992a,b); the snake, Elaphe climocophora, (Nakayama et al., 1977) and the turtle, Pseudemys scripta, (Hasegawa et al., 1984). The structure of the ANG I’s diVer only at position 9 when compared with amphibians or bird ANG I’s. Position 9 is occupied by Ala in alligators, Tyr in snakes and His in turtles but in crocodilians the chemical structure of ANG I is not known. Alligator [Asp1, Val5, Ala9]-ANG I increased arterial blood pressure in A. mississipiensis (n D 6) in a dosedependent manner but its pressor activity was not more potent than avian (quail), [Asp1, Val5, Ser9] -ANG I and rat, [ Asp1, Ile5, His9]-ANG I] or [Asp1,Val5]-ANG II The range of doses of ANG II is narrower (10,30,100 and 300 pmol kg bw¡1) than the range used in the present experiments (see Figs. 1 and 2; Table 1) but the pressor responses are similar, starting at about 7 mmHg (10 pmol kg bw¡1) and ending at approximately 30 mmHg (300 pmol kg bw¡1). In both experiments the baseline arterial pressures §SEM were also remarkably similar (Takei et al., 1993; 57.0 § 4.7 mmHg; n D 6) cf. Butler, present experiments (see, Table 1; 56.7 § 7.8 mmHg; n D 5). Pressure catheters were inserted into the femoral artery in both experiments and the animals were of approximately the same size (Takei et al., 1993). The present observations add to what is known about structure–function relationships between the ANG I molecule and the enzyme ACE leading to cleavage of the two C-terminal amino acids to yield the primary eVector peptide ANG II. l-Leu10 was replaced with d-leu10 to measure the dependency of binding on the optical form of this amino acid. Fig. 3 shows that ANG I (d-leu10) had relatively little overall pressor activity insofar as three caimans were totally unresponsive and, in the remaining

155

three animals, there was only a slight increase in MABP compared with the baseline pressures. After testing ANG I (d-leu10), this study measured the pressor response to ANG I (1–9) from which l-leu10 had been deleted. The requirement for d-leu10 to facilitate renin binding is shown by the absence of any pressor response to this analog (Fig. 3). In mammals, angiotensin converting enzyme2 (ACE2) is known to cleave the C-terminal amino acid and to form both ANG (1-9) and ANG (1–7) (Carey and Siragy, 2003; Oudit et al., 2003). Fig. 3 shows that ANG (1–9) has no vasopressor activity nor is it known to induce a cardiovascular response in mammals (Burrell et al., 2004) or Wsh (Butler and Oudit, 1995). On the other hand, ANG (1–7) has many hormonal properties in mammals. It is found in the hypothalamus and medulla oblongata and is equipotent with ANG II in vasopressin release and the induction of antidiuresis (Schiavone et al., 1988). ANG II is converted to ANG (1–7) by ACE2 which was the Wrst human homologue of ACE to be described. ACE2 is an integral membrane protein which functions as a carboxypeptidase in the generation of ANG 1–7 from ANG II (Warner et al., 2004). In mammals ANG (1–7) is vasodilatory and acts as an ACE inhibitor (Ferrario and Chappell, 1979; Schiavone et al., 1990). ANG (1–7) induced endothelium dependent relaxation and vasodilation in rat aortic strip preparations precontracted with phenylephrine. Without preconstriction, ANG 1–7 produced a signiWcant inhibition of ANG II-induced vasoconstriction (Ueda et al., 2000). ANG (1– 7) has triggered counter-regulatory actions against ANG II and blocked ANG II-induced vasodilation (Haulica et al., 2003). Chronic hypotensive eVects of Losartan in normotensive rats are mediated partly by the accumulation of ANG (1–7) during ACE blockade (Collister and Hendel, 2003). Finally, microinjection of ANG (1–7) into rat caudal ventrolateral medulla led to a vasodepressor response which was not blocked by the ANG (1–7) antagonist Ang 779 (31). ANG (1–7) induces neither centrally mediated dipsogenesis (drinking) nor peripheral vasoconstriction (Schiavone et al., 1988). In the light of these observations, it seemed worthwhile to investigate if ANG I (1–7) injections would modify cardiovascular function in spectacled caimans. In spite of these demonstrated eVects of ANG (1–7) on cardiovascular function ANG (1–7) had no measurable eVect on caiman MABP (Fig. 4) nor did it impart any cardiovascular response in Pekin ducks (Butler et al., 1998). ANG III is a pressor peptide in some vertebrate animals e.g., freshwater eels; (Butler and Oudit, 1995); and Pekin ducks, (Butler et al., 1998) and rats (Wright et al., 1985). ANG III increased aldosterone synthesis and adrenal steroid secretion from rat, calf, dog, sheep, rabbit, and man (Blair-West et al., 1971; Semple and Norton, 1976) but had no peripheral dipsogenic eVect in rats (Fitzsimons, 1979) or pigeons (Evered and Fitzsimons, 1981). An equivalent or even greater potency of ANG III compared with ANG II has been observed with respect to drinking and vasopressor

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activity in mammals (Fitzsimons, 1979). Nevertheless, ANG III failed to induce a pressor response in spectacled caimans (Fig. 3) showing that the N-terminal Wrst amino acid is required for receptor binding and the induction of a pressor response in this crocodilian. The continuous sensitivity of the caimans to other peptides throughout the test period was veriWed by similar pressor responses to ANG I and ANG II at the beginning and end of the test series (Fig. 3). The prototypical antagonists for mammalian AT1 receptors are biphenylimidazole compounds with a tetrazole ring DuP 753 (Losartan) is such a compound. AT1 receptor activation can account for nearly all of the cardiovascular responses to ANG II (Smith and Timmermans, 1994; Timmermans et al., 1993). However the mammalian AT1 blocker Losartan was ineVective and had only a small inhibitory eVect on ANG II-sensitive subfornical neurons in ducks (Schaefer et al., 1993). Furthermore, the ANG II binding sites in turkey adrenocortical cells (homologue of the mammalian cortex) were inhibited fully by dithiothreitol but minimally by Losartan (Kocsis et al., 1995). Moreover Losartan and CGP 48933 both failed to block the pressor response to ANG II in Pekin ducks (Butler et al., 1998). In the fowl (Gallus domesticus) neither the in vivo vasodepressor nor vasopressor actions of the ANG II nor its in vitro aortic relaxation were blocked by exposure to 21.7 mol kg¡1 of Losartan (Nishimura et al., 1994). African clawed frog (Xenopus laevis) hearts have two classes of receptors, both possessing a low-aYnity for Losartan which may indicate that they are pharmacologically diVerent from AT1 and AT2 receptors in mammals (Ji et al., 1991). ANG II binds to high-aYnity receptors (KD 0.3–0.4 £ 10¡10 M) in rainbow trout (Oncorhynchus mykiss) glomerulii (Cobb and Brown, 1994) although the aYnity of the receptors for Losartan is comparatively low (Cobb and Brown, 1993). Experimental evidence shows that AT1 receptors in nonmammalian vertebrates are both structurally and pharmacologically diVerent from those in mammals. Not unexpectedly, Losartan did not completely block the pressor response to a series of i.v. doses of ANG II in the spectacled caiman (Fig. 5). Acknowledgments The author acknowledges technical help from undergraduate students, W. Lam, M. Texiera, and B. Beronja. This research was supported by Grant A-2359 from the Natural Sciences and Engineering Research Council to D.G.B. References Balment, R.J., Loveridge, J.P., 1989. Endocrines and osmoregulatory mechanisms in the Nile crocodile. Crocodylus niloticus. Gen. Comp. Endocrinol. 73, 361–367. Blair-West, J.R., Coghlan, J.P., Denton, D.A., Funder, J.W., Scoggins, B.A., Wright, R.D., 1971. The eVect of heptapeptide (2–8) and the hexapeptide (3–8) fragments of angiotensin II on aldosterone secretion. J. Clin. Endocrinol. 32, 575–578.

Burrell, L.M., Johnston, C.I., Tikellis, C., Cooper, M.E., 2004. ACE2, a new regulator of the renin–angiotensin system. Trends Endocrinol. Metab. 15, 166–169. Butler, D.G., 1972. Antidiuretic eVect of arginine vasotocin in the Western painted turtle (Chrysemis picta belli). Gen. Comp. Endocrinol. 18, 121– 125. Butler, D.G., Oudit, G.Y., 1995. Angiotensin I and III-mediated cardiovascular responses in the freshwater North American eel, Anguilla rostrata: eVect of Phe8 deletion. Gen. Comp. Endocrinol. 97, 259–269. Butler, D.G., Zandevakili, R., Oudit, G.Y., 1998. EVects of ANG II and ANG III and angiotensin receptor blockers on nasal salt gland secretion and arterial blood pressure in conscious Pekin ducks (Anas platyrhynchos). J. Comp. Physiol. B 168, 213–224. Carey, R.M., Siragy, H.M., 2003. Newly recognized components of the renin–angiotensin system: potential roles in cardiovascular and renal regulation. Endocr. Rev. 24, 261–271. Cobb, C.S., Brown, J.A., 1993. Characterisation of putative glomerular receptors for angiotensin II in the rainbow trout Oncorhynchus mykiss using the antagonists losartan, PD 123177, and saralasin. Gen. Comp. Endocrinol. 92, 123–131. Cobb, C.S., Brown, J.A., 1994. Characterisation of angiotensin II binding to glomerulii from rainbow trout Oncorhynchus mykiss adapted to fresh water and seawater. Gen. Comp. Endocrinol. 94, 104–112. Collister, J.P., Hendel, M.D., 2003. The role of Ang (1–7) in mediating the chronic hypotensive eVects of losartan in normal rats. J. Renin Angiotensin Aldosterone Syst. 4, 176–179. Evered, M.D., Fitzsimons, J.T., 1981. Drinking and changes in blood pressure in response to angiotensin II in the pigeon, (Columbia livia). J. Physiol. (London) 310, 337–352. Ferrario, C.M., Chappell, M.C., 1979. What’s new in the renin–angiotensin system? Novel angiotensin peptides. Cell. Mol. Life Sci. 61, 2720–2727. Fitzsimons, J.T., 1979. The Physiology of Thirst and Sodium Appetite. Cambridge University Press, Cambridge. Fitzsimons, J.T., Kaufman, S., 1977. Cellular and extracellular dehydration and angiotensin as stimuli to drinking in the common iguana, Iguana iguana. J. Physiol. (London) 265, 443–463. Hasegawa, Y., Cipolle, M., Watanabe, T.X., Nakajima, T., Sokabe, H., Zehr, J.E., 1984. Chemical structure of angiotensin in the turtle, Pseudemys scripta. Gen. Comp. Endocrinol. 53, 159–162. Haulica, I., Bild, W., Mihaila, C.N., Ionita, T., Boisteanu, C.P., Neagu, B., 2003. Biphasic eVects of angiotensin (1–7) and its interactions with angiotensin II in rat aorta. J. Renin Angiotensin Aldosterone Syst. 4, 124–128. Ji, H., Sandberg, K., Catt, K.J., 1991. Novel angiotensin II antagonists distinguish amphibian form mammalian angiotensin II receptors expressed in Xenopus laevis oocytes. Mol. Pharmacol. 39, 120–123. Kobayashi, H., Takei, Y., 1996. The renin–angiotensin system comparative aspects. In: Zoophysiology. Springer Verlag, New York. Kocsis, J.F., Schimmel, R.J., McIlroy, P.J., Carsia, R.V., 1995. Dissociation of increases in intracellular calcium and aldosterone production induced by angiotensin II (AII): evidence for regulation by distinct AII receptor subtypes or isomorphs. Endocrinology 36, 626–634. Nakayama, T., Nakajima, T., Sokabe, H., 1977. Comparative studies on angiotensins. IV. Structure of snake (Elaphe climocophora) angiotensin. Chem. Pharm. Bull. (Tokyo) 25, 3255–3260. Nishimura, H., Walker, O.E., Patton, C.M., Madison, A.B., Chiu, A.T., Keiser, J., 1994. Novel angiotensin receptor subtypes in fowl. Am. J. Physiol. 267, R1174–R1181. Oudit, G.Y., Crackower, M.A., Backx, P.H., Penninger, J.M., 2003. The role of ACE2 in cardiovascular physiology. Trends Cardiovasc. Med. 13, 93–101. Paula, R.D., Lima, C.V., Khosla, M.C., 1995. Angiotensin (1–7) potentiates hypotensive eVect of bradykinin in conscious rats. Hypertension 26, 1154–1159. Schaefer, F., Schmid, H.A., Simon, E., 1993. Pharmacological characterization of the angiotensin II response in neurons of the subfornical organ using speciWc AT1 and AT2 antagonists (abstract). PXugers Arch. 422, R124.

D.G. Butler / General and Comparative Endocrinology 147 (2006) 150–157 Scheur, D.A., Perrone, M.H., 1993. Angiotensin type-2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am. J. Physiol. 264, R917–R923. Schiavone, M.T., Khosla, M.C., Ferrario, C.M., 1990. Angiotensin (1–7): evidence for the novel actions in the brain. J. Cardiovasc. Pharmacol. 16, S19–S24. Schiavone, M.T., Santos, R.A.S., Brosnihan, K.B., Khosla, M.C., Ferrario, C.M., 1988. Release of vasopressin from the rat hypothalamo neurohypophyseal system by angiotensin (1–7) heptapeptide. Proc. Natl. Acad. Sci. USA 85, 4095–4098. Semple, P.F., Norton, J.J., 1976. Angiotensin II and angiotensin III in rat blood. Circ. Res. 38, 122–126. Sildorf, E.P., Stephens, G.A., 1992a. EVects of converting enzyme inhibition and  receptor blockade on the angiotensin pressor response in the American alligator. Gen. Comp. Endocrinol. 87, 134–140. Sildorf, E.P., Stephens, G.A., 1992b. The pressor response to exogenous Angiotensin I and its blockade by angiotensin II analogs in the American alligator. Gen. Comp. Endocrinol. 87, 141–148. Smith, R.D., Timmermans, P.B.M.W.M., 1994. Human angiotensin receptor subtypes. Curr. Opin. Nephrol. Hypertens. 3, 112–122.

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Sokabe, H., Ogawa, M., 1974. Comparative studies of the juxtaglomerular apparatus. Int. Rev. Cytol. 37, 270–327. Takei, Y., SilldorV, E.P., Hasegawa, Y., Watanabe, T.X., Nakajima, K., Stephens, G.A., Sakakibara, S., 1993. New angiotensin I isolated from a reptile, Alligator mississippiensis. Gen. Comp. Endocrinol. 90, 214–219. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T., Herblin, W.F., BenWeld, P., Carini, D.J., Lee, R.J., Wexler, R.R., Saye, J.M., Smith, R.D., 1993. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol. Rev. 45, 205–251. Ueda, S., Masumori-Maemoto, S., Ashino, K., Naahara, T., Gotoh, E., Umemura, S., Ishii, M., 2000. Angiotensin (1–7) attenuates vasoconstriction evoked by angiotensin II but not by noradrenaline in man. Hypertension 35, 998–1001. Warner, F.J., Sith, A.I., Hooper, N.M., Turner, A.J., 2004. Angiotensinconverting enzyme-2: a molecular and cellular perspective. Cell. Mol. Life Sci. 61, 2704–2713. Wright, J.W., Morseth, S.L., Abhold, R.H., Harding, J.W., 1985. Pressor action and dipsogenicity induced by angiotensin II and III in rats. Am. J. Physiol. 249, R514–R521.