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Adrenomedullin(16–31) Has Pressor Activity in the Rat But Not the Cat
Peptides, Vol. 18, No. 1, pp. 133–136, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(96)00251-3
Adrenomedullin(16–31) Has Pressor Activity in the Rat But Not the Cat H. C. CHAMPION, D. E. FRIEDMAN, D. G. LAMBERT, W. A. MURPHY, D. H. COY AND P. J. KADOWITZ 1 Departments of Pharmacology and Medicine, Tulane University School of Medicine, New Orleans, LA 70112 Received 14 August 1996; Accepted 19 September 1996 CHAMPION, H. C., D. E. FRIEDMAN, D. G. LAMBERT, W. A. MURPHY, D. H. COY AND P. J. KADOWITZ. Adrenomedullin(16–31) has pressor activity in the rat but not the cat. PEPTIDES 18(1) 133–136, 1997.—Responses to a newly synthesized human adrenomedullin (hADM) analog, hADM(16–31), were investigated in the rat and cat. Unlike the full-sequence peptide, which has potent hypotensive activity, hADM(16–31) had pressor activity in the rat but not in the cat. Injection of hADM(16–31) in doses of 10–300 nmol/kg IV induced dose-dependent increases in systemic arterial pressure in the rat, and the peptide was approximately 10-fold less potent than norepinephrine when doses are compared on a nanomole basis. In contrast, injection of hADM(16–31) in doses up to 1000 nmol/kg IV had no significant effect on systemic arterial pressure in the cat. Increases in systemic arterial pressure in response to hADM(16–31) in the rat were significantly reduced after administration of phentolamine or reserpine. These data suggest that increases in systemic arterial pressure in response to hADM(16–31) are mediated by release of catecholamines and activation of a-adrenergic receptors in the rat. These data show that hADM(16–31) has significant pressor activity and that there are marked species differences in the response to hADM(16–31). q 1997 Elsevier Science Inc. Adrenomedullin
hADM(16–31)
a-Receptor activation
ADRENOMEDULLIN (ADM) is a hypotensive peptide first isolated from human pheochromocytoma cells (7). Human ADM (hADM) consists of 52 amino acids with a disulfide bond between cysteine residues in positions 16 and 21 that forms a six-membered ring structure similar to the ring structure found in calcitonin gene-related peptide (CGRP) and pancreatic amylin (7). ADM is present in the adrenal medulla, lung, and kidney; and plasma levels of ADM are increased in disease states, such as congestive heart failure, hypertension, and renal failure (4– 6). This peptide has been shown to possess vasodilator activity in a number of regional vascular beds in the cat, dog, and rat (1,3,8–11). Recent studies have shown that ADM ( 15 – 52 ) , an N-terminal truncated form of ADM that possesses the six-membered ring structure, retains the hypotensive and vasodilator activity of the full-sequence peptide ( 1,10,11 ) . These reports suggest that the N-terminal amino acids located before the ring structure are not necessary for the full expression of vasodilator activity ( 1,10,11 ) . Fragments of ADM, such as hADM ( 22 – 52 ) , which do not possess the ring structure, do not possess vasodilator activity ( 1,2,10 ) . It has recently been reported that N-terminal fragments of ADM, which contain the ring structure ADM ( 1 – 25 ) and ADM ( 16 – 21 ) , increase systemic arterial pressure in the rat ( 12 ) .
Vasopressor
Species differences
Whereas responses to ADM(1–25) and ADM(16–21) have been studied in the rat, little if anything is known about responses to ADM(16–31) in the systemic vascular bed. The purpose of the present study, therefore, was to investigate responses to this newly synthesized hADM analogue, which has a longer amino acid sequence after the ring structure than does ADM(1–25) or ADM(16–21), in the systemic vascular bed of the rat and cat and to determine the mechanism by which this peptide fragment increases systemic arterial pressure. METHOD
Seventeen Sprague–Dawley rats of either sex weighing 360– 520 g were anesthetized with pentobarbital sodium (50 mg/kg, IP). Supplemental doses of pentobarbital were given as needed to maintain a uniform level of anesthesia. The trachea was cannulated, and the rats breathed room air or were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.4– 2.6 ml at a rate of 30–35 breaths/min. Catheters were inserted into the external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial (aortic) pressure. Systemic arterial pressure was measured with a Statham P23 pressure transducer and was recorded on a Grass model 7 polygraph, and mean pressure was derived by electronic averaging.
1 Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.
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Four adult cats weighing 2.3–4.4 kg were sedated with ketamine hydrochloride (10–15 mg/kg, IM) and were anesthetized with pentobarbital sodium (30 mg/kg, IV). Supplemental doses of pentobarbital were given during the course of the experiment to ensure a uniform level of anesthesia. Experimental procedures were identical to the rat, except the cats were ventilated with a Harvard model 607 ventilator at a volume of 40–60 ml at 15– 22 breaths/min. The temperature of the animals was maintained at 377C with a heating blanket. In the first series of experiments, responses to IV injection of ADM(16–31) were compared in the systemic vascular bed of the rat and cat. In the second series of experiments, responses to IV injections of ADM(16–31) in the rat were compared with responses to norepinephrine (NE) when doses are expressed on a nanomole basis to take molecular weight of the pressor agents into account. In the third series of experiments, the role of the adrenergic nervous system was investigated, and responses to ADM(16– 31) were compared before and after administration of the aadrenergic receptor antagonist, phentolamine, in a dose of 1 mg/ kg, IV. Phentolamine decreased systemic arterial pressure from 118 { 6 to 109 { 8. The extent of a receptor blockade was assessed by comparing responses to NE before and after treatment with the blocking agent. In a separate series of experiments, the effects of adrenergic nerve terminal blockade on pressor responses to ADM(15–31) were investigated 20–24 h after administration of reserpine in a dose of 1.5 mg/kg, IP. The extent of adrenergic neuronal blockade was assessed by comparing responses to the indirect-acting agonist, tyramine, in reserpine-pretreated and control animals. Human synthetic adrenomedullin [hADM(16–31); H2NCys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-GlnIle-Tyr-COOHNH2 ] (Peptide Research Labs, Tulane Medical School, New Orleans, LA) was dissolved in 0.9% NaCl. Norepinephrine hydrochloride, tyramine hydrochloride, angiotensin II (Sigma Chemical Co., St. Louis, MO), and reserpine phosphate (Serpasilw ) and phentolamine mesylate (CIBA–GEIGY Corp., Summit, NJ) were dissolved in 0.9% NaCl. Injections of agonists were administered in a random order. Drug solutions were prepared on a frequent basis and kept on crushed ice. All responses were analyzed using a one-way analysis of variance (ANOVA) and Scheffe’s F-test or a paired t-test. A value of p õ 0.05 was used as the criterion for statistical significance. RESULTS
Responses to hADM(16–31) In the rat, injections of hADM(16–31) in doses of 10–300 nmol/kg IV produced dose-dependent increases in systemic arterial pressure, and these data are summarized in Fig. 1(A). However, in the cat, injections of hADM(16–31) in doses up to 1000 nmol/kg IV produced no significant change in systemic arterial pressure [Fig. 1(B)]. The increases in systemic arterial pressure in response to hADM(16–31) and NE were compared in the rat, and these data are summarized in Fig. 1(C). NE was approximately 10-fold more potent than hADM(16–31) in increasing systemic arterial pressure in the rat when doses are compared on a nanomole basis to take molecular weight of the compounds into account [Fig. 1(C)].
FIG. 1. (A) Bar graphs showing the increases in systemic arterial pressure in response to IV injection of human adrenomedullin (hADM)(16– 31) in the rat. (B) Bar graph showing the effects of IV injection of hADM(16–31) on systemic arterial pressure in the cat. (C) Dose–response curves comparing the increase in systemic arterial pressure in the rat in response to hADM(16–31) and norepinephrine (NE). n indicates number of animals.
Following treatment with the a-receptor blocking agent phentolamine in a dose of 1 mg/kg IV, the increases in systemic arterial pressure in response to hADM(16–31) were significantly reduced [Fig. 2(A)]. The increases in systemic arterial pressure in response to NE in the same animals were significantly decreased after administration of the a-receptor blocking agent [Fig. 2(A)], whereas responses to angiotensin II were not significantly altered (data not shown). The role of catecholamine release in mediating increases in systemic arterial pressure in response to hADM(16–31) in the rat was investigated, and these data are summarized in Fig. 2(B). Following treatment with the adrenergic nerve terminal-depleting agent reserpine in a dose of 1.5 mg/kg IP, pressor responses to hADM(16–31) were significantly reduced [Fig. 2(B)]. Following treatment with reserpine, increases in systemic arterial pressure in response to the indirectacting agonist, tyramine, were significantly reduced [Fig. 2(B)], and responses to NE were significantly greater than control (data not shown).
Role of the Adrenergic Nervous System The role of a-receptor activation in mediating increases in systemic arterial pressure in response to hADM(16–31) was investigated in the rat, and these data are summarized in Fig. 2.
DISCUSSION
The results of the present investigation demonstrate that hADM(16–31) increases systemic arterial pressure in the rat. However, when injected in doses up to 1000 nmol/kg IV,
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PRESSOR RESPONSES TO ADRENOMEDULLIN(16–31)
FIG. 2. (A) Influence of the a-receptor blocking agent, phentolamine, in a dose of 1 mg/kg IV on increases in systemic arterial pressure in response to hADM(16–31) and norepinephrine (NE) in the rat. (B) Influence of the adrenergic neuronal blocking agent, reserpine, in a dose of 1.5 mg/kg IP on pressor responses to hADM(16–31) and tyramine in the rat. n indicates number of animals, and the asterisk indicates that the response is significantly different (p õ 0.05).
hADM(16–31) produced no measurable change in systemic arterial pressure in the cat. These data indicate that there is a marked species difference in the effect of this ADM fragment on systemic arterial pressure in the cat and rat. In terms of relative pressor activity, hADM(16–31) was 10-fold less potent than NE, suggesting that it has considerable pressor activity in the systemic vascular bed of the rat. The increase in systemic arterial pressure in response to hADM(16–31) was attenuated by the aadrenergic receptor agent phentolamine and by the adrenergic nerve terminal-blocking agent reserpine. These data suggest that the increases in systemic arterial pressure in response to hADM(16–31) are mediated by the release of catecholamines and the activation of a-adrenergic receptors. However, the site
135 from which hADM(16–31) releases catecholamine is uncertain and further experiments are needed to define the site of catecholamine release. Although the full sequence of the ADM peptide has been shown to possess vasodilator activity in the systemic vascular bed of the rat and in a number of regional vascular beds in the cat, dog, and rat (1,3,8,11), an ADM fragment containing residues 16 to 31, hADM(16–31), was shown to possess pressor activity. Data from the present study are in agreement with a previous study in which hADM(16–21), the ring portion of the peptide, was shown to possess pressor activity in the systemic vascular bed of the rat. The data with these shortened analogues of hADM, [hADM(16–21) and hADM(16–31)], provide support for the concept that hADM fragments containing the ring structure have novel pressor activity in the rat (12). The observation that ADM(16–31) and ADM(16–21) seem to have similar pressor activity in the rat suggests that amino acid residues 22 to 31 do not contribute to the pressor response. Whereas hADM(16–31) has novel, indirectly mediated, pressor activity in the rat, the peptide has little or no effect on systemic arterial pressure in the cat when injected in doses up to 1000 nmol/kg IV. The results of experiments in the cat and rat show that there are marked differences in regard to the effects of hADM(16– 31) on systemic arterial pressure and may suggest the absence of the mechanism that mediates the pressor response to hADM(16–31) in the cat. It has been previously reported that there exists interesting species differences with regard to the mechanism by which the full-sequence peptide, hADM(1–52), induces vasodilation (9). Inhibition of nitric oxide synthase reduces vasodilator responses to hADM in the pulmonary and hindquarters vascular beds of the rat and in the renal vascular bed of the dog (3,8,9). However, vasodilator responses to hADM are not affected by nitric oxide synthase inhibitors in the feline pulmonary circulation (9) and the hindlimb vascular bed of the cat (unpublished observations, 1996). The results of the present study extend the concept that responses to ADM and analogs differ with species. The observation that N-terminal truncated forms of ADM have novel pressor activity in the rat is interesting from a structure–activity relationship point of view. However, the physiological implications of these findings are uncertain, because it is not known if these peptide fragments are formed in the body. In summary, the hADM fragment, hADM(16–31), possesses pressor activity in the systemic vascular bed of the rat, but not the cat. The pressor response to hADM(16–31) is 10-fold less potent than NE when doses were compared on a nanomole basis, and the response to hADM(16–31) is mediated by the release of catecholamine and the activation of a-adrenergic receptors. These data indicate that amino acid residues 16–31 may be required for the expression of pressor activity in the rat. ACKNOWLEDGEMENTS
The authors thank Ms. Janice Ignarro for editorial assistance. The studies were supported by NIH grants HL15580 and HL09474 and a grant from the American Heart Association-Louisiana, Inc. Hunter C. Champion was supported by NIH grant HL09474.
REFERENCES 1. Cheng, D. Y.; DeWitt, B. J.; Wegmann, M. J.; Coy, D. H.; Bitar, K.; Murphy, W. A.; Kadowitz, P. J. Synthetic human adrenomedullin and ADM(15–52) have potent short-lasting vasodilator activity in the pulmonary vascular bed of the cat. Life Sci. 55:PL-251–PL256; 1994.
2. Eguchi, S.; Hirata, Y.; Iwasaki, H.; Sato, K.; Watanabe, T. X.; Inui, T.; Nakajima, K.; Sakakibara, S.; Marumo, F. Structure–activity relationship of adrenomedullin, a novel vasodilator peptide, in cultured rat vascular smooth muscle cells. Endocrinology 135:2454–2458; 1994.
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3. Feng, C. J.; Kang, B.; Kaye, A. D.; Kadowitz, P. J.; Nossaman, B. D. L-NAME modulates responses to adrenomedullin in the hindquarters vascular bed of the rat. Life Sci. 55:PL-433 – PL438; 1994. 4. Ichiki, Y.; Kitamura, K.; Kangawa, K.; Kawamoto, M.; Matsuo, H.; Eto, T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338:6 – 10; 1994. 5. Ishimitsu, T.; Nishikimi, T.; Saito, Y.; Kitamura, K.; Eto, T.; Kangawa, K.; Matsuo, H.; Omae, T.; Matsuoka, H. Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J. Clin. Invest. 94:2158–2161; 1994. 6. Jougasaki, M.; Wei, C.; McKinley, L. J.; Burnett, J. C. Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation 92:286–289; 1995. 7. Kitamura, K.; Kangawa, K.; Kawamoto, M.; Ichiki, Y.; Nakamura, S.; Matsuo, H.; Eto, T. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192:553–560; 1993.
8. Majid, D. S. A.; Kadowitz, P. J.; Coy, D. H.; Navar, L. G. Renal responses to intra-arterial administration of adrenomedullin in dogs. Am. J. Physiol. 270:F200–F205; 1996. 9. Nossaman, D. B.; Feng, C. J.; Kaye, A. D.; DeWitt, B. J.; Coy, D. H.; Murphy, W. A.; Kadowitz, P. J. Pulmonary vasodilator responses to adrenomedullin are reduced by nitric oxide synthase inhibitors in the rat but not in the cat. Am. J. Physiol. 270:L782–L789; 1996. 10. Santiago, J. A.; Garrison, E. A.; Purnell, W. L.; Smith, R. A.; Champion, H. C.; Coy, D. H.; Murphy, W. A.; Kadowitz, P. J. Comparison of responses to adrenomedullin and adrenomedullin analogs in the mesenteric vascular bed of the cat. Eur. J. Pharmacol. 272:115–118; 1995. 11. Santiago, J. A.; Garrison, E. A.; Ventura, V. L.; Coy, D. H.; Bitar, K.; Murphy, W. A.; McNamara, D. B.; Kadowitz, P. J. Synthetic human adrenomedullin and adrenomedullin 15–52 have potent short-lived vasodilator activity in the hindlimb vascular bed of the cat. Life Sci. 55:PL-85–PL-90; 1994. 12. Watanabe, T. X.; Itahara, Y.; Inui, T.; Yoshizawa–Kumagaye, K.; Nakajima, K.; Sakakibara, S. Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rat. Biochem. Biophys. Res. Commun. 219:59–63; 1996.
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Adrenomedullin(16–31) Has Pressor Activity in the Rat But Not the Cat H. C. CHAMPION, D. E. FRIEDMAN, D. G. LAMBERT, W. A. MURPHY, D. H. COY AND P. J. KADOWITZ 1 Departments of Pharmacology and Medicine, Tulane University School of Medicine, New Orleans, LA 70112 Received 14 August 1996; Accepted 19 September 1996 CHAMPION, H. C., D. E. FRIEDMAN, D. G. LAMBERT, W. A. MURPHY, D. H. COY AND P. J. KADOWITZ. Adrenomedullin(16–31) has pressor activity in the rat but not the cat. PEPTIDES 18(1) 133–136, 1997.—Responses to a newly synthesized human adrenomedullin (hADM) analog, hADM(16–31), were investigated in the rat and cat. Unlike the full-sequence peptide, which has potent hypotensive activity, hADM(16–31) had pressor activity in the rat but not in the cat. Injection of hADM(16–31) in doses of 10–300 nmol/kg IV induced dose-dependent increases in systemic arterial pressure in the rat, and the peptide was approximately 10-fold less potent than norepinephrine when doses are compared on a nanomole basis. In contrast, injection of hADM(16–31) in doses up to 1000 nmol/kg IV had no significant effect on systemic arterial pressure in the cat. Increases in systemic arterial pressure in response to hADM(16–31) in the rat were significantly reduced after administration of phentolamine or reserpine. These data suggest that increases in systemic arterial pressure in response to hADM(16–31) are mediated by release of catecholamines and activation of a-adrenergic receptors in the rat. These data show that hADM(16–31) has significant pressor activity and that there are marked species differences in the response to hADM(16–31). q 1997 Elsevier Science Inc. Adrenomedullin
hADM(16–31)
a-Receptor activation
ADRENOMEDULLIN (ADM) is a hypotensive peptide first isolated from human pheochromocytoma cells (7). Human ADM (hADM) consists of 52 amino acids with a disulfide bond between cysteine residues in positions 16 and 21 that forms a six-membered ring structure similar to the ring structure found in calcitonin gene-related peptide (CGRP) and pancreatic amylin (7). ADM is present in the adrenal medulla, lung, and kidney; and plasma levels of ADM are increased in disease states, such as congestive heart failure, hypertension, and renal failure (4– 6). This peptide has been shown to possess vasodilator activity in a number of regional vascular beds in the cat, dog, and rat (1,3,8–11). Recent studies have shown that ADM ( 15 – 52 ) , an N-terminal truncated form of ADM that possesses the six-membered ring structure, retains the hypotensive and vasodilator activity of the full-sequence peptide ( 1,10,11 ) . These reports suggest that the N-terminal amino acids located before the ring structure are not necessary for the full expression of vasodilator activity ( 1,10,11 ) . Fragments of ADM, such as hADM ( 22 – 52 ) , which do not possess the ring structure, do not possess vasodilator activity ( 1,2,10 ) . It has recently been reported that N-terminal fragments of ADM, which contain the ring structure ADM ( 1 – 25 ) and ADM ( 16 – 21 ) , increase systemic arterial pressure in the rat ( 12 ) .
Vasopressor
Species differences
Whereas responses to ADM(1–25) and ADM(16–21) have been studied in the rat, little if anything is known about responses to ADM(16–31) in the systemic vascular bed. The purpose of the present study, therefore, was to investigate responses to this newly synthesized hADM analogue, which has a longer amino acid sequence after the ring structure than does ADM(1–25) or ADM(16–21), in the systemic vascular bed of the rat and cat and to determine the mechanism by which this peptide fragment increases systemic arterial pressure. METHOD
Seventeen Sprague–Dawley rats of either sex weighing 360– 520 g were anesthetized with pentobarbital sodium (50 mg/kg, IP). Supplemental doses of pentobarbital were given as needed to maintain a uniform level of anesthesia. The trachea was cannulated, and the rats breathed room air or were ventilated with a Harvard model 683 rodent ventilator at a tidal volume of 2.4– 2.6 ml at a rate of 30–35 breaths/min. Catheters were inserted into the external jugular vein for the IV administration of drugs and into the carotid artery for the measurement of systemic arterial (aortic) pressure. Systemic arterial pressure was measured with a Statham P23 pressure transducer and was recorded on a Grass model 7 polygraph, and mean pressure was derived by electronic averaging.
1 Requests for reprints should be addressed to Philip J. Kadowitz, Ph.D., Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112.
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Four adult cats weighing 2.3–4.4 kg were sedated with ketamine hydrochloride (10–15 mg/kg, IM) and were anesthetized with pentobarbital sodium (30 mg/kg, IV). Supplemental doses of pentobarbital were given during the course of the experiment to ensure a uniform level of anesthesia. Experimental procedures were identical to the rat, except the cats were ventilated with a Harvard model 607 ventilator at a volume of 40–60 ml at 15– 22 breaths/min. The temperature of the animals was maintained at 377C with a heating blanket. In the first series of experiments, responses to IV injection of ADM(16–31) were compared in the systemic vascular bed of the rat and cat. In the second series of experiments, responses to IV injections of ADM(16–31) in the rat were compared with responses to norepinephrine (NE) when doses are expressed on a nanomole basis to take molecular weight of the pressor agents into account. In the third series of experiments, the role of the adrenergic nervous system was investigated, and responses to ADM(16– 31) were compared before and after administration of the aadrenergic receptor antagonist, phentolamine, in a dose of 1 mg/ kg, IV. Phentolamine decreased systemic arterial pressure from 118 { 6 to 109 { 8. The extent of a receptor blockade was assessed by comparing responses to NE before and after treatment with the blocking agent. In a separate series of experiments, the effects of adrenergic nerve terminal blockade on pressor responses to ADM(15–31) were investigated 20–24 h after administration of reserpine in a dose of 1.5 mg/kg, IP. The extent of adrenergic neuronal blockade was assessed by comparing responses to the indirect-acting agonist, tyramine, in reserpine-pretreated and control animals. Human synthetic adrenomedullin [hADM(16–31); H2NCys-Arg-Phe-Gly-Thr-Cys-Thr-Val-Gln-Lys-Leu-Ala-His-GlnIle-Tyr-COOHNH2 ] (Peptide Research Labs, Tulane Medical School, New Orleans, LA) was dissolved in 0.9% NaCl. Norepinephrine hydrochloride, tyramine hydrochloride, angiotensin II (Sigma Chemical Co., St. Louis, MO), and reserpine phosphate (Serpasilw ) and phentolamine mesylate (CIBA–GEIGY Corp., Summit, NJ) were dissolved in 0.9% NaCl. Injections of agonists were administered in a random order. Drug solutions were prepared on a frequent basis and kept on crushed ice. All responses were analyzed using a one-way analysis of variance (ANOVA) and Scheffe’s F-test or a paired t-test. A value of p õ 0.05 was used as the criterion for statistical significance. RESULTS
Responses to hADM(16–31) In the rat, injections of hADM(16–31) in doses of 10–300 nmol/kg IV produced dose-dependent increases in systemic arterial pressure, and these data are summarized in Fig. 1(A). However, in the cat, injections of hADM(16–31) in doses up to 1000 nmol/kg IV produced no significant change in systemic arterial pressure [Fig. 1(B)]. The increases in systemic arterial pressure in response to hADM(16–31) and NE were compared in the rat, and these data are summarized in Fig. 1(C). NE was approximately 10-fold more potent than hADM(16–31) in increasing systemic arterial pressure in the rat when doses are compared on a nanomole basis to take molecular weight of the compounds into account [Fig. 1(C)].
FIG. 1. (A) Bar graphs showing the increases in systemic arterial pressure in response to IV injection of human adrenomedullin (hADM)(16– 31) in the rat. (B) Bar graph showing the effects of IV injection of hADM(16–31) on systemic arterial pressure in the cat. (C) Dose–response curves comparing the increase in systemic arterial pressure in the rat in response to hADM(16–31) and norepinephrine (NE). n indicates number of animals.
Following treatment with the a-receptor blocking agent phentolamine in a dose of 1 mg/kg IV, the increases in systemic arterial pressure in response to hADM(16–31) were significantly reduced [Fig. 2(A)]. The increases in systemic arterial pressure in response to NE in the same animals were significantly decreased after administration of the a-receptor blocking agent [Fig. 2(A)], whereas responses to angiotensin II were not significantly altered (data not shown). The role of catecholamine release in mediating increases in systemic arterial pressure in response to hADM(16–31) in the rat was investigated, and these data are summarized in Fig. 2(B). Following treatment with the adrenergic nerve terminal-depleting agent reserpine in a dose of 1.5 mg/kg IP, pressor responses to hADM(16–31) were significantly reduced [Fig. 2(B)]. Following treatment with reserpine, increases in systemic arterial pressure in response to the indirectacting agonist, tyramine, were significantly reduced [Fig. 2(B)], and responses to NE were significantly greater than control (data not shown).
Role of the Adrenergic Nervous System The role of a-receptor activation in mediating increases in systemic arterial pressure in response to hADM(16–31) was investigated in the rat, and these data are summarized in Fig. 2.
DISCUSSION
The results of the present investigation demonstrate that hADM(16–31) increases systemic arterial pressure in the rat. However, when injected in doses up to 1000 nmol/kg IV,
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PRESSOR RESPONSES TO ADRENOMEDULLIN(16–31)
FIG. 2. (A) Influence of the a-receptor blocking agent, phentolamine, in a dose of 1 mg/kg IV on increases in systemic arterial pressure in response to hADM(16–31) and norepinephrine (NE) in the rat. (B) Influence of the adrenergic neuronal blocking agent, reserpine, in a dose of 1.5 mg/kg IP on pressor responses to hADM(16–31) and tyramine in the rat. n indicates number of animals, and the asterisk indicates that the response is significantly different (p õ 0.05).
hADM(16–31) produced no measurable change in systemic arterial pressure in the cat. These data indicate that there is a marked species difference in the effect of this ADM fragment on systemic arterial pressure in the cat and rat. In terms of relative pressor activity, hADM(16–31) was 10-fold less potent than NE, suggesting that it has considerable pressor activity in the systemic vascular bed of the rat. The increase in systemic arterial pressure in response to hADM(16–31) was attenuated by the aadrenergic receptor agent phentolamine and by the adrenergic nerve terminal-blocking agent reserpine. These data suggest that the increases in systemic arterial pressure in response to hADM(16–31) are mediated by the release of catecholamines and the activation of a-adrenergic receptors. However, the site
135 from which hADM(16–31) releases catecholamine is uncertain and further experiments are needed to define the site of catecholamine release. Although the full sequence of the ADM peptide has been shown to possess vasodilator activity in the systemic vascular bed of the rat and in a number of regional vascular beds in the cat, dog, and rat (1,3,8,11), an ADM fragment containing residues 16 to 31, hADM(16–31), was shown to possess pressor activity. Data from the present study are in agreement with a previous study in which hADM(16–21), the ring portion of the peptide, was shown to possess pressor activity in the systemic vascular bed of the rat. The data with these shortened analogues of hADM, [hADM(16–21) and hADM(16–31)], provide support for the concept that hADM fragments containing the ring structure have novel pressor activity in the rat (12). The observation that ADM(16–31) and ADM(16–21) seem to have similar pressor activity in the rat suggests that amino acid residues 22 to 31 do not contribute to the pressor response. Whereas hADM(16–31) has novel, indirectly mediated, pressor activity in the rat, the peptide has little or no effect on systemic arterial pressure in the cat when injected in doses up to 1000 nmol/kg IV. The results of experiments in the cat and rat show that there are marked differences in regard to the effects of hADM(16– 31) on systemic arterial pressure and may suggest the absence of the mechanism that mediates the pressor response to hADM(16–31) in the cat. It has been previously reported that there exists interesting species differences with regard to the mechanism by which the full-sequence peptide, hADM(1–52), induces vasodilation (9). Inhibition of nitric oxide synthase reduces vasodilator responses to hADM in the pulmonary and hindquarters vascular beds of the rat and in the renal vascular bed of the dog (3,8,9). However, vasodilator responses to hADM are not affected by nitric oxide synthase inhibitors in the feline pulmonary circulation (9) and the hindlimb vascular bed of the cat (unpublished observations, 1996). The results of the present study extend the concept that responses to ADM and analogs differ with species. The observation that N-terminal truncated forms of ADM have novel pressor activity in the rat is interesting from a structure–activity relationship point of view. However, the physiological implications of these findings are uncertain, because it is not known if these peptide fragments are formed in the body. In summary, the hADM fragment, hADM(16–31), possesses pressor activity in the systemic vascular bed of the rat, but not the cat. The pressor response to hADM(16–31) is 10-fold less potent than NE when doses were compared on a nanomole basis, and the response to hADM(16–31) is mediated by the release of catecholamine and the activation of a-adrenergic receptors. These data indicate that amino acid residues 16–31 may be required for the expression of pressor activity in the rat. ACKNOWLEDGEMENTS
The authors thank Ms. Janice Ignarro for editorial assistance. The studies were supported by NIH grants HL15580 and HL09474 and a grant from the American Heart Association-Louisiana, Inc. Hunter C. Champion was supported by NIH grant HL09474.
REFERENCES 1. Cheng, D. Y.; DeWitt, B. J.; Wegmann, M. J.; Coy, D. H.; Bitar, K.; Murphy, W. A.; Kadowitz, P. J. Synthetic human adrenomedullin and ADM(15–52) have potent short-lasting vasodilator activity in the pulmonary vascular bed of the cat. Life Sci. 55:PL-251–PL256; 1994.
2. Eguchi, S.; Hirata, Y.; Iwasaki, H.; Sato, K.; Watanabe, T. X.; Inui, T.; Nakajima, K.; Sakakibara, S.; Marumo, F. Structure–activity relationship of adrenomedullin, a novel vasodilator peptide, in cultured rat vascular smooth muscle cells. Endocrinology 135:2454–2458; 1994.
/ av1 2x10 2287 Mp 135 Tuesday Mar 18 10:50 PM EL–PEP (v.18#1) 2287
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3. Feng, C. J.; Kang, B.; Kaye, A. D.; Kadowitz, P. J.; Nossaman, B. D. L-NAME modulates responses to adrenomedullin in the hindquarters vascular bed of the rat. Life Sci. 55:PL-433 – PL438; 1994. 4. Ichiki, Y.; Kitamura, K.; Kangawa, K.; Kawamoto, M.; Matsuo, H.; Eto, T. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338:6 – 10; 1994. 5. Ishimitsu, T.; Nishikimi, T.; Saito, Y.; Kitamura, K.; Eto, T.; Kangawa, K.; Matsuo, H.; Omae, T.; Matsuoka, H. Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure. J. Clin. Invest. 94:2158–2161; 1994. 6. Jougasaki, M.; Wei, C.; McKinley, L. J.; Burnett, J. C. Elevation of circulating and ventricular adrenomedullin in human congestive heart failure. Circulation 92:286–289; 1995. 7. Kitamura, K.; Kangawa, K.; Kawamoto, M.; Ichiki, Y.; Nakamura, S.; Matsuo, H.; Eto, T. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192:553–560; 1993.
8. Majid, D. S. A.; Kadowitz, P. J.; Coy, D. H.; Navar, L. G. Renal responses to intra-arterial administration of adrenomedullin in dogs. Am. J. Physiol. 270:F200–F205; 1996. 9. Nossaman, D. B.; Feng, C. J.; Kaye, A. D.; DeWitt, B. J.; Coy, D. H.; Murphy, W. A.; Kadowitz, P. J. Pulmonary vasodilator responses to adrenomedullin are reduced by nitric oxide synthase inhibitors in the rat but not in the cat. Am. J. Physiol. 270:L782–L789; 1996. 10. Santiago, J. A.; Garrison, E. A.; Purnell, W. L.; Smith, R. A.; Champion, H. C.; Coy, D. H.; Murphy, W. A.; Kadowitz, P. J. Comparison of responses to adrenomedullin and adrenomedullin analogs in the mesenteric vascular bed of the cat. Eur. J. Pharmacol. 272:115–118; 1995. 11. Santiago, J. A.; Garrison, E. A.; Ventura, V. L.; Coy, D. H.; Bitar, K.; Murphy, W. A.; McNamara, D. B.; Kadowitz, P. J. Synthetic human adrenomedullin and adrenomedullin 15–52 have potent short-lived vasodilator activity in the hindlimb vascular bed of the cat. Life Sci. 55:PL-85–PL-90; 1994. 12. Watanabe, T. X.; Itahara, Y.; Inui, T.; Yoshizawa–Kumagaye, K.; Nakajima, K.; Sakakibara, S. Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rat. Biochem. Biophys. Res. Commun. 219:59–63; 1996.
/ av1 2x10 2287 Mp 136 Tuesday Mar 18 10:51 PM EL–PEP (v.18#1) 2287