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Enhanced Depressor and Hyperemic Responses to Calcitonin Gene-related Peptide in Spontaneously Hypertensive Rats
Peptides, Vol. 19, No. 4, pp. 697–701, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(98)00009-6
Enhanced Depressor and Hyperemic Responses to Calcitonin Gene-related Peptide in Spontaneously Hypertensive Rats MASAMI YAMADA, TOMOHISA ISHIKAWA, AKIRA FUJIMORI, TAKASHI MIYAUCHI AND KATSUTOSHI GOTO1 Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 31 October 1997; Accepted 8 January 1998 YAMADA, M., T. ISHIKAWA, A. FUJIMORI, T. MIYAUCHI AND K. GOTO. Enhanced depressor and hyperemic responses to calcitonin gene-related peptide in spontaneously hypertensive rats. PEPTIDES 19(4) 697–701, 1998.—Intravenous (IV) bolus injection of calcitonin gene-related peptide (CGRP) caused a depressor response, which was significantly larger in 12-week-old spontaneously hypertensive rats (SHR) than in Wistar–Kyoto rats (WKY). CGRP also caused decreases in carotid and hindquarter vascular resistance, the magnitude of which was larger in the carotid than the hindquarter. In both regions, the vasodilator response to CGRP was significantly larger in SHR than WKY. Plasma CGRP level was significantly lower in SHR than WKY. These results suggest that depressor and vasodilator responses to CGRP are enhanced in SHR and that decreased plasma CGRP level in SHR may contribute to the enhanced responses. © 1998 Elsevier Science Inc. Calcitonin gene-related peptide Regional vascular resistance
Spontaneously hypertensive rat Plasma concentration
Blood pressure
Vasodilation
old SHR than age-matched normotensive Wistar–Kyoto rats (WKY; Reference 10). It has also been shown that in the basilar and pial arteries, CGRP-induced vasodilation is enhanced in SHR of hypertensive age as compared with agematched WKY (9,14). The systemic administration of CGRP results in a lowering of blood pressure and a decrease of regional vascular resistance in normotensive rats (5,8). However, it has not been elucidated whether the CGRPinduced depressor and hyperemic responses are altered in the hypertensive state. The present study was designed to obtain more information about the involvement of CGRP in essential hypertension. We carried out comparative studies in 6-week-old (normotensive) and 12-week-old (hypertensive) SHR and age-matched normotensive WKY, investigating the in vivo effects of CGRP on blood pressure and regional vascular resistance and measuring plasma concentration of CGRP in both strains of rat.
CALCITONIN gene-related peptide (CGRP) is widely distributed in the perivascular nerves and has potent vasodilator effects (2,13). CGRP causes vasodilation via stimulation of specific receptors on vascular smooth muscle cells in many species (1,3), and in several vessels such as rat aorta, the response is endothelium dependent (2). We reported previously that CGRP is released from the perivascular sensory nerves by electrical nerve stimulation and causes vasodilation in the rat mesenteric vascular bed, suggesting that CGRP-ergic nerves are involved in the regulation of vascular tonus as a vasodilator neurotransmitter (7,11). Several reports have shown that responses to CGRP are altered in spontaneously hypertensive rats (SHR), which are used as a model of human essential hypertension. In isolated perfused mesenteric vascular bed, the vasodilator response to CGRP has been shown to be larger in 15- and 30-week1
Requests for reprints should be addressed to Katsutoshi Goto, Ph.D., Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
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698
METHOD Male SHR and WKY (6 and 12 weeks of age; 18 rats each) were purchased from Charles River Japan Inc. (Kanagawa, Japan). Before the following experiments, systolic blood pressure and heart rate were determined in conscious rats with a tail-cuff sphygmomanometer (Riken Kaihatsu PS100, Kanagawa, Japan). Measurement of Blood Pressure, Heart Rate, and Regional Vascular Resistance Blood pressure, heart rate, and regional vascular resistance (RVR) were measured as described previously (19). Briefly, after each rat was anesthetized with urethane [1.5 g/kg, intraperitoneally (IP)], systemic blood pressure, mean blood pressure (MBP), and heart rate were monitored continuously via a heparinized saline (100 U/ml)filled catheter that was inserted in the right common carotid artery, and regional blood flow (RBF) of the left common carotid and femoral arteries was measured with a pulsed Doppler velocimeter (Crystal Biotech PD-20, Holliston, MA). RVR was evaluated as RBF/MBP. After surgical preparation, each animal was left for at least 20 min. Human a-CGRP (Peptide Institute, Osaka, Japan) was injected as a bolus via a catheter inserted in the right femoral vein. Measurement of Plasma CGRP Level by Radioimmunoassay Each rat was anesthetized with pentobarbital sodium (50 mg/kg IP). A catheter was inserted in the left common carotid artery, and 1.0 ml of arterial blood was collected. The collected arterial blood was mixed with aprotinin (300 IU/ml; Bayer, Germany) and EDTA-2Na (2 mg/ml; Wako Chemicals, Osaka, Japan), and centrifuged immediately (2000 3 g, 15 min). The obtained plasma was stored at -80°C until use. Plasma CGRP level was determined by radioimmunoassay according to the methods described previously (7). Briefly, the samples were preincubated with rabbit anti-human CGRP-II serum (Peninsula, Belmont, CA) at 4°C for 12 h. Then, the reaction mixture was incubated with [125I]Tyr0-rat CGRP for additional 48 h at 4°C. The antibody-bound antigen was separated from free antigens by the method of double antibody precipitation. The antibody cross-reacted completely with rat CGRP, and the standard curve was obtained using 0.625 to 640 fmol/tube of rat a-CGRP. Statistics Data are presented as mean 6 SEM. Analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was used to assess the statistical significance of the difference. A probability of p , 0.05 was accepted as the level of statistical significance.
YAMADA ET AL. TABLE 1 MEAN VALUES OF BLOOD PRESSURE AND HEART RATE OF RATS USED IN THIS STUDY
Conscious (6-week-old) SHR WKY Conscious (12-week-old) SHR WKY
Anesthetized (6-week-old) SHR WKY Anesthetized (12-week-old) SHR WKY
Systolic BP (mmHg)
HR (beats/min)
n
125 6 4.4 122 6 3.4
455 6 18 450 6 22
18 18
182 6 4.0* 135 6 3.1
380 6 21 376 6 20
18 18
Mean BP (mmHg)
HR (beats/min)
n
91 6 4.6 83 6 4.0
355 6 23 361 6 20
8 8
116 6 2.4* 88 6 2.1
353 6 13 350 6 14
8 8
Values represent mean 6 SEM. BP, blood pressure; HR, heart rate; n, number of rats. * P , 0.05 compared with age-matched WKY.
RESULTS Blood Pressure and Heart Rate in SHR and WKY In the conscious state, systolic blood pressure was significantly higher in 12-week-old SHR than age-matched WKY (Table 1), whereas there was no difference between 6-week-old SHR and WKY, indicating that 12week-old SHR had developed a hypertensive state. Heart rate was not different between SHR and WKY at both ages (Table 1). Similar results were obtained from rats anesthetized with urethane, in which MBP and heart rate were measured through a catheter in the common carotid artery (Table 1). Effects of CGRP on MBP, Heart Rate, and RBF IV bolus injection of CGRP decreased MBP in both strains of rats. The depressor response was observed immediately after the injection of CGRP and reached a peak within approximately 30 s; MBP remained below the basal level for more than 20 min. Figure 1A shows the dose–response relationship for the depressor response to CGRP. CGRP decreased MBP in a dose-dependent manner in both SHR and WKY. Percent changes in MBP in response to each dose of CGRP were significantly larger in 12-week-old SHR than age-matched WKY, whereas there was no difference between 6-week-old SHR and WKY. Heart rate was increased by IV bolus injection of CGRP in both strains of rat. As shown in Figure 1B, there was no difference in CGRP-induced changes in heart rate between SHR and WKY at either age. The evaluated resting values of carotid and femoral vas-
ENHANCED RESPONSE TO CGRP IN SHR
FIG. 1. Percent changes in mean blood pressure and heart rate in response to CGRP in anesthetized SHR and WKY at 6 and 12 weeks of age. Intravenous injection of CGRP decreased mean blood pressure (A and B) and increased heart rate (C and D) in a dose-dependent manner in SHR (F) and WKY (E) at 6 (A and C) and 12 weeks of age (B and D). Values represent the mean 6 SEM for eight experiments. **p , 0.01 compared with age-matched WKY.
FIG. 2. Percent changes in regional vascular resistance in response to CGRP in anesthetized SHR and WKY rats at 12 weeks of age. Intravenous injection of CGRP decreased regional vascular resistance of common carotid (A) and femoral (B) vascular beds in a dose-dependent manner in 12-week-old SHR (F) and WKY (E). Values represent mean 6 SEM for eight experiments. *p , 0.05 compared with WKY.
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FIG. 3. Plasma level of CGRP-like immunoreactivity in SHR and WKY. The plasma level of CGRP-like immunoreactivity (CGRPLI) was not different between 6-week-old SHR (closed column) and WKY (open column), whereas it was significantly lower in 12-week-old SHR (closed column) than in age-matched WKY (open column). Values represent the mean 6 SEM for 10 experiments. *p , 0.05 compared with age-matched WKY.
cular resistance were significantly higher in 12-week-old SHR (256 6 24 and 339 6 44 mmHg/ml/min, respectively) than age-matched WKY (146 6 9 and 197 6 16 mmHg/ ml/min, respectively). As illustrated in Figure 2, IV injection of CGRP significantly decreased RVR in both 12week-old SHR and WKY in a dose-dependent manner. The magnitude of response was larger in carotid than hindquarter. In both regions, the hyperemic response to CGRP was significantly larger in SHR than WKY. Plasma Level of CGRP The plasma level of CGRP-like immunoreactivity (CGRPLI) was significantly lower in 12-week-old SHR than agematched WKY, whereas there was no difference between 6-week-old SHR and WKY (Figure 3). DISCUSSION This paper reports comparative studies carried out in 6- and 12-week-old SHR and WKY on the effects of CGRP in vivo. It has been reported that CGRP causes depressor and vasodilator responses in SHR in vivo (12,18). However, since the effects of CGRP were examined only in aged SHR with established hypertension in those studies, whether the in vivo effects of CGRP contribute to the development of hypertension has not been elucidated. In the present study, we used two different ages of rats; 6-week-old SHR in a prehypertensive state and 12-week-old SHR with established hypertension and compared them with age-matched normotensive WKY. The data showed that IV injection of CGRP caused a dose-dependent depressor response in both
YAMADA ET AL.
strains and ages of rats. The magnitude of both the depressor and hyperemic responses to CGRP was significantly larger in 12-week-old SHR than age-matched WKY, whereas there was no difference between 6-week-old SHR and WKY. In 12-week-old SHR, the depressor response was accompanied by significant reductions in both carotid and hindquarter vascular resistance, the magnitude of which was also larger than in age-matched WKY. These results suggest that vascular responsiveness to CGRP is altered with the development of hypertension and that enhanced peripheral vasodilation induced by CGRP may be responsible for the enhanced depressor response to CGRP in SHR of hypertensive age. It has been reported that plasma CGRP-LI is higher in SHR of hypertensive age (7 weeks) than age-matched WKY (20). Conversely, Xu et al. (18) have shown that plasma CGRP-LI is lower in SHR than WKY at 10 –12 weeks of age. The present study showed that in SHR of prehypertensive age (6 weeks), plasma CGRP-LI level was not different from that in age-matched WKY, whereas it was significantly lower in 12-week-old SHR with established hypertension than in age-matched WKY. Kawasaki et al. (10) have shown that in perfused mesenteric vascular beds isolated from 15- and 30-week-old SHR but not from 8-weekold SHR, the vasodilation induced by perivascular nerve stimulation, which is mediated by the release of CGRP from perivascular nerves, is significantly smaller than in agematched WKY controls. Their study demonstrated that MBP in 8-week-old SHR is significantly higher than that in age-matched WKY but is significantly lower than that in 15and 30-week-old SHR. They have also shown that the content of CGRP-LI in perivascular nerves is significantly lower in SHR than in WKY at 30 weeks of age, but not at 8 weeks of age. Since circulating CGRP is of neuronal origin (6), the decrease in the content of CGRP-LI in perivascular nerves is considered to be responsible for the lowered plasma level of CGRP in aged SHR with established hypertension. The content of CGRP in perivascular nerves might be unchanged at an early stage of hypertension such as in 7-week-old SHR. It is expected that the concentration of CGRP might be elevated in the plasma of 7-weekold SHR to compensate for the excessive vasoconstrictor activity associated with hypertension. Supowit et al. (17) have shown that neuronal content of CGRP mRNA is significantly decreased in the dorsal root ganglia of 12- to 14-week-old SHR as compared with age-matched WKY. Taken together, it is suggested that the production of CGRP declines with the development of hypertension in SHR, which results in decreased plasma CGRP level in SHR with established hypertension. A number of studies have shown decreased responses to vasodilators such as isoproterenol, nitroprusside, and acetylcholine in a variety of hypertensive animals, including SHR (4,15,16). In contrast to these agents, the depressor and
ENHANCED RESPONSE TO CGRP IN SHR
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hyperemic responses to exogenously applied CGRP were enhanced in 12-week-old SHR as compared with agematched WKY, while such differences did not exist between 6-week-old SHR and WKY. In parallel with these changes, the plasma concentration of CGRP-LI was lowered in 12-week-old SHR as compared with age-matched WKY and 6-week-old SHR. These results support the hypothesis that CGRP-ergic vasodilator innervation is greatly decreased in SHR with established hypertension (10); the decreased plasma CGRP level could be explained by the decreased release of CGRP, and the increased vasodilator
responses to exogenously applied CGRP in SHR could be explained by up-regulation of CGRP receptors, which both would result from impaired CGRP-ergic innervation. These findings suggest that impaired CGRP-ergic innervation in SHR with established hypertension may contribute to the maintenance of hypertension. ACKNOWLEDGEMENTS This study was supported by a Grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and Uehara Memorial Foundation. We would like to thank Dr. W. A. Gray for language editing.
REFERENCES 1. Amerini, S.; Mantelli, L.; Ledda, F. Nitric oxide is not involved in the effects induced by non-adrenergic non-cholinergic stimulation and calcitonin gene-related peptide in the rat mesenteric vascular bed. Neuropeptides. 25:51–56; 1993. 2. Bell, D.; McDermott, B. J. Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor population and their (patho)physiological significance. Pharmacol. Rev. 48:253–288; 1996. 3. Champion, H. C.; Santiago, J. A.; Murphy, W. A.; Coy, D. H.; Kadowitz, P. J. Adrenomedullin-(22–52) antagonizes vasodilator responses to CGRP but not adrenomedullin in the cat. Am. J. Physiol. 272:R234 –R242; 1997. 4. Cohen, M. L.; Berkowitz, B. A. Decreased vascular relaxation in hypertension. J. Pharmacol. Exp. Ther. 196:396 – 406; 1976. 5. DiPette, D. J.; Schwarzenberger, K.; Kerr, N.; Holland, O. B. Dose-dependent systemic and regional hemodynamic effects of calcitonin gene-related peptide. Am. J. Med. Sci. 297:65– 70; 1989. 6. Emson, P. C.; Zaidi, M. Further evidence for the origin of circulating calcitonin gene-related peptide in the rat. J. Physiol. 412:297–308; 1989. 7. Fujimori, A.; Saito, A.; Kimura, S.; Watanabe, T.; Uchiyama, Y.; Kawasaki, H.; Goto, K. Neurogenic vasodilation and release of calcitonin gene-related peptide (CGRP) from perivascular nerves in the rat mesenteric artery. Biochem. Biophys. Res. Commun. 165:1391–1398; 1989. 8. Gardiner, S. M.; Compton, A. M.; Bennett, T. Regional hemodynamic effects of calcitonin gene-related peptide. Am. J. Physiol. 256:R332–R338; 1989. 9. Hong, K. W.; Yu, S. S.; Shin, Y. W.; Kim, C. D.; Rhim, B. Y.; Lee, W. S. Decreased CGRP level with increased sensitivity to CGRP in the pial arteries of spontaneously hypertensive rats. Life Sci. 60:697–705; 1997. 10. Kawasaki, H.; Saito, A.; Takasaki, K. Age-related decrease of calcitonin gene-related peptide-containing vasodilator innervation in the mesenteric resistance vessel of the spontaneously hypertensive rat. Circ. Res. 67:733–743; 1990.
11. Kawasaki, H.; Takasaki, K.; Saito, A.; Goto, K. Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature (London). 335:164 –167; 1988. 12. Lappe, R. W.; Todt, J. A.; Wendt, R. L. Regional vasodilator actions of calcitonin gene-related peptide in conscious SHR. Peptides. 8:747–749; 1987. 13. Lundberg, J. M. Pharmacology of cotransmission in the autonomic nervous system: integrative aspects on amines, neuropeptides, adenosine triphosphate, amino acids and nitric oxide. Pharmacol. Rev. 48:113–178; 1996. 14. Nishimura, Y.; Usui, H.; Suzuki, A.; Kajimoto, N.; Yamanishi, Y. Relaxant response of isolated basilar arteries to calcitonin gene-related peptide in stroke-prone spontaneously hypertensive rats. Jpn. J. Pharmacol. 59:333–338; 1992. 15. Shirasaki, Y.; Kolm, P.; Nickols, G. A.; Lee, T. J-F. Endothelium regulation of cyclic GMP and vascular responses in hypertension. J. Pharmacol. Exp. Ther. 245:53–58; 1988. 16. Shirasaki, Y.; Su, C. Endothelium removal augments vasodilation by sodium nitroprusside and sodium nitrate. Eur. J. Pharmacol. 114:93–96, 1985. 17. Supowit, S. C.; Ramana, C. V.; Westlund, K. N.; DiPette, D. J. Calcitonin gene-related peptide gene expression in the spontaneously hypertensive rat. Hypertension. 21:1010 –1014; 1993. 18. Xu, D.; Wang, X.; Wang, J.-P.; Yuan, Q.-X.; Fiscus, R. R.; Chang, J.-K.; Tang, J. Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats. Peptides. 10:309 –312; 1989. 19. Yamada, M.; Ishikawa, T.; Fujimori, A.; Goto, K. Local neurogenic regulation of rat hindlimb circulation: role of calcitonin gene-related peptide in vasodilatation after skeletal muscle contraction. Br. J. Pharmacol. 121:704 –709; 1997. 20. Zaidi, M.; Bevis, P. J. R. Enhanced circulating levels of neurally derived calcitonin gene related peptide in spontaneously hypertensive rats. Cardiovasc. Res. 25:125–128; 1991.
PII S0196-9781(98)00009-6
Enhanced Depressor and Hyperemic Responses to Calcitonin Gene-related Peptide in Spontaneously Hypertensive Rats MASAMI YAMADA, TOMOHISA ISHIKAWA, AKIRA FUJIMORI, TAKASHI MIYAUCHI AND KATSUTOSHI GOTO1 Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Received 31 October 1997; Accepted 8 January 1998 YAMADA, M., T. ISHIKAWA, A. FUJIMORI, T. MIYAUCHI AND K. GOTO. Enhanced depressor and hyperemic responses to calcitonin gene-related peptide in spontaneously hypertensive rats. PEPTIDES 19(4) 697–701, 1998.—Intravenous (IV) bolus injection of calcitonin gene-related peptide (CGRP) caused a depressor response, which was significantly larger in 12-week-old spontaneously hypertensive rats (SHR) than in Wistar–Kyoto rats (WKY). CGRP also caused decreases in carotid and hindquarter vascular resistance, the magnitude of which was larger in the carotid than the hindquarter. In both regions, the vasodilator response to CGRP was significantly larger in SHR than WKY. Plasma CGRP level was significantly lower in SHR than WKY. These results suggest that depressor and vasodilator responses to CGRP are enhanced in SHR and that decreased plasma CGRP level in SHR may contribute to the enhanced responses. © 1998 Elsevier Science Inc. Calcitonin gene-related peptide Regional vascular resistance
Spontaneously hypertensive rat Plasma concentration
Blood pressure
Vasodilation
old SHR than age-matched normotensive Wistar–Kyoto rats (WKY; Reference 10). It has also been shown that in the basilar and pial arteries, CGRP-induced vasodilation is enhanced in SHR of hypertensive age as compared with agematched WKY (9,14). The systemic administration of CGRP results in a lowering of blood pressure and a decrease of regional vascular resistance in normotensive rats (5,8). However, it has not been elucidated whether the CGRPinduced depressor and hyperemic responses are altered in the hypertensive state. The present study was designed to obtain more information about the involvement of CGRP in essential hypertension. We carried out comparative studies in 6-week-old (normotensive) and 12-week-old (hypertensive) SHR and age-matched normotensive WKY, investigating the in vivo effects of CGRP on blood pressure and regional vascular resistance and measuring plasma concentration of CGRP in both strains of rat.
CALCITONIN gene-related peptide (CGRP) is widely distributed in the perivascular nerves and has potent vasodilator effects (2,13). CGRP causes vasodilation via stimulation of specific receptors on vascular smooth muscle cells in many species (1,3), and in several vessels such as rat aorta, the response is endothelium dependent (2). We reported previously that CGRP is released from the perivascular sensory nerves by electrical nerve stimulation and causes vasodilation in the rat mesenteric vascular bed, suggesting that CGRP-ergic nerves are involved in the regulation of vascular tonus as a vasodilator neurotransmitter (7,11). Several reports have shown that responses to CGRP are altered in spontaneously hypertensive rats (SHR), which are used as a model of human essential hypertension. In isolated perfused mesenteric vascular bed, the vasodilator response to CGRP has been shown to be larger in 15- and 30-week1
Requests for reprints should be addressed to Katsutoshi Goto, Ph.D., Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
697
698
METHOD Male SHR and WKY (6 and 12 weeks of age; 18 rats each) were purchased from Charles River Japan Inc. (Kanagawa, Japan). Before the following experiments, systolic blood pressure and heart rate were determined in conscious rats with a tail-cuff sphygmomanometer (Riken Kaihatsu PS100, Kanagawa, Japan). Measurement of Blood Pressure, Heart Rate, and Regional Vascular Resistance Blood pressure, heart rate, and regional vascular resistance (RVR) were measured as described previously (19). Briefly, after each rat was anesthetized with urethane [1.5 g/kg, intraperitoneally (IP)], systemic blood pressure, mean blood pressure (MBP), and heart rate were monitored continuously via a heparinized saline (100 U/ml)filled catheter that was inserted in the right common carotid artery, and regional blood flow (RBF) of the left common carotid and femoral arteries was measured with a pulsed Doppler velocimeter (Crystal Biotech PD-20, Holliston, MA). RVR was evaluated as RBF/MBP. After surgical preparation, each animal was left for at least 20 min. Human a-CGRP (Peptide Institute, Osaka, Japan) was injected as a bolus via a catheter inserted in the right femoral vein. Measurement of Plasma CGRP Level by Radioimmunoassay Each rat was anesthetized with pentobarbital sodium (50 mg/kg IP). A catheter was inserted in the left common carotid artery, and 1.0 ml of arterial blood was collected. The collected arterial blood was mixed with aprotinin (300 IU/ml; Bayer, Germany) and EDTA-2Na (2 mg/ml; Wako Chemicals, Osaka, Japan), and centrifuged immediately (2000 3 g, 15 min). The obtained plasma was stored at -80°C until use. Plasma CGRP level was determined by radioimmunoassay according to the methods described previously (7). Briefly, the samples were preincubated with rabbit anti-human CGRP-II serum (Peninsula, Belmont, CA) at 4°C for 12 h. Then, the reaction mixture was incubated with [125I]Tyr0-rat CGRP for additional 48 h at 4°C. The antibody-bound antigen was separated from free antigens by the method of double antibody precipitation. The antibody cross-reacted completely with rat CGRP, and the standard curve was obtained using 0.625 to 640 fmol/tube of rat a-CGRP. Statistics Data are presented as mean 6 SEM. Analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was used to assess the statistical significance of the difference. A probability of p , 0.05 was accepted as the level of statistical significance.
YAMADA ET AL. TABLE 1 MEAN VALUES OF BLOOD PRESSURE AND HEART RATE OF RATS USED IN THIS STUDY
Conscious (6-week-old) SHR WKY Conscious (12-week-old) SHR WKY
Anesthetized (6-week-old) SHR WKY Anesthetized (12-week-old) SHR WKY
Systolic BP (mmHg)
HR (beats/min)
n
125 6 4.4 122 6 3.4
455 6 18 450 6 22
18 18
182 6 4.0* 135 6 3.1
380 6 21 376 6 20
18 18
Mean BP (mmHg)
HR (beats/min)
n
91 6 4.6 83 6 4.0
355 6 23 361 6 20
8 8
116 6 2.4* 88 6 2.1
353 6 13 350 6 14
8 8
Values represent mean 6 SEM. BP, blood pressure; HR, heart rate; n, number of rats. * P , 0.05 compared with age-matched WKY.
RESULTS Blood Pressure and Heart Rate in SHR and WKY In the conscious state, systolic blood pressure was significantly higher in 12-week-old SHR than age-matched WKY (Table 1), whereas there was no difference between 6-week-old SHR and WKY, indicating that 12week-old SHR had developed a hypertensive state. Heart rate was not different between SHR and WKY at both ages (Table 1). Similar results were obtained from rats anesthetized with urethane, in which MBP and heart rate were measured through a catheter in the common carotid artery (Table 1). Effects of CGRP on MBP, Heart Rate, and RBF IV bolus injection of CGRP decreased MBP in both strains of rats. The depressor response was observed immediately after the injection of CGRP and reached a peak within approximately 30 s; MBP remained below the basal level for more than 20 min. Figure 1A shows the dose–response relationship for the depressor response to CGRP. CGRP decreased MBP in a dose-dependent manner in both SHR and WKY. Percent changes in MBP in response to each dose of CGRP were significantly larger in 12-week-old SHR than age-matched WKY, whereas there was no difference between 6-week-old SHR and WKY. Heart rate was increased by IV bolus injection of CGRP in both strains of rat. As shown in Figure 1B, there was no difference in CGRP-induced changes in heart rate between SHR and WKY at either age. The evaluated resting values of carotid and femoral vas-
ENHANCED RESPONSE TO CGRP IN SHR
FIG. 1. Percent changes in mean blood pressure and heart rate in response to CGRP in anesthetized SHR and WKY at 6 and 12 weeks of age. Intravenous injection of CGRP decreased mean blood pressure (A and B) and increased heart rate (C and D) in a dose-dependent manner in SHR (F) and WKY (E) at 6 (A and C) and 12 weeks of age (B and D). Values represent the mean 6 SEM for eight experiments. **p , 0.01 compared with age-matched WKY.
FIG. 2. Percent changes in regional vascular resistance in response to CGRP in anesthetized SHR and WKY rats at 12 weeks of age. Intravenous injection of CGRP decreased regional vascular resistance of common carotid (A) and femoral (B) vascular beds in a dose-dependent manner in 12-week-old SHR (F) and WKY (E). Values represent mean 6 SEM for eight experiments. *p , 0.05 compared with WKY.
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FIG. 3. Plasma level of CGRP-like immunoreactivity in SHR and WKY. The plasma level of CGRP-like immunoreactivity (CGRPLI) was not different between 6-week-old SHR (closed column) and WKY (open column), whereas it was significantly lower in 12-week-old SHR (closed column) than in age-matched WKY (open column). Values represent the mean 6 SEM for 10 experiments. *p , 0.05 compared with age-matched WKY.
cular resistance were significantly higher in 12-week-old SHR (256 6 24 and 339 6 44 mmHg/ml/min, respectively) than age-matched WKY (146 6 9 and 197 6 16 mmHg/ ml/min, respectively). As illustrated in Figure 2, IV injection of CGRP significantly decreased RVR in both 12week-old SHR and WKY in a dose-dependent manner. The magnitude of response was larger in carotid than hindquarter. In both regions, the hyperemic response to CGRP was significantly larger in SHR than WKY. Plasma Level of CGRP The plasma level of CGRP-like immunoreactivity (CGRPLI) was significantly lower in 12-week-old SHR than agematched WKY, whereas there was no difference between 6-week-old SHR and WKY (Figure 3). DISCUSSION This paper reports comparative studies carried out in 6- and 12-week-old SHR and WKY on the effects of CGRP in vivo. It has been reported that CGRP causes depressor and vasodilator responses in SHR in vivo (12,18). However, since the effects of CGRP were examined only in aged SHR with established hypertension in those studies, whether the in vivo effects of CGRP contribute to the development of hypertension has not been elucidated. In the present study, we used two different ages of rats; 6-week-old SHR in a prehypertensive state and 12-week-old SHR with established hypertension and compared them with age-matched normotensive WKY. The data showed that IV injection of CGRP caused a dose-dependent depressor response in both
YAMADA ET AL.
strains and ages of rats. The magnitude of both the depressor and hyperemic responses to CGRP was significantly larger in 12-week-old SHR than age-matched WKY, whereas there was no difference between 6-week-old SHR and WKY. In 12-week-old SHR, the depressor response was accompanied by significant reductions in both carotid and hindquarter vascular resistance, the magnitude of which was also larger than in age-matched WKY. These results suggest that vascular responsiveness to CGRP is altered with the development of hypertension and that enhanced peripheral vasodilation induced by CGRP may be responsible for the enhanced depressor response to CGRP in SHR of hypertensive age. It has been reported that plasma CGRP-LI is higher in SHR of hypertensive age (7 weeks) than age-matched WKY (20). Conversely, Xu et al. (18) have shown that plasma CGRP-LI is lower in SHR than WKY at 10 –12 weeks of age. The present study showed that in SHR of prehypertensive age (6 weeks), plasma CGRP-LI level was not different from that in age-matched WKY, whereas it was significantly lower in 12-week-old SHR with established hypertension than in age-matched WKY. Kawasaki et al. (10) have shown that in perfused mesenteric vascular beds isolated from 15- and 30-week-old SHR but not from 8-weekold SHR, the vasodilation induced by perivascular nerve stimulation, which is mediated by the release of CGRP from perivascular nerves, is significantly smaller than in agematched WKY controls. Their study demonstrated that MBP in 8-week-old SHR is significantly higher than that in age-matched WKY but is significantly lower than that in 15and 30-week-old SHR. They have also shown that the content of CGRP-LI in perivascular nerves is significantly lower in SHR than in WKY at 30 weeks of age, but not at 8 weeks of age. Since circulating CGRP is of neuronal origin (6), the decrease in the content of CGRP-LI in perivascular nerves is considered to be responsible for the lowered plasma level of CGRP in aged SHR with established hypertension. The content of CGRP in perivascular nerves might be unchanged at an early stage of hypertension such as in 7-week-old SHR. It is expected that the concentration of CGRP might be elevated in the plasma of 7-weekold SHR to compensate for the excessive vasoconstrictor activity associated with hypertension. Supowit et al. (17) have shown that neuronal content of CGRP mRNA is significantly decreased in the dorsal root ganglia of 12- to 14-week-old SHR as compared with age-matched WKY. Taken together, it is suggested that the production of CGRP declines with the development of hypertension in SHR, which results in decreased plasma CGRP level in SHR with established hypertension. A number of studies have shown decreased responses to vasodilators such as isoproterenol, nitroprusside, and acetylcholine in a variety of hypertensive animals, including SHR (4,15,16). In contrast to these agents, the depressor and
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hyperemic responses to exogenously applied CGRP were enhanced in 12-week-old SHR as compared with agematched WKY, while such differences did not exist between 6-week-old SHR and WKY. In parallel with these changes, the plasma concentration of CGRP-LI was lowered in 12-week-old SHR as compared with age-matched WKY and 6-week-old SHR. These results support the hypothesis that CGRP-ergic vasodilator innervation is greatly decreased in SHR with established hypertension (10); the decreased plasma CGRP level could be explained by the decreased release of CGRP, and the increased vasodilator
responses to exogenously applied CGRP in SHR could be explained by up-regulation of CGRP receptors, which both would result from impaired CGRP-ergic innervation. These findings suggest that impaired CGRP-ergic innervation in SHR with established hypertension may contribute to the maintenance of hypertension. ACKNOWLEDGEMENTS This study was supported by a Grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and Uehara Memorial Foundation. We would like to thank Dr. W. A. Gray for language editing.
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