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Age-Related Decrease in Calcitonin Gene-Related Peptide mRNA in the Dorsal Root Ganglia of Spontaneously Hypertensive Rats
Jpn. J. Pharmacol. 86, 448 – 450 (2001)
Short Communication
Age-Related Decrease in Calcitonin Gene-Related Peptide mRNA in the Dorsal Root Ganglia of Spontaneously Hypertensive Rats Naka Yamaga, Hiromu Kawasaki*, Keiichi Inaizumi, Miho Shimizu, Arisa Nakamura and Yuji Kurosaki Department of Clinical Pharmaceutical Science, Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan Received April 6, 2001 Accepted May 30, 2001
ABSTRACT—Age-related changes in levels of calcitonin gene-related peptide (CGRP) mRNA of the dorsal root ganglia was studied in 8-, 12- and 15-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). CGRP mRNA levels in SHR but not in WKY decreased with age. The contents of CGRP-like immunoreactivities in the atrium and mesenteric artery of 15-week-old SHR were greater than those in age-matched WKY. These results suggest that the outflow of CGRP-containing nerves from the spinal cord and CGRP release from CGRP nerve terminals decreases in SHR. Keywords: Calcitonin gene-related peptide (CGRP) mRNA, Mesenteric arterial CGRP content, Spontaneously hypertensive rat
The rat mesenteric resistance arteries are innervated by nonadrenergic noncholinergic (NANC) vasodilator nerves in which calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, acts as the vasodilator neurotransmitter (1, 2). CGRP has been shown to distribute throughout the central and peripheral nervous system and locate in areas involved in cardiovascular function (3). A prominent site of CGRP synthesis is the dorsal root ganglia (DRG), which contains the cell bodies of primary afferent neurons that extend CGRP-containing nerves (CGRPergic nerves) to peripheral sites such as blood vessels and the central spinal cord (4). We demonstrated that the CGRPergic nerve function in spontaneously hypertensive rats (SHR) decreased with age (5). In a recent study, we showed that the levels of CGRP mRNA in DRG of 15-week-old SHR were lower than those in normotensive Wistar Kyoto rats (WKY) (6). Therefore, the present study was undertaken to clarify the age-related changes in level of CGRP mRNA in DRG of SHR and WKY and content of CGRP in the peripheral sites. Male SHR at 8, 12 or 15 weeks of age and age-matched WKY (Charles River Japan, Shizuoka) were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.). After the mean carotid blood pressure (MBP) was measured, the animals were deeply anesthetized with a large dose of sodium pentobarbital and killed by decapitation. The thoracic and
lumber DRG from each rat were immediately dissected. The isolated DRG was frozen in liquid nitrogen until subsequent analysis of CGRP mRNA as described previously (6). Total cellular RNA was prepared using the acid guanidium thyocyanate-phenol-chloroform method (7). The RNA samples were fractionated by electrophoresis on denaturing formaldehyde-agarose gels and transferred to a nylon membrane. Hybridization was done under stringent conditions (50% formamide at 42°C) with a 32P-labeled cDNA probe. Two probes for CGRP and > -actin were labeled, using a Multiprime DNA labeling system (Amersham Int., Buckinghamshire, UK) with 32P-dCTP. After hybridization, the membranes were exposed to X-ray films, and the radioactivity of each band was quantified by means of computerized densitometiric scanning (BAS 2000; Fuji Photo Film Co., Tokyo). All results are expressed relative to the >actin mRNA, serving as a control for the quality and quantity of RNA applied to the blot. The atrium and mesenteric artery isolated from 15-weekold SHR and WKY were homogenized and the supernatant solution was applied to a Bond-Elut C18 cartridge, and absorbed peptide was eluted with 60% acetonitrile in 1% trifluoroacetic acid. The elute was evaporated under vacuum and the contents of CGRP-like immunoreactivities (CGRP-LI) were determined using a commercially available enzyme-liked immunosorbent assay (ELISA) for CGRP kit (Peninsula Laboratories, Inc., San Carlos, CA, USA). All values are presented as the mean ± S.E.M. One-way analysis of variance followed by Tukey’s test was used to
*Corresponding author. FAX: +81-86-251-7970 E-mail: [email protected] 448
Short Communication
449
determine the significance between values of different ages. The unpaired Student’s t-test was used to determine the significance of differences between two means. A value of P<0.05 was considered significant. The MBP in 12-week-old SHR (188.5 ± 1.4 mmHg, n = 6, P<0.01) and 15-week-old SHR (193.0 ± 2.9 mmHg, n = 6, P<0.01) was significantly greater than that in 8week-old SHR (150.8 ± 3.2 mmHg, n = 6). The MBP of SHR at 8 (P<0.01), 12 (P<0.01) and 15 (P<0.01) weeks of age was significantly greater than that in age-matched WKY (8-week-old, 99.5 ± 1.8 mmHg, n = 4; 12-week-old, 108.0 ± 0.7 mmHg, n = 4; 15-week-old, 118.8 ± 7.8 mmHg, n = 4). Figure 1A shows a representative Northern blot analysis of CGRP mRNA. As shown in Fig. 1B, the level of CGRP
Fig. 2. CGRP-LI contents of the atrium and mesenteric artery in 15week-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Values show the mean ± S.E.M. from 4 experiments.
Fig. 1. Representative image (A) of Northern blot assays of CGRP mRNA and > -actin mRNA in the dorsal root ganglia (DRG) of spontaneously hypertensive rats (SHR) and bar graph (B) showing age-related changes in the ratio of CGRP mRNA / > -actin mRNA determined by densitometric analysis of Northern blot assays in DRG of Wistar Kyoto rats (WKY) and SHR. A: lanes 1 – 3, 4 – 6 and 7 – 9 indicate 8-, 12- and 15-week-old SHR, respectively. B: values represent the fold increase over controls (8-week-old). *P<0.05, **P<0.01, compared with 8-week-old SHR.
mRNA in SHR was decreased with age, whereas the level of CGRP mRNA in WKY was slightly increased with age. When the level of CGRP mRNA in SHR was compared with WKY as the ratio of CGRP mRNA / >-actin mRNA, a significant decrease was found in 12- and 15-week-old but not 8-week-old SHR, as shown in Fig. 1B. As shown in Fig. 2, the contents of CGRP-LI in the atrium and mesenteric artery in 15-week-old SHR were greater than those in age-matched WKY. A significant difference between SHR and WKY in CGRP-LI contents was found in the atrium (Fig. 2). The present findings demonstrated that the amount of CGRP mRNA in DRG of 12- and 15-week-old SHR decreased when compared with age-matched WKY. In addition, the decrease in the CGRP mRNA level of SHR was age-related, but the level in WKY did not alter. In in vivo studies, we previously reported that electrical stimulation of the lower thoracic spinal cord (Th9 – 12) in the pithed rat produced a frequency-dependent depressor response, which was abolished by the neurotoxin tetrodotoxin or by the CGRP-receptor antagonist CGRP(8 – 37), indicating that the response is mediated by endogenous CGRP released from the CGRPergic nerves (8). In a recent study, the depressor response to spinal cord stimulation in 15week-old pithed SHR was significantly smaller than that in age-matched WKY (6). Furthermore, the present study showed that the level of CGRP mRNA in DRG was decreased in SHR compared with WKY and the decrease was age-related. Taken together, it is very likely that the outflow of CGRPergic vasodilator nerves from the spinal cord to peripheral blood vessels via the dorsal roots decreases in SHR. In in vitro studies, we previously reported that CGRPergic nerve-mediated vasodilation in response to periarterial nerve stimulation of the mesenteric artery in SHR was smaller than that in WKY and the decrease in the response
450
Short Communication
was age-related (5). In addition, the neurogenic release of CGRP-LI in the mesenteric artery of 15-week-old SHR has been shown to be smaller than that in age-matched WKY (5). The present study demonstrated that the content of CGRP-LI in the mesenteric artery of SHR was greater than that in WKY. This finding suggests that the increased contents of CGRP-LI in SHR is due to reduced release of CGRP from CGRPergic nerve terminals, even though the levels of CGRP mRNA in SHR decreased. Since CGRPergic nerves have been shown to inhibit the function of adrenergic nerves (2), the decreased function of CGRPergic nerves in SHR enhances the sympathetic adrenergic vasoconstriction. A recent study demonstrated that =-CGRP/ calcitonin gene knockout mice displayed an elevated MBP compared with wild-type mice, indicating that =-CGRP is involved in the long-term regulation of resting blood pressure (9). Therefore, the reduced CGRPergic nerve function may have caused the increased vascular resistance and high blood pressure in SHR. In conclusion, the findings of the present study showed that the CGRP gene expression in DRG in SHR decreases with age. The present study suggests that the function of CGRPergic vasodilator nerves from the spinal cord decreases in SHR and this decrease contributes to maintenance of hypertension in SHR. Acknowledgments This study was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 09672326, No. 10557244).
REFERENCES 1 Kawasaki H, Takasaki K, Saito A and Goto K: Calcitonin gene-related peptide acts as a vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature 335, 164 – 167 (1988) 2 Kawasaki H, Nuki C, Saito A and Takasaki K: Role of calcitonin gene-related peptide-containing nerves in the vascular adrenergic neurotransmission. J Pharmacol Exp Ther 252, 403 – 409 (1989) 3 Yamamoto AY and Toyama M: Calcitonin gene-related peptide in the nervous system. Prog Neurobiol 33, 335 – 386 (1989) 4 Marti E, Gibson SJ, Polak MN, Facer P, Springall DR, Aswegen G, Aitchison M and Koltzenburg M: Ontogeny of peptide- and amine-containing neurones in motor sensory and autonomic regions of rat and human spinal cord dorsal root ganglia and rat skin. J Comp Neurol 266, 332 – 359 (1987) 5 Kawasaki H, Saito A and 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) 6 Kawasaki H, Nuki Y, Yamaga N, Kurosaki Y and Taguchi T: Decreased depressor response mediated by calcitonin generelated (CGRP)-containing vasodilator nerves to spinal cord stimulation and levels of CGRP mRNA of the dorsal root ganglia in spontaneously hypertensive rats. Hypertension Res 23, 693 – 699 (2000) 7 Chomczynski P and Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156 – 159 (1987) 8 Taguchi T, Kawasaki H, Imamura T and Takasaki K: Endogenous calcitonin gene-related peptide mediates nonadrenergic noncholinergic depressor response to spinal cord stimulation in the pithed rat. Circ Res 70, 357 – 364 (1992) 9 Gangula PR, Zhao H, Supowit SC, Wimalawansa SJ, Dipette DJ, Westlund KN, Gagel RF and Yallampalli C: Increased blood pressure in alpha calcitonin gene-related peptide / calcitonin gene knockout mice. Hypertension 35, 470 – 475 (2000)
Short Communication
Age-Related Decrease in Calcitonin Gene-Related Peptide mRNA in the Dorsal Root Ganglia of Spontaneously Hypertensive Rats Naka Yamaga, Hiromu Kawasaki*, Keiichi Inaizumi, Miho Shimizu, Arisa Nakamura and Yuji Kurosaki Department of Clinical Pharmaceutical Science, Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan Received April 6, 2001 Accepted May 30, 2001
ABSTRACT—Age-related changes in levels of calcitonin gene-related peptide (CGRP) mRNA of the dorsal root ganglia was studied in 8-, 12- and 15-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). CGRP mRNA levels in SHR but not in WKY decreased with age. The contents of CGRP-like immunoreactivities in the atrium and mesenteric artery of 15-week-old SHR were greater than those in age-matched WKY. These results suggest that the outflow of CGRP-containing nerves from the spinal cord and CGRP release from CGRP nerve terminals decreases in SHR. Keywords: Calcitonin gene-related peptide (CGRP) mRNA, Mesenteric arterial CGRP content, Spontaneously hypertensive rat
The rat mesenteric resistance arteries are innervated by nonadrenergic noncholinergic (NANC) vasodilator nerves in which calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, acts as the vasodilator neurotransmitter (1, 2). CGRP has been shown to distribute throughout the central and peripheral nervous system and locate in areas involved in cardiovascular function (3). A prominent site of CGRP synthesis is the dorsal root ganglia (DRG), which contains the cell bodies of primary afferent neurons that extend CGRP-containing nerves (CGRPergic nerves) to peripheral sites such as blood vessels and the central spinal cord (4). We demonstrated that the CGRPergic nerve function in spontaneously hypertensive rats (SHR) decreased with age (5). In a recent study, we showed that the levels of CGRP mRNA in DRG of 15-week-old SHR were lower than those in normotensive Wistar Kyoto rats (WKY) (6). Therefore, the present study was undertaken to clarify the age-related changes in level of CGRP mRNA in DRG of SHR and WKY and content of CGRP in the peripheral sites. Male SHR at 8, 12 or 15 weeks of age and age-matched WKY (Charles River Japan, Shizuoka) were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.). After the mean carotid blood pressure (MBP) was measured, the animals were deeply anesthetized with a large dose of sodium pentobarbital and killed by decapitation. The thoracic and
lumber DRG from each rat were immediately dissected. The isolated DRG was frozen in liquid nitrogen until subsequent analysis of CGRP mRNA as described previously (6). Total cellular RNA was prepared using the acid guanidium thyocyanate-phenol-chloroform method (7). The RNA samples were fractionated by electrophoresis on denaturing formaldehyde-agarose gels and transferred to a nylon membrane. Hybridization was done under stringent conditions (50% formamide at 42°C) with a 32P-labeled cDNA probe. Two probes for CGRP and > -actin were labeled, using a Multiprime DNA labeling system (Amersham Int., Buckinghamshire, UK) with 32P-dCTP. After hybridization, the membranes were exposed to X-ray films, and the radioactivity of each band was quantified by means of computerized densitometiric scanning (BAS 2000; Fuji Photo Film Co., Tokyo). All results are expressed relative to the >actin mRNA, serving as a control for the quality and quantity of RNA applied to the blot. The atrium and mesenteric artery isolated from 15-weekold SHR and WKY were homogenized and the supernatant solution was applied to a Bond-Elut C18 cartridge, and absorbed peptide was eluted with 60% acetonitrile in 1% trifluoroacetic acid. The elute was evaporated under vacuum and the contents of CGRP-like immunoreactivities (CGRP-LI) were determined using a commercially available enzyme-liked immunosorbent assay (ELISA) for CGRP kit (Peninsula Laboratories, Inc., San Carlos, CA, USA). All values are presented as the mean ± S.E.M. One-way analysis of variance followed by Tukey’s test was used to
*Corresponding author. FAX: +81-86-251-7970 E-mail: [email protected] 448
Short Communication
449
determine the significance between values of different ages. The unpaired Student’s t-test was used to determine the significance of differences between two means. A value of P<0.05 was considered significant. The MBP in 12-week-old SHR (188.5 ± 1.4 mmHg, n = 6, P<0.01) and 15-week-old SHR (193.0 ± 2.9 mmHg, n = 6, P<0.01) was significantly greater than that in 8week-old SHR (150.8 ± 3.2 mmHg, n = 6). The MBP of SHR at 8 (P<0.01), 12 (P<0.01) and 15 (P<0.01) weeks of age was significantly greater than that in age-matched WKY (8-week-old, 99.5 ± 1.8 mmHg, n = 4; 12-week-old, 108.0 ± 0.7 mmHg, n = 4; 15-week-old, 118.8 ± 7.8 mmHg, n = 4). Figure 1A shows a representative Northern blot analysis of CGRP mRNA. As shown in Fig. 1B, the level of CGRP
Fig. 2. CGRP-LI contents of the atrium and mesenteric artery in 15week-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Values show the mean ± S.E.M. from 4 experiments.
Fig. 1. Representative image (A) of Northern blot assays of CGRP mRNA and > -actin mRNA in the dorsal root ganglia (DRG) of spontaneously hypertensive rats (SHR) and bar graph (B) showing age-related changes in the ratio of CGRP mRNA / > -actin mRNA determined by densitometric analysis of Northern blot assays in DRG of Wistar Kyoto rats (WKY) and SHR. A: lanes 1 – 3, 4 – 6 and 7 – 9 indicate 8-, 12- and 15-week-old SHR, respectively. B: values represent the fold increase over controls (8-week-old). *P<0.05, **P<0.01, compared with 8-week-old SHR.
mRNA in SHR was decreased with age, whereas the level of CGRP mRNA in WKY was slightly increased with age. When the level of CGRP mRNA in SHR was compared with WKY as the ratio of CGRP mRNA / >-actin mRNA, a significant decrease was found in 12- and 15-week-old but not 8-week-old SHR, as shown in Fig. 1B. As shown in Fig. 2, the contents of CGRP-LI in the atrium and mesenteric artery in 15-week-old SHR were greater than those in age-matched WKY. A significant difference between SHR and WKY in CGRP-LI contents was found in the atrium (Fig. 2). The present findings demonstrated that the amount of CGRP mRNA in DRG of 12- and 15-week-old SHR decreased when compared with age-matched WKY. In addition, the decrease in the CGRP mRNA level of SHR was age-related, but the level in WKY did not alter. In in vivo studies, we previously reported that electrical stimulation of the lower thoracic spinal cord (Th9 – 12) in the pithed rat produced a frequency-dependent depressor response, which was abolished by the neurotoxin tetrodotoxin or by the CGRP-receptor antagonist CGRP(8 – 37), indicating that the response is mediated by endogenous CGRP released from the CGRPergic nerves (8). In a recent study, the depressor response to spinal cord stimulation in 15week-old pithed SHR was significantly smaller than that in age-matched WKY (6). Furthermore, the present study showed that the level of CGRP mRNA in DRG was decreased in SHR compared with WKY and the decrease was age-related. Taken together, it is very likely that the outflow of CGRPergic vasodilator nerves from the spinal cord to peripheral blood vessels via the dorsal roots decreases in SHR. In in vitro studies, we previously reported that CGRPergic nerve-mediated vasodilation in response to periarterial nerve stimulation of the mesenteric artery in SHR was smaller than that in WKY and the decrease in the response
450
Short Communication
was age-related (5). In addition, the neurogenic release of CGRP-LI in the mesenteric artery of 15-week-old SHR has been shown to be smaller than that in age-matched WKY (5). The present study demonstrated that the content of CGRP-LI in the mesenteric artery of SHR was greater than that in WKY. This finding suggests that the increased contents of CGRP-LI in SHR is due to reduced release of CGRP from CGRPergic nerve terminals, even though the levels of CGRP mRNA in SHR decreased. Since CGRPergic nerves have been shown to inhibit the function of adrenergic nerves (2), the decreased function of CGRPergic nerves in SHR enhances the sympathetic adrenergic vasoconstriction. A recent study demonstrated that =-CGRP/ calcitonin gene knockout mice displayed an elevated MBP compared with wild-type mice, indicating that =-CGRP is involved in the long-term regulation of resting blood pressure (9). Therefore, the reduced CGRPergic nerve function may have caused the increased vascular resistance and high blood pressure in SHR. In conclusion, the findings of the present study showed that the CGRP gene expression in DRG in SHR decreases with age. The present study suggests that the function of CGRPergic vasodilator nerves from the spinal cord decreases in SHR and this decrease contributes to maintenance of hypertension in SHR. Acknowledgments This study was supported, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 09672326, No. 10557244).
REFERENCES 1 Kawasaki H, Takasaki K, Saito A and Goto K: Calcitonin gene-related peptide acts as a vasodilator neurotransmitter in mesenteric resistance vessels of the rat. Nature 335, 164 – 167 (1988) 2 Kawasaki H, Nuki C, Saito A and Takasaki K: Role of calcitonin gene-related peptide-containing nerves in the vascular adrenergic neurotransmission. J Pharmacol Exp Ther 252, 403 – 409 (1989) 3 Yamamoto AY and Toyama M: Calcitonin gene-related peptide in the nervous system. Prog Neurobiol 33, 335 – 386 (1989) 4 Marti E, Gibson SJ, Polak MN, Facer P, Springall DR, Aswegen G, Aitchison M and Koltzenburg M: Ontogeny of peptide- and amine-containing neurones in motor sensory and autonomic regions of rat and human spinal cord dorsal root ganglia and rat skin. J Comp Neurol 266, 332 – 359 (1987) 5 Kawasaki H, Saito A and 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) 6 Kawasaki H, Nuki Y, Yamaga N, Kurosaki Y and Taguchi T: Decreased depressor response mediated by calcitonin generelated (CGRP)-containing vasodilator nerves to spinal cord stimulation and levels of CGRP mRNA of the dorsal root ganglia in spontaneously hypertensive rats. Hypertension Res 23, 693 – 699 (2000) 7 Chomczynski P and Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156 – 159 (1987) 8 Taguchi T, Kawasaki H, Imamura T and Takasaki K: Endogenous calcitonin gene-related peptide mediates nonadrenergic noncholinergic depressor response to spinal cord stimulation in the pithed rat. Circ Res 70, 357 – 364 (1992) 9 Gangula PR, Zhao H, Supowit SC, Wimalawansa SJ, Dipette DJ, Westlund KN, Gagel RF and Yallampalli C: Increased blood pressure in alpha calcitonin gene-related peptide / calcitonin gene knockout mice. Hypertension 35, 470 – 475 (2000)