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(Stroke. 1995;26:1268-1272.)
© 1995 American Heart Association, Inc.
Articles |
M1 Receptors in Blood Pressure–Controlled Ischemic Spontaneously Hypertensive Rats
From the Department of Neuroscience, Institute of Molecular and Cellular Medicine (H.H., M.A., K.T., Y.K., N.O.), and the Third Department of Internal Medicine, Okayama University Medical School (H.H., Y.K.), Okayama, Japan.
Correspondence to Norio Ogawa, MD, DMSc, Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.
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Abstract |
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Background and Purpose Hypertension is a primary aggravating factor in cerebral infarction. An acute rise in blood pressure (BP) at the time of a stroke may be harmful to the brain in a hypertensive subject because both cerebral vascular structure and function are altered by hypertension. Muscarinic M1 receptors are concerned with memory and learning. We aimed to evaluate the effect of controlling BP in hypertensive subjects at the time of stroke with a biochemical index of brain damage.
Methods We gave a single dose of either the antihypertensive
-blocker phentolamine (2 mg/kg IP) or the calcium antagonist
nicardipine (2 mg/kg IP) at the start of bilateral carotid artery
occlusion to spontaneously hypertensive rats undergoing 3 hours of
transient ischemia; we measured the time course of mean BP
(MBP) and changes in the M1 receptor and its mRNA in three
brain regions 2 weeks after the transient ischemia.
Results Administration of phentolamine or nicardipine not only significantly suppressed the ischemia-induced rise of MBP, it actually decreased MBP during ischemia. In an ischemic control group, M1 receptor binding decreased in the frontal cortex and M1 receptor mRNA increased in the hippocampus 2 weeks after the ischemia. In contrast, both phentolamine- and nicardipine-treated ischemic rats showed no changes in either index compared with sham-operated controls.
Conclusions Controlling BP during an ischemic insult attenuates ischemia-induced damage of M1 receptors in the brain of spontaneously hypertensive rats. These results suggest that a rapid intensive increase of BP at the time of a stroke may exacerbate brain damage in hypertensive individuals.
Key Words: calcium channel blockers • cerebral ischemia • hypertension • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypertension causes disorders in various organs and in the brain in particular. Hypertension is a primary cause of multiple cerebral infarction1 and often occurs in patients with cerebrovascular dementia. Recurrent strokes, particularly in the hippocampus or frontal cortex, impair mental faculties.
In a patient who has just suffered a stroke, the mean blood pressure (MBP) is much higher than normal. In spontaneously hypertensive rats (SHR) serving as a model of ischemia, a significant increase in MBP occurs during ischemia, probably due to autoregulation against a decrease in cerebral blood flow in the ischemic area.2 3 Such an acute rise in MBP at the time of stroke may be harmful, especially to the brain of a hypertensive subject, since both the structure and function of the cerebral vessels are altered by hypertension.4 5
Some agents that showed favorable results in some clinical trials and experimental studies have been used in treating acute stroke.6 7 8 9 Calcium antagonists in particular have been considered neuroprotective agents against ischemia because of their potent cerebral vasodilatory activity10 and their prevention of the accumulation of intracellular calcium, which may trigger irreversible cellular damage.11 12 13 The neuroprotective effect of a calcium antagonist is due to direct blockade of calcium channels and to amelioration of the marked increase in MBP at the time of a stroke.
Muscarinic M1 receptors are responsible for memory and learning.14 15 16 17 Previously we reported that muscarinic cholinergic receptors were gradually depleted after transient ischemia in the gerbil hippocampus.18 19 In short, changes in the M1 receptor and its mRNA constitute a useful and important biochemical index with which to evaluate ischemia-induced brain damage.
Therefore, we investigated the effect of controlling MBP in
hypertensive rats at the time of a stroke on M1 receptor
and its mRNA in several brain regions. At the beginning of bilateral
carotid artery occlusion in a 3-hour transient ischemia model
of SHR, we gave either the antihypertensive -blocker phentolamine or
the calcium antagonist nicardipine as a single
intraperitoneal dose. We then monitored the time
course of MBP and measured changes in M1 receptor binding
and M1 receptor mRNA in the frontal cortex, hippocampus,
and striatum of SHR 2 weeks after the transient ischemia, when
histological damage in brain regions was not yet complete but decrease
of M1 receptor density was observed in the frontal
cortex.20
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Materials and Methods |
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Twenty-week-old male SHR were used in the experiments. With rats under ketamine anesthesia (100 mg/kg IP), silk ligatures were placed around both common carotid arteries without interrupting carotid blood flow. On the following day, with the animals under light ether anesthesia, the common carotid arteries were exposed bilaterally and then occluded for 3 hours with surgical clips. Sham-operated control SHR were treated similarly but without occlusion. Phentolamine (2 mg/kg IP) or nicardipine (2 mg/kg IP) was injected at the beginning of the operation. Both sham-operated and ischemic control SHR received the same volume of physiological saline. The blood flow in both common carotid arteries was restored by releasing the clips after the 3-hour occlusion. MBP was routinely measured by tail-cuff plethysmography (UR-5000, Ueda Seisakusyo) in conscious, restrained, warmed rats. The SHR were decapitated 2 weeks after recirculation, and their brains were immediately removed and dissected according to the method of Glowinski and Iversen21 and then subjected to receptor binding assay and Northern blot analysis.
The M1 receptor binding assay was performed according to the method of Schudt et al,22 with minor modifications. Briefly, after being homogenized in 10 volumes of ice-cold 50 mmol/L Tris-HCl buffer (pH 7.4), samples were centrifuged at 12 000g for 20 minutes at 4°C. The resulting pellet was washed twice in the same buffer by resuspension and centrifugation before finally being suspended in 170 volumes of HEPES-NaCl buffer (20 mmol/L HEPES, 100 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L EDTA, pH 7.4). The prepared membrane (about 0.1 mg of protein) in 0.25 mL of HEPES-NaCl buffer was incubated for 120 minutes at room temperature with 0.25 mL of HEPES-NaCl buffer containing a final concentration of 1.0 nmol/L [3H]telenzepine (86.8 Ci/mmol; New England Nuclear/Dupont), with or without 5 µmol/L atropine. After incubation, the reaction was stopped by filtration through glass-fiber filters (Whatman GF/B, Whatman Paper Ltd). Nonspecific binding was determined in the presence of 5 µmol/L atropine.
For Northern blot analysis, total RNA was extracted from the dissected tissues according to the method of Chomczynski and Sacchi.23 The concentration of total RNA was measured using a spectrophotometer. Total RNA (10 µg per lane) was denatured, electrophoresed, and transblotted directly onto a nylon membrane.
Synthetic 48-mer oligonucleotide probes were purchased (New England Nuclear/Dupont) with a sequence complementary to bases 4 to 51 of the rat M1 receptor mRNA.24 As an internal control probe, a synthetic 30-mer 18S rRNA oligonucleotide probe was used for hybridization with a sequence complementary to bases 1851 to 1880.25 Probes were radiolabeled on the 3'-ends with [32P]ddATP by 3'-terminal deoxynucleotidyl transferase using a commercially available kit (Amersham).
Blots were prehybridized for 4 hours at 37°C in a hybridization solution containing 4x standard saline citrate (SSC) (1x SSC; 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH 7.0), 1x Denhardt's solution, 50% formamide, 0.5% sodium dodecyl sulfate, 5% dextran sulfate, 100 µg/mL yeast tRNA, and 500 µg/mL salmon sperm DNA. Hybridization was performed for 20 hours at 37°C in the same solution containing 106 disintegrations per minute per milliliter of 32P-labeled oligonucleotide probes (M1 receptor or 18S rRNA probes). After hybridization, the membranes were washed and exposed to x-ray film for 1 week (Hyperfilm MP, Amersham). The optical densities of signals in the autoradiograms were measured with a computerized image analysis system (RAS 1000, Amersham).
Protein concentration was determined using the Bio-Rad protein assay kit, with bovine serum albumin as the standard.
Data are presented as mean±SEM. Statistical analyses used two-way ANOVA or one-way ANOVA followed by post hoc Duncan's multiple-comparison test or the Mann-Whitney U test. A level of P<.05 was accepted as statistically significant.
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Results |
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The effects of a single dose of phentolamine (2 mg/kg IP) or of nicardipine (2 mg/kg IP) on the MBP of sham-operated SHR are shown in Fig 1
. The MBP of sham-operated SHR before the operation
was 175±9 mm Hg. Neither the sham operation nor saline injection had
any effect on MBP up to 180 minutes. The MBP fell to 148±8 mm Hg and
135±6 mm Hg, respectively, 15 minutes and 30 minutes after injection
of phentolamine. These decreases were statistically significant and
lasted up to 120 minutes. Likewise, the MBP significantly decreased
after nicardipine injection, falling to 120±8 mm Hg after 15 minutes
and to 123±5 mm Hg after 30 minutes; the decrease also lasted up to
120 minutes.
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; 2 mg/kg IP) and nicardipine (
; 2 mg/kg
IP) on mean blood pressure of sham-operated spontaneously hypertensive
rats (SHR). Sham-operated control SHR (
) were injected with the
same volume of physiological saline. Each value is mean±SEM (n=5).
*P<.05 compared with the control level before the sham
operation; +P<.05 compared with time-matched,
saline-treated, sham-operated control group, by two-way ANOVA followed
by Duncan's multiple-comparison test.
Fig 2 shows the effects of phentolamine and
nicardipine on the MBP of ischemic SHR. The MBP of
ischemic SHR increased to 200±7 mm Hg 15 minutes after
bilateral common carotid artery occlusion and to 245±6 mm Hg after 60
minutes; this significant rise of MBP lasted throughout the
ischemic period. After blood flow had been restored, the MBP
decreased rapidly to 140±5 mm Hg. This significant decrease lasted
for 90 minutes after restoration of circulation, when MBP returned to
the preischemia control level. In contrast, the MBP of
ischemic SHR that received phentolamine or nicardipine
decreased to 152±3 or 134±4 mm Hg, respectively, 15 minutes after
bilateral carotid artery occlusion. These suppressive effects of
phentolamine or nicardipine on the ischemia-induced increase of
MBP persisted during the 3-hour period of ischemia and for 90
minutes after restoration of the circulation.
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The effects of phentolamine and nicardipine on M1 receptor
binding in sham-operated or ischemic SHR brain are shown in the
Table. In the frontal cortex, specific binding for
M1 receptor was decreased significantly 2 weeks after the
transient ischemia compared with that in sham-operated
controls. In contrast, no significant change in specific binding for
M1 receptor was observed in the frontal cortex of SHR that
had been injected with phentolamine or nicardipine at the beginning of
carotid occlusion. In the hippocampus, the level of M1
receptor binding showed a small, but not significant, tendency toward
decrease in the ischemic control group. In the striatum, there
was no significant change of M1 receptor binding in any
group.
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The effects of administration of phentolamine or nicardipine on
M1 receptor mRNAs are shown in Fig 3.
Northern blot analyses utilized synthetic M1 receptor
oligonucleotide probes. Single species of RNA were labeled on each blot
(Fig 3). The size, which is approximately 3.1 kb, is in agreement with
the previously reported values,26 indicating that the mRNA
encoding M1 receptor was being detected. The levels of
M1 receptor mRNA (M1 receptor mRNA to 18S rRNA
ratios) showed a tendency toward an increase in the frontal cortex of
ischemic controls (Fig 3, top). In the hippocampus of the
ischemic control group, the level of M1 receptor
mRNA was increased significantly compared with that in the
sham-operated control group. No significant changes were observed on
levels of M1 receptor mRNA in ischemic SHR treated
with phentolamine or nicardipine at the start of carotid artery
occlusion (Fig 3, middle). In the striatum, there was no significant
change of M1 receptor mRNA in any group (Fig 3, bottom).
The 18S rRNA was unchanged in rehybridized blots with 18S rRNA probes
used as internal control probes (Fig 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The SHR, a well-known animal model of hypertension, shows functional27 and biochemical28 29 differences from normotensive rats. Consequently, some phenomena that are beneficial to the normotensive rat (for example, responsive elevation of BP) occasionally may be harmful to SHR.
In the ischemic control group, the M1 receptor
binding in the frontal cortex significantly decreased 2 weeks after
transient ischemia. A similar downward trend was seen in the
hippocampus (Table
). However, the M1 receptor mRNA
increased significantly in the hippocampus and showed an upward trend
in the frontal cortex (Fig 3). The decrease in M1 receptor
binding after ischemia might be due to the destruction of
receptors by ischemia, and the increase in M1
receptor mRNA might be a result of compensatory upregulation for loss
of these receptors. Both the phentolamine- and the nicardipine-treated
ischemic groups showed no changes in either M1
receptor binding or M1 receptor mRNA compared with the
sham-operated control group (Table and Fig 3). These results indicate
that administration of these agents at the beginning of transient
ischemia may have prevented the ischemia-induced
destruction of receptors in the brain, leaving M1 receptor
mRNA unaltered. Prevention of intracellular Ca2+
accumulation, which triggers irreversible cellular injury, may be
beneficial in attenuating ischemic damage.11 30
Both phentolamine (2 mg/kg IP) and nicardipine (2 mg/kg IP)
significantly reduced the MBP of sham-operated SHR, with
antihypertensive effects lasting up to 120 minutes (Fig 1). The
administration of phentolamine or nicardipine significantly suppressed
the ischemia-induced rise of MBP and reduced MBP during the 3
hours of ischemia versus that before ischemia. The
suppressive effect of each agent persisted for 90 minutes after the
restoration of circulation (Fig 2). Although phentolamine can reduce
MBP by blocking the -receptors in the sympathetic nervous
system31 and by exerting a considerable peripheral
vasodilatory activity,32 this agent has no effect on
either Ca2+ channels or cerebral vasodilatory
activity. Therefore, prevention of the ischemia-induced
alteration in M1 receptor binding and in M1
receptor mRNA in the present study may be attributable to the
control of MBP by the administration of an antihypertensive agent, not
to the blocking of the influx of
Ca2+. Results also suggest that a
rapid intensive increase in MBP during ischemia may be harmful
to the ischemic brain of SHR.
In conclusion, controlling the BP of SHR during an ischemic insult reduced the amount of ischemia-induced damage to M1 receptors in the frontal cortex and hippocampus. In addition, controlling BP with an antihypertensive agent may reduce the brain damage in hypertensive subjects caused by a rapid rise of BP just after ischemia.
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Acknowledgments |
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This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture and by a grant from the Ministry of Health and Welfare of the Japanese Government. The authors thank Kumiko H. Ono for technical assistance.
Received July 29, 1994; revision received February 27, 1995; accepted March 30, 1995.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Coyle P. Outcomes to middle cerebral artery occlusion in hypertensive and normotensive rats. Hypertension. 1984;6(suppl I):I-69-I-74.
2. O'Brien MD, Halsey JJ, Strong ER. The effect of hypertension on ischemic cerebral edema in spontaneously hypertensive rats. Neurol Res. 1983;5:83-93. [Medline] [Order article via Infotrieve]
3.
Ooboshi H, Sadoshima S, Yao H, Nakahara T, Uchimura H,
Fujishima M. Inhibition of ischemia-induced dopamine
release by 
-conotoxin, a calcium channel blocker, in the striatum of
spontaneously hypertensive rats: in vivo brain dialysis study.
J Neurochem. 1992;58:298-303. [Medline]
[Order article via Infotrieve]
4.
Baumbach GL, Heistad DD. Cerebral circulation
in chronic arterial hypertension.
Hypertension. 1988;12:89-95.
5. Yao H, Matumoto T, Hirano M, Kuroki T, Tsutsumi T, Uchimura H, Nakahara T, Fujishima M. Involvement of brain stem noradrenergic neurons in the development of hypertension in spontaneously hypertensive rats. Neurochem Res. 1989;14:75-79. [Medline] [Order article via Infotrieve]
6.
Fujii K, Weno BL, Baumbach GL, Heistad DD.
Effect of antihypertensive treatment on focal cerebral
infarction. Hypertension. 1992;19:713-716.
7. Gelmer HJ, Gorter CJ, Weedt D, Wierzer HJ. A controlled trial of nimodipine in acute ischemic stroke. N Engl J Med. 1988;318:203-207. [Abstract]
8. Gotoh O, Mohamed AA, McCulloch J, Graham DI, Harper AM, Teasdale GM. Nimodipine and the hemodynamic and histopathological consequences of middle cerebral occlusion in the rat. J Cereb Blood Flow Metab. 1986;6:321-331. [Medline] [Order article via Infotrieve]
9.
Kawamura S, Shirasawa M, Fukusawa H, Yasui N.
Attenuated neuropathology by nilvadipine after middle cerebral
artery occlusion in rats. Stroke. 1991;22:51-55.
10. Ohtsuka M, Ono T, Hiroi J, Esumi K, Kikuchi H, Kumada S. Comparison of the cardiovascular effect of FR34235, a new dihydropyridine, with other calcium antagonists. J Cardiovasc Pharmacol. 1983;5:1074-1082. [Medline] [Order article via Infotrieve]
11.
Hadani M, Young W, Flamm ES. Nicardipine reduces
calcium accumulation and electrolyte derangements in regional cerebral
ischemia in rats. Stroke. 1988;19:1125-1132.
12. Siesjö BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metab. 1981;1:155-185. [Medline] [Order article via Infotrieve]
13. Raichle ME. The pathophysiology of brain ischemia. Ann Neurol. 1983;13:2-10. [Medline] [Order article via Infotrieve]
14. Arnt J, Lembol HL, Meier E, Pedersen H. Discriminative stimulus properties of the muscarinic receptor agonists Lu 26-046 and O-Me-THPO in rats: evidence for involvement of different muscarinic receptor subtypes. Eur J Pharmacol. 1992;218:159-169. [Medline] [Order article via Infotrieve]
15. Boddeke EW, Enz A, Shapiro G. SDZ ENS 163, a selective muscarinic M1 receptor agonist, facilitates the induction of long-term potentiation in rat hippocampal slices. Eur J Pharmacol. 1992;222:21-25. [Medline] [Order article via Infotrieve]
16. Guzman GG, Schliebs R. Effect of kainic acid administration to prepubescent rats on cholinergic markers in selected brain regions of adult rats. Neurochem Int. 1992;21:447-453. [Medline] [Order article via Infotrieve]
17. Jerusalinsky D, Cervenansky C, Walz R, Bianchin M, Izquierdo I. A peptide muscarinic toxin from the Green Mamba venom shows agonist-like action in an inhibitory avoidance learning task. Eur J Pharmacol. 1993;240:103-105. [Medline] [Order article via Infotrieve]
18. Haba K, Ogawa N, Mizukawa K, Mori A. Time course of changes in lipid peroxidation, pre- and postsynaptic cholinergic indices, NMDA receptor binding and neuronal death in the gerbil hippocampus following transient ischemia. Brain Res. 1991;540:116-122. [Medline] [Order article via Infotrieve]
19. Ogawa N, Asanuma M, Mizukawa K, Hirata H, Chou H, Mori A. Post-ischemic administration of bifemelane hydrochloride prohibits ischemia-induced depletion of the muscarinic M1-receptor and its mRNA in gerbil hippocampus. Brain Res. 1992;591:171-175. [Medline] [Order article via Infotrieve]
20. Haba K, Ogawa N, Mizukawa K, Tanaka K, Asanuma M, Hirata H. Chronic changes in cholinergic indices following ischemia in spontaneously hypertensive rats (SHR). Neurochem Res. 1992;17:655. Abstract.
21. Glowinski J, Iversen LL. Regional studies of catecholamines in the rat brain, I: the distribution of [3H]norepinephrine, [3H]dopamine and [3H]DOPA in various regions of the brain. J Neurochem. 1966;13:655-669. [Medline] [Order article via Infotrieve]
22. Schudt C, Auriga C, Kinder B, Birdsall NJM. The binding of [3H]telenzepine to muscarinic acetylcholine receptors in calf forebrain. Eur J Pharmacol. 1988;145:87-90. [Medline] [Order article via Infotrieve]
23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]
24.
Bonner TI, Buckley NJ, Young A, Brann MR.
Identification of a family of muscarinic receptor genes.
Science. 1987;237:527-532.
25.
Torczynski R, Bollon AP, Fuke M. The complete
nucleotide sequence of the rat 18S ribosomal RNA gene and
comparison with the respective yeast and flog gene.
Nucleic Acids Res. 1983;11:4879-4890.
26. Buckley NJ, Bonner TI, Brann MR. Localization of a family of muscarinic receptor mRNAs in rat brain. J Neurosci. 1988;8:4646-4652. [Abstract]
27. Fujishima M, Omae T, Takeya Y, Takeshita M, Ogata J, Ueda K. Prognosis of occlusive cerebrovascular diseases in normotensive and hypertensive subjects. Stroke. 1976;5:472-476.
28. Lim DK, Yu ZJ, Hoskin B, Rockhold RW, Ho IK. Effects of acute and subacute cocaine administration on the CNS dopaminergic system in Wistar-Kyoto and spontaneously hypertensive rats, III: dopamine uptake. Neurochem Res. 1990;15:629-634. [Medline] [Order article via Infotrieve]
29. Linthorst ACE, Van den Buuse M, De Jong W, Versteeg DHG. Electrically stimulated [3H]dopamine and [14C]acetylcholine release from nucleus caudatus slices: differences between spontaneously hypertensive rats and Wistar-Kyoto rats. Brain Res. 1990;509:266-272. [Medline] [Order article via Infotrieve]
30. Sauter A, Rudin M. Calcium antagonists for reduction of brain damage in stroke. J Cardiovasc Pharmacol. 1990;15(suppl 1):S43-S47.
31. Walker HA, Wilson S, Heymans C, Richardson AP. Effect of C-7337 on cardiovascular system of drugs. Arch Int Pharmacodyn. 1950;82:395-415.
32.
Taylor SH, Sutherland GR, MacKenzie GJ. The
circulatory effects of intravenous phentolamine in man.
Circulation. 1965;31:741-754.
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