Marmara Medical Journal Volume 12 No: 1 January 1999
MUSCARINIC RECEPTOR - MEDIATED
PHOSPHOINOSITIDE HYDROLYSIS IN RAT BRAIN
(Received 4 October, 1998)
Hiilya Cabadak, Ph.D. / Beki Kan, Ph.D.
D e p a rtm e n t o f B io p h y s ic s , S c h o o l o f M e d ic in e , M a rm a ra U n iv e rs ity , Is ta n b u l, T urkey.
ABSTRACT
Objective: Activation of cholinergic muscarinic receptors leads to activation of membrane - bound phospholipase C, leading to generation of IP3 and diacylglycérol, The present study aimed at measuring muscarinic receptor-mediated phosphoinositide breakdown in three different regions of rat brain.
Methods: Cortical, hippocampal and striatal slices labeled with [3H] myo-inositol were incubated with muscarinic agonist carbachol (Cch) and LiCI. Reaction was terminated by the addition of chloroform/methanol and labeled inositol phosphates were extracted from tissue. Individual [3H]- inositol phosphates were separated using anion exchange chromatography.
Results: Cch caused a rise in IP, formation, where the increase over basal in the cortex, hippocampus and striatum were 98%, 73% and 45%, respectively.
Conclusion: Phosphoinositide turnover was
stimulated by carbachol in all brain structures studied, with the most pronounced effects observed in the cortex and hippocampus.
K e y W o rd s :
Muscarinic receptors, inositolphosphates, carbachol, rat brain
IN TR O D U CTIO N
Muscarinic cholinergic receptors mediate a wide array of physiological effects both in the central and peripheral nervous system. Four muscarinic receptor subtypes designated as M-,, M2, M3 and M4 have been distinguished based on receptor binding studies (1). Through molecular cloning, five distinct muscarinic receptor genes (mi-m5) have been identified to be expressed in the brain and peripheral tissues (2-5). mr m4 muscarinic acetylcholine receptor subtypes are
expressed to similar extents in whole rat brain (4). Only low levels of m5 receptors are detected in several brain regions (6). The tissue distribution of m,-m4 mRNA has been studied using in s itu hybridization and Northern blot analysis (7,8). The concentration of m, and m3 mRNA were highest the cerebral cortex and hippocampus, while m4 mRNA was present at low levels in these regions and at high levels in the striatum (7). Subtype -specific antisera have been employed to measure the relative levels of five muscarinic receptor subtypes in rat and rabbit brain (6,9,10). The largest numbers of m4 receptors were found in the striatum while the largest percentage of m2 receptors were located in the cerebellum. On the other hand, the percentage of m3 receptors did not change dramatically across the brain.
The expression of each receptor subtype in mammalian cells has established that m,, m3 and m5
receptors are preferentially coupled to
phosphoinositide metabolism via a pertussis toxin independent G-protein (Gq), while m2 and m4 receptors inhibit cAMP production through Gj and possibly through Gz (2,11-13). Brain muscarinic receptors have been found to activate PI metabolism (14-16). The present study was aimed to investigate coupling of muscarinic receptors to inositol phospholipid hydrolysis in three different regions of the brain: cortex, hippocampus and striatum.
MATERIALS A N D METHODS
Myo-[2-3H] inositol (19.1Ci/mmol) was obtained from Amersham, U.K. Dowex AG1-X8, 200-400 mesh resin (formate form) was purchased from Bio-Rad. All other reagents and drugs were from Sigma Chemical Co.
Albino rats of both sexes (200-250 g) were decapitated to obtain the cerebral cortex, hippocampus and striatum. Each brain region was chopped into slices of 0.5x0.5 mm, resuspended in Krebs buffer containing
Marmara Medical Journal Volume 12 No: 1 January 1999
120 mM NaCI, 5.5 mM KCI, 2.5 mM CaCI2, 1.2 mM NaH2P 04, 1.2 mM MgCI2, 11 mM glucose and 20 mM NaHC03, pH 7.4 and preincubated in the same buffer gassed with a mixture of 95% 0 2 and 5% C 02 for 30 min at 37°C. Inositol phosphates were determined using the method described by Berridge (18) with minor modifications. Tissue slices distributed into 12x75 mm polypropylene tubes containing 0.3 ml Krebs buffer and 10 mM LiCI were labeled with 1 pCi Myo-[3H] inositol for one hour with continuous oxygenation and gentle agitation. Subsequently, Cch (10~3 M) was added and the mixture was incubated for one hour after which reactions were terminated by the addition of 0.94 ml of cold chloroform/methanol (1:2, v/v). The samples were homogenized in an Ultraturrax tissue homogenizer and 0.31 ml of chloroform and 0.3 ml of dH20 were added. The samples were mixed and centrifuged at 3000xg for 5 min. The upper aqueous phase was applied to polypropylene columns packed with Dowex AG1-X8 formate resin and the [3H]-inositol phosphates formed were separated by anion exchange chromatography as described by Berridge (18). Free inositol was eluted with 20 ml of water and glycerophosphoinositol was eluted with 20 ml of 5 mM sodium tetraborate / 60 mM sodium formate. IP,, IP2, IP3 were eluted sequentially with 5 ml of 0.1 M formic acid and 0.2 M ammonium formate (IP4); 16 ml of 0.1
M formic acid and 0.5 M ammonium formate, (IP2); 10 ml of 0.1 M formic acid and 1 M ammonium formate (IP3). One milliliter of each fraction was added to vials containing 10 ml of scintillation fluid. The vials were vortexed and the radioactivity was counted two hr later by liquid scintillation spectrometry. The lower organic phase was evaporated and used to determine the protein content according to Lowry et al.(19)
RESULTS
Carbachol-induced phosphoinositide accumulation was determined in rat hippocampal slices. Incubation of slices with Cch increased the formation of [3H] IP4 in a dose-dependent manner (Figure 1). The relative accumulation of [3H] IP4, [3H] IP2 and [3H] IP3 induced by 1CL3 M Cch were 74.5 ± 8.65, 16.49 ± 6.31 and 8.96 ± 2.46 %, respectively (mean of 3 experiments performed in duplicate, Table I).
Subsequent experiments were performed by
incubating rat hippocampal, striatal and cerebral cortical slices with 10‘3 M Cch for 60 min. Under these conditions, Cch caused accumulation of IP-| formation, where the increases over basal values in the cortex, hippocampus and striatum were 98%, 73% and 45%, respectively (Figure 2).
F ig . 1
Carbachol-induced PI hydrolysis in rat hippocampus. Tissue slices were incubated with 1 piCi [3H] myo-inositol and increasing concentrations of Cch. Labeled inositol phosphates extracted with chloroform/methanol were separated on Dowex AG1-X8 resin as described under Materials and Methods. Data represent one experiment performed in duplicate.
Table I. Relative levels of inositol phosphates formed in response to carbachol in rat hippocampus. Rat hippocampal slices were labeled with [3H] myo-inositol and carbachol (10~3 M). [3H]-inositol phosphates formed were separated as described under Materials and Methods. Data are expressed as dpm/mg protein and relative percentage of each inositol phosphate.
Experiment
iPf IP2 IP3 Total
IP, + IP2 + IP3 dpm/mg % dpm/mg % dpm/mg % dpm/mg % 1 21036.6 75.7 3986.5 14.35 2745 9.9 27767.5 100 2 20722.0 58.95 9962.4 28.34 4465 12.7 35149.4 100 3 8371.4 88.87 641.4 6.8 406.9 4.3 9419.7 100 Mean 16709.9 74.5 4863.4 16.49 2538.9 8.96 24122.4 100 26
Marmara Medical Journal Volume 12 No: 1 January 1999
Cortex
F ig . 2 . :
PI hydrolysis in rat cerebral cortex, hippocampus and striatum. Tissue slices were incubated with 1 pCi[3H] myo-inositol and 10‘3 M Cch for 60 min. Data represent the mean ± S.E.M. of three independent experiments performed in duplicate. IP, formation in each tissue was significantly different from the control value (P<0.05).
DISCUSSION
Muscarinic receptor-mediated activation of membrane- bound phospholipase C results in hydrolysis of the membrane phospholipid, phosphatidylinositol-4,5- biphosphate (PIP2) to generate the second messengers IP3 and diacylglycérol. Molecular cloning studies have established the presence of five muscarinic receptor subytpes, all of which are expressed in the brain (2-5). The five receptors have distinct distribution in brain (5), implying possible functional differences among the subtypes. m.| and m3 receptors are found to be highest within the cortex and hippocampus, while the striatum is rich in m4 subtype (7). The present study aimed to determine regional differences in phosphoinositide hydrolysis evoked by muscarinic agonist, carbachol, in rat brain. Phosphoinositide turnover was stimulated by carbachol in all brain structures studied, with the most pronounced effects observed in the cortex and hippocampus. These findings are in agreement with those of Tonnaer et al. (20) and consistent with the presence of significant amounts of m1 and m3 receptors, subtypes which are preferentially coupled to PI hydrolysis in these regions. The increase in PI hydrolysis was 45% over basal value in striatum, compared to a 98% increase in cerebral cortex. This finding is not surprising in view of the presence of low levels of m1 and m3 receptors in striatum in contrast to high levels of m4 receptors which are preferentially coupled to adenylyl cylase.
Acknow ledgem ent
This work was supported by a grant from the Turkish Government Planning Commission. The authors are grateful to Professor Norton Neff and Maria Hadjiconstantinou.
REFERENCES
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16. F is h e r KS, F iq u e ir e d o JC , B a rtu s RT. D iffe re n tia l s tim u la tio n o f in o s it o l p h o s p h o lip id tu r n o v e r in b ra in b y a n a lo g s o f o x o tr e m o r in e . J. n e u ro c h e m . 1 9 8 4 ; 4 3 : l 171-1 179. 17. F is h e r KS, B a rtu s RT. R e g io n a l d iffe re n c e s in th e c o u p lin g o f m u s c a r in ic r e c e p t o r s t o in o s it o l p h o s p h o lip id h y d ro ly s is in g u in e a p ig b ra in . J. n e u ro c h e m 19 8 5 ; 4 5 : 1 0 8 5 -1 0 9 5 . 18. B e r rid g e MJ. R a p id a c c u m u la t io n o f in o s it o l t r ip h o s p h a t e r e v e a ls t h a t a g o n is ts h y d r o ly s e p h o s p h o in o s itid e s in s te a d o f p h o s p h a tid y lin o s ito l. B io c h e m . J. 1 9 8 3 ;2 1 2 :8 4 9 - 8 5 8 . 19. L o w ry O ft, R o s e b ro u g h n J , F a rr A L a n d R a n d a ll RJ. P ro te in m e a s u r e m e n t w ith th e fo lin p h e n o l re a g e n t. J. B io l. C h e m . 1 9 5 1 ;1 9 3 :2 6 5 - 2 7 5 . 2 0 . T o n n a e r J A D M , C h e u n g CL, D e B o e r T. cG M P F o rm a tio n a n d p h o s p h o in o s itid e tu r n o v e r in ra t b ra in s lic e s a re m e d ia te d b y p h a rm a c o lo g ic a lly d is t in c t m u s c a r in ic a c e ty lc h o lin e r e c e p to rs . Eur. J. P h a rm a c o l. 1 9 9 1 ;2 0 7 :1 8 3 - 1 8 8 . 28
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