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Inhibition of polyphosphoinositide turnover in rat cerebral cortex by clonidine
Life
Sciences,
Vol.
45.
pp.
PergamonPress
993-999
printed in the U.S.A.
INHIBITION OF POLYPHOSPHOINOSITIDE TURNOVER IN RAT CEREBRAL CORTEX BY CLONIDINE Lillian E. Dyck Neuropsychiatric Research Unit Medical Research Building University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N OWO (Received in final form July
7, 1989)
In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of aladrenergic receptors, for example, increases polyphosphoinositide turnover, but stimulation of a2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inosito1 and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an a2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of a2, 5HT2, H2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation. Clonidine is a centrally acting antihypertensive agent with high affinity for a2-receptors (1,2). It may also be useful in the treatment of depression and anxiety (3-6). Clonidine is usually thought of as a specific a2-receptor agonist; however, in addition to its adrenergic effects, clonidine activates serotonergic 5HT2 and histaminergic H2 receptors (1,7). It also affects other neurotransmitter systems indirectly by its a2-receptor agonism (1,7)_ Several different types of neurotransmitter receptors are coupled to the hydrolysis of membrane inositol phospholipids which generates two second messengers (diacylglycerol and inositol trisphosphate) (8,9). al-Receptors stimulate the breakdown of these membrane lipids, but a2-receptors appear to be inactive; 5HT2 receptors are also coupled to this transduction system (8,9). It is possible, therefore, that clonidine might affect the formation of diacylglycerol and inositol trisphosphate by stimulating these receptors. In a previous study clonidine increased the breakdown of inositol phospholipids in rat pineal gland, vas deferens and caudal artery, but its mechanism of action was not investigated 0024-3205189 $3.00+ -00 Copyright (c) 1989 Pergamon Press plc
994
Clonidine
and Phosphoinositide
Hydrolysis
Vol.
45, No.
11, 1989
(10,ll) . Several other groups, however, found that clonidine did not increase hydrolysis of inositol phospholipids in the rat cortex (12-14). The purpose of this study was to determine whether or not clonidine altered polyphosphoinositide turnover' in slices of rat cerebral cortex and, if so, to attempt to identify the receptor involved. A direct assay which measures the formation of 3H-inosito1 phosphates from inositol phospholipids labeled by preincubation with 3H-inositol was used (15).
Method8 Animals. Male Wistar rats (200-250 g., Charles River Canada) were used. They were housed in hanging wire cages with ad libitum access to food and water. mica& Myoinositol [2-3H(N)] (15 Ci/mmol) was purchased from New England Nuclear (Mississauga, Ont.). It was purified immediately before use by passing it through a column of AGl-X8 ion-exchange resin (formate form, 200-400 mesh, Biorad, Rockville, NY). The hydrochloride salts of clonidine, (-) noradrenaline (NE) and yohimbine were purchased from Sigma Chemical Co. (St. Louis, MO.). Ketanserin was obtained from Janssen Pharmaceutics, idazoxan from Reckitt and Colman, and cimetidine from Smith, Kline and French Labs. . I. oinowde hvdro sis, The accumulation of 3H-inositol phosphates was measured using a modification (16) of the method of Berridge et al., 1982 (15) as described in detail previously. Rats were killed by decapitation, their brains removat room temperature on ice-chilled petri dishes. ed, dissected Cortical slices (350 x 350 p) were incubated for 30 min at 37'C in an atmosphere of 95% 02-5% C02. Then the buffer was aspirated, 50 ~1 of the slices pipetted into tubes containing 1 PCi 3H-myoinosito1 (0.3 @l) and incubated for 60 min at 37'C to label the inositol phospholipids. The 3H-myoinositol-containing buffer was removed, and the hydrolysis of the labeled phospholipids studied by incubating the slices for 60 min under these conditions: control, Krebs buffer; experimental, NE + clonidine f antagonist, carbachol & clonidine, or antagonist. To terminate the incubation, CHC13: methanol (1:2, v/v) was added. The amount of phospholipid labeling was determined by assessing the radioactivity in the chloroform phase. The 3H-inositol phosphates present in the aqueous phase were adsorbed onto an ion-exchange column (AGl-X8). The columns were washed and then, a total 3H-inositol phosphate fraction (containing inositol mono-, bis- and tris-phosphates) was eluted with 0.1 N formic acid-l.0 M ammonium formate and added to ACS liquid scintillant (Amersham, Oakville, Ont.), and radioactivity was determined by liquid scintillation counting. In all experiments, blank samples (cortical slices maintained at 2'C throughout the procedure) were processed. Blank samples gave a count of 50-60 dpm in the chloroform aliquot and loo-160 dpm for the aqueous sample. Results were Blank values were subtracted from the other samples. expressed as dpm present in the inositol phosphate fraction divided by the total dpm present in the lipid and in the inositol phosphate fraction (17). Sometimes the data were converted to percentages of Data were analyzed by control (slices incubated in Krebs buffer).
Vol. 45,
No.
11,
1989
Clonidine and PhosphoinositideHydrolysis
l-way analysis of variance Keuls or a priori t-tests.
and
comparisons
performed
995
by Newman-
. . formatEffect of clonidlne on sH_inositolhate Clonidine was incubated with cortical slices at concentrations from It did not significantly increase the formation 0.1 j.Q.4 to 26 mM. of 3H-inositol phosphates above control levels at any of these concentrations (Table I).
TABLE I Effect of Clonidine
0.1 $ 1.0 PM 100 PM ill-M 26 mM
of 3H-Inositol Phosphate
569 563 599 594 784 694
Production
f 57 f 78 + 99 _+ 84 f 113 f 293
3H-Inositol phosphate production = [(dpm in 3H-inositol phosphate fraction) + (dpm in 3H-inositol phosphate fraction + dpm present in lipids)] x 103. Values are mean f S.D., n = 9.
I I - stimulated ’ Effects of clonldlneol %k itol ohosghate formation. NE (100 PM) stimulated 3Hphosphoinositide turnover to 383 k 17% of control activity. When clonidine was co-incubated with 100 PM NE, it dose-dependently inhibited the ability of NE to stimulate 3H-inositol phosphate formation (Fig. 1). By contrast, the ability of the muscarinic agonist, carbachol, to stimulate 3H-inositol phosphate formation was not affected by clonidine (Fig. 2). . * Effects of antwts on clonidine. Various drugs were tested to see whether they inhibited the ability of clonidine to antagonize the increased 3H-inositol phosphate formation stimulated by NE. None of them had any effect on basal or NE-stimulated 3Hinositol phosphate production (data not shown) nor did they antagonize the ability of clonidine to inhibit NE-stimulated 3H-inositol phosphate production (Table II).
996
Clonidine and Phosphoinositide Hydrolysis
Vol. 45. wo. II, 1989
80 60 -
-5 -6 log [Clonidine)
-7
-4
FIG. 1 Inhibition by clonidine of 3H-inositol phosphate production stimulated by 100 @4 NE. Values are mean f sem, n = 9.
250
1
p 200 * E 150 s
100
R
50 0
q notlon 0 + Clon
O.lmM
Car
1 .O mM Car FIG. 2
Effects of clonidine (Clan) (10 w) on carbachol (Car) - stimulated 3H-inositol phosphate formation. Values are mean f sem, n = 9.
Vol. 45, No. 11, 1989
Clonidineand Phosphoinositide Hydrolysis
997
TABLE II Effects of Antagonists on Inhibition by Clonidine of NE-induced 3H-Inositol Phosphate Formation
100 @4 Clon 11 + 0.1 w 100 w "
+
Yoh
Clon 1 p Ida
100 p.M Clon It + 1 pM Atro
100 w Clan II + 0.01 w
78 f 3% 77 f 4%
10 p Clon '1+ 100 j.04 Cim
44 f 5%
75 f 3% 80 f 3%
10 pM l1 +
40 f 5% 47 f 4%
Clon 0.01 p
Ket
48 f 5%
79 f 2% 81 f 2% Ket
71 f 4% 78 f 3%
values are mean f sem, n=6. Clon = clonidine; Yoh = yohimbine; Ida = idazoxan; Atro = atropine; Cim = cimetidine; Ket ketanserin.
=
The present results show that by itself clonidine had no effect on the hydrolysis of phosphoinositides in slices of rat cortex. It neither increased nor inhibited the basal formation of inositol phosphates. This lack of stimulation agrees with previous reports in rat cortex (12-14) and indicates that clonidine was not an agonist at al or 5HTz receptors which are coupled to phosphoinositide hydrolysis (9,lO). One might have expected clonidine to decrease basal rates of phosphoinositide hydrolysis via stimulation of ag receptors (which would feedback inhibit NE release), if basal activity was caused by unstimulated release of endogenous NE. Clonidine did not, however, decrease basal phosphoinositide hydrolysis. Though clonidine itself had no effect on the basal rate or on carbachol-stimulated 3H-inositol phosphate production (Table 1 and Fig. 21, it significantly inhibited the ability of NE to stimulate 3H-inositol phosphate formation (Fig. 1). This inhibition appears to be physiologically relevant, because NE was inhibited by concentrations of clonidine equal to or less than the concentration of NE used to stimulate phosphoinositide hydrolysis in rat cortical slices. 10 pM Clonidine inhibited 100 @4 NE by 53 f 5%, and 100 m clonidine caused an almost complete inhibition of 100 pM NE (88 f 3% inhibition). Inhibition of NE by clonidine was not blocked by the az-antagonists, yohimbine and idazoxan; the 5HT2 antagonist, ketanserin; the H2 antagonist, cimetidine; nor by atropine, a muscarinic receptor antagonist; therefore, neither a2, 5HT2, H2 nor muscarinic receptors seem to be involved.
998
Clonidine and PhosphoinositideHydrolysis
Vol. 45, No. 11. 1989
Specific binding sites for clonidine and para-aminoclonidine have been found in the bovine and rat brain; these binding sites are distinct from a2 receptors and may represent an imidazole receptor involved in central control of blood pressure (18-20). These imidazole binding sites are thought to be present in the bovine ventrolateral medulla, but not in the frontal cortex (18,22). The brain contains a clonidine-displacing substance, the identity of which is as yet unknown (19-21). Antagonists which are specific for this imidazole receptor have yet to be identified; chlorphenhowever, a number of imidazoles such as cimetidine, iramine and histamine displace clonidine from its binding sites (18). Because neither cimetidine nor idazoxan (another imidazole) inhibited the effects of clonidine on polyphosphoinositide hydrolyreceptor does not seem to be involved in sis, an imidazole mediating clonidine's actions on inositol phospholipid hydrolysis. Compounds which activate protein kinase C cause a feedback inhibition of agonist-stimulated phosphoinositide hydrolysis (9,23). For example, the tumor promoter phorbol esters are kinase C activators, and so too are benzene, toluene, chloroform, and unsaturated free fatty acids (9,23). Clonidine may be sufficiently lipophilic to partition into the cell membrane and affect the interaction of protein kinase C with some of its modulators and If clonidine were a protein kinase C activator, it substrates. would inhibit agonist-induced hydrolysis of phosphoinositides as was observed. Additional studies are required to answer this question. Clonidine has many uses clinically (1,191. In addition to its antihypertensive effects, clonidine has been used to treat opiate withdrawal, migraine, Gilles de la Tourette's syndrome and glaucoma. It may be that some of the therapeutic benefits of clonidine administration arise from its ability to modulate the generation of second messengers from inositol phospholipids induced Interestingly, though clonidine inhibited the hydrolysis of by NE. phosphoinositides induced by NE, it did not affect carbachol. In summary, this paper shows that clonidine by itself has no It had no effect on the hydrolysis of polyphosphoinositides. effect on carbachol-stimulated phosphoinositide hydrolysis, but it A concentration of 10 JLM cloninhibited NE-stimulated hydrolysis. idine decreased the effect of 100 @l NE by 50% . This effect of clonidine did not appear to involve a2-, 5HT2-, Hz- or imidazoleThe mechanism of clonidine's effects on NE-stimulated receptors. formation of inositol phosphates remains to be determined.
The author thanks Dr. A. A. Boulton for advice and encouragefor technical assistance and Saskatchewan ment, R. C. Mag-atas Health for financial support.
Vol. 45, No. 11, 1989
1. 2. 3.
Clonidine and PhosphoinositideHydrolysis
999
Trends in Pharmacological Sciences 2 194-196 W. KOBINGER, (1981). SNYDER, Mol. GREENBERG and S.H. U'PRICHARD, D.A. D.C. Pharmacol. u 454-473 (1977). D.S. CHARNEY, G.R. HENINGER, D.E. STERNBERG, K.M. HAFSTAD, S. GIDDINGS and D.H. LANDIS, Arch. Gen. Psychiatry a 290-294 (1982).
and T.W. UHDE, Biol. Psychiatry Le 131-156 L.J. SIEVER (1984). A.J. FYER, P. MCGRATH and D. F. KLEIN, 5. M.R. LIEBOWITZ, Psychopharmacol. Bull. ;c1 122-123 (1981). A.F. MERCHANT, M.L. KEYSER and V.K. SMITH, 6. R. HOEHN-SARIC, Arch. Gen. Psychiatry a 1278-1282 (1981). 7. L. BERRINO, G.A. BATTINI, A. BARRA, V. SUSANNA, M. LAMA, M. LISA, M. ANGRISANI and E. MARMO, Curr. Therap. Res. 42 790806 (1987). Biochem. KENDALL and I. BATTY, NAHORSKSI, D.A. 8. S.R. Pharmacol. a 2447-2453 (1986). 9. A.A. ABDEL-LATIF, Pharmacol. Rev. 3 227-272 (1986). 10. A.F. FOX, P.W. ABEL and K.P. MINNEMAN, Eur. J. Pharmacol. ll!j 145-152 (198 ). 11. T.L. SMITH, J. EICHBERG and G. HAUSER, Life Sci. 24 2179-2184 (1979). 12. E. BROWN, D.A. KENDALL and S.R. NAHORSKI, J. Neurochem. 42 1379-1387 (1984). S.M. KNEPPER and C.O. RUTLEDGE, J. Neurochem. 13. )&D;5:?%:P&984). MINNEMAN and R.D. JOHNSON, J. Pharmacol. Exp. Therap. 14. x-317-323 (1984). 15. M.J. BERRIDGE, C.P. DOWNES and M.R. HANLY, Biochem. J. 2_Qjj 587-595 (1982). 16. L.E. DYCK, Neurochem. Res. u 63-67 (1989). 17. C.J. FOWLER, J.A. COURT, G. TIGER, P.-E. BJORKLUND and J.M. CANDY, Pharmacol. Toxicol. 6p 274-279 (1987). 18. P. ERNSBERGER, M.P. MEELEY, J.J. MANN and D.J. REIS, Eur. J. Pharmacol. ;u4 1-13 (1987). and Y. BURSTEIN, Eur. J. Biochem. m 287-293 19. D. ATLAS (1984). A.R. GRANATA and D.J. REIS, 20. M.P. MEELEY, P.R. ERNSBERGER, Life Sci. ;28 1119-1126 (1986). 21. P. BOUSQUET, J. FELDMAN and D. ATLAS, Eur. J. Pharmacol. ;L24 167-170 1986). 22. P. ERNSBERGER, M.P. MEELEY and D.J. REIS, Brain Res. m 309318 (1988). 23. A.A. FAROOQUI, T. FAROOQUI, A.J. YATES and L.A. HORROCKS, Neurochem. Res. u 499-513 (1988). 4.
Sciences,
Vol.
45.
pp.
PergamonPress
993-999
printed in the U.S.A.
INHIBITION OF POLYPHOSPHOINOSITIDE TURNOVER IN RAT CEREBRAL CORTEX BY CLONIDINE Lillian E. Dyck Neuropsychiatric Research Unit Medical Research Building University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N OWO (Received in final form July
7, 1989)
In the rat brain, a number of receptors are linked to phospholipase C which catalyzes the hydrolysis of membrane inositol phospholipids; stimulation of aladrenergic receptors, for example, increases polyphosphoinositide turnover, but stimulation of a2-receptors does not. The hydrolysis of inositol phospholipids in rat cortical slices was investigated using a direct assay involving prelabeling these lipids with 3H-inosito1 and then measuring the formation of 3H-inositol phosphates in the presence of lithium ions. As expected, clonidine, an a2-agonist, did not stimulate the formation of 3H-inositol phosphates; however, clonidine antagonized the ability of noradrenaline to stimulate 3H-inositol phosphate formation. This effect was not blocked by antagonists of a2, 5HT2, H2, or muscarinic receptors. Clonidine did not affect carbachol-stimulated 3H-inositol phosphate formation. Clonidine is a centrally acting antihypertensive agent with high affinity for a2-receptors (1,2). It may also be useful in the treatment of depression and anxiety (3-6). Clonidine is usually thought of as a specific a2-receptor agonist; however, in addition to its adrenergic effects, clonidine activates serotonergic 5HT2 and histaminergic H2 receptors (1,7). It also affects other neurotransmitter systems indirectly by its a2-receptor agonism (1,7)_ Several different types of neurotransmitter receptors are coupled to the hydrolysis of membrane inositol phospholipids which generates two second messengers (diacylglycerol and inositol trisphosphate) (8,9). al-Receptors stimulate the breakdown of these membrane lipids, but a2-receptors appear to be inactive; 5HT2 receptors are also coupled to this transduction system (8,9). It is possible, therefore, that clonidine might affect the formation of diacylglycerol and inositol trisphosphate by stimulating these receptors. In a previous study clonidine increased the breakdown of inositol phospholipids in rat pineal gland, vas deferens and caudal artery, but its mechanism of action was not investigated 0024-3205189 $3.00+ -00 Copyright (c) 1989 Pergamon Press plc
994
Clonidine
and Phosphoinositide
Hydrolysis
Vol.
45, No.
11, 1989
(10,ll) . Several other groups, however, found that clonidine did not increase hydrolysis of inositol phospholipids in the rat cortex (12-14). The purpose of this study was to determine whether or not clonidine altered polyphosphoinositide turnover' in slices of rat cerebral cortex and, if so, to attempt to identify the receptor involved. A direct assay which measures the formation of 3H-inosito1 phosphates from inositol phospholipids labeled by preincubation with 3H-inositol was used (15).
Method8 Animals. Male Wistar rats (200-250 g., Charles River Canada) were used. They were housed in hanging wire cages with ad libitum access to food and water. mica& Myoinositol [2-3H(N)] (15 Ci/mmol) was purchased from New England Nuclear (Mississauga, Ont.). It was purified immediately before use by passing it through a column of AGl-X8 ion-exchange resin (formate form, 200-400 mesh, Biorad, Rockville, NY). The hydrochloride salts of clonidine, (-) noradrenaline (NE) and yohimbine were purchased from Sigma Chemical Co. (St. Louis, MO.). Ketanserin was obtained from Janssen Pharmaceutics, idazoxan from Reckitt and Colman, and cimetidine from Smith, Kline and French Labs. . I. oinowde hvdro sis, The accumulation of 3H-inositol phosphates was measured using a modification (16) of the method of Berridge et al., 1982 (15) as described in detail previously. Rats were killed by decapitation, their brains removat room temperature on ice-chilled petri dishes. ed, dissected Cortical slices (350 x 350 p) were incubated for 30 min at 37'C in an atmosphere of 95% 02-5% C02. Then the buffer was aspirated, 50 ~1 of the slices pipetted into tubes containing 1 PCi 3H-myoinosito1 (0.3 @l) and incubated for 60 min at 37'C to label the inositol phospholipids. The 3H-myoinositol-containing buffer was removed, and the hydrolysis of the labeled phospholipids studied by incubating the slices for 60 min under these conditions: control, Krebs buffer; experimental, NE + clonidine f antagonist, carbachol & clonidine, or antagonist. To terminate the incubation, CHC13: methanol (1:2, v/v) was added. The amount of phospholipid labeling was determined by assessing the radioactivity in the chloroform phase. The 3H-inositol phosphates present in the aqueous phase were adsorbed onto an ion-exchange column (AGl-X8). The columns were washed and then, a total 3H-inositol phosphate fraction (containing inositol mono-, bis- and tris-phosphates) was eluted with 0.1 N formic acid-l.0 M ammonium formate and added to ACS liquid scintillant (Amersham, Oakville, Ont.), and radioactivity was determined by liquid scintillation counting. In all experiments, blank samples (cortical slices maintained at 2'C throughout the procedure) were processed. Blank samples gave a count of 50-60 dpm in the chloroform aliquot and loo-160 dpm for the aqueous sample. Results were Blank values were subtracted from the other samples. expressed as dpm present in the inositol phosphate fraction divided by the total dpm present in the lipid and in the inositol phosphate fraction (17). Sometimes the data were converted to percentages of Data were analyzed by control (slices incubated in Krebs buffer).
Vol. 45,
No.
11,
1989
Clonidine and PhosphoinositideHydrolysis
l-way analysis of variance Keuls or a priori t-tests.
and
comparisons
performed
995
by Newman-
. . formatEffect of clonidlne on sH_inositolhate Clonidine was incubated with cortical slices at concentrations from It did not significantly increase the formation 0.1 j.Q.4 to 26 mM. of 3H-inositol phosphates above control levels at any of these concentrations (Table I).
TABLE I Effect of Clonidine
0.1 $ 1.0 PM 100 PM ill-M 26 mM
of 3H-Inositol Phosphate
569 563 599 594 784 694
Production
f 57 f 78 + 99 _+ 84 f 113 f 293
3H-Inositol phosphate production = [(dpm in 3H-inositol phosphate fraction) + (dpm in 3H-inositol phosphate fraction + dpm present in lipids)] x 103. Values are mean f S.D., n = 9.
I I - stimulated ’ Effects of clonldlneol %k itol ohosghate formation. NE (100 PM) stimulated 3Hphosphoinositide turnover to 383 k 17% of control activity. When clonidine was co-incubated with 100 PM NE, it dose-dependently inhibited the ability of NE to stimulate 3H-inositol phosphate formation (Fig. 1). By contrast, the ability of the muscarinic agonist, carbachol, to stimulate 3H-inositol phosphate formation was not affected by clonidine (Fig. 2). . * Effects of antwts on clonidine. Various drugs were tested to see whether they inhibited the ability of clonidine to antagonize the increased 3H-inositol phosphate formation stimulated by NE. None of them had any effect on basal or NE-stimulated 3Hinositol phosphate production (data not shown) nor did they antagonize the ability of clonidine to inhibit NE-stimulated 3H-inositol phosphate production (Table II).
996
Clonidine and Phosphoinositide Hydrolysis
Vol. 45. wo. II, 1989
80 60 -
-5 -6 log [Clonidine)
-7
-4
FIG. 1 Inhibition by clonidine of 3H-inositol phosphate production stimulated by 100 @4 NE. Values are mean f sem, n = 9.
250
1
p 200 * E 150 s
100
R
50 0
q notlon 0 + Clon
O.lmM
Car
1 .O mM Car FIG. 2
Effects of clonidine (Clan) (10 w) on carbachol (Car) - stimulated 3H-inositol phosphate formation. Values are mean f sem, n = 9.
Vol. 45, No. 11, 1989
Clonidineand Phosphoinositide Hydrolysis
997
TABLE II Effects of Antagonists on Inhibition by Clonidine of NE-induced 3H-Inositol Phosphate Formation
100 @4 Clon 11 + 0.1 w 100 w "
+
Yoh
Clon 1 p Ida
100 p.M Clon It + 1 pM Atro
100 w Clan II + 0.01 w
78 f 3% 77 f 4%
10 p Clon '1+ 100 j.04 Cim
44 f 5%
75 f 3% 80 f 3%
10 pM l1 +
40 f 5% 47 f 4%
Clon 0.01 p
Ket
48 f 5%
79 f 2% 81 f 2% Ket
71 f 4% 78 f 3%
values are mean f sem, n=6. Clon = clonidine; Yoh = yohimbine; Ida = idazoxan; Atro = atropine; Cim = cimetidine; Ket ketanserin.
=
The present results show that by itself clonidine had no effect on the hydrolysis of phosphoinositides in slices of rat cortex. It neither increased nor inhibited the basal formation of inositol phosphates. This lack of stimulation agrees with previous reports in rat cortex (12-14) and indicates that clonidine was not an agonist at al or 5HTz receptors which are coupled to phosphoinositide hydrolysis (9,lO). One might have expected clonidine to decrease basal rates of phosphoinositide hydrolysis via stimulation of ag receptors (which would feedback inhibit NE release), if basal activity was caused by unstimulated release of endogenous NE. Clonidine did not, however, decrease basal phosphoinositide hydrolysis. Though clonidine itself had no effect on the basal rate or on carbachol-stimulated 3H-inositol phosphate production (Table 1 and Fig. 21, it significantly inhibited the ability of NE to stimulate 3H-inositol phosphate formation (Fig. 1). This inhibition appears to be physiologically relevant, because NE was inhibited by concentrations of clonidine equal to or less than the concentration of NE used to stimulate phosphoinositide hydrolysis in rat cortical slices. 10 pM Clonidine inhibited 100 @4 NE by 53 f 5%, and 100 m clonidine caused an almost complete inhibition of 100 pM NE (88 f 3% inhibition). Inhibition of NE by clonidine was not blocked by the az-antagonists, yohimbine and idazoxan; the 5HT2 antagonist, ketanserin; the H2 antagonist, cimetidine; nor by atropine, a muscarinic receptor antagonist; therefore, neither a2, 5HT2, H2 nor muscarinic receptors seem to be involved.
998
Clonidine and PhosphoinositideHydrolysis
Vol. 45, No. 11. 1989
Specific binding sites for clonidine and para-aminoclonidine have been found in the bovine and rat brain; these binding sites are distinct from a2 receptors and may represent an imidazole receptor involved in central control of blood pressure (18-20). These imidazole binding sites are thought to be present in the bovine ventrolateral medulla, but not in the frontal cortex (18,22). The brain contains a clonidine-displacing substance, the identity of which is as yet unknown (19-21). Antagonists which are specific for this imidazole receptor have yet to be identified; chlorphenhowever, a number of imidazoles such as cimetidine, iramine and histamine displace clonidine from its binding sites (18). Because neither cimetidine nor idazoxan (another imidazole) inhibited the effects of clonidine on polyphosphoinositide hydrolyreceptor does not seem to be involved in sis, an imidazole mediating clonidine's actions on inositol phospholipid hydrolysis. Compounds which activate protein kinase C cause a feedback inhibition of agonist-stimulated phosphoinositide hydrolysis (9,23). For example, the tumor promoter phorbol esters are kinase C activators, and so too are benzene, toluene, chloroform, and unsaturated free fatty acids (9,23). Clonidine may be sufficiently lipophilic to partition into the cell membrane and affect the interaction of protein kinase C with some of its modulators and If clonidine were a protein kinase C activator, it substrates. would inhibit agonist-induced hydrolysis of phosphoinositides as was observed. Additional studies are required to answer this question. Clonidine has many uses clinically (1,191. In addition to its antihypertensive effects, clonidine has been used to treat opiate withdrawal, migraine, Gilles de la Tourette's syndrome and glaucoma. It may be that some of the therapeutic benefits of clonidine administration arise from its ability to modulate the generation of second messengers from inositol phospholipids induced Interestingly, though clonidine inhibited the hydrolysis of by NE. phosphoinositides induced by NE, it did not affect carbachol. In summary, this paper shows that clonidine by itself has no It had no effect on the hydrolysis of polyphosphoinositides. effect on carbachol-stimulated phosphoinositide hydrolysis, but it A concentration of 10 JLM cloninhibited NE-stimulated hydrolysis. idine decreased the effect of 100 @l NE by 50% . This effect of clonidine did not appear to involve a2-, 5HT2-, Hz- or imidazoleThe mechanism of clonidine's effects on NE-stimulated receptors. formation of inositol phosphates remains to be determined.
The author thanks Dr. A. A. Boulton for advice and encouragefor technical assistance and Saskatchewan ment, R. C. Mag-atas Health for financial support.
Vol. 45, No. 11, 1989
1. 2. 3.
Clonidine and PhosphoinositideHydrolysis
999
Trends in Pharmacological Sciences 2 194-196 W. KOBINGER, (1981). SNYDER, Mol. GREENBERG and S.H. U'PRICHARD, D.A. D.C. Pharmacol. u 454-473 (1977). D.S. CHARNEY, G.R. HENINGER, D.E. STERNBERG, K.M. HAFSTAD, S. GIDDINGS and D.H. LANDIS, Arch. Gen. Psychiatry a 290-294 (1982).
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