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The effect of soluflazine on adenosine receptors in the rat brain
Neuropharmacology Vo1.30, No.1, pp.93-95, 1991 'Printed in Great Britain
0028-3908/91 $3.00+0.00 Pergamon Press plc
THE EFFECT OF SOLUPLAZINEON ADENOSINERECEPTORS IN THE RAT BRAIN S.D.O’Connor, University
M. Hawkins and M. Radulovacki
Department of Pharmacology of Illinois at Chicago, College Chicago, IL 60612 U.S.A.
of Medicine
SUMMARY Soluflazine, a potent adenosine transport inhibitor, was intracerebroventricularly 0.5 L/hr) for 14 days and the administered to rats via ALZET mini osmotic pumps (4nmole, effect on adenosine receptors was determined in specific brain areas. Soluflasine decreased adenosine Al radioligand binding in the hippocampus as measured by [3H]R-PIA, and lowered adenosine A2 binding sites in the striatum, as estimated by the “NFCA minus R-PIA” assay. Previous work from our lab has shown the ability of diasepam and triaeolam to decrease adenosine binding in the same brain areas. The data show that a specific adenosine transport inhibitor produces the same effect on adenosine receptors as benzodiazepines, and suggest a role for adenosine in the CNS effects of bensodiasepines. Key words:
soluflazine,
adenosine
receptors,
benzodiasepines
Benzodiazepines inhibit adenosine uptake in a variety of preparations ( Bender, Wu and ‘Phillis, 1980; Phillis and Wu, 1981; Phillis, Siemans and Wu, 1980; Bender and Hertz, 1986)and ,prolonged administration of the benzodiasepines, diazepam and triasolam, produced decreases in cadioligand binding to adenosine Al receptors in hippocampus and adenosine A2 receptors in the 1988; Hawkins, Hajduk, O’Connor, Radulovacki and ,striatum (Hawkins, Pravica and Radulovacki, The effect on radioligand binding to adenosine receptors by the benzodiazepinee !Stars, 1989). was hypothesized to result from the manipulation of the uptake of adenosine and enhancement of Compounds from the lidoflazine family, such as mioflazine and .ndenosine extracellular levels. soluflazine, have been developed as potential therapeutic agents in ischemia and are potent adenosine transport inhibitors(Van Belle, 1985). Furthermore, soluflasine, the water soluble .snalog of mioflazine, was shown to potentiate the effects of adenosine in guinea pig hippocampal slices ( Ashton, Willems, Deprins, Van Relle and Waquier, 1987). The purpose of this study was to test the hypothesis that adenoslne transport inhibition, by prolonged administration of soluflasine, downregulates adenosine receptors in different regions of the rat brain and to determine whether soluflasine affects adenosine receptors in a similar manner IS bensodiazepines.
Methoda Male Sprague-Dawley rats (300-400~; King, Oregon, WI). were individually housed in cages in 12 h dark cycle. Food and water were a temperature controlled room, under a 12 h light/ Rats provided ad libicum. The animals were allowed one week for adaptation and handling. were anesthetized with a combination of ketamine hydrochloride (8Cmg/kg) and acetapromaside An L-shaped stainless steel cannula (216) was stereotaxlcally implanted into the 1:2mg/ml). :.eft lateral ventricle and connected to an ALZET mini-osmotic pump (model 2002, ALZA, Palo 200 l), containing either 0.9% saline or soluflaslne. The pump, used to Alto, CA; 0.5 L/hr; :I.nsure constant delivery of drug, was connected to the csnnula via polyvinyl tubing (PE 60, Each solution was sterilized by filtration (0.22mm filter, Bolabs, Lake Havasu, AZ). Millipore Inc.) before filling Soluflazine ( 4nmole) was the tubing and the osmotic pump. administered at a rate of 0.5 L/hr for 14 days. the brains were On the final day of pump activity (day 14). rats were sacrificed and pumps were cut open to verify that the contents had been rapidly removed. Alzet mini-osmotic on an ice cold delivered. The cortex, cerebellum, hippocampus and striatum were dissected Crude membrane fractions of metal plate and stored at -7OOC until assays were performed. each hrain structure were prepared as previously described (Hawkins et al., 1988). cerebellar and hippocampal membranes and estimations Both saturation isotherms on cortical, of Al and A2 receptor numher were made using the method of Yeung and Green, (1984). Binding Scatchard
parameters for adenosine Al receptors were determined by least square analysis transformation of the [3H]R-PIA saturation isotherm data. Adenosine A2 receptor 93
of
94
Preliminary Notes
binding was estimated by the “NECA minus R-PIA” method described Statistical analyses were perfowed with Student’s t-test.
by Yeung snd Green (1984).
Table 1 shows the results of the Scatchard analysis of [3H]R-PIA bindinq. tow dose of soluflazine (4nmole; 0.5 l/hr) produced a significant decrease (27%) in the [3H]R-PIA Bmax determined in hippocampal membranes (404 * 12, ~(0.05, N-4) as compared with control values (553 2 25, N*4). no &nificant changes ware observed in cortical and In contrast, cerebellar aembranes, which may be a reflection of an inadequate concentration reaching these areas. Furthermore, this dose of soluflazine failed to alter the receptor affinity in membranes prepared from all brain areas examined.
Table
1:[3H]R-PIA after
binding
to
14 days
of
different soluflazine
CORTEX Control
Bmax Kd
Soluflazine
Bmax Kd
573 J- 13 0.88
2 0.17
629 + 24 0.57
& 0.14
brain areas treatment
pIPPOCAMPUS 553 & 25 1.34
2 0.38
404 & 12* 1.35
+ 0.68
of
the
rat
CEREBELLUM 1225 rt 75 4.43
kO.27
1052
2 94
3.98
LO.24
Bmax ( fmol/mg protein) and Kd (nM) values (means + S.E.M.) were determined from Scatchard analysis of saturation binding Statistical analysis using Student’s t-test revealed experiments. significant decreases in Bmax in the rats treated with lnmole
soluflazine, delivered at a rate of O.SpL/hr, *=p>O.O5.
Table 2 summarizes the results of the NECA minus PIA assays. In control striatal membranes the estimate of the sum total. of Al and A2 receptors was 847 + 53 fmol/mg protein and the estimate of Al receptors was 456 + 33 fmoljmg protein. Chronic treatment with soluflazine decreased the [3H]NECA binding (720 + 27 fmol/mg protein (p
The present study showed that chronic treatment of soluflazine caused a decrease in adenosine Al binding sites in the hippocampus (Table 1) and decreases in A2 binding sites in the striatum, as determined by the [3H]NECA minus [3HlR-PIA assay (table 2). These findings are in agreement with the effects on adenosine receptors of two benzodiazepines, reported to inhibit adenosine transport, dlazepam (Phillis et al., 1980) and triazolam (O’Regan and Phillis, 1988), whose prolonged administration to rats resulted in a decrease in the number of adenosine Al radloligand binding sites in the hippocampus and adenosine A2 sites in the striatum (Hawkins et al., 1988; Hawkins et al., 1989). While it is believed that the benzodiazepines may interact with adenosine transport to produce their effect, it is not understood how blockade of this transport would affect adenosine receptor populations. In accordance, when soluflazlne was topCcally applied to cerebral. cortex it did not affect the levels of purines released in both normoxic and hypoxic rats as measured by cortical cup technique (Phillis, O’Regan and Walter, 1989). However, soluflazine has been reported to potentiate the effect of adenosine on population spikes in hippocampal slices, depressing neuronal activity (Ashton et al., 1987). Furthermore, since soluflazine has been shown to have a high specificity and nanomoIar affinity for adenosine transport inhibition, the present results indicnte that soluflazine can alter adenosine receptor binding iq specific rat brain regions in a manner similar to prolonged treatment with benzodiazepines and argue for an involvement of adenosine in the action of the benzodiazepines in the CNS.
PreliminaryNotes
95
Table 2: [3H]R-PIA and [3H]NECA binding to rat striatal membranes after 14 days of soluflazine treatment TREATMENT
[3H]NECA (Al + A2)
[3H]R-PIA (Al)
CONTROL (N=lO)
a47 f. 53
456 + 33
390 + 29
SOLUFLAZINE (N=ll)
720 + 27*
437 + 21
283 + 25*
[3H]NECA-[3H]R-PIA (A2)
Striatal membranes were simultaneously prepared from control rats and rats treated with 4nmole soluflazine, i.c.v., delivered at a rate of O.SNL/hr. The data are means + S.E.M. and represent the approximations of ligand specifically bound as determined by single saturating concentrations of [3H]NECA (200pM) and [3H]R-PIA (50NM). Statistical analysis using Student's t-test revealed significant differences between the control and soluflazine groups in binding densities at A2 receptors, *=p>O.O5.
References Hahton D., Willema R., DePrina IX.,Van Belle H. and Waquier A. (1987) The
nucleoaidetransport inhibitor soluflasine ( R 64 719) increases the effects of adenosine in the guinea-pig hippocampal slice and is antagonized by adenosine deaminaae. Eur. J. Pharmacol. 142: 403-408.
Hender A.S. and Hertz L. (1986) Similarities of adenosine uptake systems in aatrocytes and neuronea in primary cultures. Neurochem. Res. 11: 1507-1524. Bender A.S., Wu P.H. and Phillis J.W. (1980) The characterization of[3H]adenoaine uptake into rat cerebral cortical aynaptosomea. J. Neurochem. 35: 629-640. of diazepam Hawkins M. Pravica M. and Radulovacki M. (1988) Chronic administration downregulates adenoaine receptors in the rat brain. Pharmac. Biochem. Behav. 30(2): 303308. Mawkina M., Hajduk P., O'Connor S.D., Radulovacki M. and Stars K. (1989) Effects of prolonged administration on adenosine Al and A2 receptors in the brain of rats. Brain Res. 505: 141-144. O'Regan M.H. and Phillia J.W. (1988) Potentiation of adenosine evoked depression cerebral cortical neurons by triazolam. Brain Res. 445: 376-379.
of rat
Philtis J.W., Siemena R-K. and Wu P.H. (1980) Effects of diazepam on adenoaine acetylcholine release from rat cerebral cortex: further evidence for a purinergic mechanism in action of diasepam. Br. J. Pharmacol. 70: 341-348. Phillis .J.W. and Wu P.H. (1981) The role of adenosine and its nucleotidea in central synaptic transmission. Prog. Neurobiol. 16: 187-239. transport Phillis J.W., O'Regan M.H. and Walter G.A. (1989) Effects of two nucleoaide inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex. Brain Rea. 481: 309-316. Van
Belle H. (1985) Myocardial purines during protection Mol. Physiol. 8: 615-630.
ischemia,
reperfusion
and pharmacological
Yeung S.-M.H. and Green R.D. (1984) [3H]5'-N-ethylcarboxamide adenoaine binds to both R, and Ri adenosine receptors in rat striatum. Naunyn-Schmiedeherg's Arch. Pharmacol. 325: 218225.
0028-3908/91 $3.00+0.00 Pergamon Press plc
THE EFFECT OF SOLUPLAZINEON ADENOSINERECEPTORS IN THE RAT BRAIN S.D.O’Connor, University
M. Hawkins and M. Radulovacki
Department of Pharmacology of Illinois at Chicago, College Chicago, IL 60612 U.S.A.
of Medicine
SUMMARY Soluflazine, a potent adenosine transport inhibitor, was intracerebroventricularly 0.5 L/hr) for 14 days and the administered to rats via ALZET mini osmotic pumps (4nmole, effect on adenosine receptors was determined in specific brain areas. Soluflasine decreased adenosine Al radioligand binding in the hippocampus as measured by [3H]R-PIA, and lowered adenosine A2 binding sites in the striatum, as estimated by the “NFCA minus R-PIA” assay. Previous work from our lab has shown the ability of diasepam and triaeolam to decrease adenosine binding in the same brain areas. The data show that a specific adenosine transport inhibitor produces the same effect on adenosine receptors as benzodiazepines, and suggest a role for adenosine in the CNS effects of bensodiasepines. Key words:
soluflazine,
adenosine
receptors,
benzodiasepines
Benzodiazepines inhibit adenosine uptake in a variety of preparations ( Bender, Wu and ‘Phillis, 1980; Phillis and Wu, 1981; Phillis, Siemans and Wu, 1980; Bender and Hertz, 1986)and ,prolonged administration of the benzodiasepines, diazepam and triasolam, produced decreases in cadioligand binding to adenosine Al receptors in hippocampus and adenosine A2 receptors in the 1988; Hawkins, Hajduk, O’Connor, Radulovacki and ,striatum (Hawkins, Pravica and Radulovacki, The effect on radioligand binding to adenosine receptors by the benzodiazepinee !Stars, 1989). was hypothesized to result from the manipulation of the uptake of adenosine and enhancement of Compounds from the lidoflazine family, such as mioflazine and .ndenosine extracellular levels. soluflazine, have been developed as potential therapeutic agents in ischemia and are potent adenosine transport inhibitors(Van Belle, 1985). Furthermore, soluflasine, the water soluble .snalog of mioflazine, was shown to potentiate the effects of adenosine in guinea pig hippocampal slices ( Ashton, Willems, Deprins, Van Relle and Waquier, 1987). The purpose of this study was to test the hypothesis that adenoslne transport inhibition, by prolonged administration of soluflasine, downregulates adenosine receptors in different regions of the rat brain and to determine whether soluflasine affects adenosine receptors in a similar manner IS bensodiazepines.
Methoda Male Sprague-Dawley rats (300-400~; King, Oregon, WI). were individually housed in cages in 12 h dark cycle. Food and water were a temperature controlled room, under a 12 h light/ Rats provided ad libicum. The animals were allowed one week for adaptation and handling. were anesthetized with a combination of ketamine hydrochloride (8Cmg/kg) and acetapromaside An L-shaped stainless steel cannula (216) was stereotaxlcally implanted into the 1:2mg/ml). :.eft lateral ventricle and connected to an ALZET mini-osmotic pump (model 2002, ALZA, Palo 200 l), containing either 0.9% saline or soluflaslne. The pump, used to Alto, CA; 0.5 L/hr; :I.nsure constant delivery of drug, was connected to the csnnula via polyvinyl tubing (PE 60, Each solution was sterilized by filtration (0.22mm filter, Bolabs, Lake Havasu, AZ). Millipore Inc.) before filling Soluflazine ( 4nmole) was the tubing and the osmotic pump. administered at a rate of 0.5 L/hr for 14 days. the brains were On the final day of pump activity (day 14). rats were sacrificed and pumps were cut open to verify that the contents had been rapidly removed. Alzet mini-osmotic on an ice cold delivered. The cortex, cerebellum, hippocampus and striatum were dissected Crude membrane fractions of metal plate and stored at -7OOC until assays were performed. each hrain structure were prepared as previously described (Hawkins et al., 1988). cerebellar and hippocampal membranes and estimations Both saturation isotherms on cortical, of Al and A2 receptor numher were made using the method of Yeung and Green, (1984). Binding Scatchard
parameters for adenosine Al receptors were determined by least square analysis transformation of the [3H]R-PIA saturation isotherm data. Adenosine A2 receptor 93
of
94
Preliminary Notes
binding was estimated by the “NECA minus R-PIA” method described Statistical analyses were perfowed with Student’s t-test.
by Yeung snd Green (1984).
Table 1 shows the results of the Scatchard analysis of [3H]R-PIA bindinq. tow dose of soluflazine (4nmole; 0.5 l/hr) produced a significant decrease (27%) in the [3H]R-PIA Bmax determined in hippocampal membranes (404 * 12, ~(0.05, N-4) as compared with control values (553 2 25, N*4). no &nificant changes ware observed in cortical and In contrast, cerebellar aembranes, which may be a reflection of an inadequate concentration reaching these areas. Furthermore, this dose of soluflazine failed to alter the receptor affinity in membranes prepared from all brain areas examined.
Table
1:[3H]R-PIA after
binding
to
14 days
of
different soluflazine
CORTEX Control
Bmax Kd
Soluflazine
Bmax Kd
573 J- 13 0.88
2 0.17
629 + 24 0.57
& 0.14
brain areas treatment
pIPPOCAMPUS 553 & 25 1.34
2 0.38
404 & 12* 1.35
+ 0.68
of
the
rat
CEREBELLUM 1225 rt 75 4.43
kO.27
1052
2 94
3.98
LO.24
Bmax ( fmol/mg protein) and Kd (nM) values (means + S.E.M.) were determined from Scatchard analysis of saturation binding Statistical analysis using Student’s t-test revealed experiments. significant decreases in Bmax in the rats treated with lnmole
soluflazine, delivered at a rate of O.SpL/hr, *=p>O.O5.
Table 2 summarizes the results of the NECA minus PIA assays. In control striatal membranes the estimate of the sum total. of Al and A2 receptors was 847 + 53 fmol/mg protein and the estimate of Al receptors was 456 + 33 fmoljmg protein. Chronic treatment with soluflazine decreased the [3H]NECA binding (720 + 27 fmol/mg protein (p
The present study showed that chronic treatment of soluflazine caused a decrease in adenosine Al binding sites in the hippocampus (Table 1) and decreases in A2 binding sites in the striatum, as determined by the [3H]NECA minus [3HlR-PIA assay (table 2). These findings are in agreement with the effects on adenosine receptors of two benzodiazepines, reported to inhibit adenosine transport, dlazepam (Phillis et al., 1980) and triazolam (O’Regan and Phillis, 1988), whose prolonged administration to rats resulted in a decrease in the number of adenosine Al radloligand binding sites in the hippocampus and adenosine A2 sites in the striatum (Hawkins et al., 1988; Hawkins et al., 1989). While it is believed that the benzodiazepines may interact with adenosine transport to produce their effect, it is not understood how blockade of this transport would affect adenosine receptor populations. In accordance, when soluflazlne was topCcally applied to cerebral. cortex it did not affect the levels of purines released in both normoxic and hypoxic rats as measured by cortical cup technique (Phillis, O’Regan and Walter, 1989). However, soluflazine has been reported to potentiate the effect of adenosine on population spikes in hippocampal slices, depressing neuronal activity (Ashton et al., 1987). Furthermore, since soluflazine has been shown to have a high specificity and nanomoIar affinity for adenosine transport inhibition, the present results indicnte that soluflazine can alter adenosine receptor binding iq specific rat brain regions in a manner similar to prolonged treatment with benzodiazepines and argue for an involvement of adenosine in the action of the benzodiazepines in the CNS.
PreliminaryNotes
95
Table 2: [3H]R-PIA and [3H]NECA binding to rat striatal membranes after 14 days of soluflazine treatment TREATMENT
[3H]NECA (Al + A2)
[3H]R-PIA (Al)
CONTROL (N=lO)
a47 f. 53
456 + 33
390 + 29
SOLUFLAZINE (N=ll)
720 + 27*
437 + 21
283 + 25*
[3H]NECA-[3H]R-PIA (A2)
Striatal membranes were simultaneously prepared from control rats and rats treated with 4nmole soluflazine, i.c.v., delivered at a rate of O.SNL/hr. The data are means + S.E.M. and represent the approximations of ligand specifically bound as determined by single saturating concentrations of [3H]NECA (200pM) and [3H]R-PIA (50NM). Statistical analysis using Student's t-test revealed significant differences between the control and soluflazine groups in binding densities at A2 receptors, *=p>O.O5.
References Hahton D., Willema R., DePrina IX.,Van Belle H. and Waquier A. (1987) The
nucleoaidetransport inhibitor soluflasine ( R 64 719) increases the effects of adenosine in the guinea-pig hippocampal slice and is antagonized by adenosine deaminaae. Eur. J. Pharmacol. 142: 403-408.
Hender A.S. and Hertz L. (1986) Similarities of adenosine uptake systems in aatrocytes and neuronea in primary cultures. Neurochem. Res. 11: 1507-1524. Bender A.S., Wu P.H. and Phillis J.W. (1980) The characterization of[3H]adenoaine uptake into rat cerebral cortical aynaptosomea. J. Neurochem. 35: 629-640. of diazepam Hawkins M. Pravica M. and Radulovacki M. (1988) Chronic administration downregulates adenoaine receptors in the rat brain. Pharmac. Biochem. Behav. 30(2): 303308. Mawkina M., Hajduk P., O'Connor S.D., Radulovacki M. and Stars K. (1989) Effects of prolonged administration on adenosine Al and A2 receptors in the brain of rats. Brain Res. 505: 141-144. O'Regan M.H. and Phillia J.W. (1988) Potentiation of adenosine evoked depression cerebral cortical neurons by triazolam. Brain Res. 445: 376-379.
of rat
Philtis J.W., Siemena R-K. and Wu P.H. (1980) Effects of diazepam on adenoaine acetylcholine release from rat cerebral cortex: further evidence for a purinergic mechanism in action of diasepam. Br. J. Pharmacol. 70: 341-348. Phillis .J.W. and Wu P.H. (1981) The role of adenosine and its nucleotidea in central synaptic transmission. Prog. Neurobiol. 16: 187-239. transport Phillis J.W., O'Regan M.H. and Walter G.A. (1989) Effects of two nucleoaide inhibitors, dipyridamole and soluflazine, on purine release from the rat cerebral cortex. Brain Rea. 481: 309-316. Van
Belle H. (1985) Myocardial purines during protection Mol. Physiol. 8: 615-630.
ischemia,
reperfusion
and pharmacological
Yeung S.-M.H. and Green R.D. (1984) [3H]5'-N-ethylcarboxamide adenoaine binds to both R, and Ri adenosine receptors in rat striatum. Naunyn-Schmiedeherg's Arch. Pharmacol. 325: 218225.