Brain Research, 538 (1991) 193-195
193
Elsevier BRES 16221
Differential changes of retina dopamine binding sites and adenylyl cyclase responses following 6-hydroxydopamine treatment Maria Hadjiconstantinou 1'2, Zhi-Xiang Qu 2 and Norton H. Neff 2 Departments of ~Psychiatry and of 2pharmacology, The Ohio State University College of Medicine, Columbus, OH 43210 (U.S.A.) (Accepted 7 August 1990)
Key words: Retina; D 1 and D2 dopamine receptor; Cyclic AMP; Adenylyl cyclase; Supersensitivity
Both dopamine D, and D 2 receptors are present in rat retina. D~ receptors are positively coupled to adenylyl cyclase, while D 2 receptors are negatively coupled. After intraocular administration of the neurotoxin 6-hydroxydopamine (6-OHDA) there is depletion of retinal dopamine by about 90%, as well as a decrease of the number of D, and D2 receptor binding sites. Following the 6-OHDA lesion, there is an enhancement of the D~ receptor-stimulated accumulation of cyclic-AMP and a loss of D 2 receptor-inhibited accumulation of cyclic-AMP. Our results suggest that some of the retinal D2 receptors are coupled to adenylyl cyclase and some are not.
INTRODUCTION
to A C
and some
are
not,
and that
they can be
differentiated by destroying the dopaminergic neurons. D o p a m i n e ( D A ) in the rat retina is contained in a subpopulation of amacrine cells5 and in some interplexiform cells 13. D A O 1 and D 2 receptors are present as well 16. Generally, activation of D 1 receptors enhances while activation of O 2 receptors suppresses the accumulation of cyclic-AMP (cAMP) 6'1s. In some n e u r o n a l tissues O 2 receptors may be coupled to second messenger systems other than adenylyl cyclase ( A C ) 1'2'8'14'19. Following the destruction of catecholaminergic neurons with a neurotoxin, there is a loss of the amine neurotransmitter, a loss of presynaptic ligand binding sites, and there is often an increased responsiveness of postsynaptic receptors that have been deprived of neurotransmitter, i.e. supersensitivity. We have administered the neurotoxin 6-hydroxydopamine ( 6 - O H D A ) intraocularly and evaluated receptor selective ligand binding and activation of A C by D A and the selective D 2 agonists bromocriptine and quinpirole. Seven days after the administration of the neurotoxin there was a decrease in the binding for both D 1 and D E receptors, implying that both receptor subtypes are probably located on dopaminergic neurons. Concomitantly, stimulation with D A resulted in enhanced accumulation of cAMP, implying D 1 receptor supersensitivity. Interestingly, D 2 receptors lost their ability to suppress the accumulation of c A M P in the lesioned retinas. The latter finding suggests that some D E receptors are coupled
MATERIALS AND METHODS Adult male Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienople, PA), 150-200 g, were housed under a 12:12 h light:dark cycle. They were anesthetized with diethyl ether and injected intravitreously with 6-OHDA or saline. Two injections of 6-OHDA, 30 pg/4 /d of saline, were injected into the right eye. The two injections of 6-OHDA were administered 24 h apart. An equal volume of sterile saline was injected into the left eye as a control preparation. The rats were decapitated 7 days following the injections and retinae isolated and prepared for analysis. DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were analyzed by HPLC with electrochemical detection after alumina extraction3. For cAMP determination, animals were euthanized at mid-day and retinae dissected and placed in Ringer's solution, pH 7.4. After a 2 min preincubation, DA, 100 gM, bromocriptine, 100 gM, or quinpirole, 100 gM, was added and incubations continued for 10 additional min. The reaction conditions were determined to be linear with protein and time of incubation during this period. SCH 23390, when present, was added during the 2 min preincubation period. Reactions were terminated by placing the samples in a boiling water bath for 3 min. cAMP was measured in the medium by radioimmunoassay16. For receptor binding studies4, retinae were dissected and homogenized with Tris-HCI buffer 50 mM, pH 7.5, in a Tissumizer (Tekmar, Cincinnati, OH) and centrifuged at 20,000 x g for 10 min. The particulate fraction was washed twice with buffer and then suspended in the Tris-HCI buffer containing (mM):MgCl2 1, NaCl 120, KCI 5 and CaCI2 2. The ligands studied were [3H]SCH 23390 (80 Ci/mmol) and [3H]spiperone (30 Ci/mmol) for D 1 and D2 receptor binding sites, respectively. Incubationswere carried out for 15 min at 37°. The reaction was stopped by rapid filtration followed by washing using a Brandel cell harvester (Gaithersburg, MD). Radioactivity on the filters was determined using a liquid scintilla-
Correspondence: N.H. Neff, Department of Pharmacology, The Ohio State University College of Medicine, 5198 Graves Hall, Columbus, OH 43210, U.S.A. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
194 TABLE I
TABLE II
Intraoeular administration of 6-OHDA and dopamine receptor binding characteristics in rat retina
Dopaminergic receptor-induced accumulation of cAMP in the rat retina following intraocular administration o f 6-OHDA
Two doses of 6-OHDA, 30 gg in 4 gl administered 24 h apart, were injected into the right and saline into the left eye of anesthetized rats. Animals were euthanized 7 days later. The ligand for D 1 sites was [3H]SCH 23390 and for the De sites [3H]spiperone. Bma x values, Imol/mg prot, and Ka values, nM, are expressed as the mean _+ S.E.M. for 3-5 experiments performed in duplicate. The % change values refer to saline.
6-OHDA, 30gg in 4#1 saline, was administered in the right eye and saline in the left eye of anesthetized rats, 2 injections 24 h apart. The animals were euthanized V days after the injections. Retinae were dissected and cAMP determined as described in the methods, n = 12-19. cAMP values found when SCH 23390 was present alone did not differ from basal activity for saline or 6-OHDA groups. Quin, quinpirole; Bromo, bromociptine; SCH, SCH 23390. A refers to cAMP accumulation compared with basal activity.
Treatment
D 1 dopamine binding sites
D 2 dopamine
B,~
Bmax
binding sites Conditions
Saline 6-OHDA
%
98_+3 61 -+3* -38
Kd
%
0.63_+0.1 6 4 _ + 3 0.74-+0.04 36_+3* -44
cAMP (pmol/mg prot/lO rain + S. E. M.)
Kd
0.69+0.09 0.52-+0.07
*P < 0.05 compared with saline-treated retina.
tion spectrometer. Specific binding was defined as the difference between total binding and that found in the presence of 1 /~M ketanserin and sulpiride for [3H]spiperone, and in the presence of 1/~M SCH 23390 for [3H]SCH 23390. Protein was assayed by the method of Lowry et al. 9, using bovine serum albumin as the standard. Brn~xand Kd values were calculated from Scatchard curve analysis using the microcomputer program GraphPAD (I.S.I. Philadelphia, PA). Multiple group comparisons were performed using a Newman-Keuls post hoc test after analysis of variance. A t-test for paired observations was used when values obtained for the saline-treated eye was compared with the 6-OHDA-treated eye.
RESULTS
Basal DA (100/~M) SCH (10/~M) plus DA Quin (100/~M) Bromo (100/~M)
Saline
d
6-OHDA
d
49 _+ 1 116+5" 36 + 1" 36 + 1" 37 + 2*
+67 -13 -13 -12
37 + 2** 151 +6*'** 87 + 76"** 39 ___4 39 + 2
+114 +50 +2 +2
*P < 0.05 compared with appropriate basal activity. **P < 0.05 compared with corresponding saline-treated groups. significantly decrease c A M P accumulation in the 6O H D A - t r e a t e d retina. Stimulation of 6 - O H D A - t r e a t e d retinae with D A in the presence of SCH 23360 resulted in an augmentation of c A M P accumulation by about 135% over basal activity. Similar conclusions are obtained if the absolute changes of c A M P accumulation are considered (Table II). DISCUSSION
Seven days following the administration 6 - O H D A
There is now overwhelming evidence from a variety of
there was a 90% fall of retina D A and 3,4-dihydroxy-
tissues that most D 1 receptors are positively coupled to A C which upon activation leads to an increased accu-
phenylacetic acid ( D O P A C ) (data not shown). There was also a significant fall of the Bmax value, about 38 and 44%, for both D 1 and D 2 sites respectively (Table I). In contrast, the K a values were relatively unaffected. D A is a non-selective activator of D~ and D 2 receptors. In the retina the algebraic sum of activation of these receptors is to enhance c A M P accumulation, primarily a D 1 receptor function (Table II). Bromocriptine and quinpirole, selective agonists of D 2 receptors, caused a significant fall of c A M P accumulation in retina. Similarly, stimulation of D2 receptors with D A after blockade of D 1 receptors with the selective antagonist SCH 23360 resuited in a significant reduction of cAMP production (Table II). After 6 - O H D A treatment there was a significant decrease in the basal cAMP values, about 28% of the saline treated retinae. Stimulation of the 6O H D A - t r e a t e d retina with D A resulted in a greater increase of the cAMP, about 300% over the corresponding basal activity, than in the saline treated group, about 136% over the corresponding basal activity. The selective agonists bromocriptine and quinpirole both failed to
mulation of cAMP. In contrast, some D 2 receptors are negatively coupled to A C and their activation leads to a fall of cAMP 6'x8. There is also evidence suggesting that some D e receptors are not coupled to A C , and are probably linked to other second messenger systems. For example, both D 1 and D E receptors are linked to A C in the striatum, while in the limbic forebrain it appears that only D 1 receptors are coupled to A C even though D 2 receptors are found there 8. In nucleus accumbens slices, activation of O 2 receptors inhibit D A and acetylcholine release i n d e p e n d e n t of c A M P formation 19. In addition, there are reports that O 2 receptors activate potassium channels2 or inhibit inositol phosphate generation a'la. In the retina, there are guanine nucleotide sensitive and insensitive classes of D A receptors 1°. O u r studies, like many studies in the literature from other tissues, are consistent with the hypothesis that some O 2 receptors in the retina are coupled to A C and some are not. Following treatment with 6 - O H D A there was about a 40% reduction of both D 1 and D 2 bindings sites,
195 suggesting that the lost receptors were associated with lost d o p a m i n e r g i c neurons. Since amacrine cells synapse with o t h e r amacrine cells, it is difficult to d e t e r m i n e if the r e c e p t o r loss is due solely to loss of presynaptic or both pre- and postsynaptic D A receptors ~. The robust accumulation of c A M P implies that the remaining D 1 receptors are supersensitive to D A . Similar responses were found in retina following the selective neurotoxin M P T P 17. T h e loss of Dx receptor binding sites and the enhanced response of A C to D A might indicate that r e c e p t o r supersensitivity can occur without up-regulation of binding sites. Missale et a1.12 r e p o r t e d that depletion of neuronal D A with reserpine resulted in up-regulation of the transduction mechanism for generating c A M P by D 1 receptors, but not of the n u m b e r of binding sites. E n h a n c e d efficiency of the guanine nucleotide coupling protein to A C was suggested. T h e loss of about 45% of D 2 binding sites and the lack of a response to activation of A C by bromocriptine or quinpirole following 6 - O H D A implies that some D 2 receptors are not coupled to the enzyme. O u r observation that b l o c k a d e of D 1 receptors with S C H 23360
followed by activation with D A in the 6 - O H D A - t r e a t e d animals e n h a n c e d the accumulation of c A M P was unexpected. We i n t e r p r e t e d this result to indicate that S C H 23360 is only able to partially prevent the D A - i n d u c e d activation of the supersensitive D1 receptors. A l t e r n a tively, if both D 1 and D 2 receptors coupled to A C are located on the same n o n - d o p a m i n e r g i c neurons, it is possible that there is differential m o d u l a t i o n of the A C coupled system with the o u t c o m e favoring e n h a n c e m e n t of D 1 function and suppression of D 2 function 7,s. The loss of D E inhibition of c A M P accumulation in the 6O H D A - t r e a t e d retina was surprising, as we previously r e p o r t e d that muscarinic inhibition of c A M P accumulation in the retina was enhanced following the intraocular injection of the cholinotoxin A F 6 4 A ~5. In summary, our studies provide evidence that both D 1 and D 2 receptors are located pre- and postsynaptically in rat retina and support the hypothesis that some D 2 receptors are negatively coupled to A C and some receptors are not. D 2 receptors negatively coupled to A C are not evident following intraocular 6 - O H D A .
REFERENCES
Retina contains guanine nucleotide sensitive and insensitive classes of dopamine receptors, Eur. J. Pharmacol., 63 (1980) 217-222. 11 Mariani, A.P., Kolb, H. and Nelson, R., Dopamine-containing amacrine cells of Rhesus monkey retina parallel rods in spatial distribution, Brain Research, 322 (1984) 1-7. 12 Missale, C., Nisoli, E., Liberini, P., Rizzonelli, P., Memo, M., Buonamici, M., Rossi, A. and Spano, P.E, Repeated reserpine administration up-regulates the transduction mechanisms of D1 receptors without changing the density of [3H]SCH 23390 binding, Brain Research, 483 (1989) 117-122. 13 Nguyen-Legros, J., Berger, B., Vigny, A. and Alvarez, C., Presence of interplexiform dopaminergic neurons in the rat retina, Brain Res. Bull., 9 (1982) 379-381. 14 Pizzi, M., Da Prada, M., Valerio, A., Memo, M., Spano, P.E and Haefely, W.E., Dopamine D2 receptor stimulation inhibits inositol phosphate generating system in rat striatal slices, Brain Research, 456 (1988) 235-240. 15 Qu, Z.-X., Fertel, R., Neff, N.H. and Hadjiconstantinou, M., Pharmacological characterization of muscarinic receptors mediating inhibition of adenylate cyclase activity in the rat retina, J. Pharmacol. Exp. Ther., 246 (1988) 839-842. 16 Qu, Z.-X., Fertel, R., Neff, N.H. and Hadjiconstantinou, M., Pharmacological characteristics of rat retinal dopamine receptors, J. Pharmacol. Exp. Ther., 248 (1989) 621-625. 17 Qu, Z.X., Neff, N.H. and Hadjiconstantinou, M., MPP+ depletes retinal dopamine and induces D-1 receptor supersensitivity, Eur. J. Pharmacol., 148 (1988) 453-455. 18 Stool, J.C. and Kebabian, J.W., Two dopamine receptors: biochemistry, physiology, and pharmacology, Life Sci., 35 (1984) 2281-2296. i9 Stoof, J.C., Verheijden, P.F.H.M. and Leysen, J.E., Stimulation of D2-receptors in rat nucleus accumbens slices inhibits dopamine and acetycholine release but not cyclic AMP formation, Brain Research, 423 (1987) 364-368.
1 Enjaibert, A., Sladeczek, E, Guillon, G., Bertrand, P., Shu, C., Epelbaum, J., Garcia-Sainz, A., Jard, S., Lombard, C., Kordon, C. and Bockaert, J., Angiotensin II and dopamine modulate both cAMP and inositol phosphate productions in anterior pituitary cells: involvement in prolactin secretion, J. Biol. Chem., 261 (1986) 4071-4075. 2 Freedman, J.E. and Weight, EE, Single K+ channels activated by D2 dopamine receptors in acutely dissociated neurons from rat corpus striatum, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 3618-3622. 3 Hadjiconstantinou, M. and Neff, N.H., Treatment with GM1 ganglioside restores striatal dopamine in the 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine-treated mouse, J. Neurochem., 51 (1988) 1190-1196. 4 Hadjiconstantinou, M., Weihmuller, F.B. and Neff, N.H., Treatment with GM1 ganglioside reverses dopamine D-2 receptor supersensitivity induced by the neurotoxin MPTP, Eur. J. Pharmacol., 168 (1989) 261-264. 5 Haggendal, J. and Maimfors, T., Identification and cellular localization of the catecholamines in the retina and the chroid of the rabbit, Acta Physiol. Scand., 64 (1965) 58-66. 6 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature, 277 (1979) 93-96. 7 Kelly, E. and Nahorski, S.R., Dopamine receptor-effector mechanisms, Rev. Neurosci., 1 (1986) 35-54. 8 Kelly, E. and Nahorski, S.R., Dopamine D-2 receptors inhibit D-1 stimulated cyclic AMP accumulation in striatum but not limbic forebrain, Naunyn. Schmiedebergs. Arch. Pharmacol., 335 (1987) 508-512. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 Makman, M.H., Dvorkin, B., Horowitz, S.G. and Thai, L.J.,
193
Elsevier BRES 16221
Differential changes of retina dopamine binding sites and adenylyl cyclase responses following 6-hydroxydopamine treatment Maria Hadjiconstantinou 1'2, Zhi-Xiang Qu 2 and Norton H. Neff 2 Departments of ~Psychiatry and of 2pharmacology, The Ohio State University College of Medicine, Columbus, OH 43210 (U.S.A.) (Accepted 7 August 1990)
Key words: Retina; D 1 and D2 dopamine receptor; Cyclic AMP; Adenylyl cyclase; Supersensitivity
Both dopamine D, and D 2 receptors are present in rat retina. D~ receptors are positively coupled to adenylyl cyclase, while D 2 receptors are negatively coupled. After intraocular administration of the neurotoxin 6-hydroxydopamine (6-OHDA) there is depletion of retinal dopamine by about 90%, as well as a decrease of the number of D, and D2 receptor binding sites. Following the 6-OHDA lesion, there is an enhancement of the D~ receptor-stimulated accumulation of cyclic-AMP and a loss of D 2 receptor-inhibited accumulation of cyclic-AMP. Our results suggest that some of the retinal D2 receptors are coupled to adenylyl cyclase and some are not.
INTRODUCTION
to A C
and some
are
not,
and that
they can be
differentiated by destroying the dopaminergic neurons. D o p a m i n e ( D A ) in the rat retina is contained in a subpopulation of amacrine cells5 and in some interplexiform cells 13. D A O 1 and D 2 receptors are present as well 16. Generally, activation of D 1 receptors enhances while activation of O 2 receptors suppresses the accumulation of cyclic-AMP (cAMP) 6'1s. In some n e u r o n a l tissues O 2 receptors may be coupled to second messenger systems other than adenylyl cyclase ( A C ) 1'2'8'14'19. Following the destruction of catecholaminergic neurons with a neurotoxin, there is a loss of the amine neurotransmitter, a loss of presynaptic ligand binding sites, and there is often an increased responsiveness of postsynaptic receptors that have been deprived of neurotransmitter, i.e. supersensitivity. We have administered the neurotoxin 6-hydroxydopamine ( 6 - O H D A ) intraocularly and evaluated receptor selective ligand binding and activation of A C by D A and the selective D 2 agonists bromocriptine and quinpirole. Seven days after the administration of the neurotoxin there was a decrease in the binding for both D 1 and D E receptors, implying that both receptor subtypes are probably located on dopaminergic neurons. Concomitantly, stimulation with D A resulted in enhanced accumulation of cAMP, implying D 1 receptor supersensitivity. Interestingly, D 2 receptors lost their ability to suppress the accumulation of c A M P in the lesioned retinas. The latter finding suggests that some D E receptors are coupled
MATERIALS AND METHODS Adult male Sprague-Dawley rats (Zivic-Miller Laboratories, Zelienople, PA), 150-200 g, were housed under a 12:12 h light:dark cycle. They were anesthetized with diethyl ether and injected intravitreously with 6-OHDA or saline. Two injections of 6-OHDA, 30 pg/4 /d of saline, were injected into the right eye. The two injections of 6-OHDA were administered 24 h apart. An equal volume of sterile saline was injected into the left eye as a control preparation. The rats were decapitated 7 days following the injections and retinae isolated and prepared for analysis. DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were analyzed by HPLC with electrochemical detection after alumina extraction3. For cAMP determination, animals were euthanized at mid-day and retinae dissected and placed in Ringer's solution, pH 7.4. After a 2 min preincubation, DA, 100 gM, bromocriptine, 100 gM, or quinpirole, 100 gM, was added and incubations continued for 10 additional min. The reaction conditions were determined to be linear with protein and time of incubation during this period. SCH 23390, when present, was added during the 2 min preincubation period. Reactions were terminated by placing the samples in a boiling water bath for 3 min. cAMP was measured in the medium by radioimmunoassay16. For receptor binding studies4, retinae were dissected and homogenized with Tris-HCI buffer 50 mM, pH 7.5, in a Tissumizer (Tekmar, Cincinnati, OH) and centrifuged at 20,000 x g for 10 min. The particulate fraction was washed twice with buffer and then suspended in the Tris-HCI buffer containing (mM):MgCl2 1, NaCl 120, KCI 5 and CaCI2 2. The ligands studied were [3H]SCH 23390 (80 Ci/mmol) and [3H]spiperone (30 Ci/mmol) for D 1 and D2 receptor binding sites, respectively. Incubationswere carried out for 15 min at 37°. The reaction was stopped by rapid filtration followed by washing using a Brandel cell harvester (Gaithersburg, MD). Radioactivity on the filters was determined using a liquid scintilla-
Correspondence: N.H. Neff, Department of Pharmacology, The Ohio State University College of Medicine, 5198 Graves Hall, Columbus, OH 43210, U.S.A. 0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
194 TABLE I
TABLE II
Intraoeular administration of 6-OHDA and dopamine receptor binding characteristics in rat retina
Dopaminergic receptor-induced accumulation of cAMP in the rat retina following intraocular administration o f 6-OHDA
Two doses of 6-OHDA, 30 gg in 4 gl administered 24 h apart, were injected into the right and saline into the left eye of anesthetized rats. Animals were euthanized 7 days later. The ligand for D 1 sites was [3H]SCH 23390 and for the De sites [3H]spiperone. Bma x values, Imol/mg prot, and Ka values, nM, are expressed as the mean _+ S.E.M. for 3-5 experiments performed in duplicate. The % change values refer to saline.
6-OHDA, 30gg in 4#1 saline, was administered in the right eye and saline in the left eye of anesthetized rats, 2 injections 24 h apart. The animals were euthanized V days after the injections. Retinae were dissected and cAMP determined as described in the methods, n = 12-19. cAMP values found when SCH 23390 was present alone did not differ from basal activity for saline or 6-OHDA groups. Quin, quinpirole; Bromo, bromociptine; SCH, SCH 23390. A refers to cAMP accumulation compared with basal activity.
Treatment
D 1 dopamine binding sites
D 2 dopamine
B,~
Bmax
binding sites Conditions
Saline 6-OHDA
%
98_+3 61 -+3* -38
Kd
%
0.63_+0.1 6 4 _ + 3 0.74-+0.04 36_+3* -44
cAMP (pmol/mg prot/lO rain + S. E. M.)
Kd
0.69+0.09 0.52-+0.07
*P < 0.05 compared with saline-treated retina.
tion spectrometer. Specific binding was defined as the difference between total binding and that found in the presence of 1 /~M ketanserin and sulpiride for [3H]spiperone, and in the presence of 1/~M SCH 23390 for [3H]SCH 23390. Protein was assayed by the method of Lowry et al. 9, using bovine serum albumin as the standard. Brn~xand Kd values were calculated from Scatchard curve analysis using the microcomputer program GraphPAD (I.S.I. Philadelphia, PA). Multiple group comparisons were performed using a Newman-Keuls post hoc test after analysis of variance. A t-test for paired observations was used when values obtained for the saline-treated eye was compared with the 6-OHDA-treated eye.
RESULTS
Basal DA (100/~M) SCH (10/~M) plus DA Quin (100/~M) Bromo (100/~M)
Saline
d
6-OHDA
d
49 _+ 1 116+5" 36 + 1" 36 + 1" 37 + 2*
+67 -13 -13 -12
37 + 2** 151 +6*'** 87 + 76"** 39 ___4 39 + 2
+114 +50 +2 +2
*P < 0.05 compared with appropriate basal activity. **P < 0.05 compared with corresponding saline-treated groups. significantly decrease c A M P accumulation in the 6O H D A - t r e a t e d retina. Stimulation of 6 - O H D A - t r e a t e d retinae with D A in the presence of SCH 23360 resulted in an augmentation of c A M P accumulation by about 135% over basal activity. Similar conclusions are obtained if the absolute changes of c A M P accumulation are considered (Table II). DISCUSSION
Seven days following the administration 6 - O H D A
There is now overwhelming evidence from a variety of
there was a 90% fall of retina D A and 3,4-dihydroxy-
tissues that most D 1 receptors are positively coupled to A C which upon activation leads to an increased accu-
phenylacetic acid ( D O P A C ) (data not shown). There was also a significant fall of the Bmax value, about 38 and 44%, for both D 1 and D 2 sites respectively (Table I). In contrast, the K a values were relatively unaffected. D A is a non-selective activator of D~ and D 2 receptors. In the retina the algebraic sum of activation of these receptors is to enhance c A M P accumulation, primarily a D 1 receptor function (Table II). Bromocriptine and quinpirole, selective agonists of D 2 receptors, caused a significant fall of c A M P accumulation in retina. Similarly, stimulation of D2 receptors with D A after blockade of D 1 receptors with the selective antagonist SCH 23360 resuited in a significant reduction of cAMP production (Table II). After 6 - O H D A treatment there was a significant decrease in the basal cAMP values, about 28% of the saline treated retinae. Stimulation of the 6O H D A - t r e a t e d retina with D A resulted in a greater increase of the cAMP, about 300% over the corresponding basal activity, than in the saline treated group, about 136% over the corresponding basal activity. The selective agonists bromocriptine and quinpirole both failed to
mulation of cAMP. In contrast, some D 2 receptors are negatively coupled to A C and their activation leads to a fall of cAMP 6'x8. There is also evidence suggesting that some D e receptors are not coupled to A C , and are probably linked to other second messenger systems. For example, both D 1 and D E receptors are linked to A C in the striatum, while in the limbic forebrain it appears that only D 1 receptors are coupled to A C even though D 2 receptors are found there 8. In nucleus accumbens slices, activation of O 2 receptors inhibit D A and acetylcholine release i n d e p e n d e n t of c A M P formation 19. In addition, there are reports that O 2 receptors activate potassium channels2 or inhibit inositol phosphate generation a'la. In the retina, there are guanine nucleotide sensitive and insensitive classes of D A receptors 1°. O u r studies, like many studies in the literature from other tissues, are consistent with the hypothesis that some O 2 receptors in the retina are coupled to A C and some are not. Following treatment with 6 - O H D A there was about a 40% reduction of both D 1 and D 2 bindings sites,
195 suggesting that the lost receptors were associated with lost d o p a m i n e r g i c neurons. Since amacrine cells synapse with o t h e r amacrine cells, it is difficult to d e t e r m i n e if the r e c e p t o r loss is due solely to loss of presynaptic or both pre- and postsynaptic D A receptors ~. The robust accumulation of c A M P implies that the remaining D 1 receptors are supersensitive to D A . Similar responses were found in retina following the selective neurotoxin M P T P 17. T h e loss of Dx receptor binding sites and the enhanced response of A C to D A might indicate that r e c e p t o r supersensitivity can occur without up-regulation of binding sites. Missale et a1.12 r e p o r t e d that depletion of neuronal D A with reserpine resulted in up-regulation of the transduction mechanism for generating c A M P by D 1 receptors, but not of the n u m b e r of binding sites. E n h a n c e d efficiency of the guanine nucleotide coupling protein to A C was suggested. T h e loss of about 45% of D 2 binding sites and the lack of a response to activation of A C by bromocriptine or quinpirole following 6 - O H D A implies that some D 2 receptors are not coupled to the enzyme. O u r observation that b l o c k a d e of D 1 receptors with S C H 23360
followed by activation with D A in the 6 - O H D A - t r e a t e d animals e n h a n c e d the accumulation of c A M P was unexpected. We i n t e r p r e t e d this result to indicate that S C H 23360 is only able to partially prevent the D A - i n d u c e d activation of the supersensitive D1 receptors. A l t e r n a tively, if both D 1 and D 2 receptors coupled to A C are located on the same n o n - d o p a m i n e r g i c neurons, it is possible that there is differential m o d u l a t i o n of the A C coupled system with the o u t c o m e favoring e n h a n c e m e n t of D 1 function and suppression of D 2 function 7,s. The loss of D E inhibition of c A M P accumulation in the 6O H D A - t r e a t e d retina was surprising, as we previously r e p o r t e d that muscarinic inhibition of c A M P accumulation in the retina was enhanced following the intraocular injection of the cholinotoxin A F 6 4 A ~5. In summary, our studies provide evidence that both D 1 and D 2 receptors are located pre- and postsynaptically in rat retina and support the hypothesis that some D 2 receptors are negatively coupled to A C and some receptors are not. D 2 receptors negatively coupled to A C are not evident following intraocular 6 - O H D A .
REFERENCES
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