Norihisa Fujita

Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Abstract Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of 3 H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K D value of the specific 3 H-QNB binding or in ID 50 value of oxotremorine in inhibiting 3 H-QNB binding. No change in the specific binding of 3 H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.

Selective enhancement in striatal [3H]nitrendipine binding following chronic treatment with morphine

Two parameters of Ca(2+) dynamics in brain preparations ((45)Ca-uptake to slices and [(3)H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on (45)Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K(+)-stimulated (45)Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K(+)-stimulated (45)Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [(3)H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.

Effects of dopaminergic agonists and antagonists on [3H]apomorphine binding to striatal membranes: sulpiride lack of interactions with positive cooperative [3H]apomorphine binding

Effects of dopaminergic agonists and antagonists on [3H]apomorphine binding to striatal membranes of rat brain was examined. Haloperidol and spiroperidol exhibited biphasic inhibition of [3H]apomorphine binding; one of which had the Hill coefficient of 0.9, whereas the other had that of 0.4. The former accounted for 65% of [3H]apomorphine binding while the latter consisted of 35% of the binding. Furthermore, the latter disappeared after kainic acid lesions. On the other hand, sulpiride and metoclopramide reduced [3H]apomorphine binding to 31% with the Hill coefficient of 0.9. The inhibition of [3H]apomorphine binding with the Hill coefficient of 0.4 which was shown by haloperidol and spiroperidol was not observed for sulpiride and metoclopramide. Previously, we demonstrated non- and positive-cooperative [3H]apomorphine binding to stiatal membranes. In the present study, it has been also shown that sulpiride inhibits non-cooperative [3H]apomorphine binding leaving that with allosteric properties unaffected. No inhibition of dopamine-sensitive adenylate cyclase was observed by 10(-4) M sulpiride while 90% inhibition was obtained with 10(-5) M haloperidol. From those results, it is suggested that non-cooperative [3H]spomorphine binding is not coupled with dopamine-sensitive adenylate cyclase.

Desensitization of the alpha adrenergic receptor system in guinea pig vas deferens.

Desensitization induced by alpha adrenergic (alpha-Ad) stimulation was investigated in organ cultured vas deferens of guinea pig. Brief exposure (1-2 min) of the muscle to noradrenaline (NA) caused short-term desensitization to both NA and acetylcholine (ACh), but not to high K+. After removing the agonist this desensitization completely disappeared within 15 min. Prolonged exposure to NA (i.e., cultured with NA for 3-24 hr) elicited long-term desensitization to NA, ACh and K+ (50 mM), but it did not change the maximal contraction by high K+ (154 mM). After removing NA from the culture medium the response to the agonist was restored to normal within 24 hr, but not within 15 min. The number and affinity of alpha-Ad and muscarinic ACh receptors, which were measured by the binding of 3H-WB4101 and 3H-QNB, respectively, were not changed in the muscle during these treatments. Moreover, long-term desensitization, but not short-term desensitization, was depressed by the concomitant presence of cycloheximide. The possible mechanisms of desensitization were discussed in comparison with those of various receptor systems.

D-2 dopamine receptor synthesis and turnover in rat striatum

Direct injection of phenoxybenzamine into rat striatum inhibited apomorphine-induced stereotyped behavior. This inhibition corresponded well with the inhibition of D-2 dopamine receptor labelling with [3H]spiroperidol. Both the behavioral response and the receptor level were completely restored within 5 days after the injection. The recoveries of both were blocked by cycloheximide. The rate of synthesis and half-life of the D-2 receptor associated with the stereotyped behavior were calculated to be 6.9 fmol/mg protein per h and 28 h, respectively.

Binding of 3H-lisuride hydrogen maleate to striatal membranes of rat brain

Abstract Binding of 3 H-lisuride hydrogen maleate (LHM), a dopaminergic agonist, to striatal membranes was inhibited by (+)-butaclamol, whereas (-)- butaclamol at a concentration of 10 −9 -10 −7 M was without effect. The difference in the amount of 3 H-LHM bound to striatal membranes in the presence of 10 −7 M (-)-butaclamol and (+)-butaclamol was designated as the specific binding of 3 H-LHM, and its properties were examined. The specific 3 H-LHM binding to striatal membranes was saturated with an equilibrium amount of 490 fmol/mg protein and had an apparent dissociation constant (Kd) of 0.5 nM. The specific binding of 3 H-LHM to striatal membranes was inhibited by LHM, haloperidol, apomorphine and methysergide with inhibitor association constants (Ki value) of 0.79, 7.1, 100 and 180 nM, respectively. Phentolamine, dopamine, (-)-norepinephrine and serotonin were weaker inhibitors of the specific binding of 3 H-LHM to striatal membranes, with Ki values of 1,100, 3,500, 33,000 and 79,000 nM, respectively. No inhibition was observed with (±)-propranolol, dichloroisoproterenol or QNB. These results are discussed in connection with dopamine receptors.

Multiple postsynaptic dopamine receptors and behavioral manifestation

Previously we demonstrated the existence of non- and positive-cooperative dopamine receptors in rat striatum in [3H] apomorphine binding experiments. Non-cooperative sites were sensitive to the inhibition of sulpiride while cooperative sites were not. In the present study results dealing with the involvement of those two types of postsynaptic dopamine receptors in different behavior manifestations is shown employing lisuride hydrogen maleate (LHM). LHM elicited contralateral turnings in 6-hydroxydopamine lesioned rats unilaterally in the striatum whereas it caused ipsilateral turnings in kainic acid lesioned rats as was observed following the administration of apomorphine. Furthermore, the effect of LHM on rotating behavior was abolished by the pretreatment with sulpiride. On the other hand, LHM inhibited apomorphine induced stereotyped behavior whereas the sulpiride failed to block it. These results suggested the dual action of LHM on multiple postsynaptic dopamine receptors. The results also indicated that non-cooperative postsynaptic dopamine receptors are involved in rotating behavior while cooperative receptors participate in the elicitation of stereotypy.

Endogenous modulator of dopamine receptor(s)

An endogenous modulator(s) of the dopamine receptor(s) in bovine and rat brain striatum was detected by demonstrating that water extracts of the striatum inhibited [(3)H]apomorphine binding. This modulator(s) was partially purified by methanol extraction and then successive ion exchange chromatographies on SP-Sephadex C-25 and QAE-Sephadex A-25, and gel chromatography on Sephadex G-25. The partially purified (about 1,500-fold) modulator was a fluorescamine-positive substance, Mr = 500 1000, which was heat-stable (95 degrees C, 10 min), and was destroyed by acid- and alkali-treatment, but not by treatments with various peptidases. The modulator inhibited binding of the dopamine agonist, [(3)H]apomorphine non-competitively, but did not inhibit binding of the dopamine antagonist, [(3)H]spiroperidol. Direct injection of the modulator into rat brain striatum depressed apomorphine-induced locomotor activity. Moreover the modulator inhibited dopamine-sensitive adenylate cyclase activity. These findings indicate that the modulator acts at a site(s) other than the ligand binding site of the dopamine receptor(s) and modulates the activities of dopamine agonists.