Acute in vivo amphetamine produces a homologous desensitization of dopamine receptor-coupled adenylate cyclase activities and decreases agonist binding to the D1 site

We have previously reported that, 30 min after a single injection of 7.5 mg/kg d-amphetamine sulfate, there was a significant 25% decrease in the apparent Vmax for stimulation of adenylate cyclase activity by the D1 receptor-selective partial agonist SKF 38393 in rat striatal membranes, as compared with saline-injected controls. This desensitization was seen in the striatal but not the mesolimbic forebrain. In the present study this desensitization was further characterized by using various ligands that interact with the three components of the D1 receptor-coupled adenylate cyclase complex to determine the site of modification that resulted in the desensitization. The desensitization was not associated with a change in the stimulation of adenylate cyclase at the level of the catalytic subunit or the guanyl nucleotide-regulatory protein Ns. Receptor number, as assessed by the binding of the D1 selective antagonist [3H]SCH 23390, was unaltered in the desensitized state. In contrast, the number of high affinity binding sites, as measured with the agonist [3H]dopamine was decreased 30% by acute amphetamine exposure. This suggests that the amphetamine-induced desensitization may be the result of an uncoupling of the receptor from Ns. In order to further assess the effects of amphetamine on receptor/Ns coupling, we measured the ability of the guanyl nucleotide guanosine-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] to decrease high affinity [3H]dopamine binding to striatal membranes. The inclusion of 100 microM Gpp(NH)p in the assay decreased the number of receptors in the high affinity state by 40% and 52% in membranes from saline- and amphetamine-pretreated rats, respectively. These results imply that amphetamine treatment does not modify the ability of Gpp(NH)p to decrease high affinity agonist binding. It is possible that amphetamine treatment decreases the number of receptors that can couple to Ns but the remaining receptors can still form a high affinity complex and are sensitive to the effects of Gpp(NH)p. We also report that maximal D2 dopamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity was decreased in striatal membranes prepared from amphetamine-treated rats as compared with saline-injected controls, implying that the D2 pathway was desensitized by amphetamine treatment. Conversely, acute amphetamine injection did not alter the ability of either the adenosine A2 receptor to stimulate or the muscarinic cholinergic receptor to inhibit adenylate cyclase activity in the rat striatum. These results suggest that acute amphetamine treatment produces a dopamine receptor-specific or homologous desensitization.     

Effects of ceruletide on perioral movements and the dopamine receptor-adenylate cyclase system in rats chronically treated with fluphenazine

The effects of repeated administration of ceruletide (100 µg/kg/perday, IP for 3 days) on perioral movements and the striatal dopamine receptor adenylate cyclase system were examined in rats chronically treated with fluphenazine enanthate (FPZ) (25 mg/kg IM every 3 weeks for 30 weeks) and sesame oil-treated (control) rats. After the tenth injection of fluphenazine, the rats started to display five types of perioral movements (teeth chattering, chewing, tongue protrusion, mouth opening and perioral tremors). Moreover, increases in SCH23390 binding and spiperone binding to striatal membranes were found in the FPZ-treated rats. Furthermore, dopamine receptor-coupled adenylate cyclase activity was potentiated in striatal membranes. High amplitude EMG discharges (8–10 Hz), recorded from the masseter in the FPZ-treated rats occurred concurrently with perioral tremors. Repeated ceruletide (CLT) injections abolished perioral movements, and reversed both the elevated SCH23390 binding and the dopamine stimulated adenylate cyclase (AC) activity to the control level. The effect of CLT on perioral movements, D₁ receptors and dopamine-stimulated AC activity continued for 6 days after the final CLT injection. These finding suggest that systemically administered CLT affects the D₁ receptor adenylate cyclase system and that an increase of the D₁ receptor mechanism may play an important role in the pathogenesis of tardive dyskinesia.     

Repeated administration of sulpiride for three weeks produces behavioural and biochemical evidence for cerebral dopamine receptor supersensitivity

Administration of sulpiride (2 × 100 mg/kg i.p.) or haloperidol (5 mg/kg i.p.) to rats for 3 weeks with subsequent withdrawal for 3 or 4 days induced cerebral dopamine receptor supersensitivity. Apomorphine-induced stereotyped behaviour after drug withdrawal was enhanced by pretreatment with either haloperidol or sulpiride both of which increased the number of specific striatal binding sites (B_max) for [³H]spiperone, [³H]N,n-propylnorapomorphine and [³H]sulpiride. Neither drug altered the dissociation constant (K_D) for the ligand binding assays. Striatal dopamine sensitive adenylate cyclase activity was unaltered by such a pretreatment with either haloperidol or sulpiride. The data show that sulpiride, like haloperidol, is capable of inducing behavioural and biochemical supersensitivity of cerebral dopamine receptors.     

Differential effects of continuous administration for 1 year of haloperidol or sulpiride on striatal dopamine function in the rat

Administration of haloperidol (1.4–1.6 mg/kg/day) for up to 12 months or sulpiride (102–109 mg/kg/day) for between 6 and 12 months increased the frequency of purposeless chewing jaw movements in rats. N,n-propylnorapomorphine (NPA) (0.25–2.0 mg/kg SC) did not induce hypoactivity in haloperidol-treated rats at any time; sulpiride treatment for 9 and 12 months caused a reduction in the ability of NPA to induce hypoactivity. Haloperidol, but not sulpiride, treatment enduringly inhibited low dose apomorphine effects (0.125 mg/kg SC). After 12 months, sterotypy induced by high doses of apomorphine (0.5–1.0 mg/kg) was exaggerated in haloperidol-, but not sulpiride-treated rats.B_max for specific striatal ³H-spiperone binding was increased by haloperidol, but not sulpiride, treatment throughout the study. B_max for ³H-NPA binding was elevated only after 12 months of both haloperidol and sulpiride treatment. B_max for ³H-piflutixol binding was not alfered by chronic haloperidol or sulpiride treatment. Striatal dopamine-stimulated adenylate cyclase activity was inhibited for the 1st month of haloperidol treatment, thereafter returning to control levels; dopamine stimulation was increased after 12 months of sulpiride treatment. Striatal acetylcholine content was increased after 3 and 12 months of treatment with haloperidol, but was not affected by sulpiride.Chronic administration of sulpiride does not induce identical changes in striatal dopamine function to those caused by haloperidol.     

Subsensitivity of the rat striatal dopaminergic system after treatment with bromocriptine: effects on [3H]spiperone binding and dopamine-stimulated cyclic AMP formation

Summary Repeated daily administration of the dopamine (DA) agonist bromocriptine (15 mg/kg; s. cut.) to rats led to a time dependent decrease in the in vitro binding of [³H] spiperone to striatal membranes. Kinetic analysis of [³H]spiperone binding after 2 and 7 days of bromocriptine treatment showed a 25–50% reduction in the total number of binding sites with no change in their affinity for spiperone. These was also a decreased accumulation of cyclic AMP (cAMP) in striatal slices in response to DA after bromocriptine treatment. The DA-sensitive adenylate cyclase in striatal homogenates, however, remained unchanged in bromocriptine treated rats. There was also no change in cyclic nucleotide phosphodiesterase activity in striatal tissue after bromocriptine treatment. Furthermore, incubation of striatal slices in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine did not alter the decreased cAMP response to DA after 2 days of bromocriptine treatment. These results suggest that a decreased number of DA receptor sites may be responsible for the reduced cAMP response to DA in striatal slices after bromocriptine treatment.M. Q. was supported by a fellowship from the Canadian Medical Research Council     

Effects of lithium on the beta-adrenergic receptor-adenylate cyclase system in rat cerebral cortical membranes.

The effects of lithium on the beta-adrenoceptor-adenylate cyclase system in cerebral cortical membranes of rats were investigated. Lithium chloride inhibited adenylate cyclase activity in a concentration-dependent manner in vitro. However, relatively high concentrations of lithium were needed for this inhibition; and at 1 mM, no significant reduction in adenylate cyclase activity was seen under any condition. Administration of lithium carbonate for 21 days decreased the maximum number of [3H]dihydroalprenolol binding sites without changing the apparent dissociation constant. Activation of adenylate cyclase by (-)-isoproterenol in the presence of 1 microM guanyl-5'-ylimidodiphosphate (Gpp(NH)p) was significantly attenuated in lithium-treated rats compared with the controls. Lithium treatment reduced the Gpp(NH)p-stimulated adenylate cyclase activity in the presence of 10 microM (-)-isoproterenol, but not in the absence of this beta-adrenergic receptor agonist. Basal activity or adenylate cyclase activity stimulated by forskolin or manganese was not affected, whereas the activity stimulated by sodium fluoride was significantly attenuated by long-term lithium treatment. These results indicate that chronic lithium treatment induces subsensitivity in the beta-adrenoceptor-adenylate cyclase system, for which down-regulation of beta-adrenergic receptors is chiefly responsible.     

Activation of adrenal adenylate cyclase by guanine nucleotides. Promotion of nucleotide binding by calcium but not by adrenocorticotropic hormone

Calcium ion is essential for normal stimulation of adrenal cortical adenylate cyclase by adrenocorticotropic hormone (ACTH). Both ACTH and Ca2+ act to promote the activation of adenylate cyclase by guanine nucleotides such as guanyl-5'-yl imidodiphosphate [Gpp(NH)p]. To define further the mechanisms by which Ca2+ and ACTH interact with guanine nucleotides, we have correlated the binding of [3H]Gpp(NH)p to adrenal membranes and solubilized membrane proteins with activation of membrane-bound and solubilized adenylate cyclase. Ca2+ increases both the rate of reversible nucleotide binding and the rate of adenylate cyclase activation by nucleotide. This effect is accompanied by the appearance of binding sites having an 8- to 10-fold higher affinity for [3H]Gpp(NH)p. In contrast to Ca2+, ACTH increases the rate of enzyme activation but has no significant effect on nucleotide binding. In Ca2+-depleted membranes, measured nucleotide binding is low, and ACTH has no effect on enzyme activation. Once nucleotide is initially bound, both divalent cations and hormone can promote the transition of the enzyme to an activated state. Mg2+ is more effective than Ca2+ in promoting this transition, while Ca2+ is more effective than Mg2+ in promoting initial nucleotide binding. When membranes containing bound [3H]Gpp(NH)p are solubilized with Lubrol PX, adenylate cyclase activity elutes on Sepharose 4B with an apparent molecular weight of 160,000. The major fraction containing bound nucleotide elutes with an apparent molecular weight of 40,000-50,000. Nucleotide bound to this fraction is increased by pretreatment of the membranes with Ca2+ but is not affected by pretreatment with ACTH. Nucleotide bound to solubilized membrane components dissociates after treatment with EDTA. These findings suggest that Ca2+ promotes the initial binding of Gpp(NH)p to a biologically effective site that may involve a guanine nucleotide regulatory protein. ACTH activates adenylate cyclase by promoting a step subsequent to the binding of guanine nucleotide.     

Effect of 3-PPP,a putative dopamine autoreceptor agonist,on [3H]ligand binding to dopamine receptors of calf and rat striatum

3-(3-Hydroxyphenyl)-N-n-propyliperidine (3-PPP) is most effective in inhibiting [³H]apomorphine binding in rat striatal membranes, with Ki values of 63 nM. 3-PPP was six to 27 times less effective when it competed with the binding of [³H]dopamine or [³H]spiperone in calf and rat striatal membranes. At concentrations up to 10 μM, 3-PPP failed to substitute for dopamine in the activation of adenylate cyclase in rat striatal membranes. 3-PPP at 4.8-5 μM caused 50% inhibition of catecholamine uptake in synaptosomes of corpus striatum and hypothalamus, therefore appearing to be a relatively weak uptake inhibitor. The higher affinity of 3-PPP for [³H]apomorphine binding sites is consistent with its binding to a subset of dopamine receptors which are characterized by a high affinity for both the agonist and antagonist of dopamine.     

Photoaffinity labelling of beta-adrenergic receptors of C6 glioma cells. Presence of a nucleophilic group in the receptor

(±) 1-[1-(p-Nitrophenoxy), 2-methyl, 2-propylamino], 3-(α-naphtyloxy), 2-propanol (PNP) was synthesized and found to be a potent photoaffinity label for β-adrenergic receptors of C₆ glioma cells.In the dark, PNP displaced all the [³H]DHA binding sites on C₆ glioma cell membranes (K_D = 5.5 × 10^?8 M).Upon photolysis on isolated C₆ glioma cell membranes: (a) PNP reduced in a dose-dependent manner maximally stimulated β-adrenergic sensitive adenylate cyclase. After extensive washing of the membranes, the maximal β-adrenergic stimulation was reduced without change in the apparent affinity for isoproterenol. A 62% decrease in activity was obtained with 10^?5 M PNP without any change in basal and NaF stimulated adenylate cyclase activities, (b) PNP also irreversibly reduced in a dose-dependent manner the total number of [³H]DHA binding sites without changing the affinity of the remaining sites. The effects of PNP on adenylate cyclase and [³H]DHA binding were suppressed in the presence of (?)alprenolol. Upon photolysis on intact C₆ glioma cells, PNP inactivated β-adrenergic receptors coupled with adenylate cyclase without any change in basal, NaF and Gpp(NH)p stimulated adenylate cyclase activities.These results indicate that PNP photolabelling occurred on the β-adrenergic receptors. Furthermore, as PNP was shown to react with model nucleophiles upon photolysis, this labelling implies the presence of a nucleophilic group in the β-adrenergic receptor.     

Differential alteration in striatal dopaminergic and cortical serotonergic receptors induced by repeated administration of haloperidol or centbutindole in rats

Centbutindole is a new neuroleptic drug having a pharmacological profile similar to haloperidol, but it does not cause hypothermia and has a higher separation between doses causing catalepsy and neurolepsy. The interactions of centbutindole with striatal dopamine and cortical 5-HT2 receptors have been studied along with haloperidol following 3 weeks of administration. Rats received haloperidol (1.0 mg/kg, p.o.), centbutindole (0.5 mg/kg, p.o.) or saline daily for 21 days. Following drug withdrawal for 3 days, apomorphine (0.1-1.0 mg/kg, i.p.) or 5-hydroxytryptamine (5-HTP, 50-200 mg/kg, i.p.) was injected. Apomorphine-induced stereotyped behaviour was potentiated in the haloperidol-treated rats, while the 5-HTP-induced behavioural syndrome was increased in centbutindole-treated rats. Receptor binding studies indicated an increase in the maximal binding capacity Bmax of striatal dopamine receptor (29.4%) in haloperidol-treated and of cortical 5-HT2 receptor (17.8%) in centbutindole-treated animals. No change in the apparent dissociation constant Kd was observed. It is concluded that repeated treatment with haloperidol produced striatal dopamine receptor supersensitivity while centbutindole treatment produced cortical serotonergic receptor supersensitivity.     

Changes in apomorphine-induced stereotypy as a result of subacute neuroleptic treatment correlates with increased D-2 receptors, but not with increases in D-1 receptors

Administration of haloperidol (5 mg/kg i.p.), cis-flupenthixol (2.5 mg/kg i.p.) or sulpiride (2 X 100 mg/kg i.p.) daily for 21 days followed by a 3-day drug withdrawal period caused equivalent cerebral dopamine receptor supersensitivity as judged by enhanced apomorphine-induced stereotypy. These treatments also produced equivalent rises in the number of adenylate cyclase-independent dopamine receptors (D-2) in both striatal and mesolimbic tissue as assessed by specific [3H]spiperone and [3H]N,n-propylnorapomorphine (NPA) binding. No change in the dissociation constant (KD) was apparent in response to neuroleptic treatment. However, only repeated administration of cis-flupenthixol caused an increase in the number of adenylate cyclase-linked dopamine receptors (D-1) in striatum as assessed by enhanced [3H]piflutixol binding and increased dopamine-stimulated cyclic AMP formation. The dissociation constant for [3H]piflutixol binding was unchanged by cis-flupenthixol administration. No change in D-1 receptor numbers or dopamine stimulation of adenylate cyclase occurred in mesolimbic tissue. Repeated treatment with sulpiride or haloperidol was without effect on either [3H]piflutixol binding to D-1 receptors or cyclic AMP formation. In conclusion, increased apomorphine-induced stereotypy following subacute neuroleptic treatment correlates with changes in D-2 receptor numbers, but not with changes in D-1 receptors.     

Correlation between drug-induced supersensitivity of dopamine dependent striatal mechanisms and the increase in striatal content of the Ca2+ regulated protein activator of cAMP phosphodiesterase

Summary Rats were injected daily with 1.3 mol/kg of haloperidol s.c. for 10 days. From the second to the ninth day after haloperidol withdrawal the rats developed supersensitivity to the behavioral affects of apomorphine. Concomitantly, the K _a of dopamine for the activation of striatal adenylate cyclase was lowered and the striatal content of the Ca²⁺ dependent protein that activates cAMP phosphodiesterase was increased. This activator protein is stored in striatal membranes and can be released by membrane phosphorylation in cytosol. This protein increases the activity of the high K _m phosphodiesterase (Uzunov et al., 1976) but when it is bound to striatal membranes it facilitates the activation of striatal adenylate cyclase by dopamine (Gnegy et al., 1976 b). The increase in protein activator content of striatal membranes caused by haloperidol could be a primary factor in causing supersensitivity to the biochemical and behavioral effects of dopamine receptor agonists.     

Differential alterations in striatal dopamine receptor sensitivity induced by repeated administration of clinically equivalent doses of haloperidol,sulpiride or clozapine in rats

Rats received therapeutically equivalent doses of either haloperidol (1.7–1.9 mg/kg/day), sulpiride (112–116 mg/kg/day) or clozapine (30–35 mg/kg/day) continuously for 4 weeks. Treatment with haloperidol, but not sulpiride or clozapine, caused inhibition of stereotyped behaviour induced by apomorphine (0.125–0.25 mg/kg SC). Following drug withdrawal for up to 7 days, haloperidol and sulpiride, but not clozapine treatment caused an exaggeration of stereotyped behaviour induced by apomorphine.B_max values for striatal ³H-spiperione binding were erevated in animals treated for 2 and 4 weeks with haloperidol, but not with sulpiride or clozapine. Following drug withdrawal, haloperidol, but not sulpiride or clozapine, treatment caused an increase in B_max for striatal ³H-piperone binding.B_max values for striatal ³H-NPA binding revealed no change during haloperidol or clozapine treatment. Sulpiride treatment for 1 week caused an increase in B_max for ³H-NPA binding, which returned to control levels at 2 and 4 weeks. Following drug withdrawal, there was an increase in B_max for ³H-NPA binding in rats treated with haloperidol and sulpiride, but not clozapine.On continuous treatment and following withdrawal from haloperidol, sulpiride, or clozapine the ability of dopamine to stimulate striatal adenylate cyclase activity did not differ from that in control animals.Repeated administration of sulpiride or clozapine may not induce striatal dopamine receptor supersensitivity when given in clinically relevant doses, although haloperidol does.     

Altered function of adenylate cyclase in the myocardium of the spontaneously hypertensive rat

The effects of Mg²⁺, Mn²⁺, Ca²⁺, guanylylimidodiphosphate [Gpp(NH)p] and isoproterenol on adenylate cyclase activity in myocardial membrane preparations from spontaneously hypertensive rats (SHR) and Kyoto Wistar normotensive rats (WKY) were compared. The isoproterenol-stimulated adenylate cyclase activity of myocardial membranes from WKY rats responded at a lower threshold (10 vs 200 nM) and with a lower EC 50 (300 nM vs 1 μM) and a higher maximal velocity. When the isoproterenol effect was studied in the presence of 100 nM Gpp(NH)p, the threshold for isoproterenol was similar in SHR and WKY. However, the activity at each dose was significantly lower (P<0.05) in SHR. In the presence of Gpp(NH)p alone, no differences were observed between SHR and WKY. Increasing Mg²⁺ and Mn²⁺ concentrations stimulated adenylate cyclase activity but no differences were observed between SHR and WKY for Mn²⁺ stimulation. However, the SHR myocardial activity was significantly reduced at Mg²⁺ concentrations ranging from 6 to 25 mM. The effect of varying the Mg²⁺ concentration was further tested in the prsence of 1 μM isoproterenol. The adenylate cyclase activity of myocardia of SHR was significantly reduced at Mg²⁺ concentrations between 4 and 24 mM. These observations suggest that sensitivity of the adenylate cyclase to isoproterenol has been decreased in SHR.     

Chronic neuroleptic treatment increases D-2 but not D-1 receptors in rat striatum

In vitro binding of [3H]cis(Z)-flupentixol (FPT) to rat striatal membranes was evaluated as an assay for D-1 receptors. It was found that under the appropriate assay conditions [3H]FPT bound to a single saturable site which was most abundant in striatum and bound dopaminergic agonists in the potency order that these drugs demonstrate for adenylate cyclase. These data support previous work suggesting that [3H]FPT labels the D-1 receptor. Next, rats received haloperidol or fluphenazine for 54 days and striatal dopamine receptors were assayed 72 h later. The drug treatments increased the density of D-2 receptors as measured by [3H]spiperone binding by 40% and 25% respectively. However, no change was observed in D-1 receptor density. We conclude that effects of chronic neuroleptic treatment that depend upon increased dopamine receptor density are mediated via the D-2 receptor subtype.     

Serotonin-sensitive adenylate cyclase and [3H]serotonin binding sites in the CNS of the rat—II: Respective regional and subcellular distributions and ontogenetic developments

The 5-HT receptor linked to adenylate cyclase and the high affinity binding site of [³H]5-HT were compared on the basis of their localization and ontogenetic changes in the CNS of the rat. Subcellular fractionation of cerebral tissues from newborn rats showed a good correlation between the distributions of 5-HT-sensitive cyclase and [³H]5-HT binding sites, with the mitochondrial fraction exhibiting the highest specific adenylate cyclase activity and density of binding sites. There was also a good correlation between the regional distributions of the cyclase and the binding in the CNS of newborn rats. However, when the regional distribution of [³H]5-HT binding in newborns was compared to that of adults, no correlation was found, showing that large changes were occurring during ontogenesis. In the cortex and hippocampus, there was little change in the amount of 5-HT-sensitive adenylate cyclase during development whereas [³H]5-HT binding increased approximately 7-fold from birth to adulthood. Only in the striatum was there a positive correlation between the changes in the cyclase and the binding. The injection of kainic acid into the striatum of 10-day-old rats produced large decreases in both DA-and 5-HT-sensitive adenylate cyclase activities. The specific binding of [³H]5-HT was also reduced in the injected striatum but to a smaller extent. Therefore, the 5-HT-sensitive adenylate cyclase but not the [³H]5-HT-high affinity binding site appeared to be preferentially associated with neurons destroyed by kainic acid, i.e. neurons intrinsic to the striatum. The findings that the 5-HT-sensitive adenylate cyclase and the [³H]5-HT binding sites can develop independently and are localized, at least partly, on different types of cells provide additional evidence for the existence of multiple types of 5-HT receptors in the CNS of the rat.     

Further evidence for the subsensitivity of striatal AMPA receptors, induced by chronic haloperidol administration: an autoradiographic study

The aim of the present study was to investigate the influence of chronic treatment with haloperidol on the striatal N-methyl-D-aspartate (NMDA), -amino-3-hydro-xy-5-methyl-4-isoxasole-propionic acid (AMPA) and dopamine D₂ receptors using a quantitative autoradiography in rats. Haloperidol was given to animals in a dose of ca. 1 mg/kg/day in drinking water for 6 weeks or 3 months and was afterwards withdrawn for 5 days. Haloperidol increased by 20–50% the binding of [³H]spiperone in different regions of the caudate-putamen. Haloperidol decreased by ca. 30% the binding of [³H]AMPA in the ventrolateral region of intermediate part of the caudate-putamen, but did not influence the binding of [³H]MK-801. The present results suggest that, apart from supersensitivity to dopamine, chronic treatment with haloperidol also induces subsensitivity of striatal AMPA receptors.     

Behavioral stimulation without alteration of β and 5-HT receptors and adenylate cyclase activity in rat brain after chronic sertraline administration

Effects of chronic treatment with selective 5-HT reuptake inhibitors (SSRIs) on the monoaminergic functions have not been much investigated in compared with tricyclic antidepressants. Therefore, we compared the effects of 3-week treatment with sertraline, a potent SSRI, to those of imipramine (10 mg/kg, IP, twice a day), on monoamine receptors and adenylate cyclase (AC) activity in rat brain. Two-week treatment with both sertraline and imipramine reduced immobility in the water wheel test to the comparable extent. Sertraline treatment did not affect K_d and B_max of [³H]CGP12177 and [³H]ketanserin bindings or cAMP accumulation by norepinephrine, isoproterenol, 5’-guanylylimidodiphosphate [Gpp(NH)p] and forskolin in the cortical membrane compared with vehicle-treated rats. On the other hand, imipramine treatment decreased B_max of both bindings and norepinephrine- or isoproterenol-stimulated cAMP accumulation. Treatment with either antidepressant induced no apparent changes in [³H]8-OH-DPAT [2-(N, N-dipropylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene] binding in the hippocampal membrane. These results suggested that chronic treatment of sertraline induced little effect on monoamine receptors and AC activity in the brain and that the alteration of these functions may not be primarily involved in antidepressive effects of antidepressants, at least of SSRIs. Received: 19 April 1996 /Final version: 29 October 1996     

Stable Guanosine 5′-Triphosphate-Analogues Inhibit Specific (+)-[3H]Isradipine Binding in Rat Hearts by a Ca2+-Lowering,G Protein-Independent Mechanism

Abstract: We investigated if and how stable guanosine 5′-triphosphate-analogues affect (+)-[³H]isradipine binding in rat hearts. Gpp(NH)p and GTP-γ-S inhibit specific (+)-[³H]isradipine binding in membranes and cell-homogenates by reducing the binding density without changing the K_d or the k_-1. Inhibition by Gpp(NH)p was less in crude tissue homogenates than in membranes apparently due to a soluble factor. Pretreatment of cardiomyocytes with cholera toxin or the presence of the protein kinase A inhibitor, PKI_6-22, did not influence the effect of 10^-3 M Gpp(NH)p on binding. The inhibitory effect of 10^-3 M Gpp(NH)p was not significantly altered in membranes from in vivo pertussis toxin treated rats. The addition of 10^-3 M Ca²⁺ or Mg²⁺ abolished the inhibitions. Gpp(NH)p in the concentration that inhibits binding, reduced the free concentration of Ca²⁺. The Ca²⁺ -lowering effect of 10^-3 M Gpp(NH)p produced 70%, 60% and 100% of the inhibition in membranes, sonicated and unsonicated cell homogenates. Thus, Gpp(NH)p inhibited specific (+)-[³H]isradipine binding mainly by lowering the free concentration of Ca²⁺ by chelation and not by activation of cholera toxin or pertussis toxin-sensitive G proteins or protein kinase A.     

Effects of the desensitization by morphine of the opiate-dependent adenylate cyclase system in the rat striatum on the activity of the inhibitory regulatory G protein

Opiates act through a specific receptor to inhibit the striatal adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4,6.1.1] and stimulate a high-affinity GTPase (EC 3.6.1). The present study analyzes the functions of the striatal adenylate cyclase complex following chronic morphine treatment in the rat. The inhibitory effects of GTP on basal adenylate cyclase activity, between 10(-6) and 10(-4) M, were reduced. Moreover, the half-maximal inhibitory concentration of the opiate receptor agonist (D-Ala2-Met5)-enkephalinamide (DAME) on striatal adenylate cyclase activity was increased by about four times, whereas the maximal effect was reduced in membranes from treated rats. In parallel, the half-maximal stimulatory concentration of DAME on GTPase was increased by two times, and the maximal stimulation was reduced from 60 to 25%. Binding studies performed with [3,5-3H]DAME (saturation curves) and with [3H]naloxone (competition curves) did not show any change in opiate receptor numbers and affinity. Moreover, the kinetics of the activation of the inhibitory GTP binding protein (Gi) which transduces the opiate receptor effect on adenylate cyclase showed a small but significant delay. Therefore, hypofunction of Gi can be, at least in part, responsible for the observed desensitization by morphine of the opiate-dependent GTPase and adenylate cyclase.