Dopamine-stimulated Adenylyl Cyclase Activity Research Articles

Decreased striatal dopamine D1 receptor-stimulated adenylyl cyclase activity in human methamphetamine users.

It has been assumed that some behavioral changes associated with repeated exposure to dopaminergic psychostimulant drugs might be explained by changes in activity of dopamine receptors, including the dopamine D(1) receptor, which is linked by a stimulatory G protein to the effector enzyme adenylyl cyclase. To establish whether dopamine D(1) receptor function might be altered in human methamphetamine users, the authors measured dopamine-stimulated adenylyl cyclase activity in the brain of chronic human users of the drug. Adenylyl cyclase activity stimulated by dopamine and by guanylyl-imidodiphosphate (to assess G protein and adenylyl cyclase coupling) was determined in the postmortem brain tissue of 16 methamphetamine users who had used the drug both recently and chronically (i.e., at least 1 year) as well as 21 matched comparison subjects. A 25%-30% decrease in the maximal extent of dopamine stimulation of adenylyl cyclase activity was seen in the striatum (nucleus accumbens, caudate, and putamen) of the methamphetamine users. No changes were found in basal or guanylyl-imidodiphosphate-stimulated enzyme activity. These data suggest that dopamine receptor function linked to adenylyl cyclase is partially desensitized in the striatum of human methamphetamine users. Decreased dopamine D(1) receptor function might underlie part of the known (drug withdrawal syndrome) or expected (drug tolerance) consequences of methamphetamine exposure in humans.

Increased dopamine receptor signaling and dopamine receptor-G protein coupling in denervated striatum.

Chronic interruption of the nigrostriatal dopaminergic pathway leads to sensitized dopaminergic responses in striatum. We attempted to explore the mechanism(s) underlying this dopaminergic supersensitivity by assessing dopamine receptor signaling and receptor-G protein coupling in unilateral 6-hydroxydopamine-lesioned rats. Dopamine-stimulated adenylyl cyclase activity as well as dopamine-activated guanosine 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and [(3)H]palmitate incorporation by Galpha proteins were enhanced in tissues obtained from denervated striata without apparent changes in Galpha protein levels. Moreover, high-affinity binding sites of the D(1) dopamine receptor increased in lesioned compared with control striata without altering the expression level of the receptor. These denervation-mediated changes appear to correlate with the increase in D(1) dopamine receptor binding sites that co-immunoprecipitated with Galphas(olf)/q(11) proteins. In contrast, the total number of D(2) receptor binding sites was increased, yielding an increase in absolute number of high-affinity sites without significant changes in the proportion of high-affinity sites. Stimulation of the D(2) dopamine receptor enhanced coupling to Galphai protein; this was increased in the striata lesioned. The results provide an important molecular mechanism by which dopamine receptor-regulated signaling is enhanced following denervation of dopaminergic input to striatum. Although D(1) dopamine receptor supersensitivity appears to be mediated by enhanced coupling of the receptor to its G proteins, sensitization in the D(2) dopamine receptor system is mediated by increased D(2) receptor density and enhanced D(2) receptor-Gi protein coupling.

Modulation of agonist stimulated adenylyl cyclase and GTPase activity by l-pro- l-leu-glycinamide and its peptidomimetic analogue in rat striatal membranes

l-Prolyl- l-leucyl-glycinamide (PLG), also known as melanocyte-stimulating hormone release inhibiting factor (MIF-1), is an endogenous brain tripeptide. Previous studies have shown that PLG, and its peptidomimetic analogues, render dopamine D 2 receptors more responsive to agonists by maintaining the high-affinity binding state of the receptors. In the present study, we examined the effect PLG and its analogue 3( R)-[(2( S)-pyrrolidylcarbonyl)amino]-2-oxo-l-pyrrolidineacetamide (PAOPA) on dopamine-stimulated adenylyl cyclase and NPA-stimulated GTPase activity in rat striatal membranes. Dopamine-stimulated adenylyl cyclase activity was inhibited by both PLG and PAOPA in a dose-dependent manner, whereas R(−)-propylnorapomorphine (NPA)-stimulated low K m GTPase activity was significantly increased by 1 μM PLG or 1 nM PAOPA. These results suggest that PLG and PAOPA maintain the high affinity state of the D 2 receptor by increasing GTP hydrolysis through stimulation of agonist-induced GTPase activity.

Modifications of dopamine D 1 receptor complex in rats self-administering cocaine

Cocaine is spontaneously and experimentally self-administered and, when given repeatedly, it induces a stable form of sensitization to a previously assessed minimum active dose. In the present study, triads of rats chronically implanted with a jugular catheter were treated as follows: one animal was trained to self-inject cocaine, while the other two passively received either cocaine or saline whenever the self-administering rat completed the response requirement. After 30 days of stable responding, the animals were sacrificed and dopamine D 1 receptor density and adenylyl cyclase activity were measured in different brain areas. Animals receiving cocaine (both self-administering and yoked) showed a down-regulation of dopamine D 1 receptor number and of dopamine stimulated adenylyl cyclase activity in the nucleus accumbens, as compared to saline rats. In the olfactory tubercle, dopamine stimulated adenylyl cyclase activity appeared selectively and significantly down-regulated in self-administering animals.

Repeated methylphenidate treatment induces behavioral sensitization and decreases protein kinase A and dopamine-stimulated adenylyl cyclase activity in the dorsal striatum.

The behavioral effects of repeated methylphenidate (MPH) treatment were assessed in the adult rat. Protein kinase A (PKA) and adenylyl cyclase (basal and DA-stimulated) activity in the dorsal striatum (i.e., caudate-putamen) were measured to determine whether MPH-induced alterations in these enzymes correlate with the occurrence of behavioral sensitization. In two experiments, adult rats were injected (i.p.) on 5 consecutive pre-exposure days with saline or MPH (5, 10, 15, or 20 mg/kg). Sensitization was tested after a single abstinence day, with rats receiving a challenge injection of MPH prior to either a 40- or 150-min testing session (additional control groups received saline on the test day). Immediately after the 40-min testing session, rats were killed and tissue from the dorsal striatum was dissected for later analysis of PKA and adenylyl cyclase activity. Results showed that repeated MPH treatment sensitized the stereotyped sniffing, but not the locomotor activity, of adult rats. PKA activity was significantly depressed in rats treated with MPH (10 or 20 mg/kg) during both the pre-exposure and test day phases. DA-stimulated adenylyl cyclase activity was reduced after chronic MPH treatment, while basal adenylyl cyclase values were enhanced. Thus, the present study showed that MPH was able to sensitize the stereotyped behaviors of adult rats, an action that corresponded with drug-induced changes in dorsal striatal DA signal transduction mechanisms.

(+/-)-3-[4'-(N,N-dimethylamino)cinnamyl]benzazepine analogs: novel dopamine D1 receptor antagonists.

Neurochemical studies and structure-activity relationships of dopamine D1 receptor ligands suggest that their intrinsic activity may depend on the conformational state or binding site at which they interact on the receptor protein. Important differences in the modes of binding of these ligands may confer their agonist, partial agonist, or antagonist properties. In an effort to develop novel dopamine D1 antagonists and investigate the D1 antagonist pharmacophore, a series of (+/-)-(N-alkylamino)benzazepines were prepared in which (+/-)-7-chloro-8-hydroxy-3-[6-(N,N-dimethylamino)hexyl]-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzazepine (1) demonstrated the highest binding affinity (Ki = 49.3 nM) and selectivity to dopamine D1 receptors. This compound inhibited dopamine-stimulated adenylyl cyclase, in rat caudate, confirming a D1 receptor antagonist profile. From this initial series of N-alkylamino-substituted benzazepines, structure-activity relationships suggested that the terminal amino function was necessary for optimal binding affinity and selectivity at D1 vs D2 sites. Further, addition of this side chain to the D1 agonist pharmacophore (e.g., 7,8-dihydroxy-3-[4-(N,N-dimethylamino)butyl]-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine) greatly decreased binding affinity at D1 receptors. These data suggested that a binding domain that may be unique to the D1 antagonists may have been identified. In an attempt to exploit an apparent amine-accepting binding domain on the D1 receptor, a series of (+/-)-3-[4'-(N,N-dimethylamino)cinnamyl]benzazepine analogs was designed and prepared, as D1 antagonists. In this series, (+/-)-7-chloro-8-hydroxy-3-[4'-(N,N-dimethylamino)cinnamyl]-1-phenyl-2,3,4,5 -tetrahydro-1H-3-benzazepine (6a) showed the highest binding affinity (Ki = 60.3 nM) for dopamine D1 receptors. Compound 6a was a potent dopamine D1 antagonist as evidenced by its ability to block dopamine-stimulated adenylyl cyclase activity in rat caudate (predicted Ki value = 18.4 nM). Molecular modeling studies demonstrated that the most potent and selective dopamine D1 antagonists, in both series, contained terminal amino groups 8-9 A away from the 3-position benzazepine nitrogen. Compounds that lacked a terminal amine function or where this moiety was less than 7 A away from the benzazepine nitrogen demonstrated significantly lower binding affinities. Therefore, this series of (+/-)-3-[4'-(N,N-dimethylamino)cinnamyl]benzazepines also appears to be identifying an amine-accepting binding domain on the dopamine D1 receptor protein that may be further explored for the development of novel dopamine D1 antagonists.

(+/-)-(N-alkylamino)benzazepine analogs: novel dopamine D1 receptor antagonists.

(+/-)-(N-Alkylamino)benzazepine analogs were prepared as novel dopamine D1 receptor antagonists to further elucidate the role of these receptor subtypes in the pharmacology and toxicology of cocaine. In the first series of compounds, (+/-)-7-chloro-8-hydroxy-3- [6-(N,N-dimethylamino)-hexyl]-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepi ne (15) showed the highest affinity (Ki = 49.3 nM) and subtype-selectivity for dopamine D1 over dopamine D2, 5-HT2a, and 5-HT2c receptors. Compounds 7a [(+/-)-7-Chloro-8-hydroxy-3-[4-(N,N-dimethylamino)butyl]-1-phenyl- 2,3,4,5-tetrahydro-1H-3-benzazepine], 11 [(+/-)-7-chloro-8-hydroxy-3-[6-[(N,N-dimethylamino)hexyl]-1- phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-cyanoborane], and 15 were moderately potent dopamine D1 receptor antagonists as evidenced by their ability to block dopamine-stimulated adenylyl cyclase activity in rat caudate (predicted Ki values = 60, 34, and 21 nM, respectively). Compound 7a appears to be unique in that, despite its relatively potent inhibition of dopamine stimulated adenylyl cyclase, it demonstrated relatively weak binding affinity at the dopamine D1 receptors (Ki = 811 nM). Unlike previously reported N-alkylbenzazepines, where a significant loss in dopamine D1 receptor binding affinity was observed when successive increases in the alkyl side chain size at the benzazepine nitrogen were made, several of these novel N-alkylamino analogs demonstrated high-affinity binding with an optimal chain length of six carbons. This initial series of compounds appears to be identifying another binding domain on the dopamine D1 receptor protein that has not previously been characterized and that accepts an amino function. Further, these compounds may serve as templates for the design of peripherally active dopamine D1 receptor antagonists.

Cyclic AMP-Mediated Desensitization of D1 Dopamine Receptor-Coupled Adenylyl Cyclase in NS20Y Neuroblastoma Cells

Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase was investigated using NS20Y neuroblastoma cells. Pretreatment of the cells for 24 h with 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic monophosphate (CPT-cAMP), a membrane-permeable analog of cAMP, resulted in an ∼90% reduction of the maximum dopamine-stimulated adenylyl cyclase activity. In addition, there was a twofold reduction in the potency of dopamine for stimulating cAMP production that was not dependent on the concentration of Mg2+ in the assay. These effects of CPT-cAMP pretreatment were time dependent, showing a t1/2 of about 3 h and a maximum reduction after about 8 h. Receptor-binding activity, as measured using the D2-selective antagonist [3H]SCH-23390, also declined following CPT-cAMP pretreatment with a t1/2 of about 5 h and a maximum reduction of about 70% after 20 h. Saturation analysis indicated that the loss in radioligand binding was due to a reduction in maximum binding capacity (Bmax) with no alteration in receptor affinity (KD). The EC50 of CPT-cAMP for producing enzyme desensitization and D1 receptor downregulation was determined to be about 30 μM with a maximal response occurring at 1 mM. These regulatory effects of CPT-cAMP were pharmacologically specific as other analogs of cAMP, such as dibutryl-cAMP, 8-bromo-cAMP, and Sp-cAMPS, were capable of inducing D1 receptor desensitization and downregulation, whereas treatment of the cells with the cAMP antagonist Rp-cAMPS had no effect. Conversely, Rp-cAMPS was capable of blocking the regulatory effects of CPT-cAMP but was apparently without effect in blocking dopamine-induced desensitization. In addition to desensitization and receptor downregulation, CPT-cAMP treatment was found to promote functional uncoupling of the receptor as suggested by a loss of high-affinity agonist binding observed in dopamine/[3H]SCH-23390 competition assays following desensitization. Removal of CPT-cAMP resulted in recovery to control activities within 24 h. This recovery could be completely blocked through inhibition of cellular protein synthesis using cycloheximide. These data indicate that increasing the intracellular levels of cAMP can promote desensitization of the D1 receptor/adenylyl cyclase system and can induce a downregulatlon of D1 receptor-binding activity, presumably involving degradation of receptor protein.

Desensitization, phosphorylation and palmitoylation of the human dopamine D 1 receptor

The regulation and post-translational modifications of the human dopamine D 1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D 1 receptor or a DNA encoding a c-myc epitope tagged dopamine D 1 receptor (c-myc-dopamine D 1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D 1 and c-myc-dopamine D 1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D 1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D 1 receptor, the photoaffinity label [ 125 I](3- methyl-2-[4′- azidophenyl]-2,3,5- tetrahydro-2H-3- benzazepine) ([ 125I]MAB) bound specifically upon photolysis. A major broad band of ∼48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D 1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D 1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D 1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [ 3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D 1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.

β 1-Adrenergic and D 1 Dopaminergic Receptors in Human Neurotumor Cells: Differences in Spare Receptors and Desensitization of Adenylyl Cyclase

Haman SK-N-MC neurotumor cells express both β 1-adrenergic and D 1 dopaminergic receptors. Although there were sevenfold more β 1 than D 1 receptors, maximum stimulation of adenylyl cyclase activity by isoproterenol was only threefold more than that by dopamine. We had shown previously that the pattern of agonist-mediated densensitization was different for the two receptors. To compare the efficiency of the two receptors to couple to the same adenylyl cyclase and to explore the possible role of spare receptors, the cells were exposed to N -ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which irreversibly alkylates and inactivates catecholamine receptors. Loss of receptor binding activity was dependent on the concentration of EEDQ, D 1 receptors being more sensitive to EEDQ than β 1 receptors with IC 50 values of 1 and 30 μ M, respectively. β 1 receptors were more sensitive to the irreversible β-adrenergic antagonist N 8 -bromoacetyl- N 1-(3′-(4-indolyloxy)-2′-hydroxy-propyl)-[Z]-1,8-diamino- p-menthane (BIM) with an IC 50 of 1 n M. Inactivation of D 1 receptors parallelled the reduction in dopamine-stimulated adenylyl cyclase activity without a shift in K act, indicating the absence of spare D 1 receptors in SK-N-MC cells. By contrast, there appeared to be spare β1 receptors as the K act shifted before any reduction in isoproterenol-stimulated activity occurred in EEDQ- or BIM-treated cells. Inactivation of 70% of the β 1 receptors resulted in only a 20% decrease in isoproterenol-stimulated adenylyl cyclase activity, and the remaining β 1 receptors were as efficient as D 1 receptors in stimulating adenylyl cyclase. In addition, inactivation of the spare β 1 receptors did not alter the unique pattern of agonist-mediated desensitization. We conclude that human β 1 and D 1 receptors are equally efficient at stimulating adenylyl cyclase but differ in their mechanisms of agonist-mediated desensitization.

Sensitization of dopamine-stimulated adenylyl cyclase in the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys and patients with idiopathic Parkinson's disease.

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys and humans with idiopathic Parkinson's disease and compared with the activity in control tissue. No differences between parkinsonian and control tissue were found in the presence of 20 mM NaCl. However, when 120 mM NaCl was included in the assay medium, a significantly higher increase in the Vmax of dopamine-stimulated adenylyl cyclase activity was observed in the caudate of MPTP-parkinsonian rhesus monkeys and the putamen of patients with idiopathic Parkinson's disease. No such sensitization was seen in the MPTP-treated rhesus putamen or human Parkinson's disease caudate tissue. A role of D2 receptors in this sensitization could be ruled out by the concomitant use of the D2 antagonist l-sulpiride and by [3H]spiperone saturation analysis of the D2 receptor density, which was found at control level in the caudate tissue of MPTP-treated rhesus monkeys. Similarly, on the basis of saturation binding with the D1 selective ligand 125I-SCH 23982, there was no difference in caudate nucleus D1 receptor densities between control and MPTP-treated monkeys. Our results point to a region-specific functional sensitization of D1 receptors as a consequence of severe dopaminergic denervation of the striatum and suggest the possibility of a therapeutic potential of a D1 agonist with full intrinsic activity in Parkinson's disease.

Functional sensitization of striatal dopamine D 1 receptors in the 6-hydroxydopamine-lesioned rat

Dopamine-stimulated adenylyl cyclase activity was measured in striatal homogenates of rats in which the nigrostriatal pathway was lesioned by 6-hydroxydopamine 20–24 months before the experiments. In the intact (contralateral) striatum the potency and the efficacy of dopamine in stimulating adenylyl cyclase was lower in the presence of high NaCl concentrations (120 mM) compared with the effects of dopamine in an NaCl-poor assay medium (20 mM). The same effect of NaCl was observed in the striatum on the side of a weak, behaviourally ineffective 6-hydroxydopamine lesion resulting in a loss of 57% of striatal dopamine. This effect of NaCl was absent in the strongly denervated striatum, i.e. in rats having a 99.8% dopamine loss and rotating when challenged with a low dose of apomorphine. This, in denervated vs intact striatum, in the presence of a physiological concentration of NaCl, dopamine-stimulated adenylyl cyclase showed a sensitization which was absent in assays with 20 mM NaCl. The inhibition of adenylyl cyclase by dopamine via D 2 receptors, which was seen in the presence of 120 mM NaCl and the D 1 antagonist SCH 23390, was not affected by denervation. We suggest that chronic dopaminergic denervation of the striatum results in a stabilized, i.e. NaCl-insensitive,high affinity state of D 1 receptors. This may be the basis for a sensitization of the coupling mechanism of the denervated D 1 receptors to adenylyl cyclase.