The dopamine-sensitive adenylate cyclase of the rat caudate nucleus—3. The effect of aporphines and protoberberines

A series of aporphines and protoberherines were tested for activity with da and beta-type adenylate cyclases. Loss of da agonist but not antagonist activity of apomorphine was associated with the removal or methylation of the hydroxyls or the S-configuration. The effects of other alterations in structure were not as clear-cut and alterations in antagonist activity were not similar in the two enzyme systems. Surprisingly the protoherberines were fairly potent as inhibitors of the da-cyclase with little effects on the beta system. In most situations the S-isomer was much more potent than the R-antipode. None of the compounds studied possessed beta-agonist activity.     

Dopamine receptor topography. Characterization of antagonist requirements of striatal dopamine-sensitive adenylate cyclase using protoberberine alkaloids

Representative protoberberine-related alkaloids, i.e. tetrahydroprotoberberines (THPB), quaternary protoberberine salts (Quat. PB) and quaternary dehydroprotoberberine salts (Dehyd. Quat. PB), have been used to characterize the geometric and stereospecific requirements of antagonists of the dopamine receptor. The optical isomers of 2, 3, 10, 11-THPB were tested for their ability to antagonize dopamine stimulated adenylate cyclase activity. The results indicate that (±)-2. 3, 10, 11-THPB inhibited the ability of 100 μM dopamine to elevate adenylate cyclase in homogenates of the rat caudate nucleus. The ic₅₀ was observed to be 6 μM. The S-(?)-isomer of 2, 3, 10, 11-THPB was a more potent antagonist of dopaminesensitive adenylate cyclase activity than the R-(+)-isomer. The ic₅₀ for (?)-THPB was 1μM whereas that for the (+)-isomer was 50 μM. The data also show that the positional isomer, 2, 3, 9, 10-THPB, antagonized dopamine activation of adenylate cyclase with the same degree of potency as 2, 3, 10, 11-THPB. Exhaustive O-methylation of THPB at all four hydroxyl positions, the 2, 3-position of the “a” ring and the 10, 11-position of the “d” ring as in xylopinine or the 2, 3-position of the “a” ring and the 9, 10-position of the “d” ring as in tetrahydropalmatine, rendered these compounds weak antagonists of the dopamine response. Selective O-methylation of the THPB molecule markedly altered the potency of the resultant compounds as antagonists depending on the position of the O-methyl substitution. Overall, these data are consistent with the idea that the orientation of the nitrogen atom in a fixed (cis: gauche) position 2 carbon atoms from a catechol nucleus renders antagonist properties to these compounds which interact with the dopamine receptor.     

Factors affecting solubilized dopamine-sensitive adenylate cyclase

Bovine striatal adenylate cyclase was solubilized with sodium cholate and assayed for its responsiveness to a variety of agents. Magnesium ions (0–20 mM), guanylyl-imidodiphosphate (10–50 μM), and striatal lipids were effective in increasing enzyme activity. The adenylate cyclase could be stimulated by dopamine, and neuroleptic drugs inhibited the effect of dopamine (50 μM) with potencies that paralleled their clinical potencies. The ic₅₀ values for spiroperidol, haloperidol and chlorpromazine were 0.2, 3 and 500 nM respectively.     

Adenosine receptor-coupled adenylate cyclase in the caudate nucleus of the rat brain

5'-(N-Ethylcarboxamido)adenosine (NECA) and N6-[(R)-(phenylisopropyl)]adenosine (PIA) were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat, in order to examine the effect of kainic acid and a 6-hydroxydopamine-induced lesion on adenosine receptor coupled adenylate cyclase in vitro. There was an enhancement of formation of cyclic AMP induced by NECA, that was mediated by A2 adenosine receptors. Phenylisopropyl adenosine also stimulated adenylate cyclase in the striatum, with a maximum increase at 0.1 mM. At smaller concentrations, PIA inhibited the basal activity, which was previously described to be an effect mediated by A1 adenosine receptors. In caudate-putamen tissue from rats receiving a unilateral lesion, induced with kainic acid, basal and maximally NECA- and PIA-stimulated activity of adenylate cyclase was decreased. The maximum stimulatory effects of both substances were also significantly decreased, whereas no change of the inhibitory effect of PIA was observed. After unilateral lesion induced with 6-OHDA, basal and maximally NECA- and PIA-activated adenylate cyclase was increased; however, no inhibitory effect of PIA was seen. These results suggest that A2 adenosine receptor-coupled adenylate cyclase was located on neurones intrinsic to the neostriatum and probably postsynaptic to the dopaminergic input. The A1 adenosine receptors seem to be associated with the nigrostriatal pathway implying a presynaptic localization on dopaminergic afferents. In addition, since after both kainic acid- and 6-OHDA-induced lesions, respectively, in caudate-putamen tissue of the contralateral side, PIA no longer inhibited the activity of adenylate cyclase, contralateral structures also appeared to be involved in the regulation of A1 adenosine receptors.     

Dopamine-sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine or haloperidol.

The effects of morphine and haloperidol were compared on dopamine-sensitive adenylate cyclase activity of the rat caudate nucleus, an enzyme activity which has been related to the “dopamine receptor.” The response of adenylate cyclase activity to dopamine was measured in a hypo-osmotically shocked mitochondrial-synaptosomal fraction. The addition of 3–300 μM morphine, levorphanol, dextrorphan, d- and l-methadone, nalorphine and naloxone in vitro caused no significant changes in the response to dopamine, while addition of haloperidol in vitro completely inhibited the dopamine response at a concentration of 3 μM or higher. When morphine was administered subcutaneously at a dose of 60 mg/kg, significant increases were found in basal and dopamine-sensitive adenylate cyclase activity from the rat caudate from 15 to 120 min after the injection. Twenty to sixty min after the subcutaneous injection of 20 mg/kg of haloperidol, 1.7 to 2.0-fold increases were found in the dopamine-stimulated activity. The increased dopamine-sensitive cyclase activity found after acute administration of haloperidol o r morphine returned to control values 4 hr after the injection. Implantation of two morphine pellets for 2 or 3 days produced a significant increase in the dopamine sensitivity of the cyclase activity without altering basal activity. These results from experiments in vitro and in vivo suggest that, while haloperidol has a direct effect on the dopamine receptor-associated cyclase activity, morphine must act by another mechanism, and that chronic use of either drug produces enhanced dopamine sensitivity.     

Regional effects of neuroleptics on dopamine metabolism and dopamine-sensitive adenylate cyclase activity.

The effect of haloperidol, chlorpromazine, thioridazine and sulpride on the levels of DOPAC and HVA, as an index of DA turnover, and on the activity of DA-stimulated adenylate cyclase was investigated inthe striatum, the nucleus accumbens and the tuberculum olfactorium of the rat brain. Haloperidol, chlorpromazine and thioridazine caused a more marked increase in DA turnover in the striatum than in the mesolimbic areas, while the reverse was true for sulpiride. In contrast, although the relative potency of these compounds varied greatly, the Ki of each drug for the DA-sensitive adenylate cyclase was similar in three structures of rat brain. The results indicate that in the three brain structures investigated there was no correlation between the differential effects of neuroleptics on dopamine turnover in vivo and the blockade by these drug of the DA-sensitive adenylate cyclase activity in vitro.     

Dopamine-sensitive adenylate cyclase in canine renal artery.

To characterize further a putative dopamine receptor in the renal artery, the effects of dopamine on canine renal artery adenylate cyclase activity were studied. Since the femoral artery is thought to be devoid of a similar dopamine receptor, the effects of dopamine on the adenylate cyclase activity of the canine femoral artery were also studied. In tissues from dogs with or without phenoxybenzamine pretreatment, renal artery adenylate cyclase was maximally stimulated by 4 muM dopamine, compared to 20 muM required for the femoral artery enzyme. The concentrations of isoprenaline required to maximally stimulate renal and femoral artery adenylate cyclase were 0-04 and 0-2 muM, respectively. In tissue from the phenoxybenzamine-pretreated dog, the sitmulatroy effect of dopamine on the renal artery enzyme was selectively blocked by 0-01 muM haloperidol, but not by 0-2 muM propranolol. In the femoral artery, however, the dopamine stimulation was blocked by both antagonists. Stimulation by isoprenaline of renal and femoral artery adenylate cyclase was blocked by propranolol. These data suggest the concept that dopamine interacts with a specific artery receptor apparently different from alpha-and beta-adrenoceptors.     

Opioid receptors and inhibition of dopamine-sensitive adenylate cyclase in slices of rat brain regions receiving a dense dopaminergic input.

In slices of rat nucleus accumbens, olfactory tubercle, frontal cortex and mediobasal hypothalamus exposed to dopamine (DA), the activation of DA D1 receptors stimulated cyclic AMP (cAMP) formation whereas, in nucleus accumbens slices only, activation of D2 receptors appeared to inhibit D1 receptor-stimulated adenylate cyclase at the same time. Activation of mu-opioid receptors by [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO; 1 microM), but not of delta-opioid receptors by 1 microM [D-Pen2,D-Pen5]enkephalin (DPDPE), inhibited (by 35-40%) DA-stimulated cAMP production in slices of nucleus accumbens and olfactory tubercle. When adenylate cyclase was stimulated by selective D1 receptor activation, i.e. by DA in the presence of (-)-sulpiride, DPDPE reduced cAMP formation (by about 45%) in nucleus accumbens slices but not in slices of the other brain regions. The kappa-agonist, U 50,488, did not affect DA- or D1 receptor-stimulated adenylate cyclase activity in any of the brain regions. Preincubation of nucleus accumbens slices with the irreversible delta-ligand, fentanyl isothiocyanate (FIT; 1 microM), not only antagonized the inhibitory effect of DPDPE but also prevented the antagonism by naloxone of the inhibitory effect of DAMGO. Therefore, in nucleus accumbens opioids may inhibit DA-sensitive adenylate cyclase through activation of a mu/delta-opioid receptor complex, whereas in olfactory tubercle mu-receptors appear to mediate the inhibition of adenylate cyclase activity. Opioids do not seem to affect DA-stimulated cAMP formation in frontal cortex and mediobasal hypothalamus.     

Inhibition of adenylate cyclase by GTP and its modulation by opiate receptor in rat caudate nucleus

Morphine inhibited the adenylate cyclase activity of the crude synaptosomal fraction of the rat caudate nucleus in the presence of BTP, GDP, Gpp(NH)p or ITP. The purine nucleotides themselves had an inhibitory action on the enzyme. Beta-endorphin and Met-enkephalin also inhibited the enzyme in the presence of GTP. The GTP-dependent in inhibitory action of morphine was blocked by naloxone. Various opiates and opioid peptides inhibited the enzyme by up to approximately 20 per cent in the presence of GTP. The relative potency was in higher order of levorphanol greater than beta-endorphin greater than Met-enkephalin greater than morphine greater than pentazocine. Levorphanol was about 50,000 times as potent as its biologically inactive enantiomer, dextrorphan. Morphine enhanced the inhibitory actions of GTP and GTPase-resistant Gpp(NH)p on the adenylate cyclase activity. These results suggest that GTP plays an important role in the regulation of adenylate cyclase activity in the rat caudate nucleus and that the occupation of opiate receptor by agonists inhibits the enzyme through an actual increase in the inhibitory action of GTP, rather than a suppression of the enzymatic degradation of GTP.     

Tricyclic antidepressive drugs and dopamine-sensitive adenylate cyclase from rat brain striamtum

Tricyclic antidepressant drugs were examined as inhibitors of dopamine-sensitive adenylate cyclase in a cell-free homogenate of rat brain striatum. Amitriptyline (K_i 0.17 μM) and doxepin (K_i 0.24 μM) were found to be potent inhibitors of dopamine-sensitive adenylate cyclase, chlorimipramine (K_i 0.59 μM) and nortriptyline (K_i 0.50 μM) were moderate inhibitors and imipramine, desmethylimipramine, protriptyline melitracene were weak inhibitors with K_i values higher than 1 μM.     

Regional study of 3H-spiperone binding and the dopamine-sensitive adenylate cyclase in rat brain.

The inhibition of ³H-spiperone binding in vitro was determined in homogenates of a number of rat brain regions by 2-amino-6,7-dihydroxytetralin (ADTN), a rigid dopamine analog, cinanserin, a 5-hydroxytryptamine (5-HT) blocker and (+)-butaclamol, a neuroleptic drug with high affinity for both dopamine and 5-HT receptors. ADTN was a potent displacer of ³H-spiperone in striatum, was less effective in the olfactory tubercle, substantia nigra and hypothalamis and displaced ³H-spiperone from hippocampus and several cerebral cortical regions only at concentrations greater than 10 μM. The regional effects of cinanserin in inhibiting ³H-spiperone binding were essentially the converse of those observed for ADTN. (+)-Butaclamol, on the other hand, displaced ³H-spiperone only slightly less potently in regions with a poor or no dopaminergic innervation as compared to regions with a rich innervation. When ADTN and cinanserin were used in combination to displace ³H-spiperone, the results were similar to those observed with (+)-butaclamol or high concentrations of dopamine as displacer, further supporting the hypothesis that ³H-spiperone binds to both 5-HT and dopamine receptors. ADTN displaceable ³H-spiperone binding, which defines the specific binding of the radiolabel to a dopamine component, was compared to the dopamine-stimulated adenylate cyclase in the different brain regions. The poor correlation between these two parameters suggest that the receptors determined by the ³H-spiperone binding assay and the dopamine-sensitive adenylate cyclase constitute two different dopamine receptor populations.     

Effects of several ergosines on adenylate cyclase activity in synaptosomal membranes of the bovine caudate nucleus

The effects of several ergosines and different dopamine agonists and antagonists on the activity of dopamine-sensitive adenylate cyclase in synaptosomal membranes of the bovine caudate nucleus were comparatively studied. Among ergot alkaloid derivatives used, ergosinine was the most active in stimulating adenylate cyclase activity. Ergosine, bromoergosine, dihydroergosine, dihydroergocryptine and lisuride also stimulated this enzyme. Dihydroergosinine, bromodihydroergosine and bromoergocryptine did not affect adenylate cyclase activity. Saccharino derivatives of both ergosine and ergosinine were inactive. When used in higher concentrations, ergosine, ergosinine, dihydroergocryptine and lisuride inhibited dopamine-stimulated adenylate cyclase whereas other ergot alkaloid derivatives examined did not. If the extent of dopamine-sensitive adenylate cyclase stimulation is considered as a measure of dopaminergic activity, examination of the structure/dopaminergic activity relationship showed that modifications of ergot alkaloid molecules such as isomerization in position 8, hydrogenation of delta 9(10)-double bond, or introduction of bromine into position 2 of the molecule, lead to a significant decrease of stimulatory effects of adenylate cyclase. Introduction of a saccharino group into position 2 of the molecule caused a total loss of stimulatory activity of both ergosine and ergosinine, probably because of the size of the saccharino residue.