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Dopamine acts through a family of G protein-coupled receptors to exert its myriad effects. The D3 Dopamine receptor is one member of the D2-like dopamine receptors. We have previously demonstrated in human embryonic kidney (HEK293) cells that D3 receptor stimulation of phospholipase D (PLD) activity is pertussis toxin insensitive [Everett and Senogles. Neurosci. Lett. 371 (2004), 34]. We hypothesized that a low molecular weight G protein was involved in the agonist-mediated activation of PLD. When the D3 receptor was coexpressed with RhoA in HEK293 cells, agonist-induced stimulation of PLD activity was increased. However, co-expression of Rac or Cdc42 with the D3 receptor did not change the PLD activity. As well, expression of a dominant-negative construct of RhoA, N19 Rho completely ablated D3 receptor-mediated PLD activation, when co-expressed with the D3 receptor in HEK293 cells. In contrast, expression of dominant-negative constructs of Rac or Cdc42 had no effect. Treatment of HEK293 cells transfected with the D3 receptor and treated with a D3 preferring agonist R+-hydroxy(dipropylamino)tetralin hydrobromide, results in an agonist-induced physical complex of D3 receptor and either endogenous Rho or transfected hemaglutinin (HA)-RhoA that can be detected by immunoprecipitation and western blotting. Treatment of cells transfected with D3 receptor with R+-hydroxy(dipropylamino)tetralin hydrobromide also results in agonist-dependent Rho activation, as measured by a Rho effector pull-down assay. The data suggest that D3 receptor/RhoA association and activation is necessary for D3 receptor-mediated PLD activation.

Recent studies have demonstrated that D1-selective and D2-selective dopamine receptor agonists inhibit catecholamine secretion and Ca2+ uptake into bovine adrenal chromaffin cells by receptor subtypes that we have identified by PCR as D5, a member of the D1-like dopamine receptor subfamily, and D4, a member of the D2-like dopamine receptor subfamily. The purpose of this study was to determine whether activation of D5 or D4 receptors inhibits influx of Na+, which could explain inhibition of secretion and Ca2+ uptake by dopamine agonists. D1-selective agonists preferentially inhibited both dimethylphenylpiperazinium- (DMPP) and veratridine-stimulated 22Na+ influx into chromaffin cells. The D1-selective agonists chloro-APB hydrobromide (CI-APB; 100 microM) and SKF-38393 (< 00 microM) inhibited DMPP-stimulated Na+ uptake by 87.5 +/- 2.3 and 59.7 +/- 4.5%, respectively, whereas the D2-selective agonist bromocriptine (100 microM) inhibited Na+ uptake by only 22.9 +/- 5.0%. Veratridine-stimulated Na+ uptake was inhibited 95.1 +/- 3.2 and 25.7 +/- 4.7% by 100 microM CI-APB or bromocriptine, respectively. The effect of CI-APB was concentration dependent. A similar IC50 (approximately 18 microM) for inhibition of both DMPP- and veratridine-stimulated Na+ uptake was obtained. The addition of 8-bromo-cyclic AMP (1 mM) had no effect on either DMPP- or veratridine-stimulated Na+ uptake. These observations suggest that D1-selective agonists are inhibiting secretagogue-stimulated Na+ uptake in a cyclic AMP-independent manner.

Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K+ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5- tetrahydro-1H-3-benzazepine (CI-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with approximately 10 microM CI-APB and approximately 100 microM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by CI-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca2+ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification of soluble guanylate cyclase on DEAE-Sephacel yields two separate peaks of guanylate cyclase activity. After 10-fold purification of the soluble enzyme, guanylate cyclase is markedly inhibited by micromolar concentrations of dopamine (I50 = 0.2 microM). Dopamine inhibition is observed whether the reaction is conducted with Mn2+ or with Mg2+, under atmosphere or N2(g), and using enzyme from either peak from the DEAE-Sephacel column. Other catecholamines also inhibit partially purified guanylate cyclase with an order of potency at 1 microM of: dopamine = L-DOPA > norepinephrine = isoproterenol = adrenochrome > epinephrine. The structural requirements for inhibition are two free hydroxyl groups on the phenyl ring and an ethylamine side chain. Dopamine also inhibits the Triton X-100-solubilized microsomal guanylate cyclase after partial purification on DEAE-Sephacel. Neither chlorpromazine, propranolol, nor phentolamine at 20 microM effectively block the dopamine inhibition of partially purified soluble guanylate cyclase. Micromolar concentrations of the reducing agents dithiothreitol and glutathione also inhibit partially purified guanylate cyclase, but unlike these agents, catecholamines can inhibit whether added in the reduced or the oxidized forms. Inhibition of enzyme activity by micromolar concentrations of dopamine, adrenochrome, or dithiothreitol is rapidly reversed by dilution and the dopamine inhibition is competitive with MgGTP. Inhibition does not appear to involve covalent binding or to result from the ability of catecholamines to reduce the concentrations of oxygen or free radicals in solution.

The stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+-dependent guanylate cyclase activities showed no significant change over this period. Mn2+-dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine-stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine-sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.