Inhibition of <Emphasis Type="Italic">DD2R</Emphasis> gene expression in the corpus allatum activates alkaline phosphatase in female <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Tissue-specific inhibition of the expression of the D2-like dopamine receptor gene (DD2R) in the corpus allatum (CA), which is a gland that synthesizes the juvenile hormone (JH), was tested for effect on alkaline phosphatase (ALP) activity and the intensity of the ALP response to heat stress (stress reactivity) in female Drosophila melanogaster. ALP activity and ALP stress reactivity in transgenic females with lower DD2R expression in the CA were higher than in control flies. A pharmacological elevation in JH increased ALP activity in females of the control strains. DD2R was assumed to mediate the inhibitory effect of dopamine of JH synthesis in the CA of D. melanogaster.     

Role of DopR in the molecular mechanism of the dopamine control of juvenile hormone metabolism in female Drosophila

The effect of a decreased availability of the D1-like dopamine receptor (DopR) in Drosophila (caused by DopR antagonist added into food) on the juvenile hormone (JH) synthesis rate in young female D. melanogaster has been studied. The JH degradation rate and the alkaline phosphatase (ALP) and tyrosine decarboxylase (TDC) activities were used as indicators of the JH synthesis rate. Treatment of the flies with butaclamol, a specific DopR antagonist, has been demonstrated to increase the JH degradation rate, and the stress reactivity of the system of JH metabolism and to decrease the ALP activity and stress reactivity, and to increase the TDC activity and stress reactivity. As shown earlier, all this indicates a decrease in the JH synthesis rate in young female drosophila with a decreased DopR availability. It is concluded that the activating effect of dopamine on JH synthesis in Drosophila is mediated by D1-like receptors.     

The D1 family dopamine receptor,DopR, potentiates hind leg grooming behavior in Drosophila

Drosophila groom away debris and pathogens from the body using their legs in a stereotyped sequence of innate motor behaviors. Here, we investigated one aspect of the grooming repertoire by characterizing the D1 family dopamine receptor, DopR. Removal of DopR results in decreased hind leg grooming, as substantiated by quantitation of dye remaining on mutant and RNAi animals vs. controls and direct scoring of behavioral events. These data are also supported by pharmacological results that D1 receptor agonists fail to potentiate grooming behaviors in headless DopR flies. DopR protein is broadly expressed in the neuropil of the thoracic ganglion and overlaps with TH‐positive dopaminergic neurons. Broad neuronal expression of dopamine receptor in mutant animals restored normal grooming behaviors. These data provide evidence for the role of DopR in potentiating hind leg grooming behaviors in the thoracic ganglion of adult Drosophila. This is a remarkable juxtaposition to the considerable role of D1 family dopamine receptors in rodent grooming, and future investigations of evolutionary relationships of circuitry may be warranted.     

Selective degeneration of dopaminergic neurons by MPP+ and its rescue by D2 autoreceptors in Drosophila primary culture

Drosophila melanogaster is widely used to study genetic factors causing Parkinson's disease (PD) largely because of the use of sophisticated genetic approaches and the presence of a high conservation of gene sequence/function between Drosophila and mammals. However, in Drosophila, little has been done to study the environmental factors which cause over 90% of PD cases. We used Drosophila primary neuronal culture to study degenerative effects of a well‐known PD toxin MPP⁺. Dopaminergic (DA) neurons were selectively degenerated by MPP⁺, whereas cholinergic and GABAergic neurons were not affected. This DA neuronal loss was because of post‐mitotic degeneration, not by inhibition of DA neuronal differentiation. We also found that MPP⁺‐mediated neurodegeneration was rescued by D2 agonists quinpirole and bromocriptine. This rescue was through activation of Drosophila D2 receptor DD2R, as D2 agonists failed to rescue MPP⁺‐toxicity in neuronal cultures prepared from both a DD2R deficiency line and a transgenic line pan‐neuronally expressing DD2R RNAi. Furthermore, DD2R autoreceptors in DA neurons played a critical role in the rescue. When DD2R RNAi was expressed only in DA neurons, MPP⁺ toxicity was not rescued by D2 agonists. Our study also showed that rescue of DA neurodegeneration by Drosophila DD2R activation was mediated through suppression of action potentials in DA neurons.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

D2‐like dopamine receptors mediate regulation of pupal diapause in Chinese oak silkmoth Antheraea pernyi

The present study investigated the pharmacological properties of dopamine receptors that functioned in the termination of pupal diapause in the Chinese oak silkmoth, Antheraea pernyi (Lepidoptera: Saturniidae). Dopamine receptors are classified according to their structure and function into two subfamilies as D1‐ and D2‐like receptors. D1‐like receptors activate, whereas D2‐like receptors inhibit, adenylate cyclase. We examined the effects of agonists and antagonists selective for D1‐ and D2‐like receptors on the diapause state. As A. pernyi is a long‐day species, pupal diapause is maintained during short days and can be terminated by exposure to a long‐day photoperiod. The D2‐like receptor‐selective agonist quinpirole delayed the timing of adult emergence under long days, and the D2‐receptor‐selective antagonist sulpiride terminated pupal diapause even under a short‐day photoperiod. The D1‐like receptor‐selective agonist and antagonist, SKF‐38393 and SCH‐23390, respectively, caused no significant effects on diapause pupae. These results suggest that not D1‐ but D2‐like receptors mediated diapause regulation in A. pernyi. This dopamine pathway appeared to block the termination of pupal diapause. Furthermore, the actions of the cAMP analog 8‐CPT‐cAMP and dopamine receptor antagonists upon diapause pupae were similar, which supports the notion that D2‐like receptors involved in diapause of this insect prevent adenylate cyclase from producing cAMP like vertebrate D2‐like receptors. Taken together, our findings suggest that dopamine blocked diapause termination through D2‐like receptors that inhibited adenylate cyclase in A. pernyi. During short days under which diapause was maintained in pupae, the dopaminergic mechanism might be stimulated to suppress cAMP levels in cells regulating diapause.     

Drug-Induced Up-Regulation of Dopamine D2 Receptors on Cultured Cells

Abstract: Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C₆ glioma cells stably expressing the short (415-amino-acid; D2_S) and long (444-amino-acid; D2_L) forms of the receptor. Overnight treatment of C6-D2_L cells with N-propylnorapomorphine (NPA) caused a time- and concentration-dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 µM NPA was maximal after 10 h, at which time the density of D2_L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2_L receptors. The receptor up-regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30–60%) increases in receptor density. Prolonged treatment with 10 µM NPA desensitized D2_L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA-induced receptor up-regulation in each of four clonal lines of C6-D2_L cells (mean increase, 80%) was greater than in all four lines of C6-D2_S cells (33%). Inactivation of pertussis toxin-sensitive G proteins had no effect on the basal density of D2_L receptors or on the NPA-induced receptor up-regulation. Treatment with 5 µg/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA-induced increase. Chimeric D1/D2 receptors were used to identify structural determinants of dopamine receptor regulation. Treatment with the D1/D2 agonist NPA decreased the density of D1 and chimeric CH4 and CH3 receptors. The latter two receptors have D1 sequence from the amino-terminus to the amino-terminal end of transmembrane region (TM) VII and VI, respectively. CH2, with D1 sequence up to the amino-terminal end of TM V, and thus the third cytoplasmic loop of the D2 receptor, was up-regulated by NPA or the D2-selective agonist quinpirole. Quinpirole treatment decreased the density of CH3 and had no effect on CH4 or D1 receptors. The different responses of CH2 and CH3 to agonist treatment suggest a role for TM V and the third cytoplasmic loop in the direction of receptor regulation.     

Dopamine down-regulates activity of alkaline phosphatase in Drosophila: The role of D2-like receptors

The effect of a rise in dopamine (DA) level as a result of a mutation, stress or pharmacological treatment on the activity of the enzyme of its synthesis, alkaline phosphatase (ALP) in females of Drosophila virilis and Drosophila melanogaster has been studied. It has been found that regardless of its nature, a rise in DA level has a negative effect on ALP activity, which indicates that DA down-regulates activity of the enzyme. The effects of bromocriptine (an agonist of Drosophila dopamine 2-like receptor (DD2R)) on ALP activity have been studied. ALP activity was found to drop in response to bromocriptine in flies. Conversely ALP activity was increased in flies with reduced DD2R expression (i.e. Actin5C-Gal4 > UAS-ds-DD2R RNA-interference flies) vs. corresponding controls (i.e. Actin5C-Gal4 > w1118 flies). Bromocriptine treatment of RNAi flies rescues ALP activity to the level typical of Actin5C-Gal4 > w1118 flies. A change in DD2R number or availability was found not to prevent the response of ALP to heat stress, but to change the intensity of its response to the stress exposure. The role of D2-like receptors in down-regulation of ALP activity by DA and in ALP response to stressor in Drosophila is discussed.     

Mutation of Drosophila dopamine receptor DopR leads to male-male courtship behavior

In Drosophila, dopamine plays important roles in many biological processes as a neuromodulator. Previous studies showed that dopamine level could affect fly courtship behaviors. Disturbed dopamine level leads to abnormal courtship behavior in two different ways. Dopamine up-regulation induces male-male courtship behavior, while down-regulation of dopamine level results in increased sexual attractiveness of males towards other male flies. Until now, the identity of the dopamine receptor involved in this abnormal male-male courtship behavior remains unknown. Here we used genetic approaches to investigate the role of dopamine receptors in fly courtship behavior. We found that a dopamine D1-like receptor, DopR, was involved in fly courtship behavior. DopR mutant male flies display male-male courtship behavior. This behavior is mainly due to the male's increased propensity to court other males. Expression of functional DopR successfully rescued this mutant phenotype. Knock-down of D2-like receptor D2R and another D1-like receptor, DAMB, did not induce male-male courtship behavior, indicating the receptor-type specificity of this phenomenon. Our findings provide insight into a possible link between dopamine level disturbance and the induced male-male courtship behavior.     

Circadian Dysfunction Reduces Lifespan in Drosophila melanogaster

Circadian clocks regulate physiological and behavioral processes in a wide variety of organisms, and any malfunction in these clocks can cause significant health problems. In this paper, we report the results of our study on the physiological consequences of circadian dysfunction (malfunctioning of circadian clocks) in two wild‐type populations of fruit flies (Drosophila melanogaster). We assayed locomotor activity behavior and lifespan among adult flies kept under constant dark (DD) conditions of the laboratory, wherein they were categorized as rhythmic if their activity/rest schedules followed circadian (approximately 24 h) patterns, and as arrhythmic if their activity/rest schedules did not display any pattern. The rhythmic flies from both populations lived significantly longer than the arrhythmic ones. Based on these results, we conclude that circadian dysfunction is deleterious, and proper functioning of circadian clocks is essential for the physiological well being of D. melanogaster.     

Role of Neostriatal Dopamine D1 and D2 Receptors in the Organization of Neuronal Activity in the Entopeduncular Nucleus

In chronic experiments on cats, we studied the effects of selective blockade of the dopamine D1 and D2 receptors in the nucleus caudatus (NC) against the background neuronal activity (BA) in the entopeduncular nucleus (ENT) and on the responses of these neurons evoked by stimulation of the subthalamic nucleus (STN). A blocker of D1 receptors, SCH 23390, and sulpiride, a blocker of D2 receptors, when injected into the NC head, led to changes in the BA frequency. The direction of these effects was different: blockade of the D2 receptors evoked a significant decrease in the BA frequency of ENT neurons, but when the D2 receptors were blocked, a trend toward an increase in this frequency was observed. Some of the ENT cells demonstrated similar changes in the temporal organization of their impulse activity with blockade of the D1 and D2 receptors. Namely, besides single action potentials (AP), high-frequency burst discharges began to be generated. Blockade of both D1 and D2 receptors prolonged the responses of ENT neurons to STN stimulation due to the appearance of similar AP bursts, while control animals demonstrated reactions consisting of solely single AP. The question on a dual (synergic and antagonistic) involvement of the neostriatal D1 and D2 receptors in the dopaminergic regulation of the basal ganglia is under discussion.     

Molecular bases of proliferation of Francisella tularensis in arthropod vectors

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis–arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes‐derived macrophages and D. melanogaster‐derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella‐containing phagosome matures to a late endosome‐like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod‐derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster‐derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster‐derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis–arthropod vector interaction and its patho‐adaptation to infect mammals.     

Techniques and applications of three‐dimensional electron microscopy in entomological research

Structural studies using two‐dimensional (2D) images show limitations in understanding the structure and functions of cellular organelle and protein. To overcome the difficulty, over the last few years 3D reconstruction techniques using electron microscopy have been developed at extremely high speed. In this paper, currently available 3D reconstruction techniques of electron microscopy (such as electron tomography, serial section analysis and single particle analysis) are introduced using our data as examples of the application. The 3D structure of mitochondria with the defect of mitochondrial protein in round worm, Caenorhabditis elegans, through electron tomography, the cell–cell interaction in lamina of Drosophila melanogaster by serial‐section using ultramicrotome and high‐voltage electron microscopy and a thin filament related to muscle contraction in Drosophila melanogaster were used for examples of the application. These results through 3D reconstruction reveal the structural changes in a cellular organelle and protein that had not been shown by 2D structure.     

Probable mechanism of sexual dimorphism in insulin control of Drosophila heat stress resistance

Adult males of Drosophila species (Drosophila melanogaster L. and Drosophila virilis) show a lower tolerance to heat stress compared with females. The present study investigates the effects of RNA interference (RNAi) knockdown of the insulin‐like receptor in the corpus allatum of D. melanogaster males on dopamine metabolism and content, heat stress resistance and juvenile hormone metabolism. In male flies, the knockdown of insulin‐like receptor in the corpus allatum is shown to change metabolism of juvenile hormone but not dopamine. It is also shown that knockdown of the insulin‐like receptor in the corpus allatum results in a decrease of heat stress resistance in females but not males, and that juvenile hormone rescues this decrease. The results suggest that at least one of the ways in which insulin‐like growth factors affect heat stress resistance is by successive mediation through juvenile hormone and dopamine, which could lead to sexual dimorphism in the trait.     

The Drosophila dopamine 2-like receptor D2R (Dop2R) is required in the blood brain barrier for male courtship

The blood brain barrier (BBB) has the essential function to protect the brain from potentially hazardous molecules while also enabling controlled selective uptake. How these processes and signaling inside BBB cells control neuronal function is an intense area of interest. Signaling in the adult Drosophila BBB is required for normal male courtship behavior and relies on male-specific molecules in the BBB. Here we show that the dopamine receptor D2R is expressed in the BBB and is required in mature males for normal mating behavior. Conditional adult male knockdown of D2R in BBB cells causes courtship defects. The courtship defects observed in genetic D2R mutants can be rescued by expression of normal D2R specifically in the BBB of adult males. Drosophila BBB cells are glial cells. Our findings thus identify a specific glial function for the DR2 receptor and dopamine signaling in the regulation of a complex behavior.     

Locomotor activity is regulated by D2-like receptors in Drosophila: an anatomic and functional analysis

In mammals, dopamine 2-like receptors are expressed in distinct pathways within the central nervous system, as well as in peripheral tissues. Selected neuronal D2-like receptors play a critical role in modulating locomotor activity and, as such, represent an important therapeutic target (e.g. in Parkinson's disease). Previous studies have established that proteins required for dopamine (DA) neurotransmission are highly conserved between mammals and the fruit fly Drosophila melanogaster. These include a fly dopamine 2-like receptor (DD2R; Hearn et al. PNAS 2002 99(22):14554) that has structural and pharmacologic similarity to the human D2-like (D2R). In the current study, we define the spatial expression pattern of DD2R, and functionally characterize flies with reduced DD2 receptor levels. We show that DD2R is expressed in the larval and adult nervous systems, in cell groups that include the Ap-let cohort of peptidergic neurons, as well as in peripheral tissues including the gut and Malpighian tubules. To examine DD2R function in vivo, we generated RNA-interference (RNAi) flies with reduced DD2R expression. Behavioral analysis revealed that these flies show significantly decreased locomotor activity, similar to the phenotype observed in mammals with reduced D2R expression. The fly RNAi phenotype can be rescued by administration of the DD2R synthetic agonist bromocriptine, indicating specificity for the RNAi effect. These results suggest Drosophila as a useful system for future studies aimed at identifying modifiers of dopaminergic signaling/locomotor function.     

Extent of mismatch between the period of circadian clocks and light/dark cycles determines time‐to‐emergence in fruit flies

Circadian clocks time developmental stages of fruit flies Drosophila melanogaster, while light/dark (LD) cycles delimit emergence of adults, conceding only during the “allowed gate.” Previous studies have revealed that time‐to‐emergence can be altered by mutations in the core clock gene period (per), or by altering the length of LD cycles. Since this evidence came from studies on genetically manipulated flies, or on flies maintained under LD cycles with limited range of periods, inferences that can be drawn are limited. Moreover, the extent of shortening or lengthening of time‐to‐emergence remains yet unknown. In order to pursue this further, we assayed time‐to‐emergence of D. melanogaster under 12 different LD cycles as well as in constant light (LL) and constant dark conditions (DD). Time‐to‐emergence in flies occurred earlier under LL than in LD cycles and DD. Among the LD cycles, time‐to‐emergence occurred earlier under T4–T8, followed by T36–T48, and then T12–T32, suggesting that egg‐to‐emergence duration in flies becomes shorter when the length of LD cycles deviates from 24 h, bearing a strong positive and a marginally negative correlation with day length, for values shorter and longer than 24 h, respectively. These results suggest that the extent of mismatch between the period of circadian clocks and environmental cycles determines the time‐to‐emergence in Drosophila.     

Pharmacological Characterization of Mammalian D1 and D2 Dopamine Receptors Expressed in Drosophila Schneider‐2 Cells

Abstract

Mammalian D₁ and D₂ dopamine receptors were stably expressed in Drosophila Schneider‐2 (S2) cells and screened for their pharmacological properties. Saturable, dose‐dependent, high affinity binding of the D₁‐selective antagonist [³H]SCH‐23390 was detected only in membranes from S2 cells induced to express rat dopamine D₁ receptors, while saturable, dose‐dependent, high affinity binding of the D₂‐selective antagonist [³H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D₂ receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D₁ and D₂ receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D₁ receptor bound the (+)‐enantiomer of the D₁‐selective agonist SKF38393 with higher affinity than the (?)‐enantiomer, while the dopamine D₂ receptor bound the (?)‐enantiomer of the D₂‐selective agonist norpropylapomorphine with higher affinity than the (+)‐enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPγS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D₁ and D₂ receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D₁ and D₂ dopamine receptors free of promiscuous G protein contaminants.     

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: 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 (Cl-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with ～10 µM Cl-APB and ～100 µM 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 Cl-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. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1- and D2-selective agonists inhibit Ca²⁺ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.