Estimated rate of prostacyclin secretion into the circulation of normal man.

The rate of secretion of prostacyclin (PGI2) into the circulation of normal man was estimated by measurement of the 2,3-dinor-6-keto-PGF1 alpha (D) and 15-keto-13,14-dihydro-2,3-dinor-6-keto-PGF1 alpha (KDD) urinary metabolites of PGI2. Subjects received 6-h intravenous infusions of vehicle alone and PGI2 at 0.1, 0.4, and 2.0 ng/kg per min in random order. The fractional elimination of the metabolites was independent of the rate of PGI2 infusion. 6.8 +/- 0.3% of the infused PGI2 appeared as D and 4.1 +/- 0.4% as KDD. The regression of infused PGI2 upon the quantities of the two metabolites excreted in excess of control values permitted estimation of the rate of entry of endogenous PGI2 into the circulation corresponding to a given quantity of metabolite excreted. Using the quantities excreted in the 24 h from commencement of the infusions the estimated rates were 0.08 +/- 0.02 ng/kg per min from D and 0.10 +/- 0.03 from KDD. Studies with exogenous PGI2 suggest that infusion rates 2--4 ng/kg per min are required to achieve the threshold for inhibition of platelet function (ex vivo) in man. Although not precluding a role for PGI2 in local platelet-vessel wall interactions, the much lower estimates obtained in this study suggest that endogenous PGI2 is unlikely to act as a circulating antiplatelet agent in healthy man.

Recombination of endogenous D2 dopamine receptor gene with a metallothionein promoter in GH4C1 cells confers functional and inducible D2 response.

We have previously shown that expression of a functional endogenous D2 short dopamine receptor is obtained in GH4C1 cells following transfection with a plasmid that confers resistance to neomycin (pRSVNeo) (Allard et al. (1993) Biochem. Biophys. Res. Commun. 193, 801-807). In order to better understand the mechanisms responsible for such a phenomenon, we cloned and sequenced the 5' region of the D2 gene present in native GH4C1 cells as well as the cDNA of transfected cells. No homology with the published sequence of the rat D2 dopamine receptor promoter was found; however, this region has perfect homology with the mouse metallothionein promoter. In cells expressing D2 receptor, the promoter is fully functional and can regulate dopaminergic D2 receptor mRNA levels and receptor expression in a dose-dependent manner in the presence of Zn2+ or Cd2+. The receptor level is raised from 500 to 3000 fmol/mg of protein in the presence of 100 microM of Zn2+. These results suggest that in GH4C1 cells, a recombination between the mouse metallothionein promoter and the D2 dopamine receptor took place. This system provides us with a cell line expressing an endogenous dopamine D2 receptor in which the level of expression can be easily modulated.

Metabolism of 8,11,14-eicosatrienoic acid in human platelets.

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).

Dopamine receptor subtypes: beyond the D1/D2 classification.

The D1/D2 dopamine receptor classification is widely accepted. However, intense investigative efforts over the last several years using pharmacological, biochemical and behavioral approaches have produced results that are increasingly difficult to reconcile with the existence of only two dopamine receptor subtypes. Recent developments, including cloning of the cDNAs and/or genes for several members of the large family of G-protein-coupled receptors, have revealed that heterogeneity in the pharmacological or biochemical characteristics of individual receptors often indicates the presence of previously unsuspected molecular subtypes. In this article, Marc Caron and colleagues have assembled the main lines of evidence that suggest the presence of several novel subtypes for both D1 and D2 dopamine receptors and predict that molecular cloning will, in the near future, confirm their existence.

The functional state of the dopamine receptor in the anterior pituitary is in the high affinity form.

In order to determine whether the high affinity state or the low affinity state of the dopamine receptor mediated the inhibition of release of PRL by dopamine agonists, a large number of dopaminergic agonists (n = 31) and antagonists (n = 24) were tested for their potencies to inhibit the binding of [3H] spiperone to porcine anterior pituitary tissue, and for their potencies to affect the release of PRL from rat anterior pituitary cells in culture. All agonists (except bromocriptine, ergocryptine, and dihydroergocryptine) inhibited [3H]spiperone binding in two phases: one phase occurred at nanomolar or subnanomolar concentrations (representing the high affinity state of the dopamine receptor) and the other phase occurred at much higher concentrations of agonist (the low affinity state of the dopamine receptor). The dissociation constants (K) for each drug at each state were derived by computer, with the program LIGAND. It was observed that the agonist K values for the high affinity state were virtually identical with those agonist concentrations inhibiting PRL release; the K values for the low affinity state were about 2 orders higher. These data suggest that the high affinity state of the D2 dopamine receptor is the functional state which mediates the inhibition of PRL release.

In vivo production of prostaglandin I2 in Dahl salt-sensitive and salt-resistant rats.

Prostaglandin I2 (PGI2, prostacyclin), a potent vasodilator synthesized by the blood vessels, has been postulated to play a role in hypertension. The purpose of our study was to test this hypothesis by monitoring the in vivo production of PGI2 in Dahl salt-sensitive (S) and salt-resistant (R) rats under normal and high sodium intake. The 24-hour urinary excretion of two endogenous metabolites of PGI2, 2-,3-dinor-6-oxo-PGF1 alpha, an 2,3-dinor-13,14-dihydro-6,15-dioxo-PGF1 alpha was measured by combined gas chromatography-mass spectrometry (GC-MS) and used as an index of the total production of PGI2 by the animals. The pattern of urinary excretion of these two metabolites in the R and the S rats during the control period indicated that, under normal conditions, early in life the basal production of PGI2 was the same in both groups of rats. Following the chronic administration of a high sodium diet (8.1% sodium chloride, starting at 36 days of age), a significant and sustained increase in the urinary excretion of 2,3-dinor-6-oxo-PGF1 alpha was documented in the R rats (from 37 +/- 7 ng/24 hrs at age 35 days to 63 +/- 7, 52 +/- 4, and 56 +/- 10 ng/24 hrs at 50, 60, and 80 days, respectively), whereas the urinary levels of this metabolite decreased slightly in the S rats (from 41 +/- 7 ng/24 hrs at age 35 days to 25 +/- 5, 30 +/- 6, and 28 +/- 9 ng/24 hrs at 50, 60, and 80 days, respectively). During the same period, the R rats remained normotensive (103 +/- 5 mm Hg, systolic pressure) while the arterial pressure of the S rats increased gradually (to 142 +/- 8 and 180 +/- 19 mm Hg at ages 60 and 80 days, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Defective synthesis of vasodilator prostaglandins in the spontaneously hypertensive rat.

Results of in vitro studies performed in different laboratories have led to the hypothesis that in spontaneously hypertensive rats (SHR), the biosynthesis of the vasodilator prostaglandins (PG) I2 and E2 increases secondarily to the rise in the arterial pressure, as a protective mechanism for reducing the severity of hypertension. In this study, this hypothesis was tested in vivo in SHR and control Wistar-Kyoto (WKY) rats under normal and high sodium diets. The 24-hour urinary excretion of 2,3-dinor-6-oxo-PGF1 alpha, an endogenous metabolite of PGI2, was used as an index of the total production of PGI2 in the rats, whereas the urinary levels of PGE2 were used as an index of the renal production of this prostaglandin. Urinary levels of PGE2, obtained at 10 and 12 weeks of age, were always lower in SHR than in WKY rats and were not influenced by dietary sodium. In WKY and SHR on normal diets, the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha did not differ significantly. However, the chronic administration of a high sodium diet (5.9% NaCl) was accompanied by a significant and sustained rise in the urinary excretion of 2,3-dinor-6-oxo-PGF1 alpha in the order of 30% to 50% in WKY but not in SHR, a finding similar to that in Dahl rats. These results point to the existence of an impaired capacity in SHR to synthesize vasodilator prostaglandins in vivo, contrary to the situation prevailing in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostanoids and aldosterone-induced mild experimental hypertension in rats.

The goal of this study was to determine the role of prostanoids in a new model of mineralocorticoid-dependent hypertension induced by the subcutaneous infusion of aldosterone (1 micrograms/hr) to normal male Sprague-Dawley rats. This regimen caused a mild and gradual increase in systolic pressure over a period of 4 weeks (113 +/- 1 vs. 137 +/- 3 mm Hg) and was associated with an increase in the in vivo formation of prostaglandins I2 and E2 and of thromboxane A2 in the kidney. High sodium intake induced a fall in the urinary levels of prostaglandin E2 and a rise in the arterial pressure of control rats (126 +/- 1 vs. 113 +/- 1 mm Hg) but did not influence aldosterone-induced hypertension. Indomethacin (3.0 mg/kg/day) caused a profound inhibition of the in vivo synthesis of prostaglandin I2 and thromboxane A2 without modifying the renal production of prostaglandin E2. Although indomethacin exerted no effect on aldosterone-induced hypertension in rats fed a normal diet, it caused a further rise in systolic pressure in aldosterone-treated rats fed a high sodium diet (157 +/- 6 vs. 140 +/- 4 mm Hg). The results of this study in a model of aldosterone-induced mild hypertension in the rat indicate that 1) aldosterone exerts a stimulatory effect on the renal synthesis of prostanoid, particularly prostaglandin E2; 2) thromboxane A2 and prostaglandin I2 do not seem to play a role in aldosterone-induced hypertension under conditions of normal dietary salt intake, whereas the role of prostaglandin E2 is unclear; 3) there is enough sodium in a normal diet to allow for the maximal expression of the hypertensive effect of aldosterone; 4) prostaglandin I2 seems to play a significant role in modulating the cardiovascular impact of a high sodium diet in aldosterone-treated rats; and 5) the renal biosynthesis of prostaglandin E2 is particularly resistant to the inhibitory effect of indomethacin in vivo.

Neovastat, a naturally occurring multifunctional antiangiogenic drug, in phase III clinical trials.

Recent studies have indicated that bone marrow angiogenesis is increased in multiple myeloma, suggesting that treatment with an antiangiogenic agent might be useful. Among the new antiangiogenic drugs in development, Neovastat (AE-941; Aeterna Laboratories, Quebec City, Canada) can be classified as a naturally occurring multifunctional antiangiogenic agent. It has a marked inhibitory effect on the formation of blood vessels in the chicken embryo vascularization assay (EVT) and endothelial cell proliferation. Furthermore, in vivo experiments showed that oral administration of Neovastat blocks the formation of blood vessels in Matrigel implants containing basic fibroblast growth factor (bFGF). The antiangiogenic activity of Neovastat was found to be associated with two mechanisms of action. In addition to the inhibition of the matrix metalloproteinase activities (MMP-2, MMP-9, and MMP-12), Neovastat inhibits vascular endothelial growth factor (VEGF) binding to endothelial cells, VEGF-dependent tyrosine phosphorylation, and VEGF-induced vascular permeability in mice. Neovastat was also found to have a significant antitumor activity. Oral administration of Neovastat in mice with subcutaneous grafted breast cancer (DA3) cells showed a significant reduction in tumor volume. Neovastat also decreased the number of lung metastases in the Lewis lung carcinoma model. Interestingly, the effect of Neovastat was additive to cisplatin in this model. Furthermore, no treatment-related mortality or loss of body weight was observed. Also, toxicology studies in rats and monkeys demonstrate no dose-limiting toxicity or target organ damage after 1 year of chronic exposure, thus suggesting that Neovastat could be safely administered in humans. Four clinical studies have been conducted to establish the dosing, safety, and early efficacy of Neovastat administered orally. In the oncology field, 482 patients have received Neovastat, of which 146 with solid tumors were exposed to the drug for more than 6 months. Two phase III clinical trials are currently underway. A phase III double-blind placebo-controlled study is being conducted to evaluate the efficacy of Neovastat in addition to induction chemotherapy/radiotherapy combined modality treatment in patients with unresectable non-small cell lung cancer stage IIIA and IIIB. A second phase III randomized, double-blind placebo-controlled study evaluates the efficacy of Neovastat as a monotherapy in metastatic renal cell carcinoma patients who have progressed following a first-line immunotherapy. Neovastat efficacy is also being evaluated in a registration phase II trial in patients with early relapse or refractory multiple myeloma.

Behavioral and biochemical evidence for a different effect of repeated administration of L-dopa and bromocriptine on denervated versus non-denervated striatal dopamine receptors.

The effect of repeated administration of bromocriptine and L-3,4-dihydroxyphenylalanine (L-DOPA) was studied behaviorally and biochemically in rats with a unilateral lesion of the nigrostriatal pathway. Groups of rats injected eight times with bromocriptine or L-DOPA significantly increased their contraversive circling. Rats receiving only two injections of bromocriptine did not. Animals receiving two injections of L-DOPA showed a slight but significant increase in circling. The affinity of the binding of [3H]spiperone to the dopamine receptors was unchanged by the lesion or the treatments, while the density of the binding was significantly modified. Chronic treatment with bromocriptine induced a significant decrease in the density of D2 dopamine receptors in the intact striata, while on the lesioned side, it remained unchanged. By contrast, chronic administration of L-DOPA induced a significant increase in density of the striatal dopamine receptors in the lesioned striata in addition to that caused by denervation, while the decrease on the intact side was not significant. It seems that contrary to the intact striatum, the lesioned side had a defective down-regulation mechanism in response to chronic treatment with a dopamine agonist. The results also show that L-DOPA was more potent than bromocriptine in inducing agonist supersensitivity in a denervated striatum. This may explain why chronic treatment with bromocriptine has a lesser tendency to induce dyskinesia in patients with Parkinson's disease.

Behavioral and biochemical evidence of apomorphine-induced supersensitivity of the striatal dopamine receptors.

The effect of repeated administration of apomorphine was studied behaviorally and biochemically in rats bearing either a unilateral lesion of the substantia nigra with 6-OHDA or an electrolytic lesion of the entopeduncular nucleus. Rats with the nigral lesion displayed a progressive increase in contralateral circling rate. In animals with the entopeduncular lesion, the ipsiversive circling response was unchanged after the eight injection of apomorphine. [3H] Spiroperidol binding to striatal membranes did not appear modified by the apomorphine treatment on the intact side. It was however increased on the side of the 6-OHDA lesion. We propose that a denervated striatum responds to repeated stimulation by dopamine agonists with a paradoxical increase in sensitivity. This may explain in part the appearance of dyskinesia in treated parkinsonian patients.

Prostaglandin I2 and glucocorticoid-induced rise in arterial pressure in the rat.

The mechanism of glucocorticoid-induced hypertension is not known. Although glucocorticoids can exert an inhibitory effect on prostaglandin synthesis in vitro, their in vivo influence on this system is controversial. The goal of the present study was to determine whether dexamethasone-induced hypertension in Wistar rats is due to inhibition of the synthesis of the vasodilator prostaglandin I2 (PGI2) in vivo. Dexamethasone caused a profound reduction (7 +/- 1 versus 21 +/- 5 ng per 24 h) in the urinary excretion of PGI-M (PGI-M), a major metabolite of PGI2, and a sustained rise in systolic arterial pressure which was maximal after 5 days (144 +/- 9 versus 103 +/- 3 mmHg). A study of the metabolism of [3H]-labeled 6-oxo-PGF1 alpha and PGI2 revealed that dexamethasone exerted a dual action on the prostaglandin system in vivo: an inhibition of PGI2 biosynthesis and an alteration of its metabolism, both effects contributing to the observed reduction in urinary levels of PGI-M. Exogenous arachidonic acid induced a fourfold increase in urinary PGI-M in normal rats (from 14 +/- 3 to 61 +/- 6 ng per 24 h). Despite a large decrease upon addition of dexamethasone, urinary PGI-M remained in the high-normal range in arachidonic acid-treated rats (21 +/- 8 ng per 24 h). Arachidonic acid exerted antihypertensive effects which were marginal initially but significant in the later phase of dexamethasone-induced hypertension (124 +/- 8 versus 139 +/- 8 mmHg in arachidonic acid-treated versus control rats after 7 days of dexamethasone).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of vasopressin-induced formation of diradylglycerols in vascular smooth muscle cells by incorporation of eicosapentaenoic acid in membrane phospholipids.

OBJECTIVE: Eicosapentaenoic acid and linoleic acid exert antihypertensive effects by an unknown mechanism unrelated to prostanoids, a property which is not shared by arachidonic acid. This study investigated the influence of these three acids on the formation of diradylglycerols and phosphatidic acid, key intracellular messengers involved in the mediation of agonist-induced vascular smooth muscle cell contraction. DESIGN: Rat mesenteric artery vascular smooth muscle cells in culture were pre-incubated for 24 h with eicosapentaenoic acid, linoleic acid or arachidonic acid. After thorough washing the cells were then incubated for 20 min in the presence of arginine vasopressin or vehicle, either immediately or following cell labelling with 32P-orthophosphate. METHODS: The fatty acid composition of cell lipids was determined by gas chromatography after transesterification in the presence of boron trifluoride and methanol. Diradylglycerols and 32P-phosphatidic acid were purified from cell lipid extracts by thin-layer chromatography and diradylglycerols were analysed. RESULTS: Incubation of vascular smooth muscle cells with eicosapentaenoic acid, linoleic acid or arachidonic acid resulted in the incorporation of these fatty acids at the sn-2 position of membrane phospholipids, mainly phosphatidylcholine and phosphatidylethanolamine. Eicosapentaenoic acid treatment was associated with a reduction, and linoleic acid treatment with an increase in the relative proportions of arachidonic acid found in cell phospholipids. Arginine vasopressin stimulated the formation of both diradylglycerols and 32P-phosphatidic acid. The arginine vasopressin-induced stimulation of diradylglycerols accumulation was almost completely abolished in eicosapentaenoic acid-treated cells, whereas it was not modified by linoleic acid or by arachidonic acid treatment. The arginine vasopressin-stimulated formation of 32P-phosphatidic acid was significantly inhibited by linoleic acid treatment but was not influenced by eicosapentaenoic acid or arachidonic acid treatment. CONCLUSION: The incorporation of eicosapentaenoic acid or linoleic acid at the sn-2 position of membrane phospholipids leads to an inhibition of arginine vasopressin-induced formation of diradylglycerols or phosphatidic acid, respectively, in rat mesenteric artery vascular smooth muscle cells in culture. These properties may contribute to the antihypertensive effects in these fatty acids in vitro.

Continuous or pulsatile chronic D2 dopamine receptor agonist (U91356A) treatment of drug-naive 4-phenyl-1,2,3,6-tetrahydropyridine monkeys differentially regulates brain D1 and D2 receptor expression: in situ hybridization histochemical analysis.

The effect of a chronic D2 dopamine receptor agonist (U91356A) treatment on dopamine receptor gene expression in the brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys was investigated using quantitative in situ hybridization histochemistry. U91356A was administered to MPTP-monkeys for 27 days in a pulsatile (n=3) or continuous (n=3) schedule. Animals treated in a pulsatile mode showed progressive sensitization and developed dyskinesia; whereas with the continuous mode behavioural tolerance was observed but no dyskinesia developed. Untreated MPTP as well as naive control animals were also studied. The efficacy and uniformity of the MPTP effect was assessed by measures of dopamine concentrations by high performance liquid chromatography with electrochemical detection in the relevant brain areas. D1 and D2 receptor messenger RNAs levels were examined by in situ hybridization histochemistry using human complementary RNA probes. Intense specific labelling for D1 and D2 receptor messenger RNAs was measured in the caudate and putamen with a rostrocaudal gradient for D2 receptors and a lower density in the cortex for D1 receptors messenger RNA. D1 receptor mRNA levels in rostral striatum and cortex decreased whereas D2 receptor messenger RNA in caudal striatum increased in MPTP-monkeys compared to control animals. Continuous administration of U91356A reversed the MPTP-induced increase of D2 receptor messenger RNA, whereas the pulsatile administration did not significantly correct these messenger RNA changes. U91356A treatment whether continuous or pulsatile partially corrected the D1 receptor messenger RNA lesion-induced decrease in the striatum, whereas no correction was observed in the cortex. All MPTP-monkeys were extensively and similarly denervated suggesting that the D1 and D2 receptor expression changes following U91356A administration were treatment related. Our data show a lesion-induced imbalance of D1 (decrease) and D2 (increase) receptor messenger RNAs in the striatum of MPTP-monkeys. The response of these receptors to D1 agonist treatment showed receptor selectivity and was influenced by the time-course of drug delivery. Hence chronic continuous but not pulsatile administration of U91356A reversed the striatal D1 receptor messenger RNA increase.

Pituitary PRL secretion induced by tetraethylammonium is inhibited by dopamine through D2 receptors.

We have evaluated the inhibitory effect of dopamine on PRL secretion induced by blocking K+ channels. Tumor-derived GH4C1 cells and collagenase-dispersed normal anterior pituitary (AP) cells from young adult male rats were perifused with Krebs-Ringer Hepes medium. In both cell types blocking K+ channels with tetraethylammonium (TEA) induced PRL secretion but did not stimulate cyclic AMP generation. Blocking Na+ channels with 1 microM tetrodotoxin had no effect on basal or TEA-induced PRL secretion. Dopamine inhibited the TEA-induced rise in [Ca2+]i in GH4C1 cells expressing dopamine D2 short receptors. In normal AP cells, 1-100 nM dopamine blocked PRL secretion induced by 20 mM TEA in a log-linear concentration-dependent fashion, with a plateau at > 100 nM dopamine (IC50 30 nM). The D2 dopaminergic receptor agonist, quinpirole, at 100 nM completely blocked PRL secretion induced by 20 mM TEA. The D2 dopaminergic receptor antagonist, sulpiride, at 10 microM reversed the inhibitory effect of 10 microM dopamine on PRL secretion induced by 20 mM TEA. Pretreatment of cells with 100 ng/ml pertussis toxin (PTX) for 24 h prevented 100 nM dopamine inhibition of PRL secretion induced by 20 mM TEA. The data indicate that in both normal lactotroph cells and in tumor-derived cells expressing D2 receptors, PRL secretion stimulated by blocking K+ channels is inhibited by dopamine binding to D2 receptors on the plasma membrane. This inhibition involves interaction with PTX-sensitive Gi protein.

Antihypertensive properties of linoleic acid and fish oil omega-3 fatty acids independent of the prostaglandin system.

Polyunsaturated fatty acids of the omega-6 and the omega-3 series have been shown to lower arterial pressure in humans and in various models of experimental hypertension by uncharacterized mechanisms. The objectives of our study were to compare the antihypertensive properties of linoleic acid (omega-6 series) and of fish oil fatty acids (omega-3 series) in a model of hypertension induced by the continuous subcutaneous infusion of angiotensin II in the rat and to determine whether or not their antihypertensive effects were mediated by the biosynthesis of vasodilator prostaglandins of classes 2 or 3. Linoleic acid and fish oil fatty acids (administered by subcutaneous injections) were equally potent in reducing, by half, the rise in systolic arterial pressure induced by the chronic infusion of angiotensin II. These antihypertensive effects were observed in the absence of any significant influence of either linoleic acid or fish oil fatty acids on the systemic and the renal synthesis of PGI2 or on the renal formation of PGE2 in vivo. Indomethacin caused a profound inhibition of the biosynthesis of PGI2 but not of PGE2 and could only partially neutralize the antihypertensive effects of linoleic acid and of fish oil fatty acids. These results suggest that, in this model of angiotensin II-induced hypertension, linoleic acid and fish oil fatty acids exert equipotent antihypertensive effects which are mainly independent of the prostaglandin system.

Molecular characterization of dopamine receptors.

The D1 and D2 dopamine receptors have been biochemically characterized using specific probes based on the subtype selective antagonists SCH 23390 and spiperone, respectively. The D2 dopamine receptor was identified from several tissues by photoaffinity labeling and was purified from bovine anterior pituitary to homogeneity using a combination of affinity, lectin and hydroxylapatite chromatography. A complementary DNA (cDNA) encoding a rat brain D2 dopamine receptor has been cloned via low stringency hybridization using a portion of the beta 2-adrenergic receptor gene as a probe. Photoaffinity crosslinking and affinity chromatography have also been used to identify and purify the rat brain D1 dopamine receptor.

Location and molecular cloning of D1 dopamine receptor.

Recently, our laboratory has purified the D1 dopamine receptor 6600 fold to near homogeneity from digitonin solubilized rat striatal membranes using sequential affinity, ion exchange, lectin, and size exclusion chromatographies. The resulting receptor preparations still retained ligand binding activity (-11,000 pmol [3H]SCH 23390 bound per mg/protein) and appeared as a single band at 70-80 kDa on SDS-PAGE. In order to learn more about the sequence and structure of this protein, we recently cloned the gene for a human CNS D1 dopamine receptor. This gene has an open reading frame of 1388 nucleotides and encoded for a protein with a deduced amino acid sequence of 446 residues. When expressed in mammalian cells the cloned D1 receptor had all the ligand binding properties expected for a D1 receptor (SCH 23390 > cis flupenthixol > raclopride and SKF 38393 > apomorphine > dopamine > quinpirole). The cloned D1 receptor was found to stimulate adenylyl cyclase but not phospholipase C. The message for this D1 dopamine receptor was found in caudate, putamen, frontal cortex, and hippocampus, but not in substantia nigra, heart, or kidney. These accomplishments now will allow the pursuit of biochemical studies of the receptor protein as well as investigations into structure/function relationship of the receptor using a molecular biological techniques.

Autoreceptor preference of dopamine D2 receptor agonists correlates with preferential coupling to cyclic AMP.

Dopamine autoreceptors control the synaptic release and turnover of dopamine. Some dopamine agonists display a preference for modulation of autoreceptor functions rather than postsynaptic-driven behaviors. However, the nature of this apparent selectivity is still elusive. To investigate this property, we have used an heterologous expression system in which D2S receptors are coupled to both inhibition of cyclic AMP levels and stimulation of inositol triphosphate production. We show that D2-like receptor agonists display distinct potencies on these two second messenger pathways. Moreover, a strong correlation is observed between the potency of agonists to interact with adenylate cyclase and their potency to modulate autoreceptor functions. Such a correlation does not show up with the phospholipase C pathway. This suggests that autoreceptor preference of D2-like receptor agonists may be driven by a preferential interaction with a second messenger system.