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P Falardeau

Publications and source records attributed to P Falardeau.

At least 37 records · Page 2Linked to original sources
PubMed1993

Quantification of diacylglycerols by capillary gas chromatography-negative ion chemical ionization-mass spectrometry.

We describe a method for quantifying diacylglycerols as their 1-pentafluorobenzoyl-2-acyl-3-acetyl-glycerol derivatives by capillary gas chromatography-negative ion chemical ionization-mass spectrometry. The basis of the method resides in the sequential treatment of diacylglycerols with acetic anhydride, pancreatic lipase, and pentafluorobenzoyl chloride. Cultured rat mesenteric artery vascular smooth muscle cells (VSMC) were incubated for 20 min in the presence of vehicle or vasopressin (10(-7) M). The incubations were stopped by aspirating the medium and adding 2 ml of methanol containing 790 pmol of internal standard 1-stearoyl-2-(10,13)-nonadecadienoyl- glycerol. After extraction, diacylglycerols were isolated by thin-layer chromatography, acetylated, and treated with pancreatic lipase. The resulting 2-acyl-3-acetylglycerols were then purified by thin-layer chromatography, transformed into their 1-pentafluorobenzoyl-derivatives, and monitored by capillary gas chromatography-negative ion chemical ionization-mass spectrometry on the selected ion-monitoring mode (m/z 614 and 604 for 2-arachidonoyl and 2-nonadecadienoyl species, respectively). The levels of diacylglycerols bearing an arachidonoyl moiety were 128 +/- 26 pmol/100 nmol lipid phosphorus in resting cells and 333 +/- 28 in stimulated cells (mean +/- SD, n = 3, P < 0.01). The presence of diacylglycerol species bearing an oleoyl or a linoleoyl group at the second position could also be detected in VSMC preparations by this approach. This new method can be applied to quantitate various diacylglycerol species bearing distinct acyl moieties at the second position of the glycerol molecule.

Acetic Anhydrides↗
PubMed1993

Norepinephrine in neonatal rat ventricular myocytes: association with the cell nucleus and binding to nuclear alpha 1- and beta-adrenergic receptors.

Chronic exposure of neonatal rat ventricular myocytes to norepinephrine (NE) has been demonstrated to induce fetal cardiac gene expression and hypertrophy. The precise signaling mechanism of NE induction, as well as the long delay for the onset of NE effect, are not well understood. To examine the possibility that the hormone may be transported into the cell and exerts its effect through an intracellular site, ventricular myocytes from neonatal rats were incubated with [3H]-labeled NE and the cytosolic and nuclear fractions of the cell were measured for radioactivity. The presence of intracellular adrenergic binding sites was also explored. Following incubation of neonatal rat ventricular myocytes with [3H]NE for different time intervals (from 30 min to 22 h), the highest proportion (more than 80%) of NE taken up by the cell was recovered in the nuclear fraction. The nuclear accumulation was slow and time-dependent, being non-detectable in the first 60 min. Furthermore, isolated nuclei from the ventricular myocytes contain binding sites for [3H]prazosin and dihydroalprenolol, suggesting the presence of alpha 1 and beta 1 adrenergic receptors. The apparent KD and Bmax were 0.6 nM and 0.6 fmol/mg protein for alpha 1-adrenergic receptors, while beta-adrenergic nuclear receptors exhibited an apparent KD of 12 nM and a Bmax of 61 fmol/mg protein. Thus, neonatal rat ventricular myocytes exposed to NE accumulate the hormone in the cell nucleus where it can bind to high affinity alpha 1- and beta-adrenergic receptors.

Animals↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Animals↗
PubMed1991

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.

Amino Acid Sequence↗
PubMed1991

D2 dopamine receptors in the human retina: cloning of cDNA and localization of mRNA.

1. We have obtained a cDNA clone encoding a human retinal D2 dopamine receptor. 2. The longest open reading frame (1242 bp) of this clone encodes a protein of 414 amino acids having a predicted molecular weight of 47,000 and a transmembrane topology similar to that of other G protein-coupled receptors. 3. Transient transfection of COS-7 cells with an expression vector containing the clone resulted in expression of a protein possessing a pharmacological profile similar to that of the D2 dopamine receptor found in striatum and retina. 4. Northern blot analysis indicated that, in rat brain and retina, the mRNA for this receptor was 2.9 kb in size. 5. In situ hybridization was performed to examine the distribution of the mRNA for this receptor in human retina. Specific hybridization was detected in both the inner and the outer nuclear layers. 6. These findings are consistent with prior physiological and autoradiographic studies describing the localization of D2 dopamine receptors in vertebrate retinas. Our observations suggest that photoreceptors as well as cells in the inner nuclear layer of human retinas may express the mRNA for this D2 dopamine receptor.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of G-protein coupled receptors: isolation and cloning of a D1 dopamine receptor.

This article summarizes the recent progress our laboratory has made in understanding the molecular characteristics of the D1 dopamine receptor. The D1 dopamine receptor from rat striatum has been purified to near homogeneity using a combination of several chromatographic steps. Furthermore, the gene for the human D1 dopamine receptor has been cloned, sequenced, and expressed. The cloned receptor has all the pharmacologic and biochemical properties of the classical D1 receptor coupled to adenylyl cyclase which has been previously described in the central nervous system.

Amino Acid Sequence↗
PubMed1991

Arachidonic acid does not share the antihypertensive properties of linoleic acid and fish oil omega-3 fatty acids in a model of angiotensin II-induced hypertension in the rat.

Linoleic acid and fish oil omega-3 fatty acids, but not arachidonic acid, exerted antihypertensive effects in a model of angiotensin II-induced hypertension in rats. Indomethacin did not influence the systolic arterial pressure of arachidonic acid-treated hypertensive rats whereas compound L-641,953, a prostaglandin H2/thromboxane A2 receptor antagonist, caused a notable but statistically nonsignificant decrease in blood pressure in these animals. Although these results do not exclude entirely the possibility that the lack of antihypertensive effect of arachidonic acid may be due, in part, to the concomitant formation of vasoconstrictor prostanoids, they do not support it. These observations, as well as those of a previous study, indicate that linoleic acid and fish oil omega-3 fatty acids exert antihypertensive effects of their own, independently of the prostanoid system, and that these properties are not shared by arachidonic acid.

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Molecular cloning and expression of the gene for a human D1 dopamine receptor.

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

Amino Acid Sequence↗
PubMed1990

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.

Animals↗
PubMed1990

Resistance of the renal biosynthesis of prostaglandin E2 to the inhibitory effect of indomethacin in the rat in vivo.

Our recent observation that the chronic administration of indomethacin (3.0 mg.kg-1.day) to hypertensive rats, while profoundly inhibiting the urinary excretion of 6-oxo-PGF1 alpha, dinor-6-oxo-PGF1 alpha and thromboxane B2, failed to reduce the urinary levels of PGE2, prompted us to study in more details the influence of indomethacin and of meclofenamate on the urinary excretion of prostaglandins in normal rats. A dose of 1.5 mg.kg-1 of indomethacin administered intraperitoneally was sufficient to cause a 70-75% reduction in the urinary excretion of dinor-6-oxo-PGF1 alpha and of 6-oxo-PGF1 alpha for a period of at least 12 hours. Doses of indomethacin lower than 2.5 mg.kg-1.12h beta 1 or a dose of meclofenamate equal to 5 mg.kg-1.12h beta 1 did not influence the urinary excretion of PGE2. Doses of indomethacin equal to or higher than 2.5 mg.kg-1 were needed to obtain a 50% reduction in the urinary levels of PGE2 for a period of 10-14 h. During these experiments, no circadian rhythm for the urinary excretion of 6-oxo-PGF1 alpha and of dinor-6-oxo-PGF1 alpha could be observed whereas the urine volume and the urinary excretion of PGE2 were found to be greater at night than during the day.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprostadil↗
PubMed1990

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.

Affinity Labels↗
PubMed1990

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.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Modulation of renal hemodynamics by renal eicosanoids during vasopressor infusions in man.

Pressor doses of norepinephrine (NE) (n = 8) and angiotensin II (A II) (n = 5) were infused in normal volunteers to determine whether the systemic administration of vasopressor hormones influence renal eicosanoid production and whether, in turn, the eicosanoids produced could modulate renal hemodynamics and electrolyte excretion. At the doses administered, both pressor substances induced the expected rise in blood pressure, a significant decrease (P less than 0.05) in renal blood flow and a proportionally smaller fall in glomerular filtration rate, resulting in a consistent augmentation in filtration fraction. Fractional sodium excretion was concomitantly reduced. NE infusion produced only slight modifications in urinary prostaglandin (PG)E2, 2,3-dinor-6-keto-PGF1 alpha and thromboxane (TX)B2, while urinary 6-keto-PGF1 alpha and PGF2 alpha were increased by 38% and 176% respectively. The increase in urinary 6-keto-PGF1 alpha (the non-enzymatic degradation product of PGI2, predominantly of cortical origin) was proportional to the level of circulating NE (r = 0.78, P less than 0.05) and to the renal vascular resistance (r = 0.85, P less than 0.01), suggesting an immediate compensatory role for PGI2 in response to the NE-induced pressor stimulus. The renal production of PGE2 and PGF2 alpha (predominantly medullary) was inversely correlated with the filtration fraction: the greater the increase in PGE2 and PGF2 alpha the lower the elevation in filtration fraction or the decline in renal blood flow upon NE administration. All infusion variably stimulated the renal eicosanoid production: PGE2, 41%; PGF2 alpha, 102%; 6-keto-PGF1 alpha, 38%; 2,3-dinor-6-keto-PGF1 alpha, 38%; and TXB2, 25%.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Omega-3 fatty acid dietary supplementation in systemic lupus erythematosus.

The effect of dietary fish oil (Omega-3 fatty acids--eicosapentenoic acid [EPA] and docosahexaenoic acid [DHA] on several mechanisms involved in immune, inflammatory and atherosclerotic vascular disease was determined in 12 subjects with systemic lupus erythematosus (SLE) and nephritis. These out-patients supplemented their usual diet for five weeks with daily doses of 6 g of fish oil, followed by a five-week washout period, then five weeks of 18 g of fish oil daily. The platelet EPA content rose six-fold with the lower and 15-fold with the higher dose of fish oil, and similar changes occurred to the platelet DHA content. The platelet arachidonic acid incorporation was reduced by 16 and 20%, respectively. These changes were associated with a reduction in collagen-induced platelet aggregation and an increase in red cell flexibility and a decrease in whole blood viscosity. Prostacyclin (PGI2) production was unaffected by the fish oil, but PGI3 formation correlated with its administration and dosage. Neutrophil leukotriene B4 release was reduced 78 and 42%, respectively, by the low and higher doses of fish oil. The higher fish oil dose induced a 38% decrease in triglyceride and a 39% reduction in VLDL cholesterol associated with a 28% rise in HDL, cholesterol. The fish oil had no effect on immune complex or anti-DNA antibody titer, albuminuria, intraplatelet serotonin or [14C]-serotonin release from platelets. We conclude that in patients with lupus nephritis, dietary supplementation with fish oil affects the mechanisms involved in inflammatory and atherosclerotic vascular disease.

Adult↗
PubMed1989

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.

Angiotensin II↗
PubMed1989

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)

Animals↗