Biomedical subjects

M Phillippe

Publications and source records attributed to M Phillippe.

At least 19 recordsLinked to original sources

PubMed2000

The role of diacylglycerol as a modulator of oxytocin-stimulated phasic contractions in myometrium from pregnant and nonpregnant rats.

OBJECTIVE: The role of diacylglycerol in the phosphatidylinositol-signaling pathway is to activate protein kinase C. In the myometrium, protein kinase C activation leads to inhibition of phasic contractions. These studies are designed to determine why stimulation of the phosphatidylinositol-signaling pathway caused by oxytocin does not cause a paradoxical suppression of contractions through diacylglycerol production and protein kinase C activation. Specifically, these studies were performed to test the hypothesis that diacylglycerol catabolism is significant in myometrial tissue, thereby precluding its availability for the activation of protein kinase C. STUDY DESIGN: For these studies, uterine tissue was obtained from Sprague-Dawley rats both nonpregnant and with timed gestations. In vitro contraction studies were performed with cumulative additions of oxytocin (8-64 nmol/L) with and without R59022 (a diacylglycerol kinase inhibitor) or RHC80267 (a diacylglycerol lipase inhibitor). The contraction data were computer-digitalized, analyzed for total contractile activity, normalized for tissue cross-sectional area, and reported as the percentage of spontaneous activity. RESULTS: In myometrium from nonpregnant animals, inhibition of diacylglycerol lipase with RHC80267 had little effect on oxytocin-stimulated contractile activity, whereas inhibition of diacylglycerol kinase with R59022, although producing an increase in contractile frequency, markedly suppressed total oxytocin-stimulated contractile activity. In contrast, in myometrium from near-term pregnant animals both RHC80267 and R59022 produced marked suppression of oxytocin-stimulated contractile activity. CONCLUSIONS: These studies have demonstrated that prevention of diacylglycerol degradation, especially in response to inhibition of myometrial diacylglycerol kinase, results in the paradoxic oxytocin-mediated suppression of total myometrial contractile activity. These observations support the hypothesis that, when its catabolism is prevented, diacylglycerol produced in response to stimulation of the phosphatidylinositol-signaling pathway by oxytocin becomes available for protein kinase C activation, resulting in inhibition of myometrial contractile activity.

Animals↗

PubMed1999

The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4, 5-trisphosphate receptor modulator on myometrial contractions.

These studies were performed to evaluate the effect of 2-aminoethoxydiphenyl borate (2-APB), a novel membrane-permeable inositol 1,4,5-trisphosphate-receptor inhibitor on agonist-induced, spontaneous, and KCl-stimulated in vitro myometrial contractions. 2-APB significantly inhibited spontaneous myometrial contractions as well as phasic contractions induced by various uterotonic agonists. Confiriming its effects on intracellular calcium release, 2-APB inhibited phasic contractions in the absence of extracellular calcium. 2-APB had little effect on the tonic response to KCl stimulation, implicating its insignificant effect on voltage-gated calcium channels. The inhibitory effect of 2-APB on phasic contractions was completely reversed by washout. In summary, 2-APB effectively penetrated uterine tissue and significantly inhibited myometrial events previously shown to be mediated through activation of the PI-signaling pathway.

Animals↗

PubMed1998

Cellular mechanisms underlying magnesium sulfate inhibition of phasic myometrial contractions.

These studies sought to test the hypothesis that magnesium inhibits extracellular calcium entry, thereby inhibiting intracellular calcium release and cytosolic calcium oscillations in myometrial smooth muscle. In vitro contraction studies were performed using oxytocin and other uterotonic agonists with and without the addition of magnesium in the presence and absence of extracellular calcium. Cytosolic calcium studies were performed using myometrial strips loaded with Fura 2. Oxytocin produced cytosolic calcium oscillations and simultaneous phasic contractions; both were inhibited by magnesium. The other uterotonic agonists tested also produced phasic contractions which were significantly inhibited by magnesium. The magnesium effect was reversible with washout and counteracted by Bay K 8644 (a calcium channel agonist). In the absence of extracellular calcium, intracellular calcium release in response to oxytocin was inhibited by magnesium. In summary, magnesium inhibited extracellular calcium entry, intracellular calcium release, cytosolic calcium oscillations, and phasic contractions of myometrial smooth muscle.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Phosphatidylinositol-specific phospholipase C isoform expression in pregnant and nonpregnant rat myometrial tissue.

OBJECTIVE: Activation of the phosphatidylinositol signaling pathway plays a significant role during the intracellular signal transduction events activated during agonist-stimulated phasic myometrial contractions. Phospholipase C is an essential molecular component of this signaling pathway. These studies sought to characterize the expression of phospholipase C isoform messenger ribonucleic acid in both pregnant and nonpregnant rat myometrium. STUDY DESIGN: Total cellular ribonucleic acid was isolated from myometrial tissue collected from Sprague-Dawley rats by use of the acidic guanidinium thiocyanate-phenol-chloroform extraction technique. After deoxyribonuclease treatment to ensure removal of genomic deoxyribonucleic acid, as well as resolution on formaldehyde-1% agarose horizontal slab gels to rule out degradation, the ribonucleic acid was used for semiquantitative competitive reverse transcriptase-polymerase chain reaction studies to evaluate the expression of five of the reported phospholipase C isoforms. These studies were performed with isoform-specific 20-mer primers and the inclusion of internal standard heterologous deoxyribonucleic acid sequences designed with ends homologous to the isoform-specific primers. The identity of the polymerase chain reaction products was confirmed with restriction endonuclease digestions and homology analysis of the sequenced polymerase chain reaction product deoxyribonucleic acid. RESULTS: These reverse transcriptase-polymerase chain reaction studies have confirmed expression of the phospholipase C-beta1a, phospholipase C-beta3, phospholipase C-gamma1, phospholipase C-beta2, and phospholipase C-delta1 isoforms in rat myometrial tissue. During pregnancy the levels of expression of the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-delta1 isoforms were increased compared with the levels of expression in myometrium from nonpregnant rats. In myometrium from both pregnant and nonpregnant animals the phospholipase C-beta1 a isoform was expressed at the highest level, the phospholipase C-beta3, phospholipase C-gamma1, and phospholipase C-gamma2 isoforms at an intermediate level, and the phospholipase C-delta1 isoform was expressed at the lowest levels. CONCLUSIONS: These studies have confirmed at the messenger ribonucleic acid level significant expression of several isoforms of phospholipase C in both pregnant and nonpregnant myometrial tissue. These observations provide additional support for the hypothesis that the phosphatidylinositol signaling pathway plays an important role in uterine smooth muscle.

Animals↗

PubMed1997

Increase in serum leptin and uterine leptin receptor messenger RNA levels during pregnancy in rats.

Pregnancy is a physiological state associated with significant changes in appetite, thermogenesis, and lipid metabolism, functions which are regulated in part by a hormone, leptin, secreted by adipocytes. Leptin has also been shown to have a role in reproduction, promoting centrally-regulated maturation of the reproductive system and signaling the presence of adequate maternal energy stores for fertility. Here we demonstrate that serum leptin levels are modulated during normal rat pregnancy with a 1.8-fold increase during pregnancy followed by a decrease just before parturition. Leptin receptor mRNA levels in the uterus are also regulated with an increase about 2.7-fold during this same period, whereas there is no change in other tissues examined. The results suggest that leptin may play a role during pregnancy, perhaps regulating energy utilization.

Animals↗

PubMed1997

(+)cis-dioxolane stimulation of cytosolic calcium oscillations and phasic contractions of myometrial smooth muscle.

These studies sought to determine the intracellular mechanisms underlying muscarinic receptor stimulated phasic contractions of myometrial smooth muscle. Utilizing cytosolic calcium imaging studies with Fura-2 loaded muscle strips, along with in vitro isometric contraction studies, we have demonstrated that phasic myometrial contractions stimulated in response to (+)cis-dioxolane, a muscarinic selective acetylcholine agonist, are mediated by activation of the M3 receptor subtype resulting in stimulation of the phosphatidylinositol signaling pathway and the generation of cytosolic calcium oscillations.

Animals↗

PubMed1997

Identification of gestationally regulated genes in rat myometrium by use of messenger ribonucleic acid differential display.

OBJECTIVE: We hypothesized that the proteins contributing to myometrial changes during gestation could be identified indirectly by analyzing the changing pattern of messenger ribonucleic acid expression in the myometrium during pregnancy. STUDY DESIGN: Ribonucleic acid was extracted from myometrium of timed pregnant Sprague-Dawley rats on days 12, 16, 20, 21, and 22 of pregnancy and on day 1 post partum. The technique of messenger ribonucleic acid differential display, a simple and sensitive polymerase chain reaction-based method for rapidly identifying messenger ribonucleic acids whose levels increase or decrease, was performed with the nine different anchoring primers (oligodeoxythymidine11 VN: V = G, A, or C; N = G, A, or C) in combination with 24 different 10-base oligonucleotides of random sequence. The polymerase chain reaction products were separated by electrophoresis on a 5% polyacrylamide sequencing gel, and those whose levels changed were then cloned, sequenced, and compared with those in the GenBank database to determine whether they corresponded to a known sequence in the database or were novel. Semiquantitative reverse transcriptase-polymerase chain reaction was used to confirm differential expression of selected products. RESULTS: Messenger ribonucleic acid differential display revealed > 500 polymerase chain reaction products that were differentially expressed during gestation, 179 of which were cloned and sequenced. Of these, 157 were from messenger ribonucleic acids whose levels increased during gestation, and 22 were from transcripts that decreased. Eighty-seven (49%) were related to sequences in the GenBank database, of which 62 (35%) were from messenger ribonucleic acids encoding known proteins and 25 (14%) corresponded to known expressed sequence tags. The technique of semiquantitative reverse transcriptase-polymerase chain reaction confirmed the increased expression of messenger ribonucleic acids encoding beta-tropomyosin, type II phosphatidyl inositol-4-phosphate 5-kinase, and a novel myometrial messenger ribonucleic acid named RPU0901AC. CONCLUSION: Messenger ribonucleic acid differential display is a simple and sensitive method for rapidly identifying myometrial messenger ribonucleic acids that are differentially regulated during pregnancy. The identification of these differentially expressed messenger ribonucleic acids may lead to a better understanding of the molecular basis of normal and abnormal parturition.

Aging↗

PubMed1997

Beta-adrenergic receptor subtype gene expression in timed-pregnant rat myometrium.

OBJECTIVE: Classic radioligand binding techniques have suggested that beta 1- and beta 2-adrenergic receptor subtype proteins are expressed in myometrial tissue; however, to date these observations have not been confirmed at the level of the messenger ribonucleic acid for these clinically important membrane receptors. The studies described in this report sought to use quantitative reverse transcriptase-polymerase chain reaction techniques to confirm expression of messenger ribonucleic acid for the beta 1- and beta 2-adrenergic receptors in myometrial tissue and to determine whether messenger ribonucleic acid expression for these two adrenergic receptors is modulated during pregnancy. STUDY DESIGN: For these studies total cellular ribonucleic acid was isolated from myometrial tissue obtained from timed-pregnant Sprague-Dawley rats by the guanidium thiocyanate-phenol-chloroform extraction technique; formaldehyde-agarose gels then confirmed isolation of intact ribonucleic acid. Random hexamers and reverse transcriptase were used to synthesize complementary deoxyribonucleic acid. Subsequently, polymerase chain reaction was performed with subtype specific 20-mer sense and antisense oligonucleotide primers specific for the rat beta 1- and beta 2-adrenergic receptors. Inclusion of internal standard deoxyribonucleic acid sequences allowed quantification of the reverse transcriptase-polymerase chain reaction results. RESULTS: By use of total cellular ribonucleic acid isolated from myometrial tissue, reverse transcriptase-polymerase chain reaction generated the expected 328 bp product for the beta 1-receptor and the expected 559 bp product for the beta 2-receptor along with internal standard deoxyribonucleic acid sequences for both. The identity of the beta 1- and beta 2-adrenergic receptor polymerase chain reaction products was confirmed on the basis of restriction endonuclease digestions producing the expected deoxyribonucleic acid fragments and by Southern blots using beta 1- and beta 2-adrenergic receptor-specific complementary deoxyribonucleic acid probes. The reverse transcriptase-polymerase chain reaction studies confirmed a gradual decline in beta 1-receptor messenger ribonucleic acid and stable expression of beta 2-receptor messenger ribonucleic acid during the second half of gestation in pregnant rat myometrial tissue. CONCLUSIONS: In summary, these studies have confirmed, at the messenger ribonucleic acid level, expression of the beta 1- and beta 2-adrenergic receptor subtypes in timed-pregnant rat myometrial tissue.

Animals↗

PubMed1997

Intracellular mechanisms underlying prostaglandin F2alpha-stimulated phasic myometrial contractions.

These studies sought to test the hypothesis that prostaglandin F2alpha (PGF2alpha)-stimulated phasic myometrial contractions are characterized by the activation of the phosphatidylinositol-signaling pathway resulting in the generation of cytosolic calcium oscillations. For the experiments described in this report rat myometrial tissue was used, after the tissue was loaded with fura 2, to perform cytosolic calcium imaging studies and to perform computer-digitalized in vitro isometric contraction studies. Consistent with the above hypothesis, the cytosolic calcium-imaging studies demonstrated PGF2alpha-stimulated cytosolic calcium oscillations occurring simultaneously with phasic contractions. The in vitro isometric contraction studies confirmed that previously reported inhibitors of the phosphatidylinositol-signaling pathway and cytosolic calcium oscillation mechanisms resulted in significant inhibition of PGF2alpha-stimulated phasic myometrial contractions. In summary, these studies have provided substantial support for the hypothesis that PGF2alpha-stimulated phasic myometrial contractions are generated by intracellular signaling mechanisms involving activation of the phosphatidylinositol-signaling pathway and the production of cytosolic calcium oscillation-like phenomena.

Animals↗

PubMed1996

The effects of ruthenium red, an inhibitor of calcium-induced calcium release, on phasic myometrial contractions.

Ruthenium red inhibits calcium-induced calcium release from the ryanodine-sensitive intracellular calcium stores; this study sought to evaluate the effects of ruthenium red on agonist-stimulated phasic myometrial contractions. Ruthenium red was found to significantly inhibit in vitro isometric contractions stimulated in response to oxytocin, prostaglandin F2 alpha, aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium-induced calcium release from ryanodine-sensitive calcium stores is an important event during agonist-stimulated phasic myometrial contractions.

Animals↗

PubMed1995

The effects of thimerosal, a sulfhydryl reagent, on phasic myometrial contractions.

Thimerosal inhibits calcium uptake and IP3-induced calcium release from IP3-sensitive endoplasmic reticulum; this study sought to evaluate the effects of thimerosal on agonist-stimulated phasic myometrial contractions. Thimerosal was found to significantly inhibit phasic contractions stimulated by oxytocin, aluminum fluoride, potassium chloride, ionomycin, and Bay K 8644. These observations provide support for the hypothesis that calcium uptake and IP3-induced calcium release are important events during agonist-stimulated phasic myometrial contractions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone, an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on agonist-stimulated phasic myometrial contractions.

Phasic myometrial contractions utilize mechanisms involving the cycling of calcium into and out of intracellular calcium stores. These studies were performed to determine the effects of 2,5-di(tert-butyl)-1,4-hydroquinone (tBHQ), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, on in vitro isometric myometrial contractions. These studies demonstrated that low concentrations of tBHQ (eg. 10 microM) appear to inhibit intracellular calcium cycling, whereas higher concentrations also inhibit extracellular calcium influx. These combined tBHQ effects markedly suppressed myometrial contractions stimulated in response to various agonists including oxytocin, PGF2 alpha, KCl, ionomycin, and Bay K 8644.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Ionomycin-stimulated phasic myometrial contractions.

Ionomycin, a calcium ionophore, facilitates the sustained entry of extracellular calcium; however, in myometrial tissue it stimulates phasic contractions. This study sought to define further this unanticipated effect of ionomycin and to begin to explore the possible mechanism(s) involved. Utilizing rat uterine strips, in vitro isometric contraction studies were performed to determine the effects of ionomycin with and without membrane-permeant inhibitors of cytosolic calcium oscillations. To determine the effects of ionomycin on phospholipase C, qualitative inositol phosphate production studies were performed. The in vitro contraction studies confirmed that ionomycin-stimulated phasic myometrial contractions were potentially dependent on stimulation of phospholipase C, calcium-induced calcium release, and additional calcium influx through dihydropyridine-sensitive membrane calcium channels. The inositol phosphate production studies confirmed that ionomycin stimulated phospholipase C in a dose-related fashion to levels comparable to oxytocin. In summary, these observations have confirmed the ability of ionomycin to generate dose-related phasic myometrial contractions through mechanisms potentially involving the phosphatidylinositol-signaling pathway.

Animals↗

PubMed1995

Potassium chloride effects on the hormonal signal transduction mechanisms underlying phasic myometrial contractions.

These studies sought to test the hypothesis that potassium-stimulated phasic myometrial contractions utilize cytosolic calcium oscillation-like mechanisms comparable to those activated in response to oxytocin. Uterine tissue was obtained from pro-oestrus/oestrus Sprague-Dawley rats. In vitro isometric contraction studies were performed using longitudinal myometrial strips; computer digitalized contraction data were analyzed for contraction area, and normalized for tissue cross-section area. Dose-response studies were performed using potassium chloride with and without inhibitors of cytosolic calcium oscillation mechanisms. Qualitative inositol-phosphate production studies were performed after preloading uterine tissue with [3H]inositol; subsequently, the individual inositol-phosphates produced in response to stimulation were isolated by anion exchange chromatography. Potassium chloride over a concentration of 10 to 30 mM produced a dose-related increase in phasic contractile activity. The potassium-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, stimulation of protein kinase C, inhibition of calcium-induced calcium release, and prevention of extracellular calcium influx. The qualitative inositol-phosphate production studies confirmed activation of phospholipase C in response to 20 mM potassium. These studies have provided support for the hypothesis that potassium-stimulated phasic myometrial contractions activate intracellular signal transduction mechanisms comparable to those activated in response to hormonal uterotonic agonists.

Analysis of Variance↗

PubMed1994

Neomycin inhibition of hormone-stimulated smooth muscle contractions in myometrial tissue.

These studies sought to determine the effects of neomycin, a phospholipase C inhibitor, on hormone-stimulated myometrial contractions. For these studies, computer digitalized in vitro isometric contraction data were analyzed for changes in contractile activity in response to oxytocin and aluminum fluoride with and without neomycin. Neomycin (1-5 mM) produced dose-related inhibition of oxytocin and aluminum fluoride-stimulated myometrial contractions. This neomycin effect was apparent within 2-3 minutes of addition and was completely reversible, with resolution of its inhibitory effects within 6-8 minutes of washout. This study is the first to demonstrate the functional effect of neomycin inhibition of the phosphatidylinositol signaling pathway in myometrial smooth muscle tissue.

Aluminum Compounds↗

PubMed1994

Mechanisms underlying phasic contractions of pregnant rat myometrium stimulated with aluminum fluoride.

OBJECTIVE: The mechanisms underlying phasic myometrial contractions are unknown at this time. Phasic contractions, however, are characterized by repetitive cycles of elevated intracellular calcium (i.e., calcium oscillations). These studies were performed to test the hypothesis that mechanisms underlying phasic myometrial contractions are similar to those producing classic cytosolic calcium oscillations. STUDY DESIGN: Uterine tissue was obtained from pregnant Sprague-Dawley rats (i.e., day 18 to 22 of gestation). In vitro isometric contraction studies were performed with longitudinal strips of myometrial tissue; computer-digitized data were analyzed for contraction area and normalized for tissue cross-section area. Dose-response studies were performed with aluminum fluoride and various inhibitors of cytosolic calcium oscillations. RESULTS: Aluminum fluoride stimulated a significant increase in phasic contractions. In contrast, the addition of 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (an inhibitor of phosphoinositide-specific phospholipase C), adenine (an inhibitor of calcium-induced calcium release), and phorbol 12,13-dibutyrate (an activator of protein kinase C) resulted in significant suppression of aluminum fluoride-stimulated contractions. Similarly, nifedipine (an L-type calcium channel blocker) and removal of extracellular calcium significantly inhibited phasic myometrial contractions. CONCLUSIONS: These studies have confirmed that phosphoinositide-specific phospholipase C, calcium-induced calcium release, protein kinase C, and transmembrane calcium influx are important components of the intracellular calcium oscillator that generates agonist-stimulated phasic contractions of pregnant myometrial tissue.

Aluminum Compounds↗

PubMed1994

Protein kinase C, an inhibitor of oxytocin-stimulated phasic myometrial contractions.

One of the roles previously reported for protein kinase C (PKC) is modulation of the activity of the phosphatidylinositol signaling pathway. Studies were performed to test the hypothesis that activation of PKC results in inhibition of agonist-stimulated phasic myometrial contractions: contractions that appear to be mediated by phosphatidylinositol signaling mechanisms comparable to those producing cytosolic calcium oscillations. In vitro isometric contraction studies were performed using myometrium from adult Sprague-Dawley rats. Oxytocin and aluminium fluoride (a G-protein activator) produced comparable increases in phasic contractile activity. Phorbol 12,13-dibutyrate (PDB) significantly suppressed agonist-stimulated phasic myometrial contractions; in contrast, phorbol 13,20-diacetate (PDA), an inactive phorbol ester, had no significant effect on myometrial contractions. Prolonged exposure of myometrial tissue to PDB failed to down-regulate myometrial PKC and had no consistent effect on spontaneous and oxytocin-stimulated phasic contractions. These studies have provided support for a role for PKC as an intracellular regulator of the phosphatidylinositol signaling pathway, which itself appears to be part of the myometrial calcium oscillator that results in agonist-stimulated phasic myometrial contractions.

Aluminum Compounds↗

PubMed1993

Adrenergic stimulation of diacylglycerol production in genital tract smooth muscle myocytes.

Diacylglycerol (DAG) production has not been reported in previous studies that have characterized inositol phosphate production during alpha-1 adrenergic receptor signal transduction in the DDT1 MF-2 genital tract myocytes. The current study sought to measure norepinephrine (NE)-stimulated DAG production in these transformed myocytes utilizing thin layer chromatography. DAG production was characterized as an alpha-1 adrenergic mediated event utilizing subtype specific adrenergic agonist and antagonists. DAG production occurred in response to physiologic concentration of NE, was apparent by 30 s and was significantly increased by 2 min. Maximal DAG production was unaffected by pretreatment of the myocytes for 96 h with testosterone, which has previously been shown to induce a doubling of alpha-1 adrenergic receptors in these cells. In contrast, testosterone pretreatment did result in a shift of the dose-response curve resulting in a significantly lower EC50 for NE in the treated cells compared to control myocytes. In conclusion, these studies have confirmed that DAG production occurs as a component of alpha-1 adrenergic signal transduction in the DDT1 MF-2 myocytes; transduction events that were modulated by testosterone resulting in increased agonist sensitivity.

Animals↗