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Bay K 8644 stimulates the phosphoionisitide pathway
422
January 1995 Am J Obstet Gynecol
SPO A b s t r a c t s
593 EFFECTS OF CALCIUM
INHIBITION
RELEASE
ON
OF PHASIC
IP_-SENSITIVE
595 AMNIOTIC FLUID GRO CONCENTRATIONS DURING LABOR AND
INTRA AMNIOTIC INFECTION. J. Cohen', R. Romero, H. Mufioz~, R. Gomezx, F. Ghezzi', L. Goncalves~, A. Ghidini, J. Lopez-Zeno', M. Galasso~, Wayne State Universi~/Hutzel Hospital, Detroit, MI, Perinatology Research Branch, NICHD, Bethesda, MD. BACKGROUND/O~BJECTIVE: GRO, a new member of the ehemokine family, is produced by fibroblnsts, endothelial cells and maerophages and is a potent chemoattrnctant for neutrophils and may participate in neutrophil recruitment during the course of intrauterine infection. This study was conducted to investigate the effect of labor and microbial invasion of the amniotie cavity (M1AC) on amniotic fluid (AF) and umbilical cord GRO concentrations. STUDY DESIGN: A cross sectional study was designed to include women with and without MIAC and with and without labor (term and preterm). Fluid was retrieved by amnincentnsis. MIAC was defined as a positive AF culture for microorganisms. Cord blood was retrieved at the time of delivery in 71 cases from similar clinical groups. AF and cord blood GRO concentrations were measured by ELISA (sensitivity 0.37 ag/ml). RESULTS: 1) GRO was detectable in all AF and cord blood samples; 2) Ale GRO concentrations increased with advancing gnstational age (At term: median 1.88 ng/ml, Range 1.17-4.21 vs. midtrimester: median I. I 1 ng/ml, range 0.44-5. p< 0.05); 3) MIAC in both term and preterm gestation was associated with higher AF GRO concentrations (Term, MIAC: median 2.68ng/ml, range 1.44-5 vs. term no MIAC: median 1.88 ng/ml; range 1.174.21, p <0.05; Preterm: MIAC median 5 ng/ml, range 0.63-5 vs. no MIAC; Preterm delivery 1.54ag/ml, range 0.69-3.46); and 4) In contrast to what was found in A1e, cord blood GRO concentrationsdid not change with either labor or infection. CONCLUSION: Intrauterine infection is associated with an increase in concentrations of GRO in AF but not in cord blood. GRO is a physiologic constituent of AF and its concentrations increase with gestational age.
M~'OMETRIAL
C O N T R A C T I O N S M. Phillipt~e. T. Saunders x Section of MFM, Dept of O b / G y n , University of Chicago, Chicago, IL. O B J E C T I V E : Thimerosal inhibits calcium uptake and I P . . . . 3 tnduced calcium release from IP_-senstttve endoplasmtc retieulum (ISER) (Biochem J 289:883. 199]). Previous studies have suggested that phasic myometrial contractions are dependent on cycles of calcium release from the ISER. The present study tests the hypothesis that inhibition of the ISER will have a marked effect on myometrial contractions. S T U D Y DE,~;IGN: Uterine strips from proestrus/estrus S-D rats were used for in vitro contraction studies. Computer digitalired isometric contraction data were analyzed for the area under the contraction curve for 5 minute intervals, normalized for tissue cross-section area, and reported as the percent of spontaneous or agonist-stimulated contractile activity. R E S U L T S : Thimerosal (10-1fl0/tM) significantly inhibited spontaneous myometrial contractions (p<0.05). Thimerosal also significantly inhibited contractions stimulated with oxytocin (0.48mU/mL) and AIF4" (l.5mM) (a direct G-protein agonist). KCI (20raM) and ionomycin ( I ~ M ) (a calcium ionophore) stimulated phasic contractions were also markedly suppressed by thimerosal. (all p<0.05) C O N C L U S I O N : Inhibifi~m of the ISER has a significant effect on sponlaneous and agonist-stimulated phasic myomctrial contraclions. Thcse observations support the hyl~thcsi~ 1hal phasic contractions are driven by mechanisms involving the utiliTation of the ISER calcium stores. (funded by NIH HD22063)
594 Bay K 8644 Stimulates The Phosphoinositide Pathway E. Chichi, T. Saunders~, M. Phillippe. Dept of Ob/Gyn, University of Chicago, Chicago, IL. OBJECTIVE: Phosphoinositide specific phospholipase appears to play an important role in intracellular signal transduction to produce phasic myometrial contractions. Previous reports have suggested Bay K 8644, an L type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. The mechanism still remains unclear. These studies were performed to test the hypothesis that Bay K 8644 stimulated contractions were mediated through mechanisms involving activation of the phosphatidyl inositol signaling pathway. METHODS: Uterine tissue was obtained from mature virgin SpragueDawley rats in estrus or proestrus phase, sacrificed under pentobarbital anesthesia. The uteri were minced and incubated in 3H-inositol for 18 hours at room temperature. The tissue was then stimulated for 15 minute periods with oxytucin and Bay K 8644 aller preincubation with LiCI= for 15 minutes. 3H-Inositol phosphates were recovered with anion exchange chromatography and quantified in a scintillation counter and standardized for protein content (Bradford Method). RESULTS: Reported as Percent Controla A~onist IP, IP. IPt fN~ Control 100_+0.0 100+_0.0 100 + 0.0 (4) Oxytoein(0.12mU/mL) 228.8b:kl10.1 182.5b:1:72,9 135.~:k36.9 (6) Bay K 8644(10uM) 15 I.sb:l:34.4 138.5:t:32.4 135.6b'/-18.5 (8) a.31Linositol phosphate production data (cpm/milligram tissue protein) reported as percent control (unstimulated tissue); data repotted as Mean + S.D. b. Mann-Whitney rank sum test (p<0.05) for agonist compared to control. CONCLUSION: The data confirm that Bay K 8644 stimulates the phosphatidyl inositol signaling pathway in rat uterine myometrial tissue. Increased cytosolic calcium may be a direct activator of phospholipase C and help explain the effect of calcium channel blockers for tocolysis.
596
TOCOMETRIC DIFFERENTIATION BElrWEEN TRUE AND FALSE LABOR D.Garrv, A.Lysikiewicz, T.Cooper,* E.Macha,* N.Tejani. Dept. Ob/Gyn New York Medical College, Valhalla, N Y. OBJECTIVE: To differentiate between true and false labor detecting fundal dominance with the use of multiple tocomett~ sensora. STUDY DESIGN: Three tocomeVlc sensors were placed over uterine fundus (fig.,A,B). and supmpubically(fig,,C) to record uterine contractions in 20 Patents at term. The study group conelated of 12 patients in true labor (TL) (6 or more cm dilatation and deliv~ed within 6 hours). The ¢ontrois were 8 patients who were contraddng but in false labor (FL) (1-2 cm dilated and undeliversd in 24 hours). A computer program estimated the 8me of the peak of contract~ns. The mean time difference between peaks oftha contractions in the uterine fundus and in the lcwer segment was calculated for each patent. The average time difference between ~ peak of contraction in the fundus and in lower segment was then calculated for each group and compared using t-tset. Example:FL A
~,._,~.~.~,~
....
~
....
RESULTS: In 16 of 20 traces contraction's peaks were identified by the computer program in the fundus and in the lower segment. In the true labor pe0ent group contractions occurred esdisr in the fundus than in the lower segment. This descending cor(oactions pattern was not found in the control group. The difference (4.2sec.+-3.8sec. vs -1.9sec.+-2.Tsec.) was sfa~tP,,ally significant at P = 0.002. CONCLUSION: The distinc0on between TL and FL can be made by differer~al tocomeb'y. This method has poten~al for diagnosis of true preterm labor.
January 1995 Am J Obstet Gynecol
SPO A b s t r a c t s
593 EFFECTS OF CALCIUM
INHIBITION
RELEASE
ON
OF PHASIC
IP_-SENSITIVE
595 AMNIOTIC FLUID GRO CONCENTRATIONS DURING LABOR AND
INTRA AMNIOTIC INFECTION. J. Cohen', R. Romero, H. Mufioz~, R. Gomezx, F. Ghezzi', L. Goncalves~, A. Ghidini, J. Lopez-Zeno', M. Galasso~, Wayne State Universi~/Hutzel Hospital, Detroit, MI, Perinatology Research Branch, NICHD, Bethesda, MD. BACKGROUND/O~BJECTIVE: GRO, a new member of the ehemokine family, is produced by fibroblnsts, endothelial cells and maerophages and is a potent chemoattrnctant for neutrophils and may participate in neutrophil recruitment during the course of intrauterine infection. This study was conducted to investigate the effect of labor and microbial invasion of the amniotie cavity (M1AC) on amniotic fluid (AF) and umbilical cord GRO concentrations. STUDY DESIGN: A cross sectional study was designed to include women with and without MIAC and with and without labor (term and preterm). Fluid was retrieved by amnincentnsis. MIAC was defined as a positive AF culture for microorganisms. Cord blood was retrieved at the time of delivery in 71 cases from similar clinical groups. AF and cord blood GRO concentrations were measured by ELISA (sensitivity 0.37 ag/ml). RESULTS: 1) GRO was detectable in all AF and cord blood samples; 2) Ale GRO concentrations increased with advancing gnstational age (At term: median 1.88 ng/ml, Range 1.17-4.21 vs. midtrimester: median I. I 1 ng/ml, range 0.44-5. p< 0.05); 3) MIAC in both term and preterm gestation was associated with higher AF GRO concentrations (Term, MIAC: median 2.68ng/ml, range 1.44-5 vs. term no MIAC: median 1.88 ng/ml; range 1.174.21, p <0.05; Preterm: MIAC median 5 ng/ml, range 0.63-5 vs. no MIAC; Preterm delivery 1.54ag/ml, range 0.69-3.46); and 4) In contrast to what was found in A1e, cord blood GRO concentrationsdid not change with either labor or infection. CONCLUSION: Intrauterine infection is associated with an increase in concentrations of GRO in AF but not in cord blood. GRO is a physiologic constituent of AF and its concentrations increase with gestational age.
M~'OMETRIAL
C O N T R A C T I O N S M. Phillipt~e. T. Saunders x Section of MFM, Dept of O b / G y n , University of Chicago, Chicago, IL. O B J E C T I V E : Thimerosal inhibits calcium uptake and I P . . . . 3 tnduced calcium release from IP_-senstttve endoplasmtc retieulum (ISER) (Biochem J 289:883. 199]). Previous studies have suggested that phasic myometrial contractions are dependent on cycles of calcium release from the ISER. The present study tests the hypothesis that inhibition of the ISER will have a marked effect on myometrial contractions. S T U D Y DE,~;IGN: Uterine strips from proestrus/estrus S-D rats were used for in vitro contraction studies. Computer digitalired isometric contraction data were analyzed for the area under the contraction curve for 5 minute intervals, normalized for tissue cross-section area, and reported as the percent of spontaneous or agonist-stimulated contractile activity. R E S U L T S : Thimerosal (10-1fl0/tM) significantly inhibited spontaneous myometrial contractions (p<0.05). Thimerosal also significantly inhibited contractions stimulated with oxytocin (0.48mU/mL) and AIF4" (l.5mM) (a direct G-protein agonist). KCI (20raM) and ionomycin ( I ~ M ) (a calcium ionophore) stimulated phasic contractions were also markedly suppressed by thimerosal. (all p<0.05) C O N C L U S I O N : Inhibifi~m of the ISER has a significant effect on sponlaneous and agonist-stimulated phasic myomctrial contraclions. Thcse observations support the hyl~thcsi~ 1hal phasic contractions are driven by mechanisms involving the utiliTation of the ISER calcium stores. (funded by NIH HD22063)
594 Bay K 8644 Stimulates The Phosphoinositide Pathway E. Chichi, T. Saunders~, M. Phillippe. Dept of Ob/Gyn, University of Chicago, Chicago, IL. OBJECTIVE: Phosphoinositide specific phospholipase appears to play an important role in intracellular signal transduction to produce phasic myometrial contractions. Previous reports have suggested Bay K 8644, an L type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. The mechanism still remains unclear. These studies were performed to test the hypothesis that Bay K 8644 stimulated contractions were mediated through mechanisms involving activation of the phosphatidyl inositol signaling pathway. METHODS: Uterine tissue was obtained from mature virgin SpragueDawley rats in estrus or proestrus phase, sacrificed under pentobarbital anesthesia. The uteri were minced and incubated in 3H-inositol for 18 hours at room temperature. The tissue was then stimulated for 15 minute periods with oxytucin and Bay K 8644 aller preincubation with LiCI= for 15 minutes. 3H-Inositol phosphates were recovered with anion exchange chromatography and quantified in a scintillation counter and standardized for protein content (Bradford Method). RESULTS: Reported as Percent Controla A~onist IP, IP. IPt fN~ Control 100_+0.0 100+_0.0 100 + 0.0 (4) Oxytoein(0.12mU/mL) 228.8b:kl10.1 182.5b:1:72,9 135.~:k36.9 (6) Bay K 8644(10uM) 15 I.sb:l:34.4 138.5:t:32.4 135.6b'/-18.5 (8) a.31Linositol phosphate production data (cpm/milligram tissue protein) reported as percent control (unstimulated tissue); data repotted as Mean + S.D. b. Mann-Whitney rank sum test (p<0.05) for agonist compared to control. CONCLUSION: The data confirm that Bay K 8644 stimulates the phosphatidyl inositol signaling pathway in rat uterine myometrial tissue. Increased cytosolic calcium may be a direct activator of phospholipase C and help explain the effect of calcium channel blockers for tocolysis.
596
TOCOMETRIC DIFFERENTIATION BElrWEEN TRUE AND FALSE LABOR D.Garrv, A.Lysikiewicz, T.Cooper,* E.Macha,* N.Tejani. Dept. Ob/Gyn New York Medical College, Valhalla, N Y. OBJECTIVE: To differentiate between true and false labor detecting fundal dominance with the use of multiple tocomett~ sensora. STUDY DESIGN: Three tocomeVlc sensors were placed over uterine fundus (fig.,A,B). and supmpubically(fig,,C) to record uterine contractions in 20 Patents at term. The study group conelated of 12 patients in true labor (TL) (6 or more cm dilatation and deliv~ed within 6 hours). The ¢ontrois were 8 patients who were contraddng but in false labor (FL) (1-2 cm dilated and undeliversd in 24 hours). A computer program estimated the 8me of the peak of contract~ns. The mean time difference between peaks oftha contractions in the uterine fundus and in the lcwer segment was calculated for each patent. The average time difference between ~ peak of contraction in the fundus and in lower segment was then calculated for each group and compared using t-tset. Example:FL A
~,._,~.~.~,~
....
~
....
RESULTS: In 16 of 20 traces contraction's peaks were identified by the computer program in the fundus and in the lower segment. In the true labor pe0ent group contractions occurred esdisr in the fundus than in the lower segment. This descending cor(oactions pattern was not found in the control group. The difference (4.2sec.+-3.8sec. vs -1.9sec.+-2.Tsec.) was sfa~tP,,ally significant at P = 0.002. CONCLUSION: The distinc0on between TL and FL can be made by differer~al tocomeb'y. This method has poten~al for diagnosis of true preterm labor.