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B M Sanborn

Publications and source records attributed to B M Sanborn.

At least 19 recordsLinked to original sources
PubMed2003

Assessment of analysis by gender in the Cochrane reviews as related to treatment of cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) is the leading killer of women in the United States, yet medical care is often based on evidence from clinical trials performed predominantly with men. Numerous studies show that CVD risk factors, clinical presentation, treatment, and treatment outcomes can vary between men and women. METHODS: The Cochrane Library maintains a large database of critically appraised evidence including meta-analyses of clinical trials, called Systematic Reviews. There were 30 Systematic Reviews pertaining to the treatment of CVD published collectively by the Cochrane Heart Group, Hypertension Group, and Peripheral Vascular Diseases Groups at the time of our study. We examined these 30 Systematic Reviews and the great majority of the clinical trials used for their meta-analyses for inclusion of women and gender-based data analyses. Women comprised only 27% of the pooled population of 258 clinical trials. RESULTS: Of those trials that included both men and women (n = 196), only 33% examined outcomes by gender. In trials that performed a gender-based analysis, 20% reported significant (p < 0.05) differences in cardiovascular-related outcomes by gender. CONCLUSIONS: We conclude that (1) there are not enough large-scale clinical trials or meta-analyses concerning CVD in women to determine if their medical treatment should differ from that of men, (2) all clinical trials relating to CVD treatment should have significantly more female participants, and gender-based analyses should be performed, as currently recommended for National Institutes of Health (NIH)-sponsored research by the NIH Revitalization Act of 1993, and (3) the Cochrane Library would be a more useful tool for the evidence-based healthcare of women if the Systematic Reviews used all available gender-specific information in their analyses.

Bibliometrics↗
PubMed2001

KN-93 inhibition of G protein signaling is independent of the ability of Ca2+/calmodulin-dependent protein kinase II to phosphorylate phospholipase Cbeta3 on 537-Ser.

Stimulation of the phospholipase Cbeta (PLC) signaling pathway results in intracellular Ca2+ release and subsequent activation of calmodulin (CaM) and CaM kinase II (CaMK II). KN-93, an inhibitor of CaMK II, reduced the stimulation of phosphatidylinositide (PI) turnover by Galphai-coupled (formyl-Met-Leu-Phe, fMLP) or Galphaq-coupled [M1 muscarinic and oxytocin (OT)] receptors. The inhibitory effect of KN-93 was also observed when PLCbeta3 was stimulated directly by Galphaq or Gbetagamma in overexpression assays. CaMK II phosphorylated PLCbeta3 but not PLCbeta1 in vitro. Phosphorylation occurred exclusively on 537Ser in the X-Y linker region of PLCbeta3. 537Ser was also phosphorylated in the basal state in cells and phosphorylation was enhanced by ionomycin treatment. However, mutation of 537Ser to Glu had no effect on inhibition of Galphaq or Gbetagamma-stimulated PLCbeta3 activity by KN-93. KN-93 also inhibited Galphaq -stimulated PLCbeta1 activity, even though this enzyme is not a substrate for CaMK II. These data indicate that phosphorylation of PLCbeta3 by CaMK II is not directly involved in the inhibitory effect of KN-93 on phosphatidylinositide turnover.

Amino Acid Substitution↗
PubMed2001

Phosphorylation and regulation of G-protein-activated phospholipase C-beta 3 by cGMP-dependent protein kinases.

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca(2+)] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca(2+) by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca(2+). In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.

Animals↗
PubMed2001

Hormones and calcium: mechanisms controlling uterine smooth muscle contractile activity. The Litchfield Lecture.

The regulation of myometrial contraction is of paramount importance for the maintenance of pregnancy and for parturition. Understanding this regulation involves delineating the pathways that control myometrial contraction and relaxation and defining the regulation of these pathways. The pathways can be broken down further into those signalling cascades controlling the concentration of intracellular free calcium (Ca(2+)(i)) and those controlling the contractile apparatus itself. This discussion focuses primarily on the former and their regulation during pregnancy. In particular, cross-talk between the contractant and relaxant signalling pathways mediated through cyclic AMP is markedly changed at the end of pregnancy. Experimental Physiology (2001) 86.2, 223-237.

Animals↗
PubMed2000

Molecular mechanism of the inhibition of phospholipase C beta 3 by protein kinase C.

Activation of protein kinase C (PKC) can result from stimulation of the receptor-G protein-phospholipase C (PLCbeta) pathway. In turn, phosphorylation of PLCbeta by PKC may play a role in the regulation of receptor-mediated phosphatidylinositide (PI) turnover and intracellular Ca(2+) release. Activation of endogenous PKC by phorbol 12-myristate 13-acetate inhibited both Galpha(q)-coupled (oxytocin and M1 muscarinic) and Galpha(i)-coupled (formyl-Met-Leu-Phe) receptor-stimulated PI turnover by 50-100% in PHM1, HeLa, COSM6, and RBL-2H3 cells expressing PLCbeta(3). Activation of conventional PKCs with thymeleatoxin similarly inhibited oxytocin or formyl-Met-Leu-Phe receptor-stimulated PI turnover. The PKC inhibitory effect was also observed when PLCbeta(3) was stimulated directly by Galpha(q) or Gbetagamma in overexpression assays. PKC phosphorylated PLCbeta(3) at the same predominant site in vivo and in vitro. Peptide sequencing of in vitro phosphorylated recombinant PLCbeta(3) and site-directed mutagenesis identified Ser(1105) as the predominant phosphorylation site. Ser(1105) is also phosphorylated by protein kinase A (PKA; Yue, C., Dodge, K. L., Weber, G., and Sanborn, B. M. (1998) J. Biol. Chem. 273, 18023-18027). Similar to PKA, the inhibition by PKC of Galpha(q)-stimulated PLCbeta(3) activity was completely abolished by mutation of Ser(1105) to Ala. In contrast, mutation of Ser(1105) or Ser(26), another putative phosphorylation target, to Ala had no effect on inhibition of Gbetagamma-stimulated PLCbeta(3) activity by PKC or PKA. These data indicate that PKC and PKA act similarly in that they inhibit Galpha(q)-stimulated PLCbeta(3) as a result of phosphorylation of Ser(1105). Moreover, PKC and PKA both inhibit Gbetagamma-stimulated activity by mechanisms that do not involve Ser(1105).

Cyclic AMP-Dependent Protein Kinases↗
PubMed1999

Oxytocin-stimulated capacitative calcium entry in human myometrial cells.

OBJECTIVE: Our purpose was to investigate the relative contribution of extracellular calcium recruitment and release of calcium from intracellular stores in an immortalized myometrial cell line derived from a pregnant woman (PHM1-41) and to determine the importance of capacitative calcium entry in the oxytocin-stimulated rise in intracellular free calcium. STUDY DESIGN: The PHM1-41 immortalized myometrial cell line, which retains smooth muscle phenotype, estrogen, and oxytocin receptors and responds to oxytocin with an increase in intracellular free calcium, was used for this study. Intracellular free calcium was measured directly in cells loaded with Fura 2-AM. RESULTS: The oxytocin-stimulated rise in intracellular free calcium decreased in the absence of extracellular calcium or in the presence of phospholipase C inhibitors, suggesting mobilization of calcium from both extracellular and intracellular sources to increase intracellular free calcium. Phospholipase C inhibitors resulted in greater inhibition of the oxytocin-stimulated rise in intracellular free calcium than expected on the basis of experiments performed in the absence of extracellular calcium. This implies interdependence of the intracellular and extracellular pathways for elevation of intracellular free calcium and suggests some capacitative entry of calcium as a consequence of depletion of intracellular stores. The oxytocin-stimulated intracellular free calcium increase resulting from calcium entry was blocked by store depletion by thapsigargin or cyclopiazonic acid, consistent with a capacitative calcium entry mechanism. CONCLUSION: Oxytocin stimulates both capacitative and noncapacitative calcium entry in a pregnant human myometrium cell line.

Calcium↗
PubMed1999

Oxytocin-induced Ca2+ responses in human myometrial cells.

Complex spatiotemporal changes in intracellular Ca2+ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1. 0-1000 nM). Laser cytometry revealed intracellular Ca2+ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca2+ transients by 10-15 min that lasted for at least 90 min. The amplitude of the initial Ca2+ spike was dose dependent, while the frequency of Ca2+ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca2+ involved in oscillations indicated that the major contribution to oscillation frequencies of </= 6 mHz in cells was from the inositol 1,4,5-trisphosphate-sensitive pool, accounting for about 60% of the frequencies. Most of the remaining frequencies were attributable to extracellular Ca2+, which presumably comes from plasma membrane channels other than L-type channels. When oscillation frequencies exceeded 6 mHz, a significant contribution from a ryanodine-sensitive Ca2+ pool was detected. Eight-bromo-cAMP suppressed both the initial Ca2+ spike and the long-term oscillations. Prostaglandin E1 and E2 caused a significant increase in the frequency of oxytocin-induced Ca2+ oscillations. FFT analysis may be of considerable value for study of the mechanisms of rhythmic Ca2+ transients and their function in myometrial cells, as well as the mechanisms by which uterotonins and tocolytic agents impact myometrial Ca2+ regulation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Protein kinase A anchoring to the myometrial plasma membrane is required for cyclic adenosine 3',5'-monophosphate regulation of phosphatidylinositide turnover.

The importance of the localization of protein kinase A (PKA) to the plasma membrane for cAMP-mediated inhibition of phosphatidylinositide turnover was tested in an immortalized pregnant human myometrial (PHM1-41) cell line, and the putative A kinase anchoring protein (AKAP) involved was identified. Preincubation in PHM1-41 cells with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited the ability of oxytocin to stimulate phosphatidylinositide turnover. The addition of a peptide that specifically disrupts interactions of PKA RII subunits with AKAPs (S-Ht31) reversed the effects of these agents, whereas a control peptide was ineffective. The pharmacology of S-Ht31 on this particular membrane event was further characterized. A 10-min incubation with S-Ht31 at a concentration of 1 microM completely reversed the inhibitory effect of relaxin on phosphatidylinositide turnover. S-Ht31 inhibited cAMP-stimulated PKA activity in PHM1-41 cell plasma membranes and decreased the concentration of PKA. Overlay analysis detected a single AKAP of approximately 86 kDa associated with the plasma membrane of PHM1-41 cells, suggesting that the association of PKA with this AKAP is important for the cAMP inhibitory mechanism. The mol wt of this AKAP was similar to that of an AKAP associated with the plasma membrane in the human brain, AKAP79. Antibodies against AKAP79 recognized a band at 86 kDa in purified plasma membranes from the PHM1-41 cells, indicating similar determinants in these proteins. These data suggest that PKA is anchored to the myometrial plasma membrane through association with an AKAP similar to AKAP79, and that this anchoring is required for the cAMP-mediated inhibition of phosphatidylinositide turnover in PHM1-41 cells.

A Kinase Anchor Proteins↗
PubMed1999

A role for AKAP (A kinase anchoring protein) scaffolding in the loss of a cyclic adenosine 3',5'-monophosphate inhibitory response in late pregnant rat myometrium.

During pregnancy in the rat, there is a change in the ability of chlorophenylthio (CPT)-cAMP to inhibit myometrial phosphatidylinositide turnover. This is accompanied by a change in the association of proteins with a plasma membrane A kinase anchoring protein (AKAP). Both CPT-cAMP and isoproterenol inhibited oxytocin-stimulated phosphatidylinositide turnover on days 12 through 20 of gestation, whereas neither agent had an effect on day 21. Accompanying this change was a dramatic decrease in the concentration and activity of cAMP-dependent protein kinase [protein kinase A (PKA)] and an increase in the concentration of protein phosphatase 2B (PP2B) in plasma membranes from day 21 compared with day 19 pregnant rats. In contrast, both PKA and PP2B concentrations and activities increased in total myometrial homogenates. Both PKA and PP2B coimmunoprecipitated with an antibody against the 150-kDa AKAP found in rat myometrial plasma membranes. More PKA was associated with AKAP150 on day 19 than on day 21, while the reverse was true for PP2B. Disruption of PKA/AKAP association in day 19 pregnant rat myometrial cells with the specific interaction inhibitor peptide S-Ht31 resulted in the loss of the cAMP-inhibitory effect on phosphatidylinositide turnover. PP2B activity in myometrial homogenates dephosphorylated PLCbeta3, a PKA substrate targeted in the inhibition of Galphaq-stimulated phosphatidylinositide turnover. The dramatic loss of the cAMP-inhibitory effect on day 21 of pregnancy may alter the balance between uterine contraction and relaxation near parturition. The changes in the relative concentrations of PKA and PP2B associated with AKAP150 are consistent with a functional role for AKAP150 scaffolding in the alteration of cellular signaling.

Animals↗
PubMed1998

Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq.

The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.

Animals↗
PubMed1998

Molecular mechanisms regulating the effects of oxytocin on myometrial intracellular calcium.

Oxytocin stimulates an increase in intracellular calcium in uterine myometrium by several mechanisms. Several lines of evidence indicate that the oxytocin receptor is functionally coupled to GTP-binding proteins of the G alpha q/11 class which stimulate phospholipase C activity. The IP3 generated as a result of phospholipase C activation can trigger release of calcium from intracellular stores. The finding that the oxytocin-stimulated increase in intracellular calcium in myometrial cells is greater in the presence of extracellular calcium than that in its absence indicates that oxytocin also has effects on calcium entry. This action is nifedipine-insensitive but may involve indirect stimulation of calcium entry through release-operated channels. An anti-G alpha q/11 antibody inhibits both oxytocin-stimulated GTPase activity and phospholipase C activity in myometrial membranes. The stimulation by oxytocin of phosphoinositide turnover in COS cells transfected with a plasmid expressing the oxytocin receptor is enhanced by cotransfection of G alpha q. Co-transfection of intracellular domains of the oxytocin receptor causes varying degrees of interference with oxytocin-stimulated phosphoinositide turnover. The data suggest that more than one intracellular domain is involved in oxytocin receptor/G-protein coupling. Oxytocin receptor stimulation of phospholipase C is inhibited by cAMP. This occurs in myometrial cells and in COS cells transfected with a plasmid expressing the receptor. The inhibitory mechanism involves the action of protein kinase A and is probably targeted indirectly at the G alpha q/11 /phospholipase C coupling step.

Animals↗
PubMed1998

Evidence for the involvement of several intracellular domains in the coupling of oxytocin receptor to G alpha(q/11).

In order to probe the nature of oxytocin receptor (OTR)/G alpha(q/11) protein coupling, we examined the effect of co-expression of OTR intracellular domains on oxytocin-stimulated phosphoinositide turnover in COSM6 cells overexpressing OTR and G alpha(q). Co-expression of G alpha(q) enhanced the oxytocin response maximally at a pOTR/pG alpha(q) plasmid transfection ratio of 1:0.16. In cells co-expressing OTR and G alpha(q/11), oxytocin stimulated phosphoinositide turnover with an EC50 of 48 nM. Co-transfection with plasmids expressing OTR intracellular domains inhibited oxytocin-stimulated phosphoinositide turnover by 23 +/- 6% (1i), 37 +/- 4% (2i), 55 +/- 6% (3i), and 40 +/- 6% (4i), respectively (P < 0.01). Expression of the 3i loop of the alpha(1B)-adrenergic receptor, which also couples to G alpha(q/11), inhibited phosphoinositide turnover by 35 +/- 2% (P < 0.01), while expression of the 3i loop of the dopamine 1A receptor, which couples to G alpha(s), had no effect. While these data indicate a functional role for the OTR 3i loop, they also suggest that interactions with more than one intracellular domain probably mediate the coupling of OTR to the G alpha(q/11) class of GTP-binding proteins.

Amino Acid Sequence↗
PubMed1998

Evidence for inhibition by protein kinase A of receptor/G alpha(q)/phospholipase C (PLC) coupling by a mechanism not involving PLCbeta2.

The effects of cAMP on the oxytocin-stimulated increase in phosphatidylinositide turnover and the possible pathways involved were investigated in a human myometrial cell line (PHM1-41) and in COS-M6 cells overexpressing the oxytocin receptor. Preincubation with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited oxytocin-stimulated phosphatidylinositide turnover in PHM1-41 cells, and the inhibition was reversed by H-89, a relatively specific protein kinase A inhibitor. Both CPT-cAMP and transiently expressed protein kinase A catalytic subunit inhibited stimulation by oxytocin and carbachol of [3H]inositol 1,3,4-trisphosphate formation in COS-M6 cells expressing oxytocin or muscarinic M1 receptors, respectively. CPT-cAMP also inhibited phosphatidylinositide turnover stimulation by endothelin-1 in PHM1-41 cells, further demonstrating the generality of the cAMP-inhibitory mechanism. Since G betagamma activation of phospholipase Cbeta2 (PLCbeta2) is a suggested target of protein kinase A, the possibility that the oxytocin receptor couples to PLCbeta2 via G alpha(i)G betagamma activation was explored. Western blot analysis of PHM1-41 cells and COS-M6 cells detected PLCbeta1 and PLCbeta3, but not PLCbeta2. In PHM1-41 cells, pertussis toxin reduced the oxytocin-stimulated increase in [3H]inositol 1,3,4-trisphosphate by 53%, and this was reversed completely by H-89. Thus, the inhibitory effect of pertussis toxin may result from an indirect effect of cAMP elevation. These data suggest that receptor/G alpha(q)-coupled stimulation of PLCbeta1 or PLCbeta3 can be inhibited by cAMP through a phosphorylation mechanism involving protein kinase A that does not involve PLCbeta2. In smooth muscle, this mechanism could constitute potentially important cross-talk between pathways regulating contraction and relaxation.

Animals↗
PubMed1998

G protein signalling pathways in myometrium: affecting the balance between contraction and relaxation.

Heterotrimeric G proteins are actively involved in intracellular signalling in the myometrium and play important roles in regulating myometrial contraction and relaxation. Increases in intracellular calcium can be induced by agents that stimulate uterine contractions. In a number of instances, these increases in intracellular calcium are attributed to stimulation of phospholipase C by either G alpha or G betagamma subunits as a result of activation of G protein-coupled plasma membrane receptors. This mechanism also stimulates calcium entry through calcium release-activated channels, either directly or indirectly. Thus, while phospholipase C can be activated by other pathways and calcium can enter myometrial cells through other channels, G proteins play a major role in these processes. Similarly, activation of protein kinase A and protein kinase C are consequences of G protein activation. Protein kinase A and protein kinase C exert a number of regulatory influences on phospholipase C, ion channel activity and other processes in the myometrium. The mitogen-activated protein kinase pathway can also be activated directly or indirectly by the action of G proteins in myometrium. Responsiveness to G proteins can be altered during pregnancy and depends on the relative expression of all of the components of the signalling pathways involved. The balance between G protein-mediated stimulatory and inhibitory signalling pathways has important consequences for the control of myometrial contractile activity.

Adenylyl Cyclases↗
PubMed1996

Epidermal growth factor increases phosphoinositide turnover and intracellular free calcium in an immortalized human myometrial cell line independent of the arachidonic acid metabolic pathway.

OBJECTIVES: The objectives of this study were to determine whether epidermal growth factor increases intracellular calcium and phosphoinositide turnover in human myometrial cells by a tyrosine kinase-mediated mechanism, to evaluate an obligatory role for arachidonic acid metabolites in these actions, and to compare the actions of epidermal growth factor and oxytocin. STUDY DESIGN: Intracellular calcium and phosphoinositide turnover were measured in a myometrial cell line after stimulation with epidermal growth factor (0.1 to 100 nmol/L) or oxytocin (20 nmol/L). The effects of nifedipine, thapsigargin, genestein and tyrphostin, the guanosine triphosphate binding protein antagonist GPA-7, indomethacin, and nordihydroguaiaretic acid were determined. Data were analyzed by analysis of variance and Duncan's multiple-range test. RESULTS: Epidermal growth factor stimulated phosphoinositide turnover and increased intracellular calcium in a dose-dependent manner (median effective concentration 2.6 nmol/L). In contrast to oxytocin, the effects of epidermal growth factor were inhibited by tyrosine kinase inhibitors but not by GPA-7. Indomethacin and nordihydroguaiaretic acid did not inhibit the epidermal growth factor-stimulated increase in intracellular calcium. CONCLUSIONS: The acute epidermal growth factor-stimulated increase in intracellular calcium in this myometrial cell line is primarily derived from release of calcium from intracellular stores, and it involves the activation of a tyrosine kinase, presumably the epidermal growth factor receptor. Arachidonic acid metabolites are not obligatory intermediates. Oxytocin increases phosphoinositide turnover and intracellular calcium by a distinctly different pathway.

Amino Acid Sequence↗
PubMed1996

Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line.

Smooth muscle cells isolated from the myometrium of a pregnant woman at term were infected with a replication-defective adenovirus vector expressing the E6/E7 proteins of human papilloma virus 16. A clonal line, PHM1-41, was selected by resistance to Geneticin and examined for maintenance of smooth muscle phenotype and response to oxytocin. The immortalized cell line retained morphological characteristics of proliferating smooth muscle cells in culture for up to 22 passages and has been used for over 2 years. The cells expressed smooth muscle-specific alpha-actin and retained estrogen receptors. Oxytocin receptors were present, as measured by whole cell binding assay using the oxytocin antagonist 125I-d(CH2)5[Tyr-(Me)2,Thr4,-Orn8,Tyr9-NH2] as ligand and oxytocin as competitor. The data were best described by a one-site binding model, with a Kd of 0.36 nM and a binding site concentration of 37 fmol/microgram DNA. PHM1-41 cells responded to oxytocin with an increase in intracellular free calcium (EC50 15 nM) and an increase in phosphatidylinositol turnover. Oxytocin-stimulated phosphatidylinositol turnover was inhibited by preincubation with the cAMP analog CPT-cAMP. This immortalized myometrial cell line should prove useful for studies relating to human myometrial function.

Actins↗
PubMed1996

Correlation between connexin43 expression, cell-cell communication, and oxytocin-induced Ca2+ responses in an immortalized human myometrial cell line.

The effects of 8-bromo-cAMP on gap junction expression and intracellular Ca2+ ([Ca2+]i) were examined in an immortalized human myometrial cell line (PHM1-41) generated from myometrial cells obtained from term-pregnant myometrium. PHM1-41 cells express the gap junction protein connexin43 (Cx43). Gap junction-mediated intercellular communication, monitored with a method using fluorescence recovery after photobleaching, revealed low communication rates between cells. Addition of 1.0 mM 8-bromo-cAMP to culture medium resulted in an increase in junctional communication within 5 min, while 30 microM forskolin treatment resulted in increased communication within 10 min. Cells treated with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP for 24 h revealed a dose-dependent increase in communication and an increase in immunostained gap junction plaques at cell-cell contacts. A direct correlation between the rate of dye transfer and the number of plaques was detected. No effect on communication or immunoreactive Cx43 was detected in cells exposed to 10 or 50 nM estradiol-17 beta for 24 h. In experiments analyzing [Ca2+]i, a concentration-dependent increase was induced by oxytocin (1-100 nM) with half-maximal stimulation at approximately 5 nM. Treatment of PHM1-41 cells for 24 h with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP reduced the magnitude of the oxytocin-induced [Ca2+]i response. Thus, 8-bromo-cAMP increased Cx43-containing cell surface gap junctional plaques and up-regulated junctional communication but attenuated the oxytocin-induced [Ca2+]i response. These data indicate that the action of 8-bromo-cAMP in PHM1-41 cells has potentially opposite effects on the contractile and conductive properties of cells, and suggest that up-regulation of myometrial conductivity by agents that increase cAMP could diminish the tocolytic benefits of these agents.

8-Bromo Cyclic Adenosine Monophosphate↗