Trevania Saunders

Intracellular mechanisms underlying prostaglandin F2α-stimulated phasic myometrial contractions

These studies sought to test the hypothesis that prostaglandin F2α(PGF2α)-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 PGF2α-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 PGF2α-stimulated phasic myometrial contractions. In summary, these studies have provided substantial support for the hypothesis that PGF2α-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.

Thrombin-stimulated Uterine contractions in the pregnant and nonpregnant rat

Objective: Thrombin generated during the active clotting of blood appears to be a potent uterotonic agonist; however, the mechanism underlying this effect on uterine smooth muscle is not well understood. We performed studies to confirm the uterotonic effects of thrombin and to determine whether prostaglandin production plays a role during the uterotonic effects of thrombin or clotting blood. Methods: Uterine contraction studies were performed using adult nonpregnant and near-term pregnant rats. The in vitro isometric contraction studies used uterine strips pretreatd with indomethacia or vehicle (ethanol), which were then stimulated with thrombin. Forthe in vivo contraction studies, rats were pretreated with intraperitoneal injections of indomethacia or vehicle (ethanol) then stimulated by intraluminal injection of fresh rat blood or thrombin into the uterus. The contraction data were acquired using isometric force traducers, were computer digized, normalized for spontaneous activity, and statistically analyzed. Prostaglandin (PG) F2α was measured using an enzyme-linked immunoassay. Results: The in vitro contraction studies demonstrated that both thrombin and actively clotting blood produce a significant increase in the frequency and intensity of uterine contractions. Thrombin stimulation was associated with a 54% increase in PGF2α concentration in vitro; indomethacin (1 μM) pretreatment completely inhibited that increase in PGF2α production. Despite the suppression of PGF2α production, pretreatment with indomethacin had no inhibitory effect on thrombin-stimulated contractile activity. In vivo contraction studies further confirmed that indomethacin (2 mg/kg) pretreatment had no effect on blood- or thrombin-stimulated contractile activity. Conclusions: We confirmed that thrombin andthrombin produced by actively clotting blood had a robust uterotonic effect in the rat and that prostaglandin production did not play a significant role in thrombin-stimulated contractions.

A mechanism for testosterone modulation of alpha-1 adrenergic receptor expression in the DDT1 MF-2 smooth muscle myocyte

Previous reports have confirmed that steroid hormones modulate the expression of adrenergic receptors on the surface of smooth muscle myocytes. The present study was undertaken to evaluate the mechanism by which testosterone modulates alpha-1 adrenergic receptor expression in the DDT1 MF-2 transformed smooth muscle cell. Utilizing 3H-prazosin radioligand binding studies, alpha-1 adrenergic receptors were noted to increase more than 2 fold in response to incubation with 10−8 M testosterone for 96 hours. Dihydrotestosterone similarly stimulated a significant increase in alpha-1 receptors; whereas, estradiol and hydrocortisone appeared to suppress the expression of this receptor in DDT myocytes. The testosterone effect was dose related with a maximal response observed in response to 10 −7M testosterone at both 48 and 96 hours. Kinetic experiments utilizing 10−8 M testosterone demonstrated a peak effect on alpha-1 receptor expression at 96 hours, and maintenance of the effect for at least 168 hours (7 days). The testosterone effect was completely prevented at both 48 and 96 hours by inhibition of transcription with actinomycin-D, or inhibition of translation with cycloheximide. Consistent with the receptor binding studies, RNA blotting studies have demonstrated maximal alpha-1 receptor mRNA levels at 48–96 hours of testosterone stimulation. In conclusion, these in vitro experiments have confirmed that physiologic concentrations of testosterone stimulate the increased expression of alpha-1 receptors in the DDT1 MF-2 myocyte after a delay of 48–96 hours; and that this effect appears to be mediated by transcription, translation, and synthesis of new proteins in these genital tract myocytes.

The mechanisms underlying Bay K 8644-stimulated phasic myometrial contractions.

Objective: Phasic myometrical contractions appear to be produced by calcium transients resulting from the activation of the phosphatidylinositol-signaling pathway. Bay K 8644, an L-type calcium channel activator, produces an increase in frequency and intensity of phasic myometrial contractions. These studies were performed to test the hypothesis that Bay K 8644-stimulated contractions were mediated through mechanisms involving phosphoinositide-specific phospholipase C activation and cytosolic calcium oscillation-like mechanisms. Methods: In vitro contraction studies and intracellular calcium imaging were performed on longitudinal strips of uterine tissue obtained from mature virgin Sprague-Dawley rats. Isometric contraction data were computer digitized, analyzed for contraction area, and nomalized for cross-sectional area. Dose-response studies were performed using previously reported inhibitors of cytosolic calcium oscillation mechanisms. In addition, qualitative inositol-phosphate production studies were performed after prelabeling uterine tissue in vitro with 3H-inositol. Subsequently, the labeled inositol phosphates were separated and recovered using anion exchange chromatography. Results: Bay K 8644 produced periodic calcium transients or oscillations along with a dose-related increase in contractile activity and a signfificant increase in inositol-phosphate production. In contrast, neomycin (an inhibitor of phospholipase C), adenine (an inhibitor of calcium-induced calcium release), nifedipine (an L-type calcium channel blocker), and EGTA (a calcium chelator) significantly inhibited Bay K 8644-stimulated contractile activity. Conclusions: These results are consistent with the hypothesis that Bay K 8644, through its facilitation of increased intracellular calcium, results in the activation of the phosphatidylinsitol-signaling pathway and cytosolic calcium oscillation-like phenomena, thereby resulting in the generation of phasic myometrial contractions.

Tissue-specific protein kinase C isoform expression in rat uterine tissue.

Objective: Activation of the phosphatidylinositol signaling pathway plays a key role during the generation of agonist-stimulated phasic myometrial contractions. Protein kinase C (PKC), a component of this signaling pathway, has been previously shown to produce feedback inhibition of agonist-stimulated myometrial contractions. The studies described in this report were performed to survey the tissue-specific expression of several PKC isoforms in the rat uterus. Methods: Uterine tissue was obtained from timed pregnant and normally cycling adult female Sprague-Dawley rats. Immunohistochemical studies were performed using the Vectastian ABC immunostaining technique and PKC isoform-specific polyclonal antibodies. Western blot studies were peformed using myometrial tissue separated into cytosol and membrane fractions by differential centrifugation. Results: These studies confirmed significant expression of the PKC-α, -β2, -δ, -η, and -ζ isoforms in myometrium from pregnant and estrus rats, whereas only trace or no expression of the PKC-β1, -γ, -ε, and -θ isoforms was observed. Expression of the PKC-α, -β2, and -η isoforms decreased modestly during the latter days of gestation; in contrast, PKC-δ and -ζ remained stable during this period. The immunohistochemical studies confirmed expression of the PKC-α, -β2, -δ, -η, and -ζ isoforms in both circular and longitudinal smooth-muscle layers of the near-term pregnant rat uterus. Conclusion: In summary, these studies have confirmed significant levels of expression of several isoforms of PKC in estrus and near-term pregnant rat uterine tissue, which was most prominent in the smooth-muscle cells of the myometrium.

Intrauterine Expression of Prothrombin in the Sprague-Dawley Rat

Objective: Thrombin appears to underlie myometrial contractions in response to intrauterine bleeding. In a similar fashion, thrombin generated within the uterus in the absence of active bleeding could also produce contractions. These studies sought to determine whether functionally active prothrombin is expressed in the pregnant and nonpregnant rat uterus. Methods: Uteri were obtained from proestrus/estrus and timed-pregnant Sprague-Dawley rats. Western blots were performed using antithrombin antibodies. Immunohistochemical studies were performed using the same antibodies along with the Vector Elite ABC kit. Qualitative reverse transcriptaase—polymerase chain reaction studies were performed using rat prothrombin-specific oligonucleotide primers. In vitro uterine contraction studies were performed using Taipan snake venom (an exogenous prothrombinase) and components of the plasma prothrombinase complex (Factors Xa and V) with and without pretreatment with thrombin inhibitors (heparin or hirudin). Results: The Western blots demonstrated prothrombin peptides in myometrial tissue from estrus and pregnant rats. The immunohistochemical studies confirmed prothrombin peptides in both the circular and longitudinal myometrium, along with the endometrium. The reverse transcriptase—polymerase chain reaction studies demonstrated prothrombin mRNA in the endometrium and placenta, but not in the myometrial smooth muscle. The Taipan snake venom stimulated a significant increase in contractions, which were suppressed by pretreatment within heparin and hirudin. The Factor Xa and V complex also significantly stimulated uterine contractions, which were likewise inhibited by hirudin. Conclusion: These studies provide evidence supporting the expression of functionally active prothrombin in the pregnant and nonpregnant rat uterus. Based on the presence of its mRNA, prothrombin appears to be synthesized in the endometrium and placenta; in contrast, the myometrial smooth-muscle cells appear to sequester preformed prothrombin. These results support the hypothesis that intrauterine thrombin could play an autocrine/paracrine role in the regulation of contractile activity.

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.

Tyrosine Kinase-Mediated Activation of Cytosolic Calcium Oscillations and Phastic Myometrical Contractions

These studies sought to test the hypothesis that tyrosine kinase-stimulated phasic myometrical contractions are mediated by activation of the phosphatidylinositol (PI)-signaling pathway and the generation of cytosolic calcium oscillations. For these studies, uterine tissue was obtained from adult female Sprague-Dawley white rats during the proestrus/estrus phase of the cycle. In vivo contraction studies were performed using pervanadate (a tyrosine phosphatase inhibitor) with and without inhibitors of the PI-signaling pathway, including 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (a phospholipase C inhibitor), thimerosal (an inositol-trisphosphate receptor/channel inhibitor), and Ruthenium red (a ryanodine receptor inhibitor), and with oxytocin or prostaglandin F2α (two classic uterotonic agonists). Cytosolic calcium studies were performed using Fura-2-loaded myometrial strips. During these studies, pervanadate was observed to produce cytosolid calcium oscillations and phasic contractions in myometrical tissue comparable to those produced in response to oxytocin and prostaglandin F2α. The pervandate-stimulated phasic contractions were significantly suppressed in response to inhibition of phospholipase C, the inositol-trisphosphate receptor, and the ryanodine receptor, thereby confirming the importance of the PI-signaling pathway during tyrosine kinase-associated myometrical activity. Further confirming the important and shared role for the PI-signaling pathway during pervanadate-stimulated myometrical contractions, no significant additive effects were observed when classic uterotonic agonists such as oxytocin or prostaglandin F2α were combined with pervanadate.

Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes

SummaryThe following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouths MB 751/1 medium-10% fetal bovine serum along with 10−8M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.