Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Ghalpha/tissue transglutaminase 2: an emerging G protein in signal transduction

Gh protein is an heterodimer made up of two subunits alpha and beta. Different from the traditional monomeric and heterotrimeric G proteins, Ghalpha subunit exhibits both GTPase and transglutaminase activities whereas Ghbeta was identified as calreticulin. Activation of Gh by G protein-coupled receptors (GPCR) turns off transglutaminase activity and shifts Ghalpha to signal transducer. Thereafter, Ghalpha regulates downstream effectors. All these aspects are discussed in the present review, in order to shed new light on this atypical G protein.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

人子宫平滑肌肉瘤中性鞘糖脂含量、组成及其四糖基组分糖链结构的变化

本文分析了人子宫平滑肌肉瘤组织的中性鞘糖脂,发现子宫肉瘤组织的中性鞘糖脂的含量明显低于正常组织,组成成分虽与正常子宫平滑肌相似,均含有单、双、三、四糖基及多糖基组分,但各组分的相对含量则变化显著。肉瘤中所含短糖链的组分相对减少,高极性的CPH明显增加。本文还纯化了子宫肉瘤中性糖脂中主要的四糖基组分,应用HPTLC、酸解和酶解法以及特异单抗放射免疫染色法对该组分进行鉴定。结果证明子宫平滑肌中所含主要的四糖基组分糖链结构在正常组织为Globo系列的红细胞苷脂,而在肉瘤中则转变为乳糖系列的拟红细胞糖苷脂。     

New potential regulators of uterine leiomyomata from DNA arrays: the ionotropic glutamate receptor GluR2

In the post-Genome era, new concepts emerge about the growth regulation of uterine leiomyomata. Screening of leiomyoma and myometrial tissues with DNA arrays revealed numerous genes up-regulated in leiomyomata that were not known to be expressed in the human uterus. GluR2, a subunit of a ligand-gated cation channel, is up-regulated in leiomyomata relative to myometrium by 15- to 30-fold at the protein and mRNA level and is localized in endothelial cells. GluR2 pre-mRNA in leiomyoma and myometrial tissues is nearly 100% edited at the Q/R site, indicative of low Ca(2+) permeability of the ion channels. In spontaneous leiomyomata in women or leiomyomata induced in the guinea pig model, there is a likely synergism linking increased production of estradiol and all-trans retinoic acid with up-regulation of nuclear receptor PPARgamma and RXRalpha proteins to support tumor growth. GluR2 might be coupled to this synergism directly or via interleukin-17B, kinesin KIF5 or related genes also up-regulated in leiomyomata. GluR antagonists should be tested as inhibitors of leiomyoma growth.     

An in vitro study on spontaneous myometrial contractility in the cow during estrus and diestrus

Myometrial smooth muscle strips were collected from slaughtered cows in estrus and diestrus. Longitudinal and circular smooth muscle strips were mounted in organ baths and after equilibration time and 2 g preload, their physiologic contractility was recorded for 3 h. Area under the curve (AUC), mean amplitude (MA) and frequency of contractions (F) were studied. Differences between cycle phases, between muscle layers and over the recorded time period were statistically evaluated. In the cow, physiologic contractility patterns (measured as AUC and MA) of circular versus longitudinal myometrial strips are always different during the 3 h recording. Significant differences between estrus versus diestrus are only found for circular layers, but not for longitudinal layers. Significant differences over time are only found for longitudinal layers.     

ATP-binding cassette ABCC1 is involved in the release of sphingosine 1-phosphate from rat uterine leiomyoma ELT3 cells and late pregnant rat myometrium

Sphingosine 1-phosphate (S1P), a bioactive lipid generated by sphingosine kinases (SphK1/2), initiates different signalling pathways involved in physiological and pathological processes. We previously demonstrated that in rat myometrium at late (day 19) gestation, SphK1 increases the expression of COX2 via S1P generation and release. In rat uterine leiomyoma cells (ELT3), SphK1/S1P axis controls survival and proliferation. In the present study we demonstrate that PDBu activates SphK1 but not SphK2. SphK1 activation requires PKC and MAPK ERK1/2. S1P produced by PDBu is released in the medium. PDBu-induced S1P export is abolished by Ro-318220 and BIM (PKC inhibitors), by U0126 and PD98059 (MEK inhibitors), SKI-II (SphKI/2 inhibitor) and SphK1-siRNA, suggesting the involvement of PKC, ERK and SphK1 respectively. The release of S1P is insensitive to inhibitors of ATP Binding Cassette (ABC)A1 and ABCB1 transporters, but is abolished when ABCC1 transporters are inhibited by MK571 or down-regulated by ABCC1-siRNA. PDBu increases COX2 expression that is blocked by the inhibition of PKC, ERK1/2, SphK1, and when cells are treated with MK571 or transfected with ABCC1-siRNA. The induction of COX2 by the S1P release due to PDBu or by exogenous S1P involves S1P2 receptors coupled to Gi. In myometrium from rat at late gestation, the release of S1P is also strongly reduced when SphK and ABCC1 are inhibited. The data reveal that in rat leiomyoma cells and late pregnant rat myometrium, the release of S1P involves a similar signalling pathway and occurs through ABCC1.     

人正常子宫肌肉的鞘糖脂组成及其在不同生理功能阶段的变化

本文测定了新生儿、生育期、更年期和足月妊娠人子宫肌肉的神经节苷脂(Gg)与中性鞘糖脂(N-GSL)的含量,比较了两种鞘糖脂的HPTLC谱。新生儿期Gg的总含量(以脂结合唾液酸LBSA量表示)最高,每克湿重组织约45.2μg,足月妊娠子宫肌肉中的含量最低,为10.4μg,生育期为32.8μg、更年期为39.5μg。N—GSL的含量却以足月妊娠子宫肌肉中最多,达99.4μg。按HPTLC谱分析子宫肌肉中Gg的主要组分为GD_3和GM_3,在子宫发育成熟与妊娠时,肌肉组织中这两种组分的含量变化明显:生育期样品的GD_3由新生儿的25.4％增加到56.6％(按占LBSA总量的百分比计算),GM_3则由33.2％降至16.9％。此外,GM_1和GD_(1a)也明显减少。N—GSL在生育期CMH、CDH和CTH的含量(按占含糖基量的百分比计算)成倍增加,而含五糖基以上的组分则仅为新生儿子宫的1/5。足月妊娠与新生儿子宫肌肉的两类鞘糖脂的HPTLC谱类似,但前者GT1b占19.4％,明显高于新生儿样品(6.1％)。     

Differential expression of desmin in the uterine myometrium and cervix as a possible mechanism for successful parturition in rats

Expression of desmin, an intermediate filament, in the myometrium and cervix were investigated in peripartum rats (full term day 22 of pregnancy (DP22)). Des mRNA was expressed in lesser amounts in the cervix at peripartum (DP17 and 21, and day of birth 1 (DB1)), compared to those in the cervixes of ovariectomized rats. Immunohistochemical analysis revealed that desmin protein was diffusely present in the myometrium, and locally in the epithelium of the cervix. Western blot analysis showed that desmin protein levels in the myometrium increased 4- to 6-fold at DP17, 21 and DB1, and decreased rapidly at DB2 to the basal level observed in ovariectomized or non-pregnant rats. In contrast, cervical desmin protein levels increased approximately 10-fold at DP21 compared to those in ovariectomized rats, but decreased rapidly at DB1, indicating its decrease at parturition and an inconsistency between mRNA and protein expression. The administration of 17β-estradiol to ovariectomized rats increased desmin protein levels in the myometrium and cervix after 24 h. S-nitrosylated desmin protein was detected in the myometrium and cervix at DP21. The mRNA expression of inducible nitric oxide synthase was consistent with the expression of desmin protein. Thus, desmin, which is regulated by estradiol, is differentially expressed in the myometrium and cervix at peripartum possibly for successful pregnancy and parturition. In the cervix, desmin protein expression seems to be regulated by estradiol at the translational level. S-nitrosylation of desmin may have a potential role in the peripartum uterus.