Chun-Ying Ku

Molecular signaling through G-protein-coupled receptors and the control of intracellular calcium in myometrium.

Cellular mechanisms regulating myometrial intracellular free calcium (Ca2+) are addressed in this review, with emphasis on G-protein-coupled receptor pathways. An increase in myometrial Ca2+ i results in phosphorylation of myosin light chain, an incerase in myosin light chain, an increase in myosin denosine monophosphatse (ATPase) activity and contraction. Dephosphorylation of myosin light chain and a decline in Ca2+ i are associated with relaxation. Increases in Ca2+ i are controlled by multuiple signaling pathways, including receptor-mediated activation of phospholipase Cβ (PLCβ), leading to release of Ca2+ from intracellular stores. Ca2+ also enters myometrial cells through plasma membrane Ca2+ channels. Conversely, adenosine triphosphate (ATP)-dependent Ca2+ pumps lower Ca2+ i concentrations and potassium channels promote hyperpolarization that can decrease Ca2+ entry. Receptor-coupled pathways that promote uterine relaxation primarily involve activation of cyclic adenosine monophosphate (cAMP)-or cyclic guanosine monophosphate (cGMP)-stimulated protein kinases that phosphorylate proteins regulating Ca2+ homeostasis. cAMP has inhibitory effects on myometrial contractile activity, agonist-stimulated phosphatidylinositide turnover and increases in Ca2+ i. Some of those effects require association of protein kinase A (PKA) with a plasma membraneassociated A-kinase-anchoring-protein (AKAP). Near term in the rat, there is a decline in the plasma membrane localization of PKA associated with this anchoring protein. This correlates with changes in the regulation of signaling pathways controlling Ca2+ i. L-type voltage-operated Ca2+ entry is an important regulator of myometrial contraction. In addition, putative signal-regulated or capacitative Ca2+ channel proteins, Trp Cs, are expressed in myometrium, and signal-regulated Ca2+ entry is observed in human myometrial cells. This Ca2+ entry mechanism may play a significant role in the control of myometrial Ca2+ i dynamics and myometrial contraction. The regulation of myometrial Ca2+ i is complex. Understanding the mechanisms involved may lead to design of tocolytics that target multiple pathways and achieve improved suppression of premature labor.

Progesterone Prevents the Pregnancy-Related Decline in Protein Kinase A Association with Rat Myometrial Plasma Membrane and A-Kinase Anchoring Protein

Abstract The presence of cAMP-dependent protein kinase (PKA) in the plasma membrane compartment and its association with an A-kinase anchoring protein (AKAP150) is implicated in mediating cAMP regulatory events in the rat myometrium. The association of PKA with purified myometrial plasma membrane declined gradually between Day 16 and Day 21 of gestation, with a decrease of 53% ± 11% of the catalytic subunit and of 61% ± 7% of the regulatory subunit at Day 21 compared with Day 19. To determine the role of progesterone in this association, pregnancy was prolonged by administration of progesterone or shortened by administration of the antiprogestin RU486. Progesterone treatment maintained PKA association with plasma membrane at Day 21 at 123% ± 23% (catalytic subunit) and 92% ± 4% (regulatory subunit) of Day 19 levels. In contrast, protein phosphatase 1, protein phosphatase 2B, phospholipase Cβ3, and AKAP150 concentrations in the plasma membrane did not change over this interval or with progesterone treatment. Changes in PKA coimmunoprecipitated with membrane-associated AKAP150 paralleled those in total plasma membrane on Days 19 and 21 and on Day 21 following progesterone treatment. In contrast, plasma membrane PKA catalytic and regulatory subunits decreased by 20 h after RU486 injection on Day 15 of pregnancy to levels resembling those on Day 21. These data indicate that progesterone prevents the decline in PKA associated with myometrial plasma membrane and with AKAP150 in the pregnant rat. The decrease in membrane-bound PKA between Days 19 and 21 and after RU486 treatment precedes the onset of parturition in both experimental paradigms. The loss of plasma membrane PKA may be critical for the decrease in the inhibitory effect of cAMP on oxytocin-induced phosphatidylinositide turnover that occurs near the end of pregnancy and may contribute to enhanced myometrial contractile responsiveness near term.

Reduction in TRPC4 expression specifically attenuates G-protein coupled receptor-stimulated increases in intracellular calcium in human myometrial cells.

Canonical transient receptor potential (TRPC) proteins may play a role in regulating changes in intracellular calcium ([Ca(2+)](i)). Human myometrium expresses TRPC4, TRPC1 and TRPC6 mRNAs in greatest relative abundance. Contributions of TRPC4 to increases in [Ca(2+)](i) were assessed in PHM1-41 and primary human uterine smooth muscle (UtSMC) cells using short hairpin RNAs (shRNAs). Based on a reporter assay screen, one shRNA was selected to construct an adenoviral expression vector (TC4sh1). TC4sh1 induced both mRNA and protein TRPC4 knockdown in PHM1-41 cells without affecting expression of other TRPCs. Signal-regulated Ca(2+) entry (SRCE), defined as a stimulus- and extracellular Ca(2+)-dependent increase in [Ca(2+)](i), was measured in PHM1-41 cells treated with oxytocin (G-protein coupled receptor (GPCR)-stimulated), thapsigargin (store depletion-stimulated), and OAG (diacylglycerol-stimulated), using Fura-2. Cells infected with TC4sh1 exhibited attenuated oxytocin-, ATP- and PGF2alpha-mediated SRCE, but no change in thapsigargin- or OAG-stimulated SRCE. Similar results were obtained in primary uterine smooth muscle cells. Additionally, cells expressing TC4sh1 exhibited a significantly smaller increase in channel activity in response to oxytocin administration than did cells infected with empty virus. These data show that, in human myometrial cells, knockdown of endogenous TRPC4 specifically attenuates GPCR-stimulated, but not thapsigargin- or OAG-stimulated extracellular calcium-dependent increases in [Ca(2+)](i). These data imply that, in this cellular context, the mechanisms regulating extracellular Ca(2+)-dependent increases in [Ca(2+)](i) are differentially affected by different signaling pathways.

Multiple Signals Regulate Phospholipase CBeta3 in Human Myometrial Cells

Phospholipase CB3 (PLCB3) serine(1105) (S(1105)), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S(1105) phosphorylation (P-S(1105)) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S(1105). Relaxin also stimulated P-S(1105); this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S(1105), as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S(1105), whereas the reverse was the case for the oxytocin-stimulated increase in P-S(1105). PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S(1105)A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S(1105) dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.

Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells.

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).

Pathways Used by Relaxin to Regulate Myometrial Phospholipase C

Abstract: Relaxin exhibits pleiotropic effects on reproductive and nonreproductive tissues; the signaling mechanisms underlying these functions are still not well understood. Activation of protein kinase A and several other signal‐regulated protein kinases results in the phosphorylation of phospholipase C (PLC)‐β3 and inhibit Gαq‐stimulated PLC activity. Therefore, PLCβ3 may be targeted by both contractant and relaxant signaling pathways in myometrium and play a critical role in the balance between them. PHM1 cells express mRNA for relaxin receptor LGR7, and relaxin inhibits oxytocin‐stimulated PLC activity in these cells. Thus, this model system may be useful in delineating signaling pathways used by relaxin. Here, we present evidence that relaxin stimulates phosphorylation of PLCβ3 in PHM1 cells.

Changes in Rat Myometrial Plasma Membrane Protein Kinase A Are Confined to Parturition

We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)—dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.