Increased Inhibitory Effect of Phorbol Ester on Cytosolic Ca2+ Level and Contraction in Rat Myometrium after Gestation

Abstract

ABSTRACT-Activation of voltage-dependent Ca 21 channels by high K+ (40 mM) increased the cytosolic Ca2+ level ([Ca 2+]i) (estimated by fura-PE3 fluorescence ratio) and force in myometrium isolated from pregnant (21 days after gestation) and non-pregnant (estrus) rats. 12-Deoxyphorbol 13-isobutyrate (DPB, 1 mM) decreased the high K+-stimulated [Ca 2+]i and force in a concentration-dependent manner. The inhibitory effect was stronger in the pregnant myometrium than in the non-pregnant myometrium. In the pregnant myometrium, the increase in Ca 21 permeability by ionomycin (1 uM) greatly increased [Ca 2+]i and force, which were only partially inhibited by verapamil (10 pM). DPB (1 pM) inhibited the verapamilinsensitive component of the increases in [Ca 2+]i and muscle tension. Oxytocin (100 nM) and thapsigargin (1 pM) also induced a verapamil-insensitive increase in [Ca 2+]i and force, and DPB (1 pM) inhibited these increments. Ca2+ sensitivity of contractile elements, estimated from the relationships between Ca 2+ and muscle force in intact and a-toxin permeabilized muscle, was not significantly changed by DPB (1 pM). In summary, DPB inhibits the increase in [Ca 21]i more strongly in myometrium isolated from pregnant rats than that from non-pregnant rats without any change in the [Ca 2+]i/tension relationship. Since DPB decreased [Ca 2+]i-rise induced by three different mechanisms, DPB may activate Ca 2+ extrusion, rather than to inhibit a specific influx pathway, to decrease [Ca 2+]i.Keywords: Uterine smooth muscle, Phorbol ester, Relaxation, Cytosolic Ca2+ level, Pregnancy It is well-known that uterotonic agonists, such as oxytocin, carbachol and prostaglandins, increase intracellular Ca2+ concentration ([Ca 2+]i) and contraction (1-3). These receptor agonists stimulate phosphoinositide turnover in the uterine smooth muscle (4, 5). In various types of smooth muscle, inositol 1,4,5,-trisphosphate, one of the phosphoinositide hydrolysis products, releases Ca 2+ from the intracellular Ca2+ store and induces transient contraction (6). Another phosphoinositide product, diacylglycerol, stimulates protein kinase C that phosphorylates various proteins involved in cellular function (7). It has been reported that protein kinase C activation with phorbol ester inhibits smooth muscle contraction by inhibiting Ca 2+ channel activity or inositol phosphate production (8, 9), while it induces contraction by activation of contractile proteins (10-12). In cultured cells from rat portal vein, phorbol esters increase Ca 2-7 channel activity (13). In myometrium, it has been reported that phorbol ester induces stimulatory and inhibitory effects on contraction (14-16). However, few attempts have been made to compare the role of protein kinase C before and after pregnancy. In the present studies, we compared the effect of protein kinase C activation by phorbol esters on [Ca 2+]i and muscle contraction in isolated uterine smooth muscle from non-pregnant and pregnant rats. Female Wistar rats (200-250 g; Shiraishi Laboratory Animals, Tokyo...

Possible role of the protein kinase C/CPI‐17 pathway in the augmented contraction of human myometrium after gestation

“…In the light of the awareness of species‐related differences in PKC‐mediated signaling, we attempted to examine the effects of PKC activation on [Ca 2+ ] i and contraction in myometrium isolated from pregnant and nonpregnant women. We found that PKC activation induces positive feedback control in the activation processes of the smooth muscle contractile element in human myometrium, which is distinct from findings in rat myometrium showing that PKC is involved in the negative feedback control of the contractile mechanism (Savineau & Mironneau, 1990; Kim et al , 1996 ).…”

“…In the myometrium isolated either from pregnant or nonpregnant rat, it has been reported that phorbol ester induces inhibitory effects on contraction (Savineau & Mironneau, 1990; Phillippe, 1994a,1994b). We have also observed decreases in [Ca 2+ ] i and muscle contraction upon PKC stimulation by phorbol ester in rat myometrium, showing that PDBu inhibits the increase in [Ca 2+ ] i more strongly in myometrium from pregnant than from nonpregnant rats ( Kim et al , 1996 ). In contrast to the results in rat myometrium, others have suggested the importance of PKC in the agonist‐induced contractions in human myometrium ( Morrison et al , 1996 ; Breuiller‐Fouche et al , 1998 ; Eude et al , 2000 ).…”

Role of protein kinase Cβ in rhythmic contractions of human pregnant myometrium

“…It has been reported that PKC activation by phorbol ester inhibits gastric smooth muscle contractions by inhibiting inositol phosphate production , but induces vascular and tracheal smooth muscle contractions by increasing the Ca 2C sensitivity of contractile elements (Morgan & Morgan 1984, Sato et al 1988, Ozaki et al 1990, Sato et al 1992. In rat myometrial cells, the PKC activation inhibits Ca 2C channel activity (Kusaka & Sperelakis 1995), and thus PKC activation in the rat myometrium has an inhibitory effect on contractions (Baraban et al 1985, Savineau & Mironneau 1990, Phillippe 1994, Kim et al 1996. In contrast, in cultured cells of rat portal veins, PKC activation increases Ca 2C channel activity (Loirand et al 1990).…”

A Review of Signal Transduction in Mechanisms of Smooth Muscle Contraction and Its Relevance for Specialized Physical Therapy

“…Some drugs are also known to contract smooth muscle 2,3) . Many previous studies, including our own, have reported on signal transduction mechanisms and smooth muscle contractions [4][5][6][7][8][9][10][11][12][13][14] . In spite of the importance of the smooth muscle in the fields of cardiovascular physiotherapy, obstetrics, and clinical methods, there is still a need for more research [15][16][17] .…”

“…The activated MLCK phosphorylates the 20-kDa serine residue of myosin light chain (MLC 20 ), which leads to muscle contraction by increasing the activation of myosin-Mg 2+ ATPase (Fig. 1A) [1][2][3][4] . Additionally, receptors activated by agonists such as prostaglandin F2α (PGF2α), endothelin-1 (ET-1), and norepinephrine cause conformational changes in the receptors and activate GTP-binding protein (or G-protein).…”