Sensitivity Of Contractile Elements Research Articles

Synchronous phosphorylation of CPI-17 and MYPT1 is essential for inducing Ca2+ sensitization in intestinal smooth muscle

Myosin phosphatase activity is regulated by mechanisms involving the phosphorylation of CPI-17 and MYPT1, primarily based on studies with tonic-type vascular smooth muscles. This study examined how these mechanisms contribute to the regulation of contraction of a phasic-type intestinal smooth muscle. Phosphorylation levels, tension, and Ca(2+) sensitization was detected in rat ileal smooth muscle. Key Results In rat ileal smooth muscle, phosphorylation level of CPI-17 at Thr(38) and MYPT1 at Thr(853) , but not MYPT1 at Thr(696) , were increased with carbachol (1μmolL(-1) ) accompanied with muscle contraction. The PKC inhibitor Go6976 (1μmol L(-1) ) inhibited the carbachol-induced phosphorylation of CPI-17, whereas the Rho-associated kinase (ROCK) inhibitor, Y-27632 (10μmol L(-1) ) inhibited the carbachol-induced phosphorylation of both CPI-17 and MYPT1. Application of Go6976 or Y-27632 alone inhibited the carbachol-induced contraction; however, the combined application of these inhibitors did not inhibit the contraction in an additive manner. In β-escin-permeabilized ileal strip, treatment with antiphosphorylated antibodies for CPI-17 at Thr(38) and MYPT1 at Thr(853) and Thr(696) alone almost completely abolished the Ca(2+) sensitization due to carbachol with GTP. In conclusion, receptor stimulation increases the Ca(2+) sensitivity of contractile elements through CPI-17 phosphorylation via the PKC/ROCK pathways and MYPT1 phosphorylation via the ROCK pathway, when these mechanisms operate cooperatively and/or synchronously in intestinal smooth muscle.

Mechanism of potentiation by tea epigallocatechin of contraction in porcine coronary artery: The role of protein kinase Cδ-mediated CPI-17 phosphorylation

The effects of green tea catechins, (+)- and (−)-catechins (C), (−)-epicatechin (EC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCG), on vascular contractility were investigated in porcine coronary artery. At the concentration of 200 μM, only EGC, but not other catechins, potentiated high K +-induced contraction in a concentration-dependent manner, although EGC by itself did not produce contraction. The potentiator effect of EGC was still observed in endothelium-denuded preparations. Moreover, EGC increased the translocation of protein kinase Cδ (PKCδ) from the cytosol to the plasma membrane and increased phosphorylations of 17-kDa PKC-potentiated protein phosphatase inhibitor protein (CPI-17) and myosin light chain (MLC 20). These effects of EGC were inhibited by the PKCδ inhibitor rottlerin, but not by the conventional PKC inhibitor Gö6976. These results suggest that EGC activates PKCδ, leading to the phosphorylation of CPI-17, which in turn inhibits myosin light chain phosphatase and increases MLC 20 phosphorylation. The series of events would increase Ca 2+ sensitivity of contractile elements, thereby augmenting high K +-induced vascular contraction.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca(2+) level ([Ca(2+)](i)), and Ca(2+) sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 microM) inhibited high K(+) (25 mM and 40 mM)- or histamine (3 microM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K(+)-evoked contractions. Spontaneous changes in [Ca(2+)](i) and contractions were inhibited by forskolin (1 microM) without changing the resting [Ca(2+)](i). Forskoln (10 microM) inhibited muscle tension more strongly than [Ca(2+)](i) stimulated by high K(+), and thus shifted the [Ca(2+)](i)-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 microM) inhibited both [Ca(2+)](i) and muscle tension without changing the [Ca(2+)](i)-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 microM) inhibited the 0.3 microM Ca(2+)-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca(2+)-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca(2+) sensitivity of contractile elements in high K(+)-stimulated muscle and a decrease in [Ca(2+)](i) in histamine-stimulated muscle.

Differences in the gestational pattern of mRNA expression of the Rnd family in rat and human myometria

Uterine myometrial contractility remains a poorly characterized area of research in reproductive physiology. Rnd1, a novel member of the GTP-binding Rho protein family, inhibits Ca 2+-sensitization by specifically interfering with a RhoA/Rho-activated kinases-dependent mechanism in smooth muscle. In addition to Rnd1, there are two other members, Rnd2 and Rnd3, in the Rnd family of Rho proteins. In the present comparative study of myometrial contractility in rats and humans, we found that all three Rnd mRNAs were expressed in nonpregnant rat myometrium and in nonpregnant human myometrial tissues. Although all three mRNA levels increased significantly after gestation in rat myometria, only Rnd1 expression was significantly greater after gestation in human samples. In the ovariectomized rat, administration of estrogen and/or progesterone increased the expression of all Rnd mRNAs. These results suggest that universal Rnd family up-regulation during pregnancy in rats may have an important role for negative-feedback control of uterine contraction during gestation by inhibiting RhoA-mediated increase in Ca 2+ sensitivity of contractile elements. Such increases in Rnd levels may be due to augmented levels of reproductive steroids in rats. Our data also point to gestational differences between rats and humans in Rnd isoform patterns.

Chronic Treatment with Interleukin-1β Attenuates Contractions by Decreasing the Activities of CPI-17 and MYPT-1 in Intestinal Smooth Muscle

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1beta on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1beta (10 ng/ml), contractions elicited by high K+ and carbachol were inhibited in a time-dependent manner. IL-1beta more strongly inhibited the carbachol-induced contractions than high K+ with decreasing myosin light chain phosphorylation. In the alpha-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5'-3-O-(thio)triphosphate increased the Ca2+ sensitivity of contractile elements, and this G protein-coupled Ca2+ sensitization was significantly reduced in the IL-1beta-treated ileum. Among the functional proteins involved in the smooth muscle Ca2+ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1beta, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1beta treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1beta-treated muscles. These results suggest that long term treatment with IL-1beta decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1beta to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1beta.

Up-regulation of Rnd1 during pregnancy serves as a negative-feedback control for Ca 2+ sensitization of contractile elements in rat myometrium

We investigated the role of Rnd1, a member of the small GTP-binding Rho protein family, in the change in Ca 2+ sensitivity of contractile element in rat myometrium at estrus, gestation, and postpartum stages. In the permeabilized muscles, GTPγS or carbachol with GTP increased Ca 2+ sensitivity of contractile force in non-pregnant myometrium at the estrus stage, whereas these stimuli were ineffective in pregnant myometrium at day 21. After postpartum, the reduced Ca 2+ sensitization was recovered. Semi-quantitative RT-PCR analysis indicated that the expressions of RhoA, ROCKI, and ROCKII were not significantly different between non-pregnant and pregnant myometria. In contrast, the expression of Rnd1 was increased during the course of pregnancy, reaching a maximal at day 21, and rapidly declined after the delivery. On the other hand, Ca 2+ sensitization of contractile elements was decreased during the progress in gestation. These results suggest that Rnd1 may have an important role as a negative-feedback control of uterine contraction during gestation through the inhibition of RhoA-mediated increase in the Ca 2+ sensitivity of contractile elements.

Regulation of Ca2+ sensitivity of contractile elements in cardiac muscle

Contraction of cardiac muscle is regulated by changes in the concentration of intracellular Ca(2+) . An increase in the Ca(2+) concentration results in enhanced contractility of cardiac muscle. It is also known that cardiac contractility is influenced by changes in Ca(2+) sensitivity of contractile elements. Recent studies at the molecular level have revealed the complexity of the regulation of Ca(2+) sensitivity of contractile elements.

Relaxation in Different-Sized Rat BloodVessels Mediated by Endothelium-Derived Hyperpolarizing Factor: Importance of Processes Mediating Precontractions

To clarify the mechanisms involved in relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF), acetylcholine (ACh)-induced endothelium-dependent relaxations and hyperpolarizations were examined in the rat aorta, the main branch of the mesenteric artery (MBMA) and the first branch of the mesenteric aftery (FBMA). In the presence of 100 μM N^G-nitro-L-arginine (L-NNA) and 10 μM indomethacin, ACh (1 nM to 100 μM) produced no relaxation in the phenylephrine-precontracted aorta. The L-NNA-resistant relaxations by ACh in MBMA precontracted with phenylephrine were eliminated in the presence of 1 μM nifedipine where contractions were independent of L-type Ca²⁺ channel activation. In FBMA precontracted with phenylephrine, the L-NNA-resistant relaxations were only partially inhibited by nifedipine. When vessels had been contracted with 300 nM phorbol-12,13-dibutyrate in the presence of nifedipine, ACh-induced L-NNA-resistant relaxations were observed in FBMA only. Pinacidil produced relaxations in all different-sized blood vessels, although sensitivity was inversely related to vessel size. The extent of the ACh hyperpolarizing responses was much smaller than that by pinacidil in the aorta. The membrane potential changes by ACh and pinacidil were almost the same in FBMA. These results indicate that the contribution of EDHF to endothelium-dependent relaxations increases as the vessel size decreases. This may be partly explained by precontractile processes dependent on Ca²⁺ entry through L-type Ca²⁺ channels, because Ca²⁺ channel deactivation seems to be involved as a major mechanism of EDHF-mediated vasorelaxations. However, EDHF may also generate vasorelaxations by an additional mechanism, probably a reduced Ca²⁺ sensitivity of contractile elements, as proposed for ATP-sensitive K⁺ channel openers.

Possible involvement of K+ channel opening to the interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

We have previously shown that interleukin-1 beta relaxes vascular smooth muscle by the NO-dependent and independent mechanisms (Takizawa et al.: Eur. J. Pharmacol. 330: 143-150, 1997). In this study, we investigated the mechanism of NO-independent relaxation. Treatment of the rat aorta with interleukin-1 beta for 24 hr inhibited the high-K+ induced contraction by decreasing cytosolic Ca2+ level ([Ca2+]i). The relationship between [Ca2+]i and tension in intact muscle and the pCa-tension curves in permeabilized muscle suggested that Ca2+ sensitivity of contractile element was not changed after the interleukin-1 beta-treatment. After a treatment with interleukin-1 beta for 24 hr, contractile effects of phenylephrine (1 microM-10 microM) were markedly inhibited in the presence of L-NMMA (100 microM) applied to inhibit NO synthesis. A blocker of ATP-sensitive K+ channel, glibenclamide (1 microM), partially recovered the interleukin-1 beta-induced inhibition. In contrast, a blocker of Ca(2+)-activated K+ channel, charybdotoxin (0.1 microM), was ineffective. These results suggest that membrane hyperpolarization due to activation of ATP-sensitive K+ channels may partly be responsible for the NO-independent mechanism of interleukin-1 beta-induced inhibition of vascular smooth muscle contraction.

内皮細胞由来過分極因子と血管弛緩

Properties of endothelium-derived hyperpolarizing factor (EDHF) have been reviewed briefly. The production of EDHF requires an increase in endothelial [Ca2+]i, the properties being similar to those of nitric oxide (NO). EDHF activates K(+)-channels and hyperpolarizes vascular smooth muscle. The EDHF-induced hyperpolarization is greatly inhibited by charybdotoxin (ChTX) and partially inhibited by apamin, but not by K(+)-channel inhibitors such as Ba2+, glibenclamide, 4-aminopyridine, suggesting that the K(+)-channels involved are mainly the Ca(2+)-sensitive type. Membrane hyperpolarization induces vasodilation by unidentified mechanisms. Experiments using K(+)-channel openers and electrophysiology suggest that hyperpolarization may reduce (i) influx of Ca2+ through voltage-sensitive Ca(2+)-channels, (ii) production of InsP3 in the case of agonist-induced contraction, (iii) Ca(2+)-sensitivity of contractile elements and (iv) agonist-stimulated ion channel activities. In the endothelium-dependent vasodilation, the EDHF/EDRF ratio is larger in peripheral vessels than in the proximal ones, indicating significant importance of EDHF mainly in peripheral arteries. The chemical nature of EDHF remains undetermined, although some candidates such as arachidonic acid metabolites or endogenous cannabinoids are proposed. As the inhibition of gap junctions in artrerial tissues reduces the amplitude of EDHF-induced relaxation, the possible involvement of electrical communication between endothelial and smooth muscle cells has also been considered.

Effects of acidosis on Ca2+ sensitivity of contractile elements in intact ferret myocardium.

We investigated the effects of acidosis on the intracellular Ca2+ concentration ([Ca2+]i) and contractile properties of intact mammalian cardiac muscle during tetanic and twitch contractions. Aequorin was injected into ferret papillary muscles, and the [Ca2+]i and tension were simultaneously measured. Acidosis was attained by increasing the CO2 concentration in the bicarbonate (20 mM)-buffered Tyrode solution from 5% (pH 7.35, control) to 15% (pH 6.89, acidosis). Tetanic contraction was produced by repetitive stimulation of the preparation following treatment with 5 microM ryanodine. The relationship between [Ca2+]i and tension was measured 6 s after the onset of the stimulation and was fitted using the Hill equation. Acidosis decreased the maximal tension to 81 +/- 2% of the control and shifted the [Ca2+]i-tension relationship to the right by 0.18 +/- 0.01 pCa units. During twitch contraction, a quick shortening of muscle length from the length at which developed tension became maximal (Lmax) to 92% Lmax produced a transient change in the [Ca2+]i (extra Ca2+). The magnitude of the extra Ca2+ was dependent on the [Ca2+]i immediately before the length change, suggesting that the extra Ca2+ is related to the amount of troponin-Ca complex. Acidosis decreased the normalized extra Ca2+ to [Ca2+]i immediately before the length change, which indicates that the amount of Ca2+ bound to troponin C is less when [Ca2+]i is the same as in the control. The decrease in the Ca2+ binding to troponin C explains the decrease in tetanic and twitch contraction, and mechanical stress applied to the preparation induced less [Ca2+]i change in acidosis.

Norepinephrine- and K+-Induced Mn2+-Dependent Contractions and the Dynamics of Intracellular Mn2+ Changes in Dispersed Smooth Muscle Cells From Ca2+-Depleted Mn2+-Loaded Vas Deferens of the Guinea Pig

The cellular mechanisms of norepinephrine (NE)-induced Mn2+-dependent contractions were investigated in dispersed smooth muscle cells from guinea pig vas deferens by characterizing the effects of NE and K+ on cell length observed by videotape microscopy and on intracellular Mn2+ and Ca2+ concentration ([Mn2+]i and [Ca2+]i) observed by confocal microscopy. Both stimulants induced slow sustained contraction in Ca2+-depleted, Mn2+-accumulated cells (Mn2+-loaded cells), whereas they induced biphasic contractions in normal cells that were neither Ca2+-depleted nor Mn2+-loaded. In both conditions, the number of cells responding to NE as well as the magnitude of NE-induced contractions increased in a dose-dependent manner. Contractions induced by K+ in Mn2+-loaded strip preparations were markedly smaller than those induced by NE. Although individual K+-induced contractions in responsive Mn2+-loaded cells were as large as those induced by NE, a much smaller percentage of Mn2+-loaded cells was responsive to K+ than to NE. These results are consistent with the idea that the contractions of strip preparations may reflect the magnitude of the contractions of individual cells as well as the percentage of responsive cells in the preparations. Inconsistent with the contractions, the [Mn2+]i rise induced by K+ was larger than that induced by NE, and the percentage of cells responsive to K+ was larger than that responsive to NE. These results suggest that NE may increase the Mn2+ sensitivity of contractile elements.

Increased Ca2+ sensitivity of contractile elements via protein kinase C in alpha-toxin permeabilized SMA from young spontaneously hypertensive rats.

The purpose of the present investigation was to examine the Ca2+ sensitivity of the contractile elements via protein kinase C (PKC) in superior mesenteric artery (SMA) from young (5-6 weeks old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Staphylococcal aureus alpha-toxin, which produces pores in the plasma membrane too small to allow passage of proteins such as PKC, was used to investigate the signal transduction system in vascular smooth muscle cells. We investigated the Ca2+ sensitivity of the contractile apparatus via PKC in intact and alpha-toxin skinned SMA from young SHR and WKY. In intact SMA, high K+ responses were not different between SHR and WKY. However, phorbol 12,13-dibutyrate (PDBu, a PKC activator) augmented high K(+)-evoked contractions and PKC inhibitors, such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, suppressed them more in SHR as compared with WKY. In alpha-toxin skinned SMA, the [Ca2+]i-force relationship curve was not significantly different between SHR and WKY. However, PDBu augmented [Ca2+]i-evoked contractions and PKC inhibitors suppressed them more in SHR than in WKY. These results suggest that the Ca2+ sensitivity of the contractile elements via PKC is significantly greater in prehypertensive SHR than in age-matched WKY. This abnormality in small muscular arteries may be involved in the pathogenesis of hypertension in SHR.

CGMP-kinase mediates cGMP- and cAMP-induced Ca 2+ desensitization of skinned rat artery

(Rp)-8-Bromo-guanosine 3′,5′-cyclic monophosphorothioate (Rp-8-Br-cGMPS) inhibited competitively both isozymes of type Iα and Iβ cGMP-dependent protein kinase (cGMP-kinase) purified from porcine aorta with apparent K i values (μM) of 3.7 for Iα and 1.8 for Iβ. The compound also inhibited bovine heart type II cAMP-dependent protein kinase (cAMP-kinase), but with a K i of 25 μM. Thus, it is a selective inhibitor of cGMP-kinase. In α-toxin-skinned smooth muscle preparations from rat mesenteric artery, 8-Br-cGMP (10 −7 M) and 8-Br-cAMP (10 −6 M) produced a rightward shift of the concentration-contraction curves for Ca 2+, denoting a decrease in Ca 2+ sensitivity of the contractile elements. The shift by 8-Br-cAMP as well as by 8-Br-cGMP was completely reversed by Rp-8-Br-cGMPS, while a selective inhibitor of activation of cAMP-kinase, (Rp)-adenosine-3′,5′-cyclic monophosphorothioate (Rp-cAMPS), was without effects on the shift produced by these two compounds. These findings indicate the pivotal role that the activation of cGMP-kinase plays in the production of a decrease in Ca 2+ sensitivity of contractile elements. © 1997 Elsevier Science B.V. All rights reserved.

Calcium independent contraction of bladder smooth muscle.

Recently, it has been suggested that in vascular smooth muscle a Ca(2+)-independent mechanism or Ca(2+)-sensitization of contractile elements may participate in smooth muscle contraction. In this study, we evaluate this mechanism in detrusor muscle. Strips of smooth muscle from rabbit aorta, rabbit bladder and human bladder were evaluated by in vitro contraction studies. The results show that (1) in Ca(2+)-free solution containing ethyleneglycol bis (-aminothylether)-N,N,-tetraacetic acid (EGTA), carbachol and phorbol ester produced sustained contractions in detrusor muscle (Ca(2+)-free contraction); (2) depletion of Ca2+ stores by caffeine did not affect Ca(2+)-free contraction induced by carbachol; and (3) W-7 (calmodulin inhibitor) and ML-9 (myosin light chain kinase [MLCK] inhibitor) did not show inhibitory effects on Ca(2+)-free contraction, while H-7 (protein kinase C. [PKC] inhibitor) abolished this contraction. These results suggest that neither stored Ca2+ nor the Ca(2+)-calmodulin-MLCK system is involved in the carbachol-induced Ca(2+)-free contraction of detrusor muscle. This Ca(2+)-independent contraction seems to be mediated by the activation of PKC coupled with agonist stimulation of the muscarinic receptor.

Effects of the potassium channel openers KRN4884 and levcromakalim on the contraction of rat aorta induced by A23187, compared with nifedipine.

We examined the different vasodilatory effects of the K+ channel openers levcromakalim and 5-amino-N- [2-(2-chlorophenyl)ethyl]-N'-cyano-3-pyridinecarboxamidine (KRN4884), and the Ca2+ channel blocker nifedipine in the rat aorta. KRN 4884 (10(-10)-10(-5) M) and nifedipine (10(-10)-10(-5) M) produced concentration-dependent relaxation in the rat aorta precontracted by 25 mM KCl. The K+ channel blocker glibenclamide (1 microM) inhibited the relaxation induced by KRN4884 but did not influence nifedipine-induced relaxation. KRN4884 had almost no effect on contraction induced by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl, whereas nifedipine completely relaxed the muscle precontracted by 80 mM KCl. These results indicate that KRN4884 is a K+ channel opener. We investigated the relaxant effects of KRN4884 (10(-10)-10(-5) M), levcromakalim (10(-9)-10(-5) M) and nifedipine (10(-9)-10(-5) M) on A23187 (1 microM)-induced contraction. KRN4884 and levcromakalim had a potent relaxant effect but nifedipine only a weak effect on the smooth muscle contracted by A23187. Glibenclamide (1 microM) inhibited the relaxation induced by KRN4884 and levcromakalim, but did not influence the nifedipine-induced relaxation. KRN4884 (1 microM) produced a larger relaxation of A23187-induced contraction but had little effect on the increase in intracellular [Ca2+] induced by A23187. These results suggest that KRN4884 is a specific K+ channel opener and its vasodilating mechanisms involve not only deactivation of Ca2+ channels but also a decrease in the Ca2+ sensitivity of contractile elements.

MCI-154-induced relaxation in vascular smooth muscles of guinea pig.

We investigated the vasorelaxant effects of MCI-154, a cardiotonic agent designed to target thin filaments in cardiac muscles in intact and skinned vessels from guinea pigs. In normal Krebs-Henseleit solution, MCI-154 (10(-7)-10(-4) M) inhibited the contractions induced by angiotensin II, (Ang II), endothelin-1 (ET-1), phenylephrine, and phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner in guinea pig aorta. In Ca(2+)-free solutions, ET-1 and PMA caused slowly developing and sustained contractions in guinea pig aorta, whereas phenylephrine and caffeine induced transient contractions due to Ca2+ release from the sarcoplasmic reticulum (SR). MCI-154 (10(-7)-10(-4) M) inhibited the contractile responses to ET-1 and PMA. MCI-154 also reduced the contraction induced by Ca2+ release from phenylehrine- and caffeine-sensitive Ca2+ store sites. On the other hand, the relaxation response to MCI-154 was not affected by the presence of methylene blue, a guanylate cyclase inhibitor or by the removal of endothelial cells. MCI-154 decreased the Ca(2+)-activated tension development in saponin-treated skinned fibers from guinea pig femoral arteries. The effects of MCI-154 were not potentiated in the presence of protein kinase A (PKA), whereas those of cyclic AMP were potentiated, possibly because of lack of protein kinase A. The present experiments demonstrate that MCI-154 inhibits vascular contraction when the contractions are produced by any of three mechanisms: protein kinase C (PKC) activation, Ca2+ mobilization from store sites, or sensitization of contractile elements by Ca2+.

Agonists increase the sensitivity of contractile elements for CA2+ in the pregnant rat myometrium.

Agonists increase the sensitivity of contractile elements for CA2+ in the pregnant rat myometrium.

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

Activation of voltage-dependent Ca2+ channels by high K+ (40 mM) increased the cytosolic Ca2+ level ([Ca2+]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 [Ca2+]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 Ca2+ permeability by ionomycin (1 μM) greatly increased [Ca2+]i and force, which were only partially inhibited by verapamil (10 //M). DPB (1 μM) inhibited the verapamil-insensitive component of the increases in [Ca2+]i and muscle tension. Oxytocin (100 nM) and thapsigargin (1 μM) also induced a verapamil-insensitive increase in [Ca2+]i and force, and DPB (1 μM) inhibited these increments. Ca2+ sensitivity of contractile elements, estimated from the relationships between Ca2+ and muscle force in intact and α-toxin permeabilized muscle, was not significantly changed by DPB (1 μM). In summary, DPB inhibits the increase in [Ca2+]i more strongly in myometrium isolated from pregnant rats than that from non-pregnant rats without any change in the [Ca2+]i/tension relationship. Since DPB decreased [Ca2+]i-rise induced by three different mechanisms, DPB may activate Ca2+ extrusion, rather than to inhibit a specific influx pathway, to decrease [Ca2+]i.

Effects of a Prostaglandin I2 Analog Iloprost on Cytoplasmic Ca2+ Levels and Muscle Contraction in Isolated Guinea Pig Aorta

In the isolated guinea pig aorta, the prostaglandin I2 analog iloprost (0.01 – 10 μM) inhibited the contractions induced by the thromboxane A2 analog U46619 (9,11-dideoxy-11α,9α-epoxymethano-prostaglandin F2α; 30 nM) and prostaglandin F2α (PGF2α, 1 μM) in a concentration-dependent manner. In contrast, iloprost only partially inhibited the high K+ (65.4 mM)-induced contraction. In the muscle stimulated with high K+, verapamil (0.3 and 10 μM) inhibited [Ca2+]i and muscle tension in parallel, whereas iloprost (1 μM) inhibited muscle tension with only a small decrease in [Ca2+]i. In the muscle stimulated with U46619 (30 nM), verapamil and iloprost decreased both [Ca2+]i and muscle tension. However, as compared with the effect of verapamil, iloprost more strongly inhibited muscle tension than [Ca2+]i. The iloprost (0.1 – 1 μM)-induced relaxation was accompanied by a concentration-dependent increase in cAMP content. It was further demonstrated that inhibition of the U46619-contractions was augmented in the presence of cycloxygenase inhibitors, such as indomethacin (10 μM), ibuprofen (10 μM) and aspirin (10 μM). In contrast, the inhibition of PGF2α-induced contraction was not affected by indomethacin. Similarly, the inhibitory effect of forskolin on U46619-induced contractions, but not on PGF2α-induced contraction, was enhanced by indomethacin. These results suggest that iloprost inhibits vascular smooth muscle contraction by decreasing [Ca2+]i and the Ca2+ sensitivity of contractile elements through a cAMP-dependent mechanism. The results also suggest that in U46619-stimulated muscle, vasoactive prostaglandins that counterbalance the relaxing action of cAMP may be generated.