Chain Phosphorylation In Smooth Muscle Research Articles

The Effects of Placental Corticotrophin Releasing Hormone on the Physiology and Psychology of the Pregnant Woman

In response to stress corticotropin releasing hormone (CRH) is secreted from the hypothalamus and travels to the anterior pituitary where it stimulates the release of proteins derived from the precursor protein, proopiomelanocortin (POMC), including adrenocorticotropic hormone (ACTH) and β-endorphin. β-endorphin interacts with opioid receptors and relieves stress while ACTH travels through the blood stream to the adrenal cortex where it stimulates the release of cortisol. Cortisols actions include releasing glucose from stored glycogen and stimulating breakdown of fat from adipose tissue, induction of gluconeogenesis and modulation of the immune system. As a negative feedback control, cortisol inhibits CRH release from the hypothalamus and ACTH from the anterior pituitary. During pregnancy the placenta synthesizes and secretes CRH into the systemic circulation causing a dramatic increase in the circulating levels of CRH throughout pregnancy that peaks at delivery and unlike the situation in the hypothalamus, cortisol has a positive effect on placental CRH production. While the CRH binding protein (CRH-BP) may attenuate the action of placental CRH on the pregnant womans pituitary, there is evidence that the CRH-BP does not completely cut off access of placental CRH to the pituitary. For example, CRH-BP levels decrease in late pregnancy lowering the ability to decrease CRH activity. In late pregnancy when circulating CRH levels rise dramatically and superimpose over falling levels of the CRH-BP, the anterior pituitary may desensitize to placental CRH so that ACTH and cortisol levels do not become pathologically elevated. In non-pregnant individuals, disturbances in the hypothalamic CRH - pituitary ACTH axis are associated with mood disturbances and in pregnant women who have elevated CRH levels during pregnancy an association with prenatal depression has been shown. More work needs to be done to investigate the possible roles of CRH in pregnancy related depression. Besides effects on the pituitary, placental CRH may exert physiological regulation on the uterus. Higher than normal levels of CRH in mid-pregnancy are associated with preterm pregnancy suggesting a role for CRH in the timing of parturition. In preterm pregnancy when the uterus has to be kept quiescent, CRH receptors are coupled to various cell signaling systems including the cyclic AMP system that inhibits myosin light chain phosphorylation in smooth muscle thereby dampening uterine contraction. As the time for labour approaches, CRH receptors may alter coupling to cellular signaling systems that phosphorylate myosin light chain and initiate rhythmic contraction of the uterus. The possibility of using modulators of CRH action such as CRH receptor agonists to correct pathological conditions in the expectant mother provides a powerful motivation for future studies to fully elucidate the roles of CRH in pregnancy. Keywords: Corticotrophin releasing hormone (CRH), CRH binding protein (CRH-BP), CRH receptors, placenta, uterus, pregnancy, depression

Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle

The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AIF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca(2+)-free conditions. In ileum, PDBu translocated PKCs alpha, beta 1, beta 2, epsilon and theta to the membrane fraction, and GTP[S] caused a small translocation of PKC-epsilon. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-epsilon was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs alpha, beta 1, beta 2, epsilon and theta do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.

Propofol-induced changes in myosin light chain phosphorylation in smooth muscle are reversed by Okadaic Acid

Propofol-induced changes in myosin light chain phosphorylation in smooth muscle are reversed by Okadaic Acid

Cooperative activation of myosin by light chain phosphorylation in permeabilized smooth muscle.

The purpose of this study was to determine the quantitative relationship between the number of myosin molecules that increase their ATPase activity and the degree of myosin light chain phosphorylation in smooth muscle. Single turnover experiments on the nucleotide bound to myosin were performed in the permeabilized rabbit portal vein. In the resting muscle, the rate of exchange of bound nucleoside diphosphate was biphasic and complete in approximately 30 min. When approximately 80% of the myosin light chain was thiophosphorylated, the nucleoside diphosphate exchange occurred at a much faster rate and was almost complete in 2 min. Thiophosphorylation of 10% of the myosin light chains caused an increase in the rate of ADP exchange from much more than 10% of the myosin subfragment-1. Less than 20% thiophosphorylation of the total myosin light chains resulted in the maximum increase in ADP exchanged in 2 min. It appears that a small degree of myosin light chain phosphorylation cooperatively turns on the maximum number of myosin molecules. Interestingly, even though less than 20% thiophosphorylation of the myosin light chain caused the maximum exchange of ADP within 2 min, higher degrees of thiophosphorylation were associated with further increases in the ATPase rates. We conclude that a small degree of myosin light chain thiophosphorylation cooperatively activates the maximum number of myosin molecules, and a higher degree of thiophosphorylation makes the myosin cycle faster. A kinetic model is proposed in which the rate constant for attachment of unphosphorylated cross bridges varies as a function of myosin light chain phosphorylation.

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.

Regulation of myosin light chain phosphorylation in smooth muscle: James Stull, Daniel Fitzsimons, Patricia Gallagher, Paul Herring, Kristine Kamm, Da-Chun Tang, Malu Tansey, Ann Word. Department of Physiology, UT Southwestern, Dallas, TX 75235 USA

Regulation of myosin light chain phosphorylation in smooth muscle: James Stull, Daniel Fitzsimons, Patricia Gallagher, Paul Herring, Kristine Kamm, Da-Chun Tang, Malu Tansey, Ann Word. Department of Physiology, UT Southwestern, Dallas, TX 75235 USA

O-061 - Effects of HA1077 on tension development and myosin light chain phosphorylation in smooth muscle

O-061 - Effects of HA1077 on tension development and myosin light chain phosphorylation in smooth muscle

Stimulus-specific patterns of myosin light chain phosphorylation in smooth muscle of rabbit thoracic artery

When the rabbit thoracic artery was stimulated with submaximal concentrations of agonist [40 mM K+, 30 microM prostaglandin F2 alpha (PGF2 alpha) or 7 microM histamine], about 90% of a maximal contraction occurred. Each agonist induced a rapid development of contraction followed by a sustained response. The maximal rate of force generation stimulated with PGF2 alpha was twice that seen with K+ or histamine. Stimulation with 40 mM K+ increased the extent of monophosphorylated 20 kDa myosin light chain (MLC-P) for up to 1 min to a maximal value of 38.8 +/- 1.0%, there was a subsequent rapid decrease and the MLC-P level remained just above the basal value for 40 min (6.8 +/- 3.0%). In the case of stimulation with 7 microM histamine, MLC-P level increased rapidly and was sustained for up to 40 min (28.0 +/- 4.9%). In contrast to the stimulation with K+ or histamine, PGF2 alpha induced both mono- and diphosphorylated MLC20 (MLC-P and MLC-P2 respectively) at a low concentration (3 microM). The monophosphorylation of MLC20 induced by 30 microM PGF2 alpha reached the maximal value of 32.8 +/- 5.2%, and was sustained for up to 40 min (15.2 +/- 5.4%). The diphosphorylation of MLC20 increased rapidly (7.4 +/- 4.0% at 5 min), then decreased to the basal value within 40 min. These results suggest that different modes of stimulation of smooth muscle contraction produce different profiles of MLC20 phosphorylation. The implications of these observations are that the diphosphorylated form, specifically induced by certain agents, may modify the mode of contraction of the aortic artery.

Phosphatase inhibition with okadaic acid does not alter the relationship between force and myosin light chain phosphorylation in permeabilized smooth muscle

The phosphatase inhibitor, okadaic acid, has been used to test the hypothesis that myosin light chain phosphatase activity plays a central role in latchbridge formation in smooth muscle. In the permeabilized rabbit portal vein there is a non-linear relationship between myosin light chain phosphorylation and force production such that maximum force output occurs with about 50% phosphorylation. Treatment of the muscle with okadaic acid does not change this relationship even though there is a profound inhibition of phosphatase activity. The data suggest that dephosphorylation of the myosin light chain while the myosin is in the force producing state does not account for the high force output with low levels of light chain phosphorylation in smooth muscle.

Myosin light chain and heavy chain phosphorylation in smooth muscle: potential regulatory roles for calcium, phospholipids and cyclic nucleotides.

Myosin light chain and heavy chain phosphorylation in smooth muscle: potential regulatory roles for calcium, phospholipids and cyclic nucleotides.

6] Myosin light chain phosphorylation in smooth muscle and nonmuscle cells as a probe of calmodulin function

The function of Ca2+-calmodulin-dependent phosphorylation of phosphorylatable myosin light chain (P-light chain) is currently an area of immense interest and research. In the smooth muscle, P-light chain phosphorylation is a requisite for the interaction of myosin with actin. One major problem in working with intact smooth muscle or nonmuscle cells has been the lack of a sensitive technique for quantitating the extent of P-light chain phosphorylation in preparations that are routinely used in physiological and pharmacological investigations. This chapter discusses methodology for quantitating P-light chain phosphorylation during changes in isometric tension development of the bovine tracheal smooth muscle. The applicability of the methodology for quantitating P-light chain phosphorylation in various types of vascular smooth muscles, cardiac muscle, and skeletal muscles has also been determined in the chapter. This methodology should also be adaptable to other tissues or nonmuscle cells, such as platelets and macrophages, where myosin P-light chain phosphorylation may be involved in the regulation of motile processes. The combined use of pyrophosphate gel electrophoresis, isoelectric focusing, and ammoniacal-silver staining offers a relatively rapid, accurate, and sensitive technique for quantitating the extent of P-light chain phosphorylation during changes in contractile activity in the smooth muscle.

Force-velocity characteristics of respiratory airway smooth muscle.

Force-velocity characteristics of respiratory airway smooth muscle.