The signal transduction pathways within corporal smooth muscle cells are the intracellular molecular mechanisms of corporal smooth muscle tone regulation. Various neurotransmitters activate the membrane receptor proteins or intracellular enzyme pathways and result in the production of extracellular chemical signals. Second messenger molecules and ions transmit and amplify the signals, and subsequently induce the relaxation of smooth muscle cells and penile erection. Therefore, the study of signal transduction in cavernous smooth muscle play an important role in understanding the physiology of erection and the pathophysiology of erectile dysfunction as well as in developing new selective drugs for the treatment of erectile dysfunction.
Calcium ; metabolism ; Humans ; Male ; Muscle, Smooth ; metabolism ; Myosin-Light-Chain Kinase ; physiology ; Penis ; metabolism ; Signal Transduction ; physiology

OBJECTIVETo compare the effect of high frequency oscillatory ventilation (HFOV) and conventional mandatory ventilation (CMV) on the myocardial function of rabbits with inhalation injury.

METHODSSteam inhalation injury model was reproduced in 16 New Zealand albino rabbits. They were randomly divided into CMV group (n = 8) and HFOV group (n = 8) by drawing lots, and they received ventilation in metered volume and HFOV treatment respectively. Heart blood was drawn from rabbits before they were sacrificed 4 hours after treatment to determine the plasma activity of lactate dehydrogenase 1 (LDH1) and creatine phosphorylated kinase (CPK-MB). Myocardial tissue from left ventricle was harvested and homogenized to determine the concentration of TNF-α and IL-8, the activity of caspase-1, and the activity of myosin-light-chain kinase (MLCK) and the ATPase of myosin light chain (MLC-ATPase) by enzyme-linked immunosorbent assay, spectrophotometry, and the nuclide liquid scintillation technique respectively. Part of the myocardial tissue sample was examined pathologically. Data were processed with analysis of variance.

RESULTS(1) The activities of LDH1 and CPK-MB in plasma were obviously higher in CMV group than in HFOV group [(643 ± 108), (342 ± 48) U vs. (233 ± 92), (186 ± 36) U, with F value respectively 10.326 and 9.846, P values all below 0.01]. (2) The contents of TNF-α, IL-8 and the activity of caspase-1 in myocardial tissue homogenate were obviously higher in CMV group than in HFOV group [(181 ± 35), (89 ± 19) pg/g, and (0.56 ± 0.27) g/g protein vs. (94 ± 21), (43 ± 11) pg/g, and (0.24 ± 0.12) g/g protein, with F value respectively 8.239, 7.826, 5.716, P values all below 0.01]. (3) The activities of MLC-ATPase and MLCK were lower in CMV group than in HFOV group [(0.24 ± 0.12) µmol×mg(-1)×min(-1), (3.3 ± 1.1) mmol×mg(-1)×min(-1) vs. (0.48 ± 0.16) µmol×mg(-1)×min(-1), (7.7 ± 1.7) mmol×mg(-1)×min(-1), with F value respectively 4.125, 4.766, P values all below 0.01]. (4) No obvious necrosis, degeneration or inflammatory cell infiltration was observed in myocardial tissue of rabbits in 2 groups under light microscope; but the myocardial fiber was slightly swollen, and it was less marked in the HFOV group.

CONCLUSIONSThe influence of HFOV on myocardial myosin phosphorylation system of rabbits with inhalation injury is less than that of CMV.

Animals ; Burns, Inhalation ; metabolism ; physiopathology ; therapy ; High-Frequency Ventilation ; Myocardium ; metabolism ; Myosin-Light-Chain Kinase ; metabolism ; Rabbits ; Respiration, Artificial

OBJECTIVETo study the role of myosin light chain kinase (MLCK) in intestinal epithelial barrier dysfunction after hypoxia.

METHODSThe Caco-2 monolayers developed with Transwell inserts were exposed to hypoxia for 0 h (NC group), 2, 6, 8, 12 and 24 h (H group), and 6 h hypoxic specimens were treated with 100 mol/L ML-9 (T group). The transepithelial electrical resistance (TER) of monolayers was measured with an ohmmeter. The tight junction protein ZO-1 of monolayers was analyzed by immunofluorescence assay. The protein expressions of phosphorylated myosin light chain (p-MLC) and MLCK were detected by Western blotting.

RESULTSThe TER of monolayers in H group at 6, 8, 12 and 24 h was 422 +/- 17, 427 +/- 27, 403 +/- 40 and 426 +/- 22 ohms respectively, which was significantly lower than that of NC group (451 +/- 27 ohms, P < 0.05). The TER of monolayers in T group was 558 +/- 110 ohms, which was significantly higher than that in H group at each time point ( P < 0.01). The ZO-1 of monolayers in H group at 6 h was irregular in arrangement, with interruptions and rugae, and sawtooth. These abnormalities were ameliorated in T group (regular in arrangement, with little or without ruga and sawtooth). The protein expressions of p-MLC and MLCK in H group at each time point were higher than those in NC group.

CONCLUSIONSIntestinal epithelial barrier dysfunction after hypoxia can be mediated by MLCK.

Caco-2 Cells ; Epithelium ; metabolism ; physiopathology ; Humans ; Hypoxia ; metabolism ; physiopathology ; Intestinal Absorption ; Intestinal Mucosa ; metabolism ; physiopathology ; Intestines ; cytology ; metabolism ; physiopathology ; Myosin Light Chains ; metabolism ; Myosin-Light-Chain Kinase ; metabolism

The objective of study was to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptor activation, and to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptor activation. Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet, and the changes of platelet aggregation and GPIb were analyzed with 100 nmol/L wortmannin (inhibitor of PI3-K) and 10 micromol/L wortmannin (inhibitor of MLCK). The results indicated that the platelet activation was influenced by either concentration of wortmannin in response to PAR stimulation. Platelet aggregation was apparently inhibited by 10 micromol/L wortmannin through both PAR peptides, and was slightly inhibited by 100 nmol/L wortmannin only under PAR1-AP activation. In addition, GPIbalpha internalization was partly inhibited by 100 nmol/L wortmannin in response to PAR1 (p < 0.05 at 1, 2, 5 min) and PAR4 (p < 0.05 at 2, 5, 10 min) activation. Meanwhile, 10 micromol/L wortmannin induced little change for GPIbalpha centralisation in the course of PAR activation, with a delayed restoration of surface GPIbalpha observed under PAR1-AP activation, and no change of GPIbalpha redistribution existed under PAR4-AP activation. It is concluded that the different roles of PI3-K and MLCK exist in the course of thrombin receptor activation. PI3-K accelerates the short course of GPIb centralisation for two PAR signal pathways, while MLCK inhibits the restoration of GPIbalpha in PAR1 pathway.
Adult ; Androstadienes ; pharmacology ; Female ; Humans ; Male ; Myosin-Light-Chain Kinase ; metabolism ; Phosphatidylinositol 3-Kinase ; metabolism ; Platelet Activation ; drug effects ; Platelet Aggregation ; Receptors, Thrombin ; metabolism ; physiology ; Signal Transduction

OBJECTIVETo investigate the effect of combination of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) on intestinal epithelial barrier function.

METHODSThe Caco-2 monolayers were cultured in DMEM nutrient solution, and then they were inoculated in 24-well or 6-well plate with Transwell inserts.They were divided into control group (ordinary treatment), IFN-γ group (with addition of 10 ng/mL IFN-γ), TNF-α group (with addition of 10 ng/mL TNF-α), and IFN-γ plus TNF-α group (with addition of 10 ng/mL TNF-α and 10 ng/mL IFN-γ). Monolayers inoculated in 24-well plate were collected for determination of transepithelial electrical resistance (TER) with an ohmmeter at post treatment hour (PTH) 0, 6, 12, 24, 36, and 48, the permeability of monolayers with fluorescein isothiocyanate-labeled dextran (FITC-dextran) tracer method at PTH 48, the distribution and morphological change of tight junction occludin with immunofluorescence assay at PTH 48. Monolayers inoculated in 6-well plate were collected for determination of protein expression of occludin, myosin light chain kinase (MLCK), and phosphorylated MLC (pMLC) with Western blot at PTH 24. Data were processed with one-way analysis of variance and t test.

RESULTS(1) There was no obvious difference in TER in control group at each time point (F = 0.86, P > 0.05). TER in IFN-γ group and TNF-α group were gradually decreased during PTH 6-48, but showed no statistical difference as compared with that at PTH 0 (with F value respectively 1.69, 2.47, P values all above 0.05). TER in IFN-γ plus TNF-α group was significantly decreased from PTH 24 as compared with that at PTH 0 (t = 4.97, P < 0.05) and that in each of the other three groups (F = 11.54, P < 0.05). (2) The permeability of monolayers in IFN-γ plus TNF-α group [(1197 ± 215)pmol] was significantly higher than that in control group, IFN-γ group, and TNF-α group [(303 ± 93), (328 ± 76), (797 ± 177) pmol, with t value respectively 4.8, 5.0, 6.9, P values all below 0.01]. (3) There was no statistical difference in occludin expression at PTH 24 among four groups (F = 0.26, P > 0.05). The occludin in control group at PTH 48 was regular in arrangement, while that in IFN-γ and TNF-α groups was irregular in arrangement. The arrangement of occludin in IFN-γ plus TNF-α group at PTH 48 was interrupted, with obvious redistribution in cytoplasm. (4) The protein expression of pMLC in IFN-γ plus TNF-α group (0.95 ± 0.05) was significantly higher than that in control group, IFN-γ group, or TNF-α group (0.57 ± 0.12, 0.56 ± 0.07, 0.59 ± 0.10, respectively, F = 17.97, P < 0.01). The protein expression of MLCK in IFN-γ plus TNF-α group (1.57 ± 0.36) was also significantly higher than that in control, IFN-γ, TNF-α groups (0.85 ± 0.18, 1.04 ± 0.23, 1.00 ± 0.07, respectively, F = 9.05, P < 0.05).

CONCLUSIONSCombination of IFN-γ and TNF-α can induce intestinal epithelial barrier dysfunction by up-regulating MLCK protein expression and promoting MLC phosphorylation.

Caco-2 Cells ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Intestinal Mucosa ; cytology ; physiopathology ; Membrane Proteins ; metabolism ; Myosin Light Chains ; metabolism ; Myosin-Light-Chain Kinase ; metabolism ; Occludin ; Tumor Necrosis Factor-alpha ; pharmacology

Massive burn trauma is characterized by hypovolemic shock induced by the loss of plasma from vessels. The major reasons for this systemic microvascular leakage in burns include an increase in vascular permeability triggered by inflammatory mediators and the increase of vascular hydrostatic pressure caused by vessel dilation. The maintenance of normal vascular permeability depends on the integrity of endothelial barrier function regulated by the interaction of intracellular junctions, cell-matrix adhesion and the cytoskeleton contractile force. This review summarizes some recent discovery in endothelial mechanisms during burn-induced vascular hyperpermeability.
Actins ; metabolism ; Burns ; metabolism ; physiopathology ; Capillary Permeability ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; metabolism ; Humans ; Myosin-Light-Chain Kinase ; metabolism ; rho-Associated Kinases ; metabolism

Severe burn injury is often accompanied by intestinal epithelial tight junction barrier dysfunction, which is believed to be closely associated with postburn shock, inflammation, hypermetabolism, infection, organ dysfunction etc. Recent studies have documented the critical role of tight junction-associated protein regulation in intestinal epithelial barrier dysfunction induced by severe burn injury. Myosin light chain (MLC) phosphorylation regulated by both myosin light chain kinase, which can phosphorylate MLC directly, and Rho-associated kinase, which can inhibit MLC phosphatase and then induce MLC phosphorylation indirectly, play a critical role in intestinal epithelial tight junction barrier dysfunction which occurs in severe burn injury. Recent advances have provided new insights into the mechanisms and the therapeutic strategies of intestinal epithelial tight junction barrier dysfunction following severe burn injury.
Burns ; metabolism ; physiopathology ; Humans ; Intestinal Mucosa ; metabolism ; physiopathology ; Myosin-Light-Chain Kinase ; metabolism ; Permeability ; Phosphorylation ; Tight Junctions ; metabolism ; physiology ; rho-Associated Kinases ; metabolism

OBJECTIVETo evaluate the functional regulation of endothelial Myosin light chain kinase (MLCK) in extravascular migration of fibrosarcoma HT1080 cells.

METHODSAn in vitro model of fibrosarcoma cell transmigration across a monolayer of HUVEC cultured on collagen gel was applied to observe extravascular migration of HT1080 cells,and were the electrical resistance of HUVEC monolayer and endothelial MLC phosphorylation in extravascular migration of HT1080 cells.

RESULTHT1080 cells migrated through endothelial cells into collagen gel, the electrical resistance of a HUVEC monolayer was reduced and endothelial MLC phosphorylation was enhanced in extravascular migration of fibrosarcoma cells. Endothelial MLCK inhibitor (ML-7) blocked extravascular migration of HT1080 cells and inhibited reduction of electrical resistance of a HUVEC monolayer and enhancement of endothelial MLC phosphorylation in extravascular migration of HT1080 cells in a dose-dependent manner.

CONCLUSIONEndothelial MLCK regulates fibrosarcoma cell transendothelial migration through MLC phosphorylation, leading to cytoskeletal reorganization and endothelial cell constriction, then fibrosarcoma cells migrate into extravascular tissue through the gaps between endothelial cells.

Cell Line, Tumor ; Cell Movement ; drug effects ; Endothelial Cells ; cytology ; enzymology ; Fibrosarcoma ; pathology ; physiopathology ; Humans ; Myosin-Light-Chain Kinase ; metabolism ; physiology ; Neoplasm Invasiveness ; Umbilical Veins ; cytology

Contraction of smooth muscle is initiated by an increase in cytosolic Ca2+ leading to activation of Ca2+/calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of Ca2+ and the kinetic of Ca2+ mobilization. Ca2+ mobilizing agonists stimulate different phospholipases (PLC-beta, PLD and PLA2) to generate one or more Ca2+ mobilizing messengers (IP3 and AA), and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of PLC-beta, PLA2 and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of PIP2 and generation of IP3 and IP3-dependent Ca2+ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated Ca2+ influx, cADP ribose formation and Ca2+/-induced Ca2+ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by Ca2+/-independent mechanism involving activation of PKC- epsilon by DAG derived form PLD. A functional linkage between G13, RhoA, ROCK, PKC- epsilon, CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the Gq/11 type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1, 4, 5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca2+ and formation of a Ca2+/-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.
Acetylcholine ; Arachidonic Acid ; Cyclic ADP-Ribose ; Cytosol ; Esophageal Sphincter, Lower ; GTP-Binding Proteins ; Hydrolysis ; Inositol ; Intestines ; Metabolism ; Molecular Weight ; Muscle, Smooth* ; Myocytes, Smooth Muscle ; Myosin Light Chains ; Myosin-Light-Chain Kinase ; Negotiating ; Phosphatidylcholines ; Phosphatidylinositols ; Phospholipase D ; Phospholipases ; Phospholipases A2 ; Phosphorylation ; Phosphotransferases ; Protein Kinase C ; Receptor, Muscarinic M3 ; Receptors, Muscarinic ; Second Messenger Systems ; Type C Phospholipases ; Whooping Cough

OBJECTIVETo investigate the function of myosin light chain kinase (MLCK) in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing.

METHODSCross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response (ABR) to evaluate mice hearing function at different frequencies.

RESULTSMlck knockout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones (8 kHz, 16 kHz, 32 kHz), and the rise was over 20 dB at high-frequency(32 kHz). Further analyses of waveforms showed that wave-I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone (16 kHz) were less than control group(P < 0.05) on average, but the ratio of wave I/II and I/III were not difference (P > 0.05).

CONCLUSIONSMlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high-frequencies.

Animals ; Audiometry, Pure-Tone ; Auditory Threshold ; Evoked Potentials, Auditory, Brain Stem ; Hair Cells, Auditory, Inner ; metabolism ; Hearing Loss ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Myosin-Light-Chain Kinase ; genetics