Dimethyl sulphoxide relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of the contractile apparatus.

BACKGROUND AND PURPOSE: The intravesical administration of dimethyl sulphoxide (DMSO) is used to alleviate the symptoms of interstitial cystitis. We investigated the relaxant effect of DMSO and its underlying mechanism in the detrusor muscle. EXPERIMENTAL APPROACH: The effects of DMSO on contraction, on Ca2+ sensitivity of myofilaments, and on myosin light chain (MLC) phosphorylation were investigated in both intact and alpha-toxin-permeabilized strips of rabbit detrusor muscle. KEY RESULTS: In fura-PE3-loaded strips, DMSO (>1%) induced a significant relaxation during sustained contractions induced by 60 mM K+-depolarization or 10 microM carbachol, while having no effect on the [Ca2+](i) level. DMSO decreased the level of MLC phosphorylation during the contractions induced by 60 mM K+ and 10 microM carbachol. DMSO also inhibited both the contraction and MLC phosphorylation induced by calyculin-A in intact strips. In the alpha-toxin-permeabilized preparations, DMSO relaxed the Ca2+-induced contraction and also inhibited the tension development induced by a stepwise increment of Ca2+ concentrations. Such a relaxant effect of DMSO was enhanced in the presence of phosphate. CONCLUSIONS AND IMPLICATIONS: DMSO relaxes rabbit detrusor muscle by decreasing the Ca2+ sensitivity of myofilaments. Inhibition of the kinase activities involved in myosin phosphorylation may play a major role in DMSO-induced Ca2+ desensitization. Inhibition of the cross-bridge cycling at the step of phosphate release may also contribute to the relaxant effect of DMSO. Such relaxant effects of DMSO could be linked to the therapeutic effect of DMSO in interstitial cystitis.

Peri-prosthetic tuberculous infection of the hip in a patient with no previous history of tuberculosis.

We report a case of peri-prosthetic tuberculous infection nine years after total hip arthroplasty in a patient with no history of tuberculosis before the procedure. Further investigation revealed active pulmonary tuberculosis which was thought to have spread haematogeneously to the arthroplasty. The infection did not respond to standard antituberculous drugs. Removal of the prosthesis and insertion of an antibiotic spacer were required.

Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation.

The inhibition of myosin phosphatase evokes smooth muscle contraction in the absence of Ca(2+), yet the underlying mechanisms are not understood. To this end, we have cloned smooth muscle zipper-interacting protein (ZIP) kinase cDNA. ZIP kinase is present in various smooth muscle tissues including arteries. Triton X-100 skinning did not diminish ZIP kinase content, suggesting that ZIP kinase associates with the filamentous component in smooth muscle. Smooth muscle ZIP kinase phosphorylated smooth muscle myosin as well as the isolated 20-kDa myosin light chain in a Ca(2+)/calmodulin-independent manner. ZIP kinase phosphorylated myosin light chain at both Ser(19) and Thr(18) residues with the same rate constant. The actin-activated ATPase activity of myosin increased significantly following ZIP kinase-induced phosphorylation. Introduction of ZIP kinase into Triton X-100-permeabilized rabbit mesenteric artery provoked a Ca(2+)-free contraction. A protein phosphatase inhibitor, microcystin LR, also induced contraction in the absence of Ca(2+), which was accompanied by an increase in both mono- and diphosphorylation of myosin light chain. The observed sensitivity of the microcystin-induced contraction to various protein kinase inhibitors was identical to the sensitivity of isolated ZIP kinase to these inhibitors. These results suggest that ZIP kinase is responsible for Ca(2+) independent myosin phosphorylation and contraction in smooth muscle.

Mechanisms underlying the neurokinin A-induced contraction of the pregnant rat myometrium.

1. Using fura-PE3 fluorimetry and alpha-toxin permeabilization, the characteristics of the contractile responses to neurokinin A (NKA) were determined in the pregnant rat myometrium. 2. NKA induced contractions in rat myometrium in a concentration-dependent manner. There were no significant differences in the maximum contractions and EC(50) values between the pregnant and non-pregnant myometrium, however, the contraction of only the former was greatly enhanced in the presence of phosphoramidon (PPAD), an endopeptidase inhibitor. 3. In the pregnant myometrium, NKA induced sustained increases in [Ca(2+)](i) and tension in normal physiological saline solution, while only small transient increases in [Ca(2+)](i) and tension were observed in Ca(2+)-free solution. 4. Both diltiazem (10 microM) and SK-F 96365 (10 microM) significantly inhibited the NKA-induced elevations of [Ca(2+)](i) and tension. The effects were additive when these drugs were used together. 5. NKA induced a significant leftward shift of the [Ca(2+)](i)-tension curve obtained by changing the external Ca(2+) (0 - 2.5 mM) during depolarization with high K(+) solution. This Ca(2+)-sensitizing effect by NKA was also observed in the alpha-toxin permeabilized myometrium. 5. These results indicated that in the pregnant rat myometrium: (1) the responsiveness to NKA increased, although it was masked by the increase in the endopeptidase activity; (2) NKA induced contractions of the myometrium by increasing both [Ca(2+)](i) and the myofilament Ca(2+) sensitivity and (3) The NKA-induced [Ca(2+)](i) elevation was partly due to the intracellular Ca(2+) release and mainly due to the Ca(2+) influx, which was thought to be through both voltage dependent calcium channels and non-specification channels.

Expression, subcellular localization, and cloning of the 130-kDa regulatory subunit of myosin phosphatase in porcine aortic endothelial cells.

In endothelial cells in situ and in primary culture, immunoblot analysis revealed an expression of the 130-kDa subunit of myosin phosphatase, similar to the myosin phosphatase targeting subunit (MYPT) of smooth muscle. Screening of an endothelial cell cDNA library yielded a clone encoding an NH2-terminal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. Two isoforms differing by a central insert of 56 residues were detected. In growing cells, MYPT1 was localized on stress fiber, but at confluence the localization pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. The membrane localization of MYPT1 suggested a target other than myosin and raised the possibility that MYPT1 may be involved in dephosphorylation of alternative substrate(s). These distinct mechanisms would also be dependent on the growth state of the endothelial cells, i.e., regulation of actin-myosin interactions in growing cells and an unknown function in cells at confluence.

First dorsal interosseous muscle of the foot and its innervation.

During dissection of the foot region, it is frequently found that some nerve branches run close to the tibial surface of the second metatarsal bone. To investigate the nerve branches, detailed dissection of the first dorsal interosseous muscle was performed in 10 Japanese adult feet with special reference to its innervation. In all specimens the muscle was clearly separated into lateral and medial parts. The branches ran between these parts and innervated the parts. The small dorsal region of the medial part of the muscle was also innervated by a branch of the deep peroneal nerve. Based on its innervation, the muscle appears to be composed of two elements from the flexores breves profundi and an element from the dorsal primordium. A possible schematic model of the origins of this muscle is proposed.

Mechanisms of galanin-induced contraction in the rat myometrium.

A neuropeptide, galanin, regulates the reproductive process and directly induces myometrial contraction. The aim of this study was to determine the mechanism of galanin-induced myometrial contraction. For this purpose, we simultaneously measured intracellular Ca2+ concentration ([Ca2+]i) and tension using fura-PE3-fluorometry and the rat longitudinal myometrium. The effect of galanin on the Ca2+ sensitivity of the contractile apparatus was examined in beta-escin permeabilized strips. The expression of galanin and the galanin receptors mRNAs in the rat myometrium were determined by reverse transcription polymerase chain reaction (RT-PCR). Galanin (10-300 nM) induced phasic contraction with or without oscillation in the pregnant rat myometrium in a concentration-dependent manner. The maximal response was obtained at 100 nM. There was no significant difference either in the maximal responses or EC50 values for galanin-induced myometrial contractions among myometriums from non-pregnant and pregnant (day 4, day 11, day 20, day 22) rats. In the day 20 and 22 pregnant myometriums, assigning the levels of [Ca2+]i and tension at 40 mM K+-depolarization to be 100%, galanin increased the [Ca2+]i and tension to 126.9+/-2.9% and 116.3+/-2.7%, respectively. Diltiazem (10 microM) inhibited the galanin-induced elevation of [Ca2+]i and tension to 71.9+/-2.4% and 16.2+/-0.7%, respectively. Ni2+, by itself, decreased the basal [Ca2+]i to -50.2+/-3.9% without affecting resting tension. After Ni2+ treatment, galanin-induced increases in [Ca2+]i and tension were -19.6+/-3.4% and 0.9+/-0.1%, respectively. In myometrium treated with diltiazem, no oscillation in [Ca2+]i and tension was observed. In Ca2+-free solution with 0.1 mM EGTA, galanin increased [Ca2+]i from -40.2+/-2.7% to -18.0+/-2.6% and induced transient contraction (3.6+/-0.8%). In beta-escin permeabilized myometrium, galanin enhanced the contraction induced by 0.3 microM Ca2+ in the presence of GTP. In the presence of GDPbetaS (1 mM) instead of GTP, galanin failed to increase the Ca2+ sensitivity of the contractile apparatus. RT-PCR revealed that galanin mRNA was hardly expressed in the non-pregnant rat myometrium and increased to reach a maximal level at mid pregnancy (day 11), but decreased to the same level as in the non-pregnant myometrium at term (day 22). Type 2 galanin receptor (GALR2) mRNA was found to be expressed in the rat myometrium whereas type 1 galanin receptor (GALR1) mRNA expression was not detected. In conclusion, galanin induces contraction of the rat myometrium by increasing [Ca2+]i as well as by increasing Ca2+ sensitivity of the contractile apparatus. Galanin-induced increases in [Ca2+]i are caused by both intracellular Ca2+ release and Ca2+ influx from extracellular space. The responsiveness of the rat myometrium to galanin does not change during pregnancy. The galanin mRNA is expressed in the rat myometrium and it is upregulated during mid-pregnancy. Rat myometrium expresses GALR2 but not GALR1 mRNA. By changing mRNA expression in the myometrium during pregnancy, galanin may act as a paracrine or autocrine mediator in the regulation of myometrial contractility.

Expression of beta3-adrenoceptors in rat detrusor smooth muscle.

PURPOSE: To investigate the expression of beta-adrenoceptor (AR) subtypes responsible for detrusor smooth muscle relaxation. MATERIALS AND METHODS: Isolated rat detrusor smooth muscle was examined by tension measurement and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Norepinephrine (NE), epinephrine (EP) and isoproterenol (ISO) were found to relax the detrusor muscle pre-contracted by 6 x 10(-7) M carbachol in the presence of 10(-6) M phentolamine. NE relaxed the detrusor muscle as potently as EP. This potency order (NE=EP) thus indicated the beta-ARs of the rat detrusor muscle to possibly be a beta1 subtype. However, in the presence of 10(-6) M propranolol, beta1- and beta2- but not beta3-AR antagonist, NE showed a more potent relaxation than EP. This observation indicated that the rat detrusor muscle also possesses beta3-AR. RT-PCR revealed all three subtypes of beta-AR mRNA, namely beta1-, beta2- and beta3-AR mRNA, to be expressed in rat detrusor smooth muscle cells. CONCLUSION: We concluded that beta3-ARs exist in rat detrusor smooth muscle based on both pharmacological and molecular biological studies. Based on these findings, beta-ARs of rat detrusor smooth muscle are considered to be mixed populations consisting of three subtypes which play an important role in relaxing smooth muscle in response to catecholamines.

Up-regulation of rho A and rho-kinase mRNAs in the rat myometrium during pregnancy.

It has recently been suggested that rho A and rho kinase (ROK) play a role in the increase in the Ca2+ sensitivity of the smooth muscle myofilaments. In the present study, we investigated the mRNA expressions of rho A and two types of ROK (alpha and beta) in the myometrium obtained from both non-pregnant and pregnant rats. Reverse transcription polymerase chain reaction (RT-PCR) experiments using total RNA from these tissue specimens and the specific primers revealed rho A and both types of ROK mRNAs to be expressed in the rat myometrium. The mRNA expressions of rho A, ROK alpha and ROK beta in the pregnant myometrium were found to increase in comparison to those in the non-pregnant myometrium. These results thus support the idea that the up-regulation of these proteins might be involved in the mechanism underlying the increased contractility of the pregnant myometrium.