A 35-70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from Phosphorylation of a light chain of myosin has been shown to occur in myosins isolated from both muscle and cytoplasmic sources (1, 2). In each case, covalent phosphorylation occurs on a specific light chain of myosin in the size range of 18,000 to 20,000 daltons-e.g., white skeletal muscle, 18,000; red skeletal muscle, 20,000; cardiac muscle, 20,000; smooth muscle, 20,000; and platelet, 20,000 (1-3).Phosphorylation is catalyzed by a specific enzyme, myosin light chain kinase, which differs in the cases of cytoplasmic and muscle myosin in that the former enzyme is independent of Ca2+ for activity whereas the latter enzyme requires Ca2+ (1, 4, 5). Dephosphorylation of the myosin light chain is catalyzed by an exogenous (6) or endogenous phosphatase (7, 28).Phosphorylation of platelet myosin has been shown to control the interaction between platelet actin and myosin; phosphorylated myosin is activated by actin to a greater extent (5-to 7-fold) than is nonphosphorylated myosin (6). Dephosphorylation of platelet myosin results in a decrease in the actinactivated ATPase activity (6). Although these experiments indicated a role for phosphorylation in controlling actin-activation of myosin in nonmuscle cells, there was no evidence for such control in muscle.Recent preliminary reports from a number of laboratories have suggested that phosphorylation of smooth muscle myosin may play a role in the actin-activation and Ca2+ regulation of smooth muscle myosin ATPase activity (8)(9)(10). In this paper we report the isolation and purification of phosphorylated and METHODS All procedures were carried out at 40 unless noted as otherwise. Deionized water was used throughout. Preparation of smooth muscle actomyosin One-month-old male guinea pigs were anesthetized and their vasa deferentia were removed, collected in balanced salt solution (Hanks'), and carefully freed from connective tissue under a dissecting microscope. The cleaned specimens were pooled (usually 120 in number weighing approximately 5 g) and homogenized in 35 ml of the extraction buffer: 60 mM KCI, 40 mM imidazole-HCl (pH 7.1), 4 mM (ethylenedinitrilo)tetraacetic acid (EDTA), 10 mM ATP, and 10 mM dithiothreitol (DTT). A tissue homogenizer was used for a total of 2 min and the material being homogenized was chilled in ice. The homogenized material was sedimented at 48,000 X g for 20 min to yield a cloudy supernatant and a pellet. Immediately after addition of ATP and MgCl2 to 10 mM, the supernatant was fractionated into 0-35% and 35-70% fractions by addition of a saturated ammonium sulfat...

We demonstrate, using reverse transcriptase-polymerase chain reaction, that, whereas abdominal aorta from rabbit consists almost entirely of myosin heavy chain (MHC) mRNA with no insert at the 5'-terminal coding region, the distributing arteries (femoral and saphenous) begin to show MHC mRNA with the 21-nucleotide insert that encodes seven amino acids in the ATP-binding region located in the myosin head. The femoral/iliac artery contains > 50% inserted mRNA, whereas the more distal saphenous artery contains > 80% inserted mRNA. This insert is also present in the smooth muscle from rat tail artery but is absent in the smooth muscle from rat aorta. The actin-activated ATPase activity of myosin from the rabbit femoral/saphenous artery is 1.7-fold higher than that of the myosin from the aorta. A concomitant increase (about twofold) in the maximum shortening velocity of the saphenous artery, compared with that of the aorta, indicates that the preponderance of the inserted myosin is associated with both an increase in the actin-activated ATPase activity and a larger maximum velocity of shortening. Furthermore, analysis of the 17-kDa essential light chain from the aorta reveals near equal quantities of the 17-kDa light chain isoforms a and b, whereas the myosin from the femoral/ saphenous artery contains predominantly the 17-kDa light chain a isoform. Together, these data indicate that the smooth muscle cells from the small distributing arteries are similar to those of visceral smooth muscle with respect to the expression of myosin isoforms, actin-activated myosin ATPase activity and contractility.

The process of cervical ripening has been likened to an inflammatory reaction associated with the catabolism of cervical extracellular matrix by enzymes released from infiltrating leukocytes. We hypothesized that smooth muscle cells in the cervix also participate in this process and that pro-inflammatory cytokines act on cervical smooth muscle cells (CSMC) to provoke the expression of matrix-degrading enzymes. We treated primary cultures of human CSMC with tumor necrosis factor-␣ (TNF-␣) and examined expression of the elastinolytic enzyme , cathepsin S, the collagen metabolizing matrix metalloproteinases (MMP)-1 , -3 , -9 , and the tissue inhibitor of metalloproteinase (TIMP)-1 and -2. A time course analysis revealed that 10 ng/ml of TNF-␣ induced cathepsin S, MMP-1 , -3 , and -9 mRNA expression with the maximal response observed after 24 -48 hours. TNF-␣ induced cathepsin S , MMP-1 , -3 , and -9 mRNA expression in a dose-dependent manner: the maximal effect was observed at a concentration of 10 ng/ml , with appreciable increases observed at concentrations of 0.1 to 1.0 ng/ml. In contrast , TIMP-1 and -2 mRNAs were not significantly increased by TNF-␣ treatment. Interleukin-1␤ produced a pattern of gene expression in the CSMC similar to that observed following TNF-␣ treatment. Western blot analysis and zymography confirmed the induction of proMMP-1 , -3 , and -9 in response to TNF-␣, but MMP-2 immunoreactivity and zymographic activity were unaffected. TNF-␣ increased secretion of procathepsin S , but did not affect TIMP-1 and reduced TIMP-2 production. We conclude that CSMC are targets of pro-inflammatory cytokines, which induce a repertoire of enzymes capable of degrading the cervical extracellular matrix. The induction of these enzymes may facilitate the normal ripening of the cervix at term and participate in the The human cervix is composed primarily of connective tissue consisting mainly of fibrillar collagens, elastin, and glycosaminoglycans.

Clinical data provides evidence of high level of co-morbidity among genitourinary and gastrointestinal disorders characterized by chronic pelvic pain. The objective of this study was to test the hypothesis that colonic inflammation can impact the function of the urinary bladder via activation of TRPV1 signaling pathways followed by alterations in gene and protein expression of Substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory neurons and in the bladder. Inflammation was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS, 12.5 mg/kg) and desensitization of TRPV1 receptors was evoked by intracolonic resiniferatoxin (RTX, 10 −7 M). mRNA and protein concentrations of CGRP and SP were measured at 3, 5 and 30 days. RTX instillation in the colon caused 3-fold up-regulation of SP mRNA in the urinary bladder at day 5 (n=7, p≤0.05) followed by 35-fold increase at day 30 (n=5, p≤0.05). Likewise, TNBS colitis triggered 15.8-fold up-regulation of SP mRNA one month after TNBS (n=5, p≤0.05). Desensitization of colonic TRPV1 receptors prior to TNBS abolished SP increase in the urinary bladder. RTX led to 4.3-fold increase of CGRP mRNA at day 5 (n=7, p≤0.05 to control) in the bladder followed by 28-fold increase at day 30 post-RTX (n=4, p≤0.05). Colitis did not alter CGRP concentration during acute phase, however, at day 30 mRNA level was increased by 17.8±6.9 fold (n=5, p≤0.05) in parallel with 4-fold increase in CGRP protein (n=5, p≤0.01) in the detrusor. Protein concentration of CGRP in the spinal cord was diminished by 45-65% (p≤0.05) during colitis. RTX pretreatment did not affect CGRP concentration in the urinary bladder, however, caused a reduction in CGRP release from lumbosacral DRG neurons during acute phase (3 and 5 days post-TNBS). Our results clearly demonstrate that colonic inflammation triggers the release of pro-inflammatory neuropeptides SP and CGRP in the urinary bladder via activation of TRPV1 signaling mechanisms enunciating the neurogenic nature of pelvic organ cross-sensitization.