Changes In Smooth Muscle Function Research Articles

Remodeling of murine vaginal smooth muscle function with reproductive age and elastic fiber disruption

Advanced maternal age during pregnancy is associated with increased risk of vaginal tearing during delivery and maladaptive postpartum healing. Although the underlying mechanisms of age-related vaginal injuries are not fully elucidated, changes in vaginal microstructure may contribute. Smooth muscle cells promote the contractile nature of the vagina and contribute to pelvic floor stability. While menopause is associated with decreased vaginal smooth muscle content, whether contractile changes occur before the onset of menopause remains unknown. Therefore, the first objective of this study was to quantify the active mechanical behavior of the murine vagina with age. Further, aging is associated with decreased vaginal elastin content. As such, the second objective was to determine if elastic fiber disruption alters vaginal contractility. Vaginal samples from mice aged 2–14 months were used in maximum contractility experiments and biaxial extension-inflation protocols. To evaluate the role of elastic fibers with age, half of the vaginal samples were randomly allocated to enzymatic elastic fiber disruption. Contractile potential decreased and vaginal material stiffness increased with age. These age-related changes in smooth muscle function may be due, in part, to changes in microstructural composition or contractile gene expression. Furthermore, elastic fiber disruption had a diminished effect on smooth muscle contractility in older mice. This suggests a decreased functional role of elastic fibers with age. Quantifying the age-dependent mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties provides a first step towards better understanding how age-related changes in vaginal structure may contribute to tissue integrity and healing. Statement of significanceAdvanced maternal age at the time of pregnancy is linked to increased risks of vaginal tearing during delivery, postpartum hemorrhaging, and the development of pelvic floor disorders. While the underlying causes of increased vaginal injuries with age and associated pathologies remain unclear, changes in vaginal microstructure, such as elastic fibers and smooth muscle cells, may contribute. Menopause is associated with fragmented elastic fibers and decreased smooth muscle content; however, how reproductive aging affects changes in the vaginal composition and the mechanical properties remains unknown. Quantifying the mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties with age will advance understanding of the potential structural causes of age-related changes to tissue integrity and healing.

Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor.

BackgroundMyosin light-chain phosphatase is a trimeric protein that hydrolyses phosphorylated myosin II light chains (MYLII) to cause relaxation in smooth muscle cells including those of the uterus. A major component of the phosphatase is the myosin targeting subunit (MYPT), which directs a catalytic subunit to dephosphorylate MYLII. There are 5 main MYPT family members (MYPT1 (PPP1R12A), MYPT2 (PPP1R12B), MYPT3 (PPP1R16A), myosin binding subunit 85 MBS85 (PPP1R12C) and TIMAP (TGF-beta-inhibited membrane-associated protein (PPP1R16B)). Nitric oxide (NO)-mediated smooth muscle relaxation has in part been attributed to activation of the phosphatase by PKG binding to a leucine zipper (LZ) dimerization domain located at the carboxyl-terminus of PPP1R12A. In animal studies, alternative splicing of PPP1R12A can lead to the inclusion of a 31-nucleotide exonic segment that generates a LZ negative (LZ-) isovariant rendering the phosphatase less sensitive to NO vasodilators and alterations in PPP1R12ALZ- and LZ+ expression have been linked to phenotypic changes in smooth muscle function. Moreover, PPP1R12B and PPP1R12C, but not PPP1R16A or PPP1R16B, have the potential for LZ+/LZ- alternative splicing. Yet, by comparison to animal studies, the information on human MYPT genomic sequences/mRNA expressions is scant. As uterine smooth muscle undergoes substantial remodeling during pregnancy we were interested in establishing the patterns of expression of human MYPT isovariants during this process and also following labor onset as this could have important implications for determining successful pregnancy outcome.ObjectivesWe used cross-species genome alignment, to infer putative human sequences not available in the public domain, and isovariant-specific quantitative PCR, to analyse the expression of mRNA encoding putative LZ+ and LZ- forms of PPP1R12A, PPP1R12B and PPP1R12C as well as canonical PPP1R16A and PPP1R16B genes in human uterine smooth muscle from non-pregnant, pregnant and in-labor donors.ResultsWe found a reduction in the expression of PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA in late pregnancy (not-in-labor) relative to non-pregnancy. PPP1R12ALZ+ and PPP1R12ALZ- mRNA levels were similar in the non-pregnant and pregnant not in labor groups. There was a further reduction in the uterine expression of PPP1R12ALZ+, PPP1R12CLZ+ and PPP1R12ALZ- mRNA with labor relative to the pregnant not-in-labor group. PPP1R12A, PPP1R12BLZ+, PPP1R16A and PPP1R16B mRNA levels were invariant between the not in labor and in-labor groups.ConclusionsMYPT proteins are crucial determinants of smooth muscle function. Therefore, these alterations in human uterine smooth muscle MYPT isovariant expression during pregnancy and labor may be part of the important molecular physiological transition between uterine quiescence and activation.

Interleukin-13 Receptor α1-Dependent Responses in the Intestine Are Critical to Parasite Clearance.

Nematode infection upregulates interleukin-4 (IL-4) and IL-13 and induces STAT6-dependent changes in gut function that promote worm clearance. IL-4 and IL-13 activate the type 2 IL-4 receptor (IL-4R), which contains the IL-13Rα1 and IL-4Rα chains. We used mice deficient in IL-13Rα1 (IL-13Rα1(-/-)) to examine the contribution of IL-13 acting at the type 2 IL-4R to immune and functional responses to primary (Hb1) and secondary (Hb2) infections with the gastrointestinal nematode parasite Heligmosomoides bakeri There were differences between strains in the IL-4 and IL-13 expression responses to Hb1 but not Hb2 infection. Following Hb2 infection, deficient mice had impaired worm expulsion and higher worm fecundity despite normal production of Th2-derived cytokines. The upregulation of IL-25 and IL-13Rα2 in Hb1- and Hb2-infected wild-type (WT) mice was absent in IL-13Rα1(-/-)mice. Goblet cell numbers and resistin-like molecule beta (RELM-β) expression were attenuated significantly in IL-13Rα1(-/-)mice following Hb2 infections. IL-13Rα1 contributes to the development of alternatively activated macrophages, but the type 1 IL-4R is also important. Hb1 infection had no effects on smooth muscle function or epithelial permeability in either strain, while the enhanced mucosal permeability and changes in smooth muscle function and morphology observed in response to Hb2 infection in WT mice were absent in IL-13Rα1(-/-)mice. Notably, the contribution of claudin-2, which has been linked to IL-13, does not mediate the increased mucosal permeability following Hb2 infection. These results show that activation of IL-13Rα1 is critical for key aspects of the immune and functional responses to Hb2 infection that facilitate expulsion.

Differences in longitudinal smooth muscle response along the length of the rabbit small intestine

To determine whether there is a gradient in the ability of longitudinal smooth muscle along the length of the small intestine to develop tension we prepared longitudinal muscle strips from proximal (P), medial (M) and distal (D) segments of small intestine removed from 900–1200 g New Zealand white rabbits (n = 9). Isometric tension was recorded in standard tissue baths perfused with oxygenated Krebs solution and 10−6m tetrodotoxin. Basal and active (the response to 10−5m carbachol) length-tension curves were generated, and the length at which tissues developed maximal active tension (the optimum length or Lo) was determined for P, M and D tissues. Then the dose-response to carbachol (over the range 10−2-10−4m) was determined for each of the P, M and D tissues at Lo. Non-linear regression was used to determine the dose at which the contractile response was half maximal (the ED so), and the maximum tension generated (Tm) by the tissue at each location. The basal tension generated in response to graded stretch increased exponentially between 100 and 140% of initial length and was not significantly different for P, M and D tissues. The active tension generated in response to 10−5m carbachol peaked at 125% of initial length; the Lo for P, M and D tissues was identical. With tissues equilibrated at Lo, dose-response curves for carbachol and graded concentrations of KCl showed a significant increment in maximal tension generated, but no significant difference in ED50 in the more distal compared to proximal segments. Thus, the passive elastic properties and Lo were identical for the three regions of small intestine examined. However, there was a significant increase in active tension generated in distal compared to more proximal regions of the small intestine, which was not associated with a change in the ED50 value for carbachol. As the response occurs in the presence of tetrodotoxin and can be reproduced with KCl depolarization, it probably reflects post-receptor changes in smooth muscle function.

Mast Cell-Mediated Changes in Smooth Muscle Contractility during Mouse Giardiasis

Giardia intestinalis is a significant cause of diarrheal disease worldwide. Infections in animal models have been shown to cause changes in gastrointestinal transit that depend on adaptive immune responses and are mediated, in part, through neuronal nitric oxide synthase. Nitric oxide is an inhibitory neurotransmitter, and we therefore investigated potential excitatory pathways that might be involved in the response to Giardia infection. Infected mice exhibited increased spontaneous and cholecystokinin (CCK)-induced contractions of longitudinal smooth muscle. In contrast, enhanced contractile responses were not observed in response to acetylcholine, 5-hydroxytryptamine, or the protease-activated receptor-1 agonist peptide TFFLR. Giardia-induced changes in smooth muscle function appear to be mediated primarily by mast cells, as both spontaneous and CCK-induced contractions were blocked by pretreatment with either ketotifen or compound 48/80. Together, these data support a model in which CCK release triggers mast cell degranulation, leading to increases in smooth muscle contractility. These contractions, coupled with nitric oxide-mediated muscle relaxation, promote intestinal transit and parasite elimination.

Contribution of 5-HT 2A Receptor in Nematode Infection-Induced Murine Intestinal Smooth Muscle Hypercontractility

Enteric nematode infection induces a smooth muscle hypercontractility that depends on interleukin (IL)-4 and IL-13 activation of the signal transducer and activator of transcription (STAT) 6. Serotonin (5-HT) is involved in the physiologic regulation of gut function. The present study investigated the contribution of 5-HT and its receptors in nematode-induced intestinal smooth muscle hypercontractility. Mice were infected with Nippostrongylus brasiliensis (N brasiliensis) or Heligmosomoides polygyrus (H polygyrus) or injected intravenously with IL-13. Segments of jejunum were suspended in organ baths, and smooth muscle responses to 5-HT were determined in the presence or absence of specific 5-HT antagonists. IL-4, IL-13, and 5-HT receptor messenger RNA expressions were determined by real-time quantitative polymerase chain reaction. 5-HT evoked a modest contraction of smooth muscle in wild-type (WT) mice that was unaltered by the 5-HT2A antagonist ketanserin. N brasiliensis infection induced a smooth muscle hypercontractility to 5-HT that was abolished by 5-HT(2A) antagonists but not by other 5-HT antagonists. Infection-induced up-regulation of 5-HT2A expression was correlated with the smooth muscle hypercontractility to 5-HT. The infection-induced up-regulation of 5-HT2A in WT mice was observed also in IL-4(-/-) mice but was not seen in IL-13(-/-) or STAT6(-/-) mice. In addition, smooth muscle responses to 5-HT and 5-HT2A expression in WT mice were also enhanced by IL-13 or H polygyrus infection. These data show that 5-HT2A is one of the molecules downstream from STAT6 activation that mediates changes in smooth muscle function. 5-HT2A represents a novel therapeutic target for modulating immune-mediated effects on intestinal motility.

Bladder Overactivity in Mice After 1 Week of Outlet Obstruction. Mainly Afferent Dysfunction?

Partial bladder outlet obstruction (BOO) is an established way to create bladder overactivity in animals. However, the micturition pattern achieved can vary considerably. We investigated the correlation between different micturition patterns, post-void residual urine, bladder capacity, bladder weight and in vitro contractility in a conscious mouse model of BOO. Female MNRI mice with moderate (urethral) outlet obstruction were used. After 7 days of BOO cystometry without anesthesia was performed with the animal in a metabolic cage. Results were compared with those in unoperated controls and sham operated animals. In vitro contractility studies were performed. The majority of obstructed animals had an overactive voiding pattern with increased nonvoiding detrusor activity and without increased bladder weight. The remaining obstructed animals had significantly increased bladder weight and normal micturition volume but increased threshold pressure, bladder capacity and post-void residual urine. Subtle in vitro differences were found among the groups. The urodynamic characteristics of BOO mice correlate with bladder weight. However, detrusor overactivity in obstructed mice may develop without an increase in bladder weight and with only subtle changes in smooth muscle function, as investigated in vitro. This observation suggests that major disturbances caused by BOO may lie in the afferent arm of the signaling pathway.

Perimenstrual asthma: A syndrome without known cause or cure

Perimenstrual worsening of asthma has been documented in 30% to 40% of asthmatic women. This increase in symptoms has been backed up by increased health care use perimenstrually, as well as by cyclic variation in peak expiratory flows. The cause of perimenstrual asthma (PMA) remains unclear. Fluctuations in hormone levels, their ratios, or both are a plausible explanation but have not been demonstrated with any consistency. Influences of sex hormones on inflammation is an area of future research, as are hormone-induced changes in smooth muscle function and β-adrenergic receptors, prostaglandin levels, and fluid retention in the bronchial mucosa. In the light of the high prevalence of PMA, it is difficult to understand why there has been no randomized controlled trial of hormone therapy. Nevertheless, several case reports have suggested beneficial effects of estrogens, pro-gestins, and their combination. In light of these positive case reports, well-designed, double-blind studies of sufficient sample size should now be performed to give treatment of PMA an evidence base. (J Allergy Clin Immunol 2003;112:271-82.)

Beta-adrenergic relaxation of smooth muscle: differences between cells and tissues

The present studies were carried out in an attempt to resolve the controversy about the Na+ dependence of beta-adrenergic relaxation in smooth muscle. Previous studies on isolated smooth muscle cells from the toad stomach had suggested that at least some of the actions of beta-adrenergic agents, including a stimulatory effect on 45Ca efflux, were dependent on the presence of a normal transmembrane Na+ gradient. Studies by other investigators using tissues derived from mammalian sources had suggested that the relaxing effect of beta-adrenergic agents was Na+ independent. Uncertainty remained as to whether these discrepancies reflected differences between cells and tissues or differences between species. Thus, in the present studies, we utilized both tissues and cells from the same source, the stomach muscle of the toad Bufo marinus, and assessed the Na+ dependence of beta-adrenergic relaxation. We found that elimination of a normal Na+ gradient abolished beta-adrenergic relaxation of isolated cells. In tissues, however, similar manipulations had no effect on relaxation. The reasons for this discrepancy are unclear but do not appear to be attributable to changes in smooth muscle function following enzymatic dispersion. Rather they may be attributable to unique properties of tissues, such as the presence of pacemaker cells, nerves, and so on. Thus the controversy concerning the mechanism of beta-adrenergic relaxation may reflect inherent differences between tissues and cells.