Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via K(Ca) 1.1 upregulation.

Aging Cell (2015) 14, pp982–994 Doi: 10.1111/acel.12388 Altering sphingolipid composition with aging induces contractile dysfunction of gastric smooth muscle via KCa1.1 upregulation Aging Cell Shinkyu Choi,1 Ji Aee Kim,1 Tae Hun Kim,3 Hai-yan Li,1 Kyong-Oh Shin,4 Yong-Moon Lee,4 Seikwan Oh,2 Yael Pewzner-Jung,5 Anthony H. Futerman5 and Suk Hyo Suh1 Abbreviations [Ca2+]i intracellular Ca2+ concentration Departments of 1Physiology, 2Molecular Medicine, 3Internal Medicine, Medical School, Ewha Womans University, Seoul, Korea 4 College of Pharmacy and MRC, Chungbuk National University, Chongju, Korea 5 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel CerS ceramide synthase(s) IBTx iberiotoxin ICC interstitial cells of the Cajal KCa1.1 large conductance Ca2+-activated K+ channels Summary MLC myosin light chain KCa1.1 regulates smooth muscle contractility by modulating membrane potential, and age-associated changes in KCa1.1 expression may contribute to the development of motility disorders of the gastrointestinal tract. Sphingolipids (SLs) are important structural components of cellular membranes whose altered composition may affect KCa1.1 expression. Thus, in this study, we examined whether altered SL composition due to aging may affect the contractility of gastric smooth muscle (GSM). We studied changes in ceramide synthases (CerS) and SL levels in the GSM of mice of varying ages and compared them with those in young CerS2-null mice. The levels of C16- and C18-ceramides, sphinganine, sphingosine, and sphingosine 1-phosphate were increased, and levels of C22, C24:1 and C24 ceramides were decreased in the GSM of both aged wild-type and young CerS2null mice. The altered SL composition upregulated KCa1.1 and increased KCa1.1 currents, while no change was observed in KCa1.1 channel activity. The upregulation of KCa1.1 impaired intracellular Ca2+ mobilization and decreased phosphorylated myosin light chain levels, causing GSM contractile dysfunction. Additionally, phosphoinositide 3-kinase, protein kinase Cf, c-Jun N-terminal kinases, and nuclear factor kappa-B were found to be involved in KCa1.1 upregulation. Our findings suggest that age-associated changes in SL composition or CerS2 ablation upregulate KCa1.1 via the phosphoinositide 3-kinase/protein kinase Cf/c-Jun N-terminal kinases/nuclear factor kappa-B-mediated pathway and impair Ca2+ mobilization, which thereby induces the contractile dysfunction of GSM. CerS2-null mice exhibited similar effects to aged wild-type mice; therefore, CerS2null mouse models may be utilized for investigating the pathogenesis of aging-associated motility disorders. NF-jB nuclear factor kappa-B NPo the open probability times the number of channels in the patch p21CIP cyclin-dependent kinase inhibitor p21 PGF2a prostaglandin F2a p-JNK phosphorylated JNK PKI PKCf pseudosubstrate inhibitor p-MLC phosphorylated MLC p-p85 phosphorylated p85 p-PKCf phosphorylated PKCf S1P sphingosine 1-phosphate SLs sphingolipids SMCs smooth muscle cells VOCCs voltage-operated Ca2+ channels Key words: aging; Ca2+-activated K+ channel; ceramide synthases; contractile dysfunction; smooth muscle; sphingolipids. Correspondence Suk Hyo Suh, Department of Physiology, Medical School, Ewha Womans University, 911-1 Mok-5-dong, Yang Chun-gu, Seoul 158-710, Korea. Tel.: +82 2 2650 5722; fax: +82 2 2653 7891; e-mail: Accepted for publication 19 July 2015 982 Introduction Aging causes structural changes in the gastrointestinal tract, which cause a significant decline in gastrointestinal function and increase the prevalence of several gastrointestinal motor disorders such as dyspepsia and constipation. Both a decrease in the number and volume of interstitial cells of Cajal (ICC; Gomez-Pinilla et al., 2011) and the degeneration of the enteric nervous system of the gastrointestinal tract, such as the myenteric plexus, occur in an age-related manner (ElSalhy et al., 1999; Phillips & Powley, 2001). As the ICC and the enteric nervous system play key roles in the control of gastrointestinal motility, the decrease in ICC and the degeneration of the enteric nervous system may contribute to changes in gastrointestinal motility with aging; however, little is known about the effect of aging on smooth muscle. Gastric smooth muscle (GSM) generates regular contractions, which are triggered by long-lasting waves of depolarization, or slow waves. The ICC generate slow waves (Hirst & Edwards, 2006) and thereby depolarize nearby smooth muscle cells (SMCs). The depolarization activates ª 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Age-associated dysfunction of smooth muscle, S. Choi et al. 983 voltage-operated Ca2+ channels (VOCCs), and Ca2+ entry through VOCCs increases intracellular Ca2+ concentration ([Ca2+]i), which induces smooth muscle contraction. Intracellular Ca2+ then activates KCa1.1, which causes the hyperpolarization of SMCs. The hyperpolarization inactivates the VOCCs and thereby induces smooth muscle relaxation by inhibiting Ca2+ entry through the VOCCs. Thus, VOCCs and KCa1.1 act as important regulators of gastrointestinal motility by modulating membrane potential and Ca2+ influx, and changes in these ion channels greatly affect the contractility of smooth muscle. As the level of KCa1.1 is increased with advancing age (Tricarico et al., 1997; Oshiro et al., 2005), KCa1.1 upregulation might contribute to the development of age-associated gastrointestinal motility disorder. Many different types of ion channels localize to cholesterol and sphingolipid (SL)-enriched regions of the plasma membrane, known as lipid microdomains or rafts (Dart, 2010). As SLs are important structural components of cellular membranes, they might affect ion channel gating and expression on the cell surface. The short-acyl chain ceramide analog, C6-ceramide, has been reported to modulate HERG K+ channel gating by causing its translocation into caveolin-enriched lipid rafts (Ganapathi et al., 2010) and to induce the channel downregulation via ubiquitin-mediated lysosomal degradation (Chapman et al., 2005). C6-Ceramide also inhibited voltage-gated K+ currents and induced vasoconstriction in rat and human pulmonary arteries (Moral-Sanz et al., 2011). Ceramide, the backbone of all complex SLs, is synthesized by the N-acylation of a sphingoid longchain base by a family of six ceramide synthases (CerS; Levy & Futerman, 2010), each generating ceramides with a different acyl chain length. C18-ceramide is generated by CerS1 and CerS4, and C16ceramide is generated by CerS5 and CerS6. CerS2 generates very long acyl chain (C22 and C24) ceramides. Ceramides can also be formed by the degradatio (...truncated)