Phosolamban and CaM Kinase II in Stomach Smooth Muscle Plasticity

Project 2: COBRE Smooth Muscle

Led by Brian Perrino, Ph.D.
Mentors: Kenton M. Sanders and Sean Ward, Ph.D.


We have found that phospholamban modulates nitric oxide-induced sarcoplasmic reticulum (SR) Ca2+ release and CaM kinase II activity in gastric fundus and antrum smooth muscles [1;2]. In the proposed studies we intend to elucidate the role of SR Ca2+ release, CaM kinase II activation, and phospholamban phosphoryl-ation in regulating the excitability of antrum smooth muscles. We will also investigate how these mechanisms participate in the down-regulation of excitability in the antrum in diabetic gastroparesis [3]. Since SR intra-cellular Ca2+ signaling plays a key role in gastrointestinal (GI) smooth muscle contractility, alterations in SR Ca2+ handling proteins may be involved in the dystrophy of diabetic antrum smooth muscle. Thus, we will characterize changes in expression of SR Ca2+ handling proteins and contractile proteins during the develop-ment of diabetes. We hypothesize that changes in SR Ca2+ handling and excitability may be important early events in the pathogenic remodeling of diabetic antrum smooth muscle cells. The Specific Aims of the Project are: 1. Test the hypothesis that regulation of intracellular Ca2+ waves by phospholamban and CaM kinase II modulates the excitability of gastric antrum smooth muscles. The contractile activities of antrum smooth muscle strips from wild-type CD-1 mice and phospholamban-/- mice will be compared. CaM kinase II activities from wild-type and phospholamban-/- antrum smooth muscle cells will be determined. Ca2+ transients, membrane potentials, STOCS, A-type current, and intracellular Ca2+ waves will be compared using sharp electrode recordings, perforated patch whole cell recordings, and fluorescence microscopy. 2. Test the hypothesis that SR-targeted CaM kinase II phosphorylates phospholamban and modulates intracellular Ca2+ wave frequency in wild-type gastric antrum smooth muscles. CaM kinase II will be measured by Western blot and kinase assays of cytosolic and SR fractions from caffeine-treated antrum smooth muscles. Phospholamban phosphorylation will be measured by Western blot analysis using phospho-lamban-PO4-Thr17 (P-PLBThr17) antibodies. The role of phospholamban phosphorylation by SR-associated CaM kinase II in modulating intracellular Ca2+ wave frequency will be determined by comparing the Ca2+ wave frequencies before and after CaM kinase II autonomous activity, SR-association, and phospholamban phos-phorylation levels are elevated. The role of autophosphorylation in the mechanism of CaM kinase II association with the SR will be investigated by alkaline phosphatase treatment of the cytosolic fraction and Western blot analysis of the SR fraction with anti-CaM kinase II and anti- PO4 -Thr287 (P-Thr287) antibodies. 3a. Test the hypothesis that altered expression of contractile proteins, and myosin light chain phosphorylation due to loss of sensitivity to IGF-1/insulin is responsible for diabetic antral smooth muscle dystrophy. 3b. Test the hypothesis that altered phospholamban and SERCA expression, and phospholamban phosphorylation by CaM kinase II trigger the dystrophy of diabetic antrum smooth muscles by disrupting intracellular Ca2+ wave activity. Dystrophic antrum smooth muscles will be obtained from db/db mice, a model of Type 2 diabetes. Non-diabetic control, and diabetic db/db antral smooth muscles will be used to measure time-dependent changes in protein expression and phosphorylation levels of myosin light chain (MLC), MLCK, MLCP, CPI-17, MYPT-1 by Western blot analysis. Time-dependent changes in the phasic contractile activity of diabetic antral smooth muscles will be measured compared to controls. Phospholamban, phospholamban phosphorylation, SERCA, and CaM kinase II expression levels will be monitored by Western blot analysis of control and diabetic smooth muscle cells. Cytosolic and SR-associated CaM kinase II will be measured by Western blot analysis and kinase assays. Intracellular Ca2+ waves in control and diabetic antral smooth muscle cells will be analyzed using fluorescent Ca2+ indicators.

  • Video 1: Control Ca2 + Waves
  • Video 2: Colitis Ca2 + Waves

Selected Publications:

  1. Bhetwal BP, Sanders KM, An C, Trappanese DM, Moreland RS, Perrino BA. Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus. J Physiol. 591.12:2971-86. 2013. [Epub ahead of print May 27] PMID:23613531.
  2. Bhetwal BP, An C, Baker SA, Lyon KL, Perrino BA. Impaired contractile responses and altered expression and phosphorylation of Ca2+ sensitization proteins in gastric antrum smooth muscles from ob/ob mice. J Muscle Res Cell Motil. 2013. 34(2):137-49. [Epub 2013 Apr 11]. PMID: 23576331; PMCID: PMC3651903.7
  3. Bhetwal BP, An CL, Fisher SA, Perrino BA. Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles. Neurogastro-enterol Motil. 2011. 23(10):e425-36. [Epub 2011 Aug 24]. PMID: 21883701; PMCID: PMC3173524.
  4. Perrino BA. Regulation of gastrointestinal motility by Ca2+/calmodulin-stimulated protein kinase II. Arch Biochem Biophys. 2011. 510(2):174-81. [Epub 2011 Apr 3]. Review. PMID: 21443856; PMCID: PMC3134147.
  5. Qureshi S, Song J, Lee HT, Koh SD, Hennig GW, Perrino BA. CaM kinase II in colonic smooth muscle contributes to dysmotility in murine DSS-colitis. Neurogastroenterol Motil. 2010. 22(2):186-95, e64. [Epub 2009 Sep 7]. PMID: 19735476; PMCID: PMC2806503.
  6. Kim M, Hennig GW, Park K, Han IS, Smith TK, Koh SD, Perrino BA. Modulation of murine gastric antrum smooth muscle STOC activity and excitability by phospholamban. J Physiol. 2008. 586 (Pt20): 4977-91. [Epub 2008 Aug 28]. PMID: 18755751; PMCID: PMC2614062.
  7. Chen J, Chen H, Sanders KM, Perrino BA. Regulation of SRF/CArG-dependent gene transcription during chronic partial obstruction of murine small intestine. Neurogastroenterol Motil. 2008. 20(7):829-42. PMID: 18557893. Editorial Viewpoint: MacDonald JA. Smooth muscle phenotypic plasticity in mechanical obstruction of the small intestine. Neurogastroenterol Motil. 2008. 20(7): 737-40.
  8. Kim M, Hennig GW, Smith TK, Perrino BA. Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum. Am J Physiol Cell Physiol. 2008. 294(2):C432-41. [Epub 2007 Nov 28]. PMID: 18045856.
  9. Kim M, Perrino BA. CaM kinase II activation and phospholamban phosphorylation by SNP in murine gastric antrum smooth muscles. Am J Physiol Gastrointest Liver Physiol. 2007. 292(4):G1045-54. [Epub 2006 Dec 21]. PMID: 17185633.
  10.  Kim M, Han IS, Koh SD, Perrino BA. Roles of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscles. Am J Physiol Cell Physiol. 2006. 291(2):C337-47. [Epub 2006 Mar 1]. PMID: 16510846.
  11. Lorenz JM, Riddervold MH, Beckett EA, Baker SA, Perrino BA. Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues. Am J Physiol Cell Physiol. 2002. 283(5): C1399-413. PMID: 12372801.