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Brian Perrino, Ph. D.Associate Professor IMPAIRED GASTRIC ACCOMODATION The inability of the stomach to properly expand and hold food (gastric accommodation) plays an important role in causing the symptoms of dyspepsia. Dyspepsia affects roughly one in four Americans, leading to nausea, bloating, and abdominal pain or discomfort after a meal. The overall goals of the studies being undertaken in my lab are to provide a more thorough understanding of how the stomach relaxes to accommodate food and aid in the design of improved therapeutic strategies to restore normal stomach function and relieve the symptoms of impaired gastric accommodation. We are doing this by carrying out a variety of experiments whose objectives are to define in more detail a novel pathway contributing to nitrergic relaxation in gastric fundus smooth muscles involving sarcoplasmic reticulum Ca2+ release and phospholamban phosphorylation by CaM kinase II. Impaired gastric accommodation is present in a high percentage of patients with functional dyspepsia, and restoration of accommodation is a desired therapeutic goal. A thorough investigation of the nitric oxide-activated signaling pathways that relax fundus smooth muscles will help to achieve this goal. To do this we are determining how phospholamban affects nitric oxide-induced relaxation and CaM kinase II activation in gastric fundus smooth muscles using phospholamban knockout mice. We are investigating the function and mechanism of CaM kinase II-sarcoplasmic reticulum association in gastric fundus smooth muscle relaxation. Finally, we are determining the role of interstitial cells of Cajal in nitric oxide-induced activation of CaM kinase II and phospholamban phosphorylation in gastric fundus smooth muscles. We employ several methodologies, including mechanical measurements of gastric fundus smooth muscles, SDS-PAGE and Western blotting of phosphorylated phospholamban, CaM kinase II assays in smooth muscle lysates and sarcoplasmic reticulum fractions, perforated-patch whole cell recordings of STOCs, sharp electrode recordings of membrane potential, and fluorescent Ca2+ indicator dyes to measure intracellular Ca2+ transients and Ca2+ waves in gastric fundus smooth muscle cells. ULCERATIVE COLITIS Ulcerative colitis is a debilitating disease affecting over a half million Americans for which there is no cure. Patients with chronic ulcerative colitis or Crohn’s disease exhibit reduced colonic contractility, reductions in basal colonic intraluminal pressure, and reductions in postprandial motility. In both human and animal models of colitis there is evidence that inflammatory mediators produced in the mucosa and submucosa reach the muscle layer and alter colonic muscle contractility. Current treatments for ulcerative colitis revolve around the use of anti-inflammatory drugs and immunosuppressants that cause severe side effects, and do not induce a long-term benefit in a significant percentage (40%-60%) of patients. Therefore, new therapies and approaches are needed that induce remission, alter the natural course of the disease, or relieve symptoms in ulcerative colitis patients. One approach to this end would be to understand the molecular changes occurring within the smooth muscle cells as a consequence of colonic inflammation. This approach may lead to improvements in therapies utilizing motility drugs for cases of ulcerative colitis. Regulation of intracellular Ca2+ signaling by sarcoplasmic reticulum Ca2+ cycling plays a key role in GI smooth muscle contractility. In cardiac and vascular smooth muscles, many of the signaling pathways involved in the induction of pathological remodeling are linked to SR Ca2+ cycling. Similarly, emerging evidence implicates dysregulation of intracellular Ca2+ cycling in the dysmotility of ulcerative colitis. These recent findings are providing important clues to the biochemical and molecular basis for the functional changes in colon smooth muscles that give rise to the physiological findings of colonic dysmotility of ulcerative colitis.
The overall goal of these studies being undertaken in my lab is to show that phospholamban phosphorylation by CaM kinase II regulates intracellular Ca2+ signaling in colon smooth muscle cells and that this novel pathway regulating myogenic excitability in the colon is disrupted by ulcerative colitis. To pursue this goal, we are investigating how altered phospholamban and SERCA expression, and phospholamban phosphorylation by CaM kinase II trigger the molecular remodeling that leads to the dysmotility of colon smooth muscles in ulcerative colitis. Experimental colitis is induced in wild-type C57BL/6 mice or phospholamban knockout mice with dextran sodium sulfate to analyze the intracellular and myogenic responses. We employ several techniques, including mechanical measurements of colon smooth muscles, SDS-PAGE and Western blotting, CaM kinase II assays, sharp electrode recordings of membrane potential, and fluorescent Ca2+ indicator dyes to measure intracellular Ca2+ waves in colon smooth muscle cells. These studies will lead to a better understanding of how intracellular Ca2+ handling via novel regulatory pathways is involved in colon smooth muscle excitability and how these pathways become disrupted in diseases such as ulcerative colitis. Data obtained from this proposal will aid in the design of improved therapeutic strategies to restore the normal coordinated contractile activity of the colon and relieve the symptoms associated with ulcerative colitis. Substrate Selectivity of Calcineurin Catalytic Isoforms An emerging question in Ca2+-dependent signaling concerns the physiological roles of the two catalytic subunits of the multifunctional Ca2+/calmodulin (CaM)-dependent Ser/Thr protein phosphatase calcineurin (CaN). The major goals of this project are to elucidate the mechanisms underlying the substrate selectivity, sub-cellular distribution, and NFAT-binding of the calcineurin a and b catalytic subunit isoforms in vascular smooth muscles. Selected Publications Kim, M., and Perrino, B.A. CaM kinase II activation and phospholamban phosphorylation by SNP in murine gastric antrum smooth muscles. Am J Physiol Gastrointest Liver Physiol. 292.g1045-54, 2007 Jabr, R.I., R.I., Wilson, A.J., Riddervold, M.H., Jenkins, A.H., Perrino, B.A., and Clapp. Nuclear translocation of calcineurin Ab but not Aa by platelet-derived growth factor in rat aortic smooth muscle. Am. J. Physiol. Cell Physiol. 292:C2213-25, 2007 Hicks, G., Perrino, B.A., Marrion, N.V. Inhibition of BKCa channel activity by association with calcineurin in rat brain. Eur. J. Neuroscience. 24:433-41, 2006 Gary-Gouy, H., Sainz-Perez, A., Bismuth, G., Ghadiri, A., Perrino, B.A., and Dalloul, A. Cyclosporin-A inhibits ERK phosphorylation in B cells by modulating the binding of Raf protein to Bcl2. BBRC 344:134-139, 2006 Kim, M., Han, I.S., Koh, S.D., and Perrino, B.A., Role of CaM kinase II and phospholamban in SNP-induced relaxation of murine gastric fundus smooth muscle. Am. J. Physiol. Cell Physiol. 291: C337-47, 2006 Kim, M., Cho, S.Y., Han, I.S., Koh, S.D., and Perrino, B.A., CaM kinase II and phospholamban contribute to caffeine-induced relaxation of murine gastric fundus smooth muscle. Am. J. Physiol. Cell Physiol. 288:C1202-C1210, 2005 Sergeant, G.P., Ohya, S., Rehill, J.R., Perrino, B.A., Koh, S.D., Sanders, K.M., and Horowitz, B. Regulation of Kv4.3 channels by Ca2+/calmodulin-dependent protein kinase II. . Am. J. Physiol. Cell Physiol. 288:C304-C313, 2005 Leblanc, N., Ledoux, J., Saleh, S., Sanguinetti, A., Angermann, J., O’Driscoll, K., Britton, F., Perrino, B.A., and Greenwood, I.A. Regulation of Calcium-Activated Chloride Channels in Smooth Muscle Cells–A Complex Picture Is Emerging. Can. J. Physiol. Pharmacol. 83:541-546, 2005 Greenwood, I.A., Ledoux, J., Perrino, B.A., and Leblanc, N. Calcineurin-dependent dephosphorylation augments IClCa in pulmonary artery smooth muscle cells. J. Biol. Chem. 279:38830-38837, 2004 Riddervold, M.H., Lorenz, J.M., Beckett, E.A.H., Baker, S.A., and Perrino, B.A. Differential autophosphorylation of Ca2+/calmodulin-dependent protein kinase II from phasic and tonic smooth muscle tissues. AJP Cell Physiol. 283: C1399-C1413, 2002 Perrino, B.A., Wilson, A.J., Ellison, P., and Clapp, L.H. Substrate selectivity and sensitivity to inhibition by immunophilin/immunosuppressant complexes of calcineurin heterodimers composed of the ? or ? catalytic subunit. Eur. J. Biochem. 269:3540-3548, 2002 Amberg, G., Koh, S.D., Perrino, B.A., Hatton, W.J., and Sanders, K.M. Regulation of A-type potassium channels in murine colonic myocytes by phosphatase activity. Am. J. Physiol. Cell Physiol. 281:C2020-2028, 2001 Jun, J.Y., Kong, I.D., Koh, S.D., Wang, X.Y., Perrino, B.A., Ward, S.M., and Sanders, K.M. Regulation of ATP-sensitive K+ channels by protein kinase C in murine colonic myocytes. Am. J. Physiol. Cell Physiol. 281:C857-C864, 2001. Perrino, B.A. and Martin, B.A. Ca2+-and myristoylation-dependent association of calcineurin with phosphatidylserine. J. Biochem. (Tokyo)129:835-841, 2001. Martin, B.A.; Oxhorn, B.C.; Rossow, C.R.; and Perrino, B.A. A cluster of basic residues in calcineurin B participates in the binding of calcineurin to phosphatidylserine vesicles. J. Biochem. (Tokyo)129:843-849, 2001. Perrino, B.A. Regulation of calcineurin-dependent signaling pathways by protein targeting. Recent Research Developments in Endocrinology. 1:209-232, 2000. Perrino, B.A. Regulation of calcineurin phosphatase activity by its autoinhibitory domain. Arch. Biochem. Biophys. 372:159-165, 1999 Koh, S. D.; Perrino, B. A.; Hatton, W. J.; Kenyon, J. L.; and Sanders, K. M. Novel regulation of the A-type current in murine proximal colon by calcium/calmodulin-dependent protein kinase II. J. Physiol. 517 (Part1):75-84, 1999 Perrino, B.A. and Soderling, T.R. Biochemistry and Pharmacology of the Calmodulin-Regulated Phosphatase Calcineurin. Invited chapter for “Calmodulin and Signal Transduction”, Academic Press, Eds: Linda van Eldik and D. Martin Watterson. 169-236, 1998 |
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