Our group is involved in determining how smooth muscle function is regulated. We are currently examining the regulation of vascular and urinary bladder smooth muscle. Our work can be divided into three complementary areas of study: Signal Transduction, Modulation of Contraction, and Pathophysiology.
Smooth muscle contraction is believed to be initiated by a cascade of events beginning with stimulation-induced increases in cellular calcium and ending with phosphorylation of a protein called the myosin light chain. Our signal transduction studies have centered on the roles of various isoforms of kinases and phosphatase and how they initiate or modulate contraction by effects on myosin or actin binding proteins. Our experiments are designed to quantitatively determine the magnitude of kinase catalyzed phosphorylation of specific cellular substrates and the role of this phosphorylation in cellular function. We are also investigating how receptor and G-protein activation alters the calcium sensitivity of the contractile filaments and if either protein kinase C, MAP kinase or Rho kinase are involved. Our long term goal is to determine the precise steps involved in the coupling of cellular excitation and contraction and to determine the precise steps of modulatory pathways that alter the contractile response to excitation. The following cartoon provides a glimpse into the involved signal pathways important in smooth muscle.
Our work on modulation of contraction is focused on determining if thin filament proteins and specifically caldesmon are important in the development and maintenance of force. To pursue this goal, we use intact and permeabilized (a technique that allows us to control the intracellular environment) strips of smooth muscle to measure force of contraction and velocity of shortening (index of crossbridge cycling rates) as well as biochemical analysis of enzyme activities and protein phosphorylation levels. In addition, we use antisense oligonucleotides and siRNA techniques to "knock-out" specific proteins within a physiologically viable tissue to determine the role that protein plays in regulation. The following shows immunohistochemically stained strips of the intact swine carotid media cultured in the presence and absence of an siRNA against caldesmon. Organ culture on intact vascular strips in the presence of the siRNA produces a tissue with a caldesmon specific knock-down throughout the entire arterial strip.
Smooth muscle is the final common pathway for many diseases. Therefore a complete understanding of how smooth muscle changes during the genesis and maintenance of a disease is an important step toward the development of therapeutic approaches. We are currently involved in studies in collaboration with the Division of Urology at The University of Pennsylvania School of Medicine aimed at understanding how urinary bladder smooth muscle cells are altered following partial outlet obstruction similar to that which occurs during benign prostatic hyperplasia. It is known that significant changes occur in the contractile protein content and isoform profile, but how this impacts the physiology and regulation of the tissue is not understood. Recent studies from our lab have shown that bladder outlet obstruction alters the relationship between force and myosin light chain phosphorylation, decreases crossbridge cycling rates, abolishes contractions in response to activation of protein kinase C, and diminishes the G-protein dependent pathways that increase contractile protein sensitivity to calcium. We are currently investigating the precise cellular components involved in these alterations. The goal of these studies is to obtain a better understanding of the role the smooth muscle cell plays in the pathology of the tissue.
Selected references:
"Bladder smooth muscle organ culture preparation maintains the contractile phenotype"
Wang TC, Kendig DM, Chang S, Trappanese DN, Chacko S, and Moreland RS
Am. J. Physiol. Renal Physiol., in review.
"Convenient one pot procedure for the selective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones and evaluation of their calcium channel blocking activity"
Singh K, Arora D, Poremsky E, Lowery J, and Moreland RS
Eur. J. Med. Chem. 44: 1997-2001, 2009.
"Functional and molecular consequences of ionizing irradiation on large conductance Ca2+ activated K+ channels in rat aorta smooth muscle cells"
Soloviev A, Tishki, S, Ivanova I, Zelensky S, Dosenko V, Kyrychenko S, and Moreland RS
Life Sci. 84: 164-171, 2009.
"siRNA mediated knock down of h-caldesmon in vascular smooth muscle"
Smolock EM*, Falkowski DM*, Chang S, Wang TC, Titchenell P, and Moreland RS (* = equal contribution)
Am. J. Physiol. Heart Circ. Physiol. 297: H1930-H1939, 2009.
"Roles of protein kinase C and Rho kinase in carbachol induced contraction of rabbit bladder smooth muscle"
Wang TC, Kendig DM, Smolock EM, and Moreland RS
Am. J. Physiol. Renal Physiol. 297: F1534-F1542, 2009.
"Unaided addition of carbon nucleophiles to pyrimidinones. An efficacious protocol for C-4 substituted 3,4-dihydropyrimidinones. Synthesis and calcium channel binding studies"
Kamaljit S, Arora D, Falkowski D, Liu Q, and Moreland RS
Eur. J. Org. Chem. 2009: 3258-3264, 2009.
"siRNA knock down of casein kinase 2 increases force and crossbridge cycling rates in vascular smooth muscle"
Smolock EM, Wang TC, Nolt JC, and Moreland RS
Am. J. Physiol. Cell Physiol. 292:C876-C885, 2007. (featured in the March 2007 NAVBO Vascular Biology Publications Alert)
"Ionizing non-fatal whole-body irradiation inhibits Ca2+-dependent K+ current in endothelial cells of rat coronary artery: Possible contribution to depression of endothelium-dependent vascular relaxation"
Tishkin SM, Rekalov VV, Ivanova IV, Moreland RS and Soloviev AI
International J. Rad. Biol, 83: 161-169, 2007.
"Regional differences in myosin isoform expression and maximum shortening velocity in the vaginal muscularis"
Basha M, Chang S, Smolock E, Moreland RS, Wein A, and Chacko S.
Am. J. Physiol. Reg. Integr. Physiol. 291:R1076-R1084, 2006.
"Partial bladder outlet obstruction selectively abolishes protein kinase C induced contraction of rabbit detrusor smooth muscle"
Stanton MC, Austin JC, Delaney D, Gosfield A, Zderic SA, Chacko S, and Moreland RS
J. Urology 176:2716-2721, 2006 (chosen for an editorial highlight).
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