The long term objective of the applicant's research program is to unravel the ionic mechanisms that are involved in the control of electrical and mechanical activities of the smooth muscle cells of large conduit and resistance-sized vessels in health and disease. In smooth muscle cells, chloride ions (Cl-) are not passively distributed across the cell membrane. It is thought that this anion is actively accumulated in the cytoplasm by various anion transporters. Such a high internal level of Cl- (~ 40 mM) results in a predicted equilibrium for Cl- (ECl) that is more positive (-25 mV) than the resting membrane potential (RMP) of vascular smooth muscle cells (~ -40 to -60 mV). Because of this deviation between ECl and RMP, any increase in permeability to Cl- would result in passive Cl"""""""" efflux, membrane depolarization and increased vascular tone. In spite of our knowledge about high resting membrane permeability to Cl- in smooth muscle cells, the nature of this basal anion conductance still remains undefined. This proposal is focused on elucidating the mechanisms involved in the regulation by phosphorylation mechanisms involving calmodulin-dependent kinase II (CaMKII) and serine/threonine phosphatases (Calcineurin and PP2A) of a Cl- channel activated by intracellular Ca2+ (ClCa ) in pulmonary arterial smooth muscle cells, and how this might impact on the electrical and vasoactive properties of the pulmonary circulation.
4 Specific Aims are proposed in this grant: (1) determine the effect of general phosphorylation status on the biophysical properties of Clca channels in rabbit pulmonary artery smooth muscle cells; (2) determine the relative role of CaMKII and serine/threonine phosphatases in the regulation of ClCa channels in rabbit pulmonary artery smooth muscle cells; (3) determine the physiological impact of phosphatase regulation of ClCa channels on membrane potential, Ca2+ transients and tone in pulmonary arterial smooth muscle cells and intact pulmonary arteries; and (4) clone and express 3 candidate genes encoding Ca2+-activated Cl- channels in rabbit pulmonary artery and evaluate their physiological relevance to the native channels. A strong team of collaborating investigators will use a wide array of electrophysiological, biochemical and molecular biology techniques, as well as confocal imaging technology and computer simulations to accomplish the above goals. The etiology of pulmonary hypertension (PH) in humans is still poorly understood although it is becoming increasingly clear that defective ionic mechanisms may play a role in this disease. Because of their potential importance as an excitatory mechanism in pulmonary arteries, impaired regulation and/or expression of Clca channels could potentially participate in PH. The proposed studies will not only advance our knowledge about their basic properties, but should also pave the way for the development of future therapies to treat PH.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075477-02
Application #
7079430
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Lin, Michael
Project Start
2005-07-01
Project End
2010-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
2
Fiscal Year
2006
Total Cost
$318,584
Indirect Cost
Name
University of Nevada Reno
Department
Pharmacology
Type
Schools of Medicine
DUNS #
146515460
City
Reno
State
NV
Country
United States
Zip Code
89557
Davis, Alison J; Shi, Jian; Pritchard, Harry A T et al. (2013) Potent vasorelaxant activity of the TMEM16A inhibitor T16A(inh) -A01. Br J Pharmacol 168:773-84
Forrest, Abigail S; Joyce, Talia C; Huebner, Marissa L et al. (2012) Increased TMEM16A-encoded calcium-activated chloride channel activity is associated with pulmonary hypertension. Am J Physiol Cell Physiol 303:C1229-43
Angermann, Jeff E; Forrest, Abigail S; Greenwood, Iain A et al. (2012) Activation of Ca2+-activated Cl- channels by store-operated Ca2+ entry in arterial smooth muscle cells does not require reverse-mode Na+/Ca2+ exchange. Can J Physiol Pharmacol 90:903-21
Forrest, Abigail S; Angermann, Jeff E; Raghunathan, Rajesh et al. (2010) Intricate interaction between store-operated calcium entry and calcium-activated chloride channels in pulmonary artery smooth muscle cells. Adv Exp Med Biol 661:31-55
Neveux, Iva; Doe, Jinger; Leblanc, Normand et al. (2010) Influence of the extracellular matrix and integrins on volume-sensitive osmolyte anion channels in C2C12 myoblasts. Am J Physiol Cell Physiol 298:C1006-17
Davis, Alison J; Forrest, Abigail S; Jepps, Thomas A et al. (2010) Expression profile and protein translation of TMEM16A in murine smooth muscle. Am J Physiol Cell Physiol 299:C948-59
Sones, William R; Davis, Alison J; Leblanc, Normand et al. (2010) Cholesterol depletion alters amplitude and pharmacology of vascular calcium-activated chloride channels. Cardiovasc Res 87:476-84
Ayon, Ramon; Sones, William; Forrest, Abigail S et al. (2009) Complex phosphatase regulation of Ca2+-activated Cl- currents in pulmonary arterial smooth muscle cells. J Biol Chem 284:32507-21
Wiwchar, M; Ayon, R; Greenwood, I A et al. (2009) Phosphorylation alters the pharmacology of Ca(2+)-activated Cl channels in rabbit pulmonary arterial smooth muscle cells. Br J Pharmacol 158:1356-65
O'Driscoll, Kate E; Leblanc, Normand; Hatton, William J et al. (2009) Functional properties of murine bestrophin 1 channel. Biochem Biophys Res Commun 384:476-81

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