Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) are the major regulators of cross-bridge cycling and force generation in vascular smooth muscle. The overall goal of the proposed research is to gain new information on the role of these molecules (particularly MLCK) in controlling arterial contraction in normal function, and in a model of salt dependent hypertension (DOCA-salt model, deoxycorticosterone acetate and high dietary NaCl intake). Direct examination of MLCK activity in isolated arteries and in the arteries of living animals (i.e. in vivo) will be achieved through the use of (transgenic) 'biosensor'mice that express an optical (FRET) MLCK activity sensor. Regulation of MLCP in isolated arteries will be studied by quantifying threonine-855 phosphorylation of myosin phosphatase targeting subunit (MYPT1). Initial fluorescence imaging studies in isolated arteries (Aims 1 &2) will reveal the activation of MLCK and regulation of MLCP in relation to 1) myogenic tone and 2) certain G-protein coupled receptors (GPCR) that are known to be important in hypertension. Myogenic tone (MT) is a key smooth muscle function that is involved in maintenance of arterial pressure, and in the response to tissue over-perfusion in initial stages of salt-induced hypertension. Therefore, Aim 1 is to quantify the dynamic and long-term (hours) activation of MLCK and regulation of MLCP as pressure is changed over the range of 10 to 150 mm Hg in isolated arteries.
Aim 2 is to quantify MLCK activation, and MLCP inhibition, accomplished by two key classes of GPCR: 1) those coupled primarily to Gq/11, and 2) those also coupled strongly to G12/13. The latter have been implicated particularly in salt-induced hypertension and may utilize strong inhibition of MLCP, in addition to activation of MLCK. The influence of MT on GPCR induced signaling will also be studied since new data indicates that it affects contractile signaling of GPCR in ways not yet fully appreciated.
Aim 3 will build on the knowledge gained in the isolated arteries , but will utilize in vivo imaging (i.e. intravital FRET microscopy) of arteries in anesthetized biosensor animals to quantify the role of MLCK in the increased vasoconstriction that occurs in DOCA-salt hypertension. In this final Aim, two current, competing, hypotheses will be examined: 1) that DOCA-salt hypertension is importantly maintained by circulating factors acting through G12/13 coupled GPCR and therefore involves strong inhibition of MLCP, rather than exclusive activation of MLCK, and 2) that salt-dependent hypertension involves mainly endogenous Na+ pump ligands (natriuretic factors) that contract smooth muscle by increasing [Ca2+] and thus act mainly through MLCK, rather than inhibition of MLCP. Summary: The research is intended to provide a detailed, quantitative, dynamic description of the activation and regulation of MLCK and MLCP in normal and hypertensive arteries in response to physiological stimuli, including transmural pressure and GPCR signaling. It will provide the first direct evidence, from arteries in the living animal, on the role of MLCK in salt-induced hypertension.

Public Health Relevance

The proposed research is intended to provide basic information on the activity and regulation of an enzyme (myosin light chain kinase, MLCK) that is critical to contraction of arteries, both in normal physiology and in high blood pressure (hypertension). Arteries exist in a contracted state in order to maintain blood pressure. The amount of contraction changes rapidly in response to activity of the nervous system and hormones. This research will utilize a mouse model of salt-induced hypertension to provide specific new information on the role of MLCK in high blood pressure.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL091969-03
Application #
8235851
Study Section
Electrical Signaling, Ion Transport, and Arrhythmias Study Section (ESTA)
Program Officer
Gao, Yunling
Project Start
2010-03-22
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$371,250
Indirect Cost
$123,750
Name
University of Maryland Baltimore
Department
Physiology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Wier, Withrow Gil; Mauban, Joseph R H (2017) Imaging sympathetic neurogenic Ca2+ signaling in blood vessels. Auton Neurosci 207:59-66
Blaustein, Mordecai P; Chen, Ling; Hamlyn, John M et al. (2016) Pivotal role of ?2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease. J Physiol 594:6079-6103
Mauban, Joseph R H; Zacharia, Joseph; Fairfax, Seth et al. (2015) PC-PLC/sphingomyelin synthase activity plays a central role in the development of myogenic tone in murine resistance arteries. Am J Physiol Heart Circ Physiol 308:H1517-24
Wier, W Gil (2014) More in vivo experimentation is needed in cardiovascular physiology. Am J Physiol Heart Circ Physiol 307:H121-3
Mauban, Joseph R H; Fairfax, Seth T; Rizzo, Mark A et al. (2014) A method for noninvasive longitudinal measurements of [Ca2+] in arterioles of hypertensive optical biosensor mice. Am J Physiol Heart Circ Physiol 307:H173-81
Mauban, Joseph R H; Zacharia, Joseph; Zhang, Jin et al. (2013) Vascular tone and Ca(2+) signaling in murine cremaster muscle arterioles in vivo. Microcirculation 20:269-77
Wang, Youhua; Chen, Ling; Wier, W Gil et al. (2013) Intravital Förster resonance energy transfer imaging reveals elevated [Ca2+]i and enhanced sympathetic tone in femoral arteries of angiotensin II-infused hypertensive biosensor mice. J Physiol 591:5321-36
Zacharia, Joseph; Mauban, Joseph R H; Raina, Hema et al. (2013) High vascular tone of mouse femoral arteries in vivo is determined by sympathetic nerve activity via ?1A- and ?1D-adrenoceptor subtypes. PLoS One 8:e65969
Linde, Cristina I; Karashima, Eiji; Raina, Hema et al. (2012) Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats. Am J Physiol Heart Circ Physiol 302:H611-20
Reimann, Katrin; Krishnamoorthy, Gayathri; Wier, Withrow Gil et al. (2011) Gender differences in myogenic regulation along the vascular tree of the gerbil cochlea. PLoS One 6:e25659

Showing the most recent 10 out of 11 publications