V a s c u l a r   B i o l o g y

The Vascular Biology Division pioneered the characterization of contractility and Ca²⁺ signaling pathways in smooth muscle tissues from mice. Our data bank includes: aorta, portal vein, mesenteric arterioles, esophagus, trachea, bladder, gall bladder, intestinal and uterine smooth muscle Over 50 different gene-altered mice have been characterized to date, examples shown in the table; for details, see Publications:
Sutliff, R.L. and Paul, R.J. (1998) Smooth muscle studies using gene-altered mouse models: a users guide. In Hoit, B.D. and Walsh, R.A. (eds) Cardiovascular Physiology in the Genetically Engineered Mouse. Kluwer Academic, MA pp. 247-257.

F u n c t i o n a l   G e n o m i c s

Ca²⁺-Homeostasis:
We use gene-altered mice to understand the roles of pumps in the regulation of intracellular calcium. We have utilized gene-ablated mice and developed transgenic mice using smooth muscle specific promoters:
-With Evangelia Kranias, characterization of the role of Phospholamban in smooth muscle and endothelial cell function.
-With Gary Shull, characterization of SERCA and PMCA isoforms in regulation of smooth muscle [Ca²⁺]_i.
-With Jerry Lingrel, characterization of the distinct roles of the a1 and a2 isoforms of Na⁺-K⁺ ATPase in smooth muscle.

  Okunade, G.W., Miller, M.L., Pyne, G.J., Sutliff, R.L., O'Connor, K. T., Neumann, J., Andringa, A., Miller, D.A., Prasad, V., Doetschman, T., Paul, R.J. and Shull, G.E. (2004). Targeted ablation of plasma membrane Ca²⁺-ATPase isoforms 1 and 4 Indicates a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem.: 279, 33742-33750.

  Liu, L., Ishida, I., Okunade, G., Shull, G.E. and Paul, R.J. (2005) Role of plasma membrane Ca²⁺-ATPase in contraction-relaxation processes of the bladder: evidence from PMCA gene-ablated mice. Am. J. Physiol. Cell (in press)

  Shelly, D, He, S., Moseley, A, Weber, C, Stegemeyer, M., Lynch, R.M., Lingrel, J. and Paul, R.J. (2004) Specific coupling of the Na+-pump a2-isoform to excitation-contraction coupling in vascular smooth muscle: evidence from gene-targeted mice. Am. J. Physiol. Cell 286: C813-820.

  Sutliff, R.L., Conforti, L., Weber, C., Kranias, E.G. and Paul, R.J. (2004) Regulation of the spontaneous contractile activity of the portal vein by the sarcoplasmic reticulum: Evidence from the phospholamban gene-ablated mouse. Vascul Pharmacol 41: 197-204.

Smooth Muscle Motor Protein Function:
We investigate the role of isoforms Molecular motor proteins and specific contractile properties of smooth muscle.
-With Anne Martin, characterization of myosin heavy chain isoforms using smooth muscle specific expression of SM1 & SM2 isoforms
-With Muthu Periasamy, characterization of myosin heavy chain insert isoforms using SMb KO mouse.
-With James Lessard, characterization of smooth muscle a and g isoforms of actin.

  Crawford, K., Flick, R., Shelly, D., Paul, R., Bove, K., Kumar, A., and Lessard, J. (2002) Mice lacking skeletal muscle actin show reduced muscle strength, growth deficits, and neonatal death. Mol Cell Biol 22, 5887-96.

  Babu, G.J., Loukianov, E., Loukianova, T., Pyne, G.J., Huke, S., Osol, G., Low, R.B., Paul, R.J. and Periasamy, M. (2001) Loss of SM-B myosin isoform results in a decrease.of maximal force development and velocity of shortening Nature Cell Biology 3(11): 1025-29.

Compartmentation, Energetics and Metabolism:
The use of continuous measurements of oxygen consumption to monitor contractile ATP requirements under controlled mechanical conditions was developed in my laboratory and downsized to take advantage of gene-altered mice models. This is of special interest in terms of compartmentation of NKA isoforms and their link to aerobic glycolysis.

  Hardin, C.D., Allen, T.J. and Paul, R.J. (2000) Metabolism and energetics of vascular smooth muscle. In Physiology and Pathophysiology of the Heart, Sperelakis, N., Kurachi, Y., Terzic, A. and Cohen, M. (eds.), 4th edition, San Diego, CA, Academic Press, pp. 571-598.

M e c h a n i s m s   o f   V a s c u l a r  O x y g e n - S e n s i n g

The relaxation of systemic arteries to hypoxia is a vital protective response, leading to increased tissue perfusion. Most of the current theories invoke mechanisms that lower [Ca²⁺]_i. We have shown that in addition, there is an additional pathway that rather than leading to a decrease in [Ca²⁺]_i , this hypoxic mechanism operates through Ca²⁺ desensitization. We have used organ culture and proteomics to unravel these novel O₂ sensing pathways. Hypoxic relaxation is lost during organ culture and MALDI and TOF mass spectrometry used to identify target proteins. This enabled us to show that Rho Kinase was a key player in this Ca²⁺-desensitizing hypoxic relaxation. We continue to search for the oxygen-sensor & are expanding into studies on human cerebral and coronary vessels.

  Gu, M., Thorne, G.D., Wardle, Robert L, Ishida, Y. and Paul, R.J. (2005) Ca²⁺ independent hypoxic vasorelaxation in porcine coronary artery. J Physiol 562: 839-846.

  Thorne, G.D., Hilliard, G.M. and Paul, R.J. (2004) Vascular oxygen sensing: detection of novel candidates by Proteomics and organ culture J. Appl. Physiol. 96: 802-808.

  Thorne, G.D., Ishida, Y. and Paul, R.J. (2004) Hypoxic vasorelaxation: Ca²⁺ dependent and Ca²⁺-independent mechanisms. Cell Calcium 36: 201-208.

N o n - M u s c l e   C o n t r a c t i l i t y

3T3 fibroblasts cultured in collagen form a 3 dimensional gel, then shrink to form a fiber. Quantitative measurements of mechanical properties of this reconstituted fiber can be studied using the sophisticated mechanical analysis developed for muscle. Measurements of [Ca²⁺]_i on these preparation so that [Ca²⁺]_i can be correlated with mechanics and used to investigate the underlying signaling relations. Our recent studies suggest that “tensegrity” theory likely do not apply to fibroblasts and the Rho-kinase plays a major role in signaling, but not through the myosin regulatory light chain.

  Nobe, H., Nobe., K. and Paul, R.J. (2003) Fibroblast Fiber Contraction: Role of C- and Rho Kinase in Activation by Thromboxane A2. Am J. Physiol. 285: C1411-C1419.

  Nobe, H., Nobe, K., Fazal F, de Lanerolle, P., and Paul, R.J. (2003) Rho-associated kinase mediates calf serum-induced contraction in fibroblast fibers without increasing myosin LC20 phosphorylation. Am. J. Physiol. 284: C599-C606.

Contractile fiber composed of 3T3 cells grown in a collagen matrix. Shown is the fiber mounted between a glass rod and a silicon strain gage for measurements of isometric force and fiber stiffness.