The central hypothesis of this proposal is that in vascular smooth muscle cells (VSMCs) of the spontaneously hypertensive rat (SHR), elevated intracellular Ca2+ (Cai2+) or increased Ca2+ turnover rate lead to increased Na+/H+ antiport activity. These perturbations in Ca2+ result in: a) increased metabolic demands leading to accelerated H+ production, and b) increased Ca2+-ATPase activity (Ca2+-H+ exchange) and the consequent enhanced H+ entry. Thus, increased Na+/H+ exchange in VSMCs of the SHR represents a regulatory mechanism that attenuates intracellular pH (pHi) acidification. Increased Na+/H+ antiport activity can be accomplished without cytosolic acidifications by an alkaline shift in the pH set point for activation of the exchanger. This shift in the pH set point and augmented transport by the exchanger may relate to elevated Cai2+, altered protein kinase C (PKC) activity, and/or effects of second messengers such as cyclic nucleotides and diacylglycerol (DAG). To investigate the role of these variables in the increased activity of the Na+/H+ antiport, the relation between intracellular signals and the exchanger in primary cultures of VSMCs will be examined in the adult SHR and two normotensive strains - Wistar Kyoto (WKY) and Wistar (WIS) rats. In some studies, Na+/H+ exchange in VSMCs from prehypertensive rats will be assessed to determine genetic and age related effects on antiport activity. Na+/H+ antiport activity in monolayers of VSMCs will be measured with the pH-sensitive fluorescent probe 2',7'-bis (carboxyethyl)-5,6- carboxyfluorescein (BCECF). Kinetic parameters of this transport system will be estimated from experiments that monitor the initial pHi recovery of Na+/H+ exchange. The effects of second messengers will be assessed during activation of the Na+/H+ antiport by cellular acidification, osmotic shrinkage, and alkalinization resulting from stimulation of PKC-dependent and -independent mechanisms. The profiles of Cai2+ and pHi will be compared in VSMCs from the three strains following treatment with agents that alter cellular cAMP, cGMP and DAG concentrations. These second messengers have been selected because of their ability to modulate Na+/H+ antiport activity. Cai2+ will be monitored with the fluorescent probe fura-2, under experimental conditions designed to manipulate Cai2+ levels. PKC activity will be correlated with Cai2+ and Na+/H+ antiport activity under the aforementioned conditions and experimental maneuvers. The main objective is to identify the specific factors that cause increased Na+/H+ antiport activity in primary hypertension. This information is required for initiating strategies for the prevention and/or management of essential hypertension.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HL044196-03
Application #
3472995
Study Section
Experimental Cardiovascular Sciences Study Section (ECS)
Project Start
1991-05-01
Project End
1996-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Type
Schools of Medicine
DUNS #
605799469
City
Newark
State
NJ
Country
United States
Zip Code
07107
Gardner, J P; Zhang, L (1999) Glucocorticoid modulation of Ca2+ homeostasis in human B lymphoblasts. J Physiol 514 ( Pt 2):385-96
Wang, X; Gardner, J P; Kheir, A et al. (1997) Synergistic induction of HL60 cell differentiation by ketoconazole and 1-desoxy analogues of vitamin D3. J Natl Cancer Inst 89:1199-206
Brzustowicz, L M; Gardner, J P; Hopp, L et al. (1997) Linkage analysis using platelet-activating factor Ca2+ response in transformed lymphoblasts. Hypertension 29:158-64
Gardner, J P; Balasubramanyam, M; Studzinski, G P (1997) Up-regulation of Ca2+ influx mediated by store-operated channels in HL60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3. J Cell Physiol 172:284-95
Fekete, Z; Yang, X Y; Kimura, M et al. (1995) The endothelin receptor profile in L6 myotubes. J Pharmacol Exp Ther 275:215-8
Balasubramanyam, M; Gardner, J P (1995) Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells. Cell Calcium 18:526-41
Balasubramanyam, M; Rohowsky-Kochan, C; Reeves, J P et al. (1994) Na+/Ca2+ exchange-mediated calcium entry in human lymphocytes. J Clin Invest 94:2002-8
Yang, X Y; Fekete, Z; Gardner, J et al. (1994) Endothelin mobilizes calcium and enhances glucose uptake in cultured human skeletal myoblasts and L6 myotubes. Hypertension 23:1075-81
Gardner, J P; Cho, J H; Skurnick, J H et al. (1994) Blood pressure inversely correlates with thrombin-evoked calcium rise in platelets. Hypertension 23:703-9
Balasubramanyam, M; Kimura, M; Aviv, A et al. (1993) Kinetics of calcium transport across the lymphocyte plasma membrane. Am J Physiol 265:C321-7