Cysteinyl leukotrienes dependent [Ca2+]i responses to Angiotensin II in rat cardiomyocytes and aortic smooth muscle cells
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Angiotensin II (Ang II) plays a very important role in regulating cardiac and vascular contraction and proliferation/hypertrophy via stimulation of AT1 receptors. A few studies have demonstrated that 5-lipoxygenase (5-LO) derived cysteinyl leukotrienes (CysLT) contribute to Ang II evoked tension responses in rat aortic rings. Whether CysLT would contribute to Ang II evoked Ca2+ mobilization in neonatal rat cardiomyocytes (NRC) and rat aortic smooth muscle cells (ASMC) has not been investigated. In the present study, using primary cultures of NRC and minimally passaged cultures of rat ASMC, an effort was made to address this key issue. The agonists evoked increase in cytosolic free calcium ([Ca2+]i) level was determined by fura-2 fluorescence measurement in NRC and ASMC. Total CysLT levels in the culture medium were determined using an ELISA kit. CysLT1/CysLT2 receptor mRNA levels of NRC and ASMC were quantified by Northern blot analysis. In NRC, the AT1 but not the AT2 selective antagonist, attenuated the elevations in [Ca2+]i and CysLT levels evoked by Ang II. Vasopressin (AVP) and endothelin-1 (ET-1) increased [Ca2+]i but not CysLT levels. The 5-LO inhibitor, AA861, and the CysLT1 selective antagonist, MK-571, reduced the maximal [Ca2+]i responses (Emax) to Ang II but not to AVP and ET-1. While CysLT1 antagonist reduced the Emax to leukotriene D4, (LTD4), the dual CysLT1/CysLT2 antagonist, BAY u9773, completely blocked the [Ca2+]i elevation to both LTD4 and leukotriene C4 (LTC4). Both CysLT1 and CysLT2 mRNA were detected in NRC. The inositol 1,4,5 triphosphate (InsP3) antagonist, 2-aminoethoxyphenyl borate (2-APB), attenuated the [Ca2+]i responses to Ang II and LTD4. In ASMC, Ang II, ET-1 and AVP evoked [Ca2+]i responses were significantly higher in the cultured ASMC isolated from spontaneously hypertensive rats (SHR) compared to ASMC derived from age-matched normotensive Wistar-Kyoto (WKY) strain. Addition of either MK571 or BAY u9773, reduced the Emax values to Ang II (but not to ET-1and AVP) in both strains. While BAY u9773 abolished the [Ca2+]i responses evoked by both LTD4 and LTC4, MK571, the CysLT1 antagonist reduced the responses evoked by LTD4 but not LTC4. The basal CysLT levels were higher in the ASMC of SHR. Ang II but not ET-1 and AVP evoked time and concentration dependent increases in CysLT levels in ASMC of both WKY and SHR strains. The AT1 selective antagonist, losartan, but not the AT2 antagonist, PD123319, attenuated the increases in [Ca2+]i and CysLT levels evoked by Ang II. The InsP3 antagonist, attenuated the [Ca2+]i responses to Ang II, LTD4 and LTC4. Both CysLT1 and CysLT2 mRNA were detected in the ASMC of either strain; but they were significantly higher in SHR. These data suggest that AT1 mediated CysLT production contributes to Ang II evoked Ca2+ mobilization in NRC and that elevated CysLT production along with increased expression of both CysLT1/CysLT2 receptors may account for the exaggerated [Ca2+]i responses to Ang II in ASMC of SHR due to enhanced mobilization of Ca2+ from InsP3 sensitive intracellular Ca2+ stores.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeWilson, Thomas W.; Wang, Rui; Richard, Darren E.; McNeill, J. Robert; Hickie, Robert A.
Copyright DateFebruary 2005
Ang II; CysLT