The entry of cations (positively charged ions) into vascular smooth muscle cells (VSMCs) fuels the contraction of small arteries and arterioles. Indeed, the plasma membrane of VSMCs expresses a medley of receptor- and store- operated "nonselective" ion channels that mediate Na+ and Ca2+ influx, as well as voltage-gated Ca2+ channels that selectively conduct the Ca2+ ion. These ion channels function like a symphony to tightly regulate Na+ and Ca2+ influx, vascular tone and blood pressure. However, their accentuated function may enhance Na+ and Ca2+ influx into VSMCs to cause excessive vasoconstriction and hypertension. In this regard, our knowledge of the contribution of specific cation -conducting channels to the contraction of VSMCs and the development of hypertension is incomplete, but recognition of the importance of cation -conducting channels distinct from the traditional voltage-dependent Ca2+ channels continues to grow, as evidenced by recent reports (20-37). One gene family of cation-permeable channels recently explored in VSMCs codes for canonical transient receptor potential (TRPC) channels, which include seven subtypes (TRPC1, 2, 3, 4, 5, 6, 7). The TRPC channels are opened by vasoconstrictor agonists, Ca2+ store depletion, membrane stretch and other stimuli associated with VSMC activation. Recently a link between one subtype of the TRPC gene family, TRPC3, and hypertension was suggested by the findings that TRPC3 is over-expressed in the systemic arteries of genetically hypertensive rats and in the preglomerular arterioles and renal tissues of patients with hypertension. In the studies of this project, we used TRPC3 knockout (Trpc3-/-) mice to define the role of TRPC3-contaning channels in the &agr;1-adrenergic receptor (&agr;1AR) signaling cascade that activates VSMCs. Initially, we performed multi-cell PCR using VSMCs freshly isolated from mouse mesenteric arteries (MA) to confirm the expression of the TRPC3 in this cell type. We found transcripts coding for three of the seven TRPC family members in mesenteric VSMCs, namely TRPC1, TRPC3 and TRPC6. Real-time PCR confirmed the absence of TRPC3 transcript in MA of Trpc3-/- mice, whereas transcript and protein levels of TRPC1 and TRPC6 were not significantly different between arteries of Trpc3-/- and wild-type (WT) mice. Diameter reductions in response to KCl (60 mM) were similar between cannulated, pressurized (60 mmHg) MA of WT and (47±3%, n=6) and Trpc3-/- (48±5%, n=6) mice. However, concentration-dependent contractions to the &agr;1AR agonist, phenylephrine (PE, 0.01 to 100 µM) were impaired in MA from Trpc3-/- mice (n=7) compared to WT mice (n=7). Although resting levels of blood pressure and heart rate were not different between WT and Trpc3-/- mice, the increase in mean arterial pressure (MAP) in response to PE (30 &mgr;g/kg, i.p.) administration was significantly less in Trpc3-/- mice (21.6±7.0 mmHg, n=7) compared to WT mice (44.5±3.2 mmHg, n=5). Collectively, these findings demonstrate a contribution of the TRPC3-containing channel to &agr;1AR -mediated vasoconstriction and blood pressure elevation. The impact of TRPC3 deletion on the density of whole-cell cation current in mouse mesenteric VSMCs was explored by patch-clamp studies recording whole-cell cation current under basal conditions and after application of 1-oleoyl-2-acetyl-sn--glycerol (OAG), a stable analog of diacylglycerol and a direct activator of cation -conducting channels composed of TRPC3, 6 and 7. OAG activated a large cation current in VSMCs from WT mice (n=14), which was attenuated 58±7% at -60 mV in mesenteric VSMCs from Trpc3-/- mice (n=10). Similarly, PE-activated cation current at -60 mV averaged 81±5% less in VSMCs from Trpc3-/- (n=11) compared to WT (n=12) mice. Despite this evidence that the TRPC3-containing channel contributes significantly to the PE-activated cation current in mouse mesenteric VSMCs, TRPC3 deletion did not significantly alter VSMC resting membrane potential (Em), PE-induced depolarization or L-type Ca2+ channel-mediated vasoconstriction in cannulated, pressurized MA from Trpc3-/- mice. However, additional studies revealed that SN-6, an inhibitor of the reverse mode Na+/Ca2+ exchanger (NCX), dilated PE pre-constricted MA of WT mice by 41.1±4.7% (n=5) compared to only 17.9±1.1% (n=4) in MA from Trpc3-/- mice, suggesting that functional coupling between TRPC3-containing and the NCX contributes to PE-induced vasoconstriction. Next, we explored the possibility that TRPC3-containing channels contribute to the development of hypertension based on earlier reports that the TRPC3 protein is over-expressed in arteries of hypertensive animals and humans. Since the lack of a selective TRPC3 blocker has thwarted efforts to determine the precise role of TRPC3-containing channels in the pathogenesis of hypertension, we used Trpc3-/- mice as a tool to accomplish this goal. In this series of studies, basal blood pressure was recorded using biotelemetry in WT and Trpc3-/- mice; then Ang II was infused at a dose of 2 ng/g/min for two weeks to establish hypertension. These studies revealed similar expression levels of TRPC3 mRNA between MA of saline- and Ang II- infused C57BL/6 mice, but TRPC3 protein expression was ~5.7-fold higher in MA of the latter animals. The increased expression of TRPC3 was associated with a 34% enhancement of PE -induced vasoconstriction in pressurized MA of Ang II-infused WT mice (n=8) compared to saline-infused WT mice (n=8). PE -induced constrictions were 29% less in MA of Ang II-infused Trpc3-/- mice (n=7) compared to Ang II-infused WT mice (n=8). Corresponding PE-evoked cation current showed a 55% less density in freshly isolated mesenteric VSMCs from Ang II -infused Trpc3-/- mice (n=9) compared to Ang II-infused WT mice (n=7). The resting MAP was not significantly different between WT (124±3 mmHg; n=8) and Trpc3-/- mice (119±4 mmHg; n=10). However, Ang II infusion (2 ng/g/min, s.c.) for 14 days increased MAP in WT mice to 169±3 mmHg compared to only 145±4 mmHg in Trpc3-/- mice. In summary, our findings suggest that the Ang II -induced hypertension is associated with an over-abundance of TRPC3 protein in mouse MA, and the absence of the TRPC3 gene blunts development of Ang II -dependent hypertension in mice. Thus, interventions designed to reduce the expression or function of TRPC3-containing channels in VSMCs may be potentially useful as antihypertensive drug therapies. Funding: NIH R01 HL064806 (NJR), AHA 12PRE11850002 (ARP) and AHA Grant 13GRNT14590001 (NJR).
|Advisor:||Rusch, Nancy J.|
|Commitee:||Birnbaumer, Lutz, Crooks, Peter A., Marsh, James D., Rhee, Sung W., Zheng, Fang|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Pharmacology, Pharmacy sciences, Physiology|
|Keywords:||Blood pressure, Hypertension, Ion channel, Trpc, Trpc3, Vascular tone|
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