Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes - PubMed (original) (raw)

Comparative Study

Diverse properties of store-operated TRPC channels activated by protein kinase C in vascular myocytes

Sohag N Saleh et al. J Physiol. 2008.

Abstract

In vascular smooth muscle, store-operated channels (SOCs) contribute to many physiological functions including vasoconstriction and cell growth and proliferation. In the present work we compared the properties of SOCs in freshly dispersed myocytes from rabbit coronary and mesenteric arteries and portal vein. Cyclopiazonic acid (CPA)-induced whole-cell SOC currents were sixfold greater at negative membrane potentials and displayed markedly different rectification properties and reversal potentials in coronary compared to mesenteric artery myocytes. Single channel studies showed that endothelin-1, CPA and the cell-permeant Ca(2+) chelator BAPTA-AM activated the same 2.6 pS SOC in coronary artery. In 1.5 mM [Ca(2+)](o) the unitary conductance of SOCs was significantly greater in coronary than in mesenteric artery. Moreover in 0 mM [Ca(2+)](o) the conductance of SOCs in coronary artery was unaltered whereas the conductance of SOCs in mesenteric artery was increased fourfold. In coronary artery SOCs were inhibited by the protein kinase C (PKC) inhibitor chelerythrine and activated by the phorbol ester phorbol 12,13-dibutyrate (PDBu), the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) and a catalytic subunit of PKC. These data infer an important role for PKC in activation of SOCs in coronary artery similar to mesenteric artery and portal vein. Anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in coronary and mesenteric arteries and portal vein but anti-TRPC6 blocked SOCs only in coronary artery and anti-TRPC7 blocked SOCs only in portal vein. Immunoprecipitation showed associations between TRPC1 and TRPC5 in all preparations but between TRPC5 and TRPC6 only in coronary artery and between TRPC5 and TRPC7 only in portal vein. Finally, flufenamic acid increased SOC activity in coronary artery but inhibited SOCs in mesenteric artery and portal vein myocytes. These data provide strong evidence that vascular myocytes express diverse SOC isoforms, which are likely to be composed of different TRPC proteins and have different physiological functions.

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Figures

Figure 1

Figure 1. CPA-evoked whole-cell cation conductances in coronary and mesenteric artery myocytes

Aa and b, original traces showing that 10 μ

m

CPA activated whole-cell SOC currents in coronary and mesenteric arteries. Vertical deflections represent current responses to voltage ramps from −100 mV to +100 mV. Ba and b, individual I–V relationships before (dashed line) and during (continuous line) CPA-induced whole-cell SOC currents in coronary and mesenteric arteries. C, mean I–V relationships of whole-cell SOC currents (n = 10 of each).

Figure 2

Figure 2. Activation of single channel currents by ET-1 and CPA in cell-attached patches from coronary artery

Aa and b, bath application of 100 n

m

ET-1, and 10 μ

m

CPA activated channel currents at −80 mV. Ac, mean I–V relationships of ET-1- and CPA-evoked channel currents in coronary artery showing similar conductances of 2.6 pS and 2.7 pS and _E_r of about 0 mV. Ba and Bb, comparison of CPA- induced single channel currents in coronary and mesenteric arteries recorded with 0 m

m

[Ca2+]o illustrating that SOCs in mesenteric artery have larger unitary amplitudes at −80 mV. Bc, mean I–V relationships of CPA-evoked SOCs recorded in 0 m

m

[Ca2+]o showing SOCs had conductances of 2.7 pS and 7.3 pS in, respectively, coronary and mesenteric arteries and _E_r of about 0 mV in both preparations.

Figure 3

Figure 3. SOCs in coronary artery are activated by PKC

A, BAPTA-AM (50 μ

m

) evoked channel activity in a cell-attached patch held at −80 mV, which was reversibly inhibited by chelerythrine (3 μ

m

). B and C, show that PDBu (1 μ

m

) and a PKC catalytic subunit (0.05 U ml−1) activated single channel currents in cell-attached and inside-out patches, respectively. D, mean I–V relationships of channel currents induced by a PKC catalytic subunit in coronary and mesenteric arteries.

Figure 4

Figure 4. Differential sensitivity of SOCs in coronary and mesenteric arteries and portal vein to anti-TRPC antibodies

Aa, in coronary artery bath application of anti-TRPC1, -TRPC5 and -TRPC6 antibodies at 1: 200 dilution significantly inhibited SOC activity in inside-out patches in a reversible manner whereas anti-TRPC7 had no effect. Ab, mean data showing the effect of antibodies raised against all TRPC proteins on SOC activity in coronary artery. Note that in the presence of their antigenic peptides (Ag) the inhibitory action of anti-TRPC1, -TRPC5 and -TRPC6 were blocked. Ba and b, anti-TRPC1 and -TRPC5 antibodies inhibited SOCs in mesenteric artery whereas anti-TRPC6 and -TRPC7 had no effect. Ca and b, anti-TRPC1, -TRPC5 and -TRPC7 antibodies inhibited SOCs in portal vein whereas anti-TRPC6 had no effect. Note anti-TRPC3 and -TRPC4 antibodies had no effect in any preparation (Ab, Bb and Cb).

Figure 5

Figure 5. Coimmunoprecipitation of TRPC channel proteins in coronary and mesenteric arteries and portal vein

A, total tissue lysates from coronary artery were immunoprecipitated and then Western blotted (WB) using the corresponding anti-TRPC antibodies. Note RT (rat testes) relates to positive control WB using anti-TRPC1 giving bands of 90 kDa. Bands of 90 kDa were observed when Western blots were carried out with an anti-TRPC1 antibody in samples immunoprecipitated with TRPC1 and TRPC5 but not TRPC1 and antigenic peptide (Ag, Aa) showing TRPC1 and TRPC5 protein can associate together. Western blots conducted using an anti-TRPC5 antibody detected a band of 100 kDa when immunoprecipitation was carried out using anti-TRPC5 or anti-TRPC6 antibodies demonstrating TRPC5 and TRPC6 protein can also associate in coronary artery. In mesenteric artery TRPC1 and TRPC5 proteins are shown to coimmunoprecipitate with each other (Ba and b) whereas TRPC5 and TRPC6 protein do not (Bc and d). In portal vein TRPC1 and TRPC5 and TRPC5 and TRPC7 were shown to associate with each other (Ca_–_c).

Figure 6

Figure 6. Differential effects of FFA on SOCs in outside-out patches from coronary and mesenteric arteries and portal vein

A, PDBu-evoked SOCs at −80 mV were significantly enhanced in a reversible manner by FFA (100 μ

m

) in coronary artery. B and C, FFA decreased PDBu-induced SOC activity in mesenteric artery and portal vein. D, mean data of the effect of FFA on SOC activity in three vascular preparations (n = 5 of each).

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