Role of TRPC1 and TRPC3 channels in contraction and relaxation of mouse thoracic aorta

Abstract

Background/aims: Canonical transient receptor potential (TRPC) channels modulate membrane potential and intracellular Ca(2+). We examined the role of TRPC1 and TRPC3 channels in vasocontraction and relaxation in mouse aorta.

Methods: Vasocontraction and relaxation of aorta from wild-type (WT), TRPC1 KO and TRPC3 knockout (KO) mice were measured for phenylephrine (Phe) and carbachol (CCh). Intracellular Ca(2+) was measured in primary aorta endothelial cells (EC) and whole cell K(+) current in freshly isolated smooth muscle cells (SMC).

Results and conclusion: TRPC1 KO aorta showed increased vasocontraction to Phe compared to WT and TRPC3 KO aorta due to diminished role of BK(Ca) channels. BK(Ca) mRNA (aorta) and whole cell current (SMC) were reduced versus WT. Contraction in WT aorta was increased to TRPC1 KO level by BK(Ca) channel inhibition. Relaxation to CCh was reduced in TRPC1 KO and TRPC3 KO aortas with concomitant reduction in EC Ca(2+) response. Pyr3 (TRPC3 blocker) reduced the Ca(2+) response to CCh in EC from WT, but not TRPC3 KO mice. In summary, TRPC1 attenuates receptor-mediated contraction through activation and/or expression of SMC BK(Ca) channels while TRPC3 does not contribute to receptor-mediated constriction. Both TRPC1 and TRPC3 participate in EC Ca(2+) influx and vasorelaxation of aorta.

Fig 1

The effect of TRPC1 and TRPC3 gene KO on isometric contraction of mouse aorta: (A) Representative traces demonstrating changes in aorta tension after application of 3 μM phenylephrine (Phe) and (B) Average tension developed to Phe in aorta from WT (n = 23), TRPC1 KO (n = 17) or TRPC3 KO (n = 12) mice. * - p<0.05.

Fig 2

The role of large conductance Ca activated potassium channels (BK) in modulation of aorta contractile response in WT and TRPC1 KO mice. (A) Average contraction to phenylephrine (3 μM) in aorta pretreated with penitrem A (0.2 μM, pen) or vehicle (0.2 % DMSO, con) for 5 min (n=6 for each WT and KO groups). (B, left panel) Membrane current in aorta smooth muscle cells activated by command voltage changes shown on the upper panel. Scatter plots – baseline current in untreated cells from WT (black circles) and TRPC1 KO (gray circles) mice. Line plots – representative traces acquired after application of phenyephrine (25 uM) to WT (black) or TRPC1 KO (gray) cells. (B, right panel) Average density of phenylephrine activated outward current at 60 mV in WT (n=5) and TRPC1 KO (n=6) smooth muscle cells. (C) Expression of BK_Ca channel alpha subunit mRNA by real time PCR in aorta from WT and TRPC1 KO mice (n=6 each). Expression is reported relative to β-actin.

Fig 3

The effect of TRPC1 and TRPC3 gene KO on isometric relaxation of mouse aorta. (A) Representative relaxation recordings of mouse aorta in response to carbachol (CCh, 10 μM) from WT(black), TRPC1 KO (gray) and TRPC3 KO (white circles) mice. (B) Average relaxation to CCh normalized to the maximal constriction to phenylephrine (3 μM). * - p < 0.05 in TRPC1 KO (n = 16) or TRPC3 KO (n = 12) vs. WT group (n = 20).

Fig 4

Dependence of EC CCh-induced Ca response on extracellular Ca and PLC-IP₃ signaling pathway. (A) EC were superfused with the regular bath saline (1.6 mM Ca, control) or “0 Ca” saline (Ca-free, 4 mM BAPTA). CCh (100 uM) was applied at the point indicated by the arrow. (B) CCh induced increase in Fura-2 quenching. Fura-2 fluorescence at the F₃₆₀ was recorded in the presence of 1 mM Mn²⁺ for 4.5 min before application of CCh to obtain baseline quenching rate. Then CCh (100 μM) was applied (arrow) in the same Mn-containing saline. Dashed line reflects an extension of the basal quenching rate before CCh. (C) Cells were pretreated with 0.1 % DMSO or PLC inhibitor U73122 for 5 min before application of CCh (100 μM). (D) Cells were preincubated with 50 μM XeC of vehicle (5 % DMSO) for 30 min before application of CCh (100 μM).

Fig 5

Intracellular Ca²⁺ responses to CCh in EC from TRPC1 (left) and TRPC3 KO mice (right). Average Ca response to CCh (100 μM) was determined from 22–41 coverslips derived from EC of 3 to 17 mice per group. All cells per a given coverslip were averaged to obtain a single Ca response. All coverslip responses were then averaged and presented as mean±SE. The region where statistical difference between individual points exists between groups (Holm-Sidak test) is indicated by the horizontal line.

Fig 6

The effect of a TRPC3 channel blocker (Pyr3) on the CCh-induced Ca response in EC from WT, TRPC1 KO, and TRPC3 KO mice. Cells were pretreated with Pyr3 for 10 min before the recording after which Pyr3 was continuously present in the bath. DMSO (0.1 %) was used as a vehicle control. CCh (100 μM) was applied at the time indicated by arrow.