An elevation in physical coupling of type 1 inositol 1,4,5-trisphosphate (IP3) receptors to transient receptor potential 3 (TRPC3) channels constricts mesenteric arteries in genetic hypertension

Abstract

Hypertension is associated with an elevation in agonist-induced vasoconstriction, but mechanisms involved require further investigation. Many vasoconstrictors bind to phospholipase C-coupled receptors, leading to an elevation in inositol 1,4,5-trisphosphate (IP(3)) that activates sarcoplasmic reticulum IP(3) receptors. In cerebral artery myocytes, IP(3) receptors release sarcoplasmic reticulum Ca(2+) and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP(3) receptors control vascular contractility in systemic arteries and IP(3)R involvement in elevated agonist-induced vasoconstriction during hypertension. Total and plasma membrane-localized TRPC3 protein was ≈2.7- and 2-fold higher in mesenteric arteries of spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP(3)R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP(3)-induced and endothelin-1 (ET-1)-induced fluorescence resonance energy transfer between IP3R1 and TRPC3 was higher in SHR than WKY myocytes. IP3-induced cation current was ≈3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and calmodulin and IP(3) receptor binding domain peptide, an IP(3)R-TRP physical coupling inhibitor, reduced IP(3)-induced cation current and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca(2+)-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP(3)R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP(3)R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.

Figure 1. IP₃R1, TRPC3 and cav-1 are located in a macromolecular complex in rat mesenteric arteries

Monoclonal mouse anti-IP₃R1 antibody co-immunoprecipitated IP₃R1 (~270 kDa), TRPC3 (~90 kDa), and cav-1 (~ 22 kDa). Lysate supernatant (~40 µg protein) was used as the input control and mouse IgG as the negative control. Arteries pooled from ~ 8 rats were used in this experiment.

Figure 2. Hypertension is associated with an elevation in TRPC3, but not TRPC1, TRPC6, IP₃R1 or cav-1, protein in arteries

A–E, Western blots illustrating that TRPC3, but not TRPC1, TRPC6, IP₃R1 or cav-1 protein, is higher in mesenteric arteries of hypertensive (13 week old) SHR than age-matched WKY rats. F, Western blot of TRPC3 in 6 week old pre-hypertensive SHR and age-matched WKY rat arteries. G, mean data. N=4–5; *P<0.05.

Figure 3. Plasma membrane-localized TRPC3 channel protein is higher in hypertensive SHR than WKY rat arteries

A, Western blot and B, mean data (n=4) illustrating cellular distribution of TRPC3 in SHR and WKY rat arteries. C, mean data indicating that surface TRPC3 channel protein is higher in hypertensive SHR than WKY arteries (n=4). Protein samples were run on the same gel, but lanes were not contiguous. *P<0.05

Figure 4. Hypertension is associated with an elevation in spatial localization of IP₃R1 and TRPC3 channels in mesenteric artery myocytes

A, Exemplar images illustrating imunofluorescence and N-FRET for IP₃R1 and TRPC3 channels in hypertensive SHR and WKY rat isolated myocytes. Shown are fluorescent images generated by Alexa 488- and 546-conjugated antibodies, pixel overlay, and N-FRET for the same cells. B, mean N-FRET data for IP₃R1 and TRPC3 channels in 13 week old rat myocytes (untreated control: WKY, n=9; SHR, n=6, Bt-IP₃, 10 nM; WKY, n=7; SHR, n=11, and ET-1, 10 nM; WKY, n=6; SHR, n=6). C, mean N-FRET data for IP₃R1 and TRPC3 channels in 6 week old rat myocytes (untreated control: WKY, n=6; SHR, n=6) and ET-1 (10 nM; WKY, n=6; SHR, n=7). *P<0.05 versus WKY basal N-FRET; ^#P<0.05 versus SHR basal N-FRET; ^†P<0.05 versus WKY in the same condition. Scale bar, 10 µm.

Figure 5. IP₃-induced I_Cat is larger in hypertensive SHR than WKY rat arterial myocytes

A, Exemplar traces and B, mean data indicating that control I_Cat density is similar and IP₃ (10 µM)-induced I_Cat is larger in hypertensive SHR than WKY rat myocytes. Pyr3 (1 µM) and CIRBP-TAT (10 µM) inhibited IP₃-induced I_Cat in both WKY and hypertensive SHR myocytes. Control WKY, n=4; control SHR, n=5; IP₃ WKY, n=6; IP₃ SHR, n=6; IP₃+Pyr3 WKY, n=7; IP₃+Pyr3 SHR, n=6; IP₃+CIRBP-TAT WKY, n=5; IP₃+CIRBP-TAT SHR, n=5. *P<0.05, versus control; ^†P<0.05 versus WKY; ^#P<0.05 versus IP₃.

Figure 6. ET-1-induced vasoconstriction is larger in hypertensive SHR arteries, independent of SR Ca²⁺ release, and attenuated by 2-APB, Pyr3 and CIRB-TAT

A, ET-1 concentration-response in SHR (EC₅₀, 3.05 nM, n=8) and WKY (EC₅₀, 2.4 nM, n=6) arterial rings. B, mean data (SHR, n=6 for each; WKY, n=8 for each) in the absence and presence of thapsigargin (Thapsi; 100 nM). C, mean data illustrating that 2-APB (50 µM), Pyr3 (1 µM) and CIRBP-TAT (3 µM) more effectively inhibit ET-1 (10 nM)-induced constriction in SHR arteries (n=6–9) *P<0.05, versus WKY; ^#P<0.05 versus control.