Type 1 inositol 1,4,5-trisphosphate receptors mediate UTP-induced cation currents, Ca2+ signals, and vasoconstriction in cerebral arteries

Abstract

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP(3)R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP(3)R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP(3)R blockers, reduced Ca(2+) wave activation and global intracellular Ca(2+) ([Ca(2+)](i)) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP(3)R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP(3)R1 being the most abundant isoform at 82% of total IP(3)R message. IP(3)R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca(2+) wave frequency and global [Ca(2+)](i) but abolished UTP-induced Ca(2+) wave activation and reduced the UTP-induced global [Ca(2+)](i) elevation by approximately 61%. Antibodies targeting IP(3)R1 and IP(3)R1 knockdown reduced UTP-induced nonselective cation current (I(cat)) activation. IP(3)R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca(2+) by approximately 45%. These data indicate that IP(3)R1 is the predominant IP(3)R isoform expressed in rat cerebral artery smooth muscle cells. IP(3)R1 stimulation contributes to UTP-induced I(cat) activation, Ca(2+) wave generation, global [Ca(2+)](i) elevation, and vasoconstriction. In addition, IP(3)R1 activation constricts cerebral arteries in the absence of SR Ca(2+) release by stimulating plasma membrane I(cat).

Fig. 1.

UTP activates Ca²⁺ waves and elevates global intracellular Ca²⁺ concentration ([Ca²⁺]_i) as a result of inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) activation in cerebral artery smooth muscle cells. A: Ca²⁺ signals recorded in smooth muscle cells of an intact cerebral artery under control conditions and in the presence of UTP (30 μM) and UTP + 2-aminoethoxydiphenyl borate (2-APB, 100 μM). Top: colored boxes (2.2 × 2.2 μm, 10 × 10 pixels) indicate locations of changes in fluorescence ratio (F/F₀) measured over 10 s in arterial smooth muscle cells. Bottom: colored traces showing changes in F/F₀ for respective colored boxes over 10 s. Images were acquired at 30 Hz. B: mean data illustrating inhibition of UTP-induced Ca²⁺ waves in smooth muscle cells of cerebral artery segments by 2-APB (100 μM) and xestospongin C (XeC, 20 μM). C: UTP-induced global [Ca²⁺]_i elevations are abolished by 2-APB and attenuated by XeC. Changes in global [Ca²⁺]_i were calculated from a control value previously determined using fura-2 (10). Values are means ± SE; n = 27, 13, 4, and 9 for control, UTP, UTP + 2-APB, and UTP + XeC, respectively. *P < 0.05 vs. control. #P < 0.05 vs. UTP.

Fig. 2.

PCR, Western analysis, and immunofluorescence identify IP₃R1, IP₃R2, and IP₃R3 expression in cerebral artery smooth muscle cells. A: RT-PCR detection of IP₃R1 (285 bp), IP₃R2 (241 bp), IP₃R3 (319 bp), and β-actin (257 bp) in isolated smooth muscle cells. NC, negative control using smooth muscle cell RNA, instead of cDNA. Right: 50-bp-increment ladders are shown. B: real-time PCR data indicating percent message for IP₃R1, IP₃R2, and IP₃R3 in isolated smooth muscle cells. Values are means ± SE; n = 3 for each. C: Western blot detection of IP₃R1, IP₃R2, and IP₃R3 in cerebral arteries and brain. D: immunofluorescence results showing IP₃R1, IP₃R2, and IP₃R3 expression and localization in isolated smooth muscle cells.

Fig. 3.

IP₃R1 short hairpin RNA (shRNA) suppression vectors attenuate IP₃R1 protein in cerebral arteries. A: Western blot illustrating that IP₃R1shV1, IP₃R1shV2, and IP₃R1shV1 + IP₃R1shV2 reduce IP₃R1 protein in cerebral arteries. B: mean data illustrating that shRNAs targeting IP₃R1 reduce IP₃R1 expression, but do not alter IP₃R3 expression, in cerebral arteries. Values are means ± SE; n = 5, 4, 3, and 3 for IP₃R1shV1, IP₃R1shV2, and IP₃R1shV1 + IP₃R1shV2 (IP3R1) and IP₃R1shV1 + IP₃R1shV2 (IP3R3), respectively. *P < 0.05 vs. IP₃R1scrm.

Fig. 4.

IP₃R1 knockdown attenuates UTP- induced Ca²⁺ wave generation and global [Ca²⁺]_i elevation in smooth muscle cells of intact cerebral artery segments. A: IP₃R1 knockdown did not alter resting Ca²⁺ wave frequency but altered UTP-induced Ca²⁺ wave generation (n = 6). B: IP₃R1 knockdown reduced UTP-induced global [Ca²⁺]_i elevation (n = 6). Values show mean change in calibrated global [Ca²⁺]_i from a control value of 193 nM (10). C: amplitude of caffeine (10 mM)-induced [Ca²⁺]_i transients were similar in IP₃R1scrm- and IP₃R1shV-treated cerebral arteries. D: mean change in fura-2 ratio (Δ340/380) in cerebral arteries treated with IP₃R1scrm (n = 16) and IP₃R1shV (n = 14). Values are means ± SE. *P < 0.05 vs. control. #P < 0.05 vs. IP₃R1scrm.

Fig. 5.

Anti-IP₃R1 antibody inhibits UTP-induced cation current (I_cat) in rat cerebral artery smooth muscle cells. A: exemplar traces obtained using whole cell patch-clamp configuration with anti-IP₃R1 (1:100 dilution) or its heat-denatured control in the pipette before or after UTP (30 μM) application. B: mean data showing the effect of anti-IP₃R1 antibody on I_cat. Values are means ± SE; n = 11 (denatured control Ab) and 5 (all others). *P < 0.05 vs. control. #P < 0.05 vs. denatured Ab.

Fig. 6.

IP₃R1 knockdown inhibits I_cat activation by UTP in cerebral artery smooth muscle cells. A: exemplar traces illustrating UTP-induced I_cat activation in IP₃R1scrm- and IP₃R1shV-treated smooth muscle cells. B: mean current density data for IP₃R1scrm- and IP₃R1shV-treated cells. *P < 0.05 vs. control. #P < 0.05 vs. IP₃R1scrm.

Fig. 7.

IP₃R1 knockdown attenuates UTP-induced constriction in pressurized (20 mmHg) cerebral arteries. A: exemplar diameter measurements illustrating UTP-induced vasoconstriction in cerebral arteries treated with IP₃R1scrm or IP₃R1shV in the presence or absence of thapsigargin (Tg, 100 nM). B: mean diameter change data. Values are means ± SE; n = 6 (UTP and UTP + 100 nM Tg) and 4 (UPT + 1 μM Tg). *P < 0.05 vs. IP₃R1scrm.