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

Abstract

,4,5-trisphosphate receptors (IP3Rs) regulate diverse physiological functions, including contraction and proliferation. There are three IP3R isoforms, but their functional significance in arterial smooth muscle cells is unclear. Here, we investigated relative expression and physiological functions of IP3R isoforms in cerebral artery smooth muscle cells. We show that 2-aminoethoxydiphenyl borate and xestospongin C, membrane-permeant IP3R blockers, reduced Ca 2ϩ wave activation and global intracellular Ca 2ϩ ([Ca 2ϩ ]i) elevation stimulated by UTP, a phospholipase C-coupled purinergic receptor agonist. Quantitative PCR, Western blotting, and immunofluorescence indicated that all three IP3R isoforms were expressed in acutely isolated cerebral artery smooth muscle cells, with IP3R1 being the most abundant isoform at 82% of total IP3R message. IP3R1 knockdown with short hairpin RNA (shRNA) did not alter baseline Ca 2ϩ wave frequency and global [Ca 2ϩ ]i but abolished UTP-induced Ca 2ϩ wave activation and reduced the UTP-induced global [Ca 2ϩ ]i elevation by ϳ61%. Antibodies targeting IP3R1 and IP3R1 knockdown reduced UTP-induced nonselective cation current (Icat) activation. IP3R1 knockdown also reduced UTP-induced vasoconstriction in pressurized arteries with both intact and depleted sarcoplasmic reticulum (SR) Ca 2ϩ by ϳ45%. These data indicate that IP 3R1 is the predominant IP3R isoform expressed in rat cerebral artery smooth muscle cells. IP 3R1 stimulation contributes to UTP-induced I cat activation, Ca 2ϩ wave generation, global [Ca 2ϩ ]i elevation, and vasoconstriction. In addition, IP 3R1 activation constricts cerebral arteries in the absence of SR Ca 2ϩ release by stimulating plasma membrane Icat.cerebral artery smooth muscle cells; calcium wave; short hairpin RNA INOSITOL 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are expressed in many cell types and regulate several physiological functions, including development, muscle contraction, cell proliferation, and differentiation (1,45,47,56 Rs (7,43,54). IP 3 also activates cation channels in vascular smooth muscle cells through mechanisms that do not require the stimulation of sarcoplasmic reticulum (SR) Ca 2ϩ release (27, 54). In cerebral artery smooth muscle cells, IP 3 R activation stimulates TRPC3 channels, leading to Na ϩ influx, membrane depolarization, voltage-dependent Ca 2ϩ channel activation, and vasoconstriction (54). Thus IP 3 Rs regulate ion channel activity, Ca 2ϩ signals, and physiological functions in the vasculature. However, despite the functional importance of IP 3 Rs in arterial smooth muscle cells, specific IP 3 R isoforms that are expressed in this cell type and their functional significance are poorly understood.Three IP 3 R isoforms, designated IP 3 R1, IP 3 R2, and IP 3 R3, are encoded by distinct genes (6, 37). IP 3 R isoform expression varies widely between different cell types. Cerebellar Purkinje neurons express predominantly IP 3 R1, pancreatic ␤-cells primarily express IP 3 R3, and cardiac myocytes express IP 3 R2 (14, 48...

Local coupling of TRPC6 to ANO1/TMEM16A channels in smooth muscle cells amplifies vasoconstriction in cerebral arteries

“…An anti-ANO1 antibody pulled down ANO1 together with a strong signal for TRPC6 channels in rat cerebral artery lysate. Blots were also probed for TRPC3, TRPM4, and IP 3 R1, the principal functional IP 3 R isoform in cerebral artery myocytes (35). A very faint IP 3 R1 band was observed, but TRPC3 and TRPM4 were absent in the ANO1 immunoprecipitate.…”

TMEM16A channels generate Ca2+-activated Cl− currents in cerebral artery smooth muscle cells

“…Isolated SMCs were fixed and prepared as previously described (39). Immunofluorescence was also performed on cells using the same protocol except that cells were not permeabilized with Triton X-100.…”

“…The relative anion permeability ratio of I Ϫ to Cl Ϫ (PI/PCl) was calculated using the calculated shift in reversal potential (Erev) TMEM16A channel knockdown. Three small interfering (si)RNA sequences targeting TMEM16A or negative control siRNA (Invitrogen) were inserted into cerebral arteries using reverse permeabilization as previously described (1,3,4,25,39). Arteries were then maintained in serum-free DMEM-F-12 media supplemented with 1% penicillin-streptomycin (Sigma) for 4 days at 37°C in a sterile incubator (21% O2-5% CO2).…”

The Proteome of Mouse Cerebral Arteries

“…26 We detected three ITPRs, namely ITPR1, 2, and 3, all previously showed to be present in rat cerebral artery SMCs with ITPR1 being the most abundant. 27 In addition, the second messenger 1,2-diacylglycerol has been shown to activate PKC, which in turn increases smooth muscle contractility via several different mechanisms such as MLCP inhibition and increased actin availability. 20 Several pathways of Ca 2 þ entry from the extracellular space have been suggested, and include passive leak Ca 2 þ channels, as well as channels activated by membrane depolarization (voltage gated), agonist binding (receptor operated), depletion of Ca 2 þ from intracellular stores (store operated), or stretch.…”