Adebowale Adebiyi

IP3 Constricts Cerebral Arteries via IP3 Receptor–Mediated TRPC3 Channel Activation and Independently of Sarcoplasmic Reticulum Ca2+ Release

Vasoconstrictors that bind to phospholipase C–coupled receptors elevate inositol-1,4,5-trisphosphate (IP3). IP3 is generally considered to elevate intracellular Ca2+ concentration ([Ca2+]i) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic reticulum (SR)-localized IP3 receptors, leading to intracellular Ca2+ release. We show that IP3 also stimulates vasoconstriction via a SR Ca2+ release–independent mechanism. In isolated cerebral artery myocytes and arteries in which SR Ca2+ was depleted to abolish Ca2+ release (measured using D1ER, a fluorescence resonance energy transfer–based SR Ca2+ indicator), IP3 activated 15 pS sarcolemmal cation channels, generated a whole-cell cation current (ICat) caused by Na+ influx, induced membrane depolarization, elevated [Ca2+]i, and stimulated vasoconstriction. The IP3-induced ICat and [Ca2+]i elevation were attenuated by cation channel (Gd3+, 2-APB) and IP3 receptor (xestospongin C, heparin, 2-APB) blockers. TRPC3 (canonical transient receptor potential 3) channel knockdown with short hairpin RNA and diltiazem and nimodipine, voltage-dependent Ca2+ channel blockers, reduced the SR Ca2+ release–independent, IP3-induced [Ca2+]i elevation and vasoconstriction. In pressurized arteries, SR Ca2+ depletion did not alter IP3-induced constriction at 20 mm Hg but reduced IP3-induced constriction by ≈39% at 60 mm Hg. [Ca2+]i elevations and constrictions induced by endothelin-1, a phospholipase C–coupled receptor agonist, were both attenuated by TRPC3 knockdown and xestospongin C in SR Ca2+-depleted arteries. In summary, we describe a novel mechanism of IP3-induced vasoconstriction that does not occur as a result of SR Ca2+ release but because of IP3 receptor–dependent ICat activation that requires TRPC3 channels. The resulting membrane depolarization activates voltage-dependent Ca2+ channels, leading to a myocyte [Ca2+]i elevation, and vasoconstriction.

Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Rationale: Inositol 1,4,5-trisphosphate (IP3)-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives: Study mechanisms by which IP3Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries. Methods and Results: Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C–coupled receptor agonist, elevated immuno-FRET between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP3R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP3-induced nonselective cation current (ICat) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP3R1 and TRPC3, IP3-induced ICat activation, and ET-1 and IP3-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP3R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R N-terminal region that can interact with TRPC channels activated ICat. A TRPC3 CIRB domain peptide attenuated IP3- and ET-1–induced ICat activation and vasoconstriction. Conclusions: IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.

Caveolin-1 Assembles Type 1 Inositol 1,4,5-Trisphosphate Receptors and Canonical Transient Receptor Potential 3 Channels into a Functional Signaling Complex in Arterial Smooth Muscle Cells

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP3R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (ICat) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP3R1 and TRPC3 channels to communicate locally are unclear. Caveolae are plasma membrane microdomains that can compartmentalize proteins. Here, we tested the hypothesis that caveolae and specifically caveolin-1 (cav-1), a caveolae scaffolding protein, facilitate functional IP3R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-β-cyclodextrin (MβCD), which disassembles caveolae, reduced IP3-induced ICat activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP3-induced vasoconstriction, but did not alter TRPC3 and IP3R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82–101) also attenuated IP3-induced ICat activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP3R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP3R1 are spatially co-localized in arterial smooth muscle cells. IP3R1 and TRPC3 channel spatial localization was disrupted by MβCD and a CSD peptide. Cholesterol replenishment re-established IP3R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP3R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP3-induced physical coupling of these proteins, leading to ICat generation and vasoconstriction.

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

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca(2+)-activated Cl(-) (Cl(Ca)) channels in native cells. The molecular identity of proteins that generate Cl(Ca) currents in smooth muscle cells (SMCs) of resistance-size arteries is unclear. Similarly, whether cerebral artery SMCs generate Cl(Ca) currents is controversial. Here, using molecular biology and patch-clamp electrophysiology, we examined TMEM16A channel expression and characterized Cl(-) currents in arterial SMCs of resistance-size rat cerebral arteries. RT-PCR amplified transcripts for TMEM16A but not TMEM16B-TMEM16H, TMEM16J, or TMEM16K family members in isolated pure cerebral artery SMCs. Western blot analysis using an antibody that recognized recombinant (r)TMEM16A channels detected TMEM16A protein in cerebral artery lysates. Arterial surface biotinylation and immunofluorescence indicated that TMEM16A channels are located primarily within the arterial SMC plasma membrane. Whole cell Cl(Ca) currents in arterial SMCs displayed properties similar to those generated by rTMEM16A channels, including Ca(2+) dependence, current-voltage relationship linearization by an elevation in intracellular Ca(2+) concentration, a Nerstian shift in reversal potential induced by reducing the extracellular Cl(-) concentration, and a negative reversal potential shift when substituting extracellular I(-) for Cl(-). A pore-targeting TMEM16A antibody similarly inhibited both arterial SMC Cl(Ca) and rTMEM16A currents. TMEM16A knockdown using small interfering RNA also inhibited arterial SMC Cl(Ca) currents. In summary, these data indicate that TMEM16A channels are expressed, insert into the plasma membrane, and generate Cl(Ca) currents in cerebral artery SMCs.

Smooth Muscle Cell α2δ-1 Subunits Are Essential for Vasoregulation by CaV1.2 Channels

Rationale: Voltage-dependent L-type (CaV1.2) Ca2+ channels are a heteromeric complex formed from pore-forming &agr;1 and auxiliary &agr;2&dgr; and &bgr; subunits. CaV1.2 channels are the principal Ca2+ influx pathway in arterial myocytes and regulate multiple physiological functions, including contraction. The macromolecular composition of arterial myocyte CaV1.2 channels remains poorly understood, with no studies having examined the molecular identity or physiological functions of &agr;2&dgr; subunits. Objective: We investigated the functional significance of &agr;2&dgr; subunits in myocytes of resistance-size (100 to 200 &mgr;m diameter) cerebral arteries. Methods and Results: &agr;2&dgr;-1 was the only &agr;2&dgr; isoform expressed in cerebral artery myocytes. Pregabalin, an &agr;2&dgr;-1/-2 ligand, and an &agr;2&dgr;-1 antibody, inhibited CaV1.2 currents in isolated myocytes. Acute pregabalin application reversibly dilated pressurized arteries. Using a novel application of surface biotinylation, data indicated that >95% of CaV1.2 &agr;1 and &agr;2&dgr;-1 subunits were present in the arterial myocyte plasma membrane. &agr;2&dgr;-1 knockdown using short hairpin RNA reduced plasma membrane-localized CaV1.2 &agr;1 subunits, caused a corresponding elevation in cytosolic CaV1.2 &agr;1 subunits, decreased intracellular Ca2+ concentration, inhibited pressure-induced vasoconstriction (“myogenic tone”), and attenuated pregabalin-induced vasodilation. Prolonged (24-hour) pregabalin exposure did not alter total &agr;2&dgr;-1 or CaV1.2 &agr;1 proteins but decreased plasma membrane expression of each subunit, which reduced myogenic tone. Conclusions: &agr;2&dgr;-1 is essential for plasma membrane expression of arterial myocyte CaV1.2 &agr;1 subunits. &agr;2&dgr;-1 targeting can block CaV1.2 channels directly and inhibit surface expression of CaV1.2 &agr;1 subunits, leading to vasodilation. These data identify &agr;2&dgr;-1 as a novel molecular target in arterial myocytes, the manipulation of which regulates contractility.

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

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).

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca2+-activated K+ (BKCa) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BKCa channel regulation by IP3 and IP3Rs in rat and mouse cerebral artery smooth muscle cells. IP3 activated BKCa channels both in intact cells and in excised inside-out membrane patches. IP3 caused concentration-dependent BKCa channel activation with an apparent dissociation constant (Kd) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca2+ concentration ([Ca2+]i; 10 µM). IP3 also caused a leftward-shift in BKCa channel apparent Ca2+ sensitivity and reduced the Kd for free [Ca2+]i from ∼20 to 12 µM, but did not alter the slope or maximal Po. BAPTA, a fast Ca2+ buffer, or an elevation in extracellular Ca2+ concentration did not alter IP3-induced BKCa channel activation. Heparin, an IP3R inhibitor, and a monoclonal type 1 IP3R (IP3R1) antibody blocked IP3-induced BKCa channel activation. Adenophostin A, an IP3R agonist, also activated BKCa channels. IP3 activated BKCa channels in inside-out patches from wild-type (IP3R1+/+) mouse arterial smooth muscle cells, but had no effect on BKCa channels of IP3R1-deficient (IP3R1−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP3R1 is located in close spatial proximity to BKCa α subunits. The IP3R1 monoclonal antibody coimmunoprecipitated IP3R1 and BKCa channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP3R1 activation elevates BKCa channel apparent Ca2+ sensitivity through local molecular coupling in arterial smooth muscle cells.

Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane KATP channels

Hydrogen sulfide (H(2)S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H(2)S exist. Here, we tested the hypothesis that piglet cerebral arteriole smooth muscle cells generate ATP-sensitive K(+) (K(ATP)) currents and that H(2)S induces vasodilation by activating K(ATP) currents. Gas chromatography/mass spectrometry data demonstrated that after placing Na(2)S, an H(2)S donor, in solution, it rapidly (1 min) converts to H(2)S. Patch-clamp electrophysiology indicated that pinacidil (a K(ATP) channel activator), Na(2)S, and NaHS (another H(2)S donor) activated K(+) currents at physiological steady-state voltage (-50 mV) in isolated cerebral arteriole smooth muscle cells. Glibenclamide, a selective K(ATP) channel inhibitor, fully reversed pinacidil-induced K(+) currents and partially reversed (∼58%) H(2)S-induced K(+) currents. Western blot analysis indicated that piglet arterioles expressed inwardly rectifying K(+) 6.1 (K(ir)6.1) channel and sulfonylurea receptor 2B (SUR2B) K(ATP) channel subunits. Pinacidil dilated pressurized (40 mmHg) piglet arterioles, and glibenclamide fully reversed this effect. Na(2)S also induced reversible and repeatable vasodilation with an EC(50) of ∼30 μM, and this effect was partially reversed (∼55%) by glibenclamide. Vasoregulation by H(2)S was also studied in pressurized resistance-size cerebral arteries of mice with a genetic deletion in the gene encoding SUR2 (SUR2 null). Pinacidil- and H(2)S-induced vasodilations were smaller in arterioles of SUR2 null mice than in wild-type controls. These data indicate that smooth muscle cell K(ATP) currents control newborn cerebral arteriole contractility and that H(2)S dilates cerebral arterioles by activating smooth muscle cell K(ATP) channels containing SUR2 subunits.

Sulfonylurea Receptor-Dependent and -Independent Pathways Mediate Vasodilation Induced by ATP-Sensitive K+ Channel Openers

ATP-sensitive K+ (KATP) channel openers are vasodilators that activate both plasma membrane and mitochondrial KATP channels. Here, we investigated the molecular mechanisms by which diazoxide and pinacidil induce vasodilation by studying diameter regulation of wild-type [SUR2(+/+)] and sulfonylurea receptor (SUR) 2-deficient [SUR2(-/-)] mouse myogenic mesenteric arteries. Ryanodine (10 μM), a ryanodine-sensitive Ca2+ release (RyR) channel blocker; iberiotoxin (100 nM), a large-conductance Ca2+-activated K+ (KCa) channel blocker; 4-aminopyridine (4-AP; 1 mM), a voltage-gated K+ (KV) channel blocker; manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 100 μM), an antioxidant; and a combination of ryanodine and 4-AP reduced diazoxide (100 μM)-induced dilation in pressurized (60 mm Hg) SUR2(+/+) arteries by 45 to 77%. In contrast, these inhibitors did not alter pinacidil (5 μM)-induced dilation in SUR2(+/+) arteries. Reverse transcription-polymerase chain reaction indicated that SUR2B was the only SUR isoform expressed in SUR2(+/+) mesenteric artery smooth muscle cells, whereas SURs were absent in SUR2(-/-) cells. In SUR2(-/-) arteries, pinacidil-induced vasodilation was ∼10% of that in SUR2(+/+) arteries, whereas diazoxide-induced vasodilation was similar in SUR2(+/+) and SUR2(-/-) arteries. Atpenin (1 μM), a selective electron transport chain (ETC) complex II inhibitor, dilated arteries similarly to diazoxide, and this effect was attenuated by MnTMPyP and ryanodine + 4-AP. Atpenin also attenuated diazoxide-, but not pinacidil-induced vasodilation. In summary, data indicate that pinacidil-induced vasodilation requires SUR2B, whereas diazoxide-induced vasodilation does not require SURs. Rather, diazoxide-induced vasodilation involves ETCII inhibition; a smooth muscle cell-reactive oxygen species elevation; and RyR, KCa, and KV channel activation. These data indicate that KATP channel openers regulate arterial diameter via SUR-dependent and -independent pathways.

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

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 (IP3) that activates sarcoplasmic reticulum IP3 receptors. In cerebral artery myocytes, IP3 receptors release sarcoplasmic reticulum Ca2+ 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 IP3 receptors control vascular contractility in systemic arteries and IP3R 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, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-SHRs and WKY rats. Control, IP3-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 IP3 receptor binding domain peptide, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced cation current and ET-1–induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a sarcoplasmic reticulum Ca2+-ATPase blocker, did not alter ET-1–stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1–induced vasoconstriction during hypertension.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 (IP3) that activates sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs). In cerebral artery myocytes, IP3Rs release SR Ca2+ and can physically couple to canonical transient receptor potential 3 (TRPC3) channels in a caveolin-1-containing macromolecular complex, leading to cation current (ICat) activation that stimulates vasoconstriction. Here, we investigated mechanisms by which IP3Rs control vascular contractility in systemic arteries and IP3R 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 hypertensive spontaneously hypertensive rats (SHR) than in Wistar-Kyoto (WKY) rat controls, respectively. In contrast, IP3R1, TRPC1, TRPC6, and caveolin-1 expression was similar. TRPC3 expression was also similar in arteries of pre-hypertensive SHR and WKY rats. Control, IP3- and endothelin-1 (ET-1)-induced FRET between IP3R1 and TRPC3 was higher in hypertensive SHR than WKY myocytes. IP3-induced ICat was ~3-fold larger in SHR myocytes. Pyr3, a selective TRPC3 channel blocker, and CIRBP-TAT, an IP3R-TRP physical coupling inhibitor, reduced IP3-induced ICat and ET-1-induced vasoconstriction more in SHR than WKY myocytes and arteries. Thapsigargin, a SR Ca2+-ATPase blocker, did not alter ET-1-stimulated vasoconstriction in SHR or WKY arteries. These data indicate that ET-1 stimulates physical coupling of IP3R1 to TRPC3 channels in mesenteric artery myocytes, leading to vasoconstriction. Furthermore, an elevation in IP3R1 to TRPC3 channel molecular coupling augments ET-1-induced vasoconstriction during hypertension.