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

Transmembrane protein (TMEM)16A channels are recently discovered membrane proteins that display electrophysiological properties similar to classic Ca²⁺-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 Ca²⁺ dependence, current-voltage relationship linearization by an elevation in intracellular Ca²⁺ 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.

Acute kidney injury (AKI), a significant complication of cisplatin chemotherapy is associated with reactive oxygen species (ROS)-dependent renal cell death, but the cellular targets of ROS in cisplatin nephrotoxicity are not fully resolved. Here, we investigated cisplatin-induced oxidative renal damage and tested the hypothesis that ROS-dependent shedding of death activator Fas ligand (FasL) occurs in cisplatin nephropathy. We show that intraperitoneal injection of sulfobutyl ether-β-cyclodextrin (Captisol™)-solubilized cisplatin elevated the level of lipid peroxidation product malondialdehyde in mouse kidneys and urinary concentration of oxidative DNA damage biomarker 8-hydroxy-2′-deoxyguanosine. Cisplatin increased mouse kidney-to-body weight ratio and the plasma or urinary levels of predictive biomarkers of AKI, including creatinine, blood urea nitrogen, microalbumin, neutrophil gelatinase-associated lipocalin, and cystatin C. Histological analysis and dUTP nick end labeling of kidney sections indicated tubular injury and renal apoptosis, respectively in cisplatin-treated mice. Whereas the plasma concentration of soluble FasL (sFasL) was unaltered, urinary sFasL was increased ∼4-fold in cisplatin-treated mice. Real-time quantitative live-cell imaging and lactate dehydrogenase assay showed that cisplatin stimulated caspase 3/7 activation and cytotoxicity in a human proximal tubule epithelial cell line which were attenuated by inhibitors of the FasL/Fas system and poly [ADP-ribose] polymerase-1. Moreover, TEMPOL, an intracellular free radical scavenger mitigated cisplatin-induced renal oxidative stress and injury, AKI biomarker and urinary sFasL elevation, and proximal tubule cell death. Our findings indicate that cisplatin-induced oxidative stress triggers the shedding of membrane-bound FasL to sFasL in the kidney. We demonstrate that cisplatin elicits nephrotoxicity by promoting FasL/Fas-dependent oxidative renal tubular cell death.

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are a family of tetrameric intracellular calcium (Ca²⁺) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP₃R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca²⁺signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP₃R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP₃R channel activity. SMC IP₃Rs communicate with nearby ryanodine-sensitive Ca²⁺channels and mitochondria to influence SR Ca²⁺release and reactive oxygen species generation. IP₃R-mediated Ca²⁺release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca²⁺channels. SMC IP₃Rs also signal to other proteins via SR Ca²⁺release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca²⁺-activated potassium channels. IP₃R-mediated Ca²⁺release generates a wide variety of intracellular Ca²⁺signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP₃R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP₃R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP₃R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP₃R signaling contributing to disease.

Rationale : Inositol 1,4,5-trisphosphate (IP ₃ )-induced vasoconstriction can occur independently of intracellular Ca ²⁺ release and via IP ₃ receptor (IP ₃ R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear. Objectives : Study mechanisms by which IP ₃ Rs 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 IP ₃ Rs (IP ₃ R1) 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 IP ₃ R1 and TRPC3, but not between IP ₃ R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP ₃ R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP ₃ -induced nonselective cation current ( I _Cat ) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP ₃ R1 and TRPC3, IP ₃ -induced I _Cat activation, and ET-1 and IP ₃ -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 IP ₃ R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP ₃ R N-terminal region that can interact with TRPC channels activated I _Cat . A TRPC3 CIRB domain peptide attenuated IP ₃ - and ET-1–induced I _Cat activation and vasoconstriction. Conclusions : IP ₃ stimulates direct coupling between IP ₃ R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to I _Cat activation and vasoconstriction. Close spatial proximity between IP ₃ R1 and TRPC3 establishes this isoform-selective functional interaction.

Physical coupling of sarcoplasmic reticulum (SR) type 1 inositol 1,4,5-trisphosphate receptors (IP(3)R1) to plasma membrane canonical transient receptor potential 3 (TRPC3) channels activates a cation current (I(Cat)) in arterial smooth muscle cells that induces vasoconstriction. However, structural components that enable IP(3)R1 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 IP(3)R1 to TRPC3 coupling in smooth muscle cells of resistance-size cerebral arteries. Methyl-β-cyclodextrin (MβCD), which disassembles caveolae, reduced IP(3)-induced I(Cat) activation in smooth muscle cells and vasoconstriction in pressurized arteries. Cholesterol replenishment reversed these effects. Cav-1 knockdown using shRNA attenuated IP(3)-induced vasoconstriction, but did not alter TRPC3 and IP(3)R1 expression. A synthetic peptide corresponding to the cav-1 scaffolding domain (CSD) sequence (amino acids 82-101) also attenuated IP(3)-induced I(Cat) activation and vasoconstriction. A cav-1 antibody co-immunoprecipitated cav-1, TRPC3, and IP(3)R1 from cerebral artery lysate. ImmunoFRET indicated that cav-1, TRPC3 channels and IP(3)R1 are spatially co-localized in arterial smooth muscle cells. IP(3)R1 and TRPC3 channel spatial localization was disrupted by MβCD and a CSD peptide. Cholesterol replenishment re-established IP(3)R1 and TRPC3 channel close spatial proximity. Taken together, these data indicate that in arterial smooth muscle cells, cav-1 co-localizes SR IP(3)R1 and plasma membrane TRPC3 channels in close spatial proximity thereby enabling IP(3)-induced physical coupling of these proteins, leading to I(Cat) generation and vasoconstriction.