AB9074 Sigma-AldrichAnti-IP3 Receptor 2 Antibody

There are 3 major sweat-producing glands present in skin; eccrine, apocrine, and apoeccrine glands. Due to the high rate of secretion, eccrine sweating is a vital regulator of body temperature in response to thermal stress in humans; therefore, an inability to sweat (anhidrosis) results in heat intolerance that may cause impaired consciousness and death. Here, we have reported 5 members of a consanguineous family with generalized, isolated anhidrosis, but morphologically normal eccrine sweat glands. Whole-genome analysis identified the presence of a homozygous missense mutation in ITPR2, which encodes the type 2 inositol 1,4,5-trisphosphate receptor (InsP3R2), that was present in all affected family members. We determined that the mutation is localized within the pore forming region of InsP3R2 and abrogates Ca2+ release from the endoplasmic reticulum, which suggests that intracellular Ca2+ release by InsP3R2 in clear cells of the sweat glands is important for eccrine sweat production. Itpr2-/- mice exhibited a marked reduction in sweat secretion, and evaluation of sweat glands from Itpr2-/- animals revealed a decrease in Ca2+ response compared with controls. Together, our data indicate that loss of InsP3R2-mediated Ca2+ release causes isolated anhidrosis in humans and suggest that specific InsP3R inhibitors have the potential to reduce sweat production in hyperhidrosis.

When arteries constrict to agonists, the endothelium inversely responds, attenuating the initial vasomotor response. The basis of this feedback mechanism remains uncertain, although past studies suggest a key role for myoendothelial communication in the signaling process. The present study examined whether second messenger flux through myoendothelial gap junctions initiates a negative-feedback response in hamster retractor muscle feed arteries. We specifically hypothesized that when agonists elicit depolarization and a rise in second messenger concentration, inositol trisphosphate (IP(3)) flux activates a discrete pool of IP(3) receptors (IP(3)Rs), elicits localized endothelial Ca(2+) transients, and activates downstream effectors to moderate constriction. With use of integrated experimental techniques, this study provided three sets of supporting observations. Beginning at the functional level, we showed that blocking intermediate-conductance Ca(2+)-activated K(+) channels (IK) and Ca(2+) mobilization from the endoplasmic reticulum (ER) enhanced the contractile/electrical responsiveness of feed arteries to phenylephrine. Next, structural analysis confirmed that endothelial projections make contact with the overlying smooth muscle. These projections retained membranous ER networks, and IP(3)Rs and IK channels localized in or near this structure. Finally, Ca(2+) imaging revealed that phenylephrine induced discrete endothelial Ca(2+) events through IP(3)R activation. These events were termed recruitable Ca(2+) wavelets on the basis of their spatiotemporal characteristics. From these findings, we conclude that IP(3) flux across myoendothelial gap junctions is sufficient to induce focal Ca(2+) release from IP(3)Rs and activate a discrete pool of IK channels within or near endothelial projections. The resulting hyperpolarization feeds back on smooth muscle to moderate agonist-induced depolarization and constriction.

We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1greater than IP(3)R2greater than IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.

Dysregulation of Ca(2+) signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca(2+) signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca(2+) release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca(2+) release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca(2+) release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca(2+) dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca(2+) signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders.

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca(2+)) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP(3)Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca(2+) signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP(3)R-mediated intracellular Ca(2+) responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca(2+) signaling and its related functions.