Cyfluthrin exposure during pregnancy causes neurotoxicity in offspring-Ca2+ overload via IP3R-GRP75-VDAC1 pathway.

Cyfluthrin (Cy) is a widely used pyrethroid insecticide. There is growing evidence that Cy can cause damage to the nervous, reproductive, and immune systems, but there is limited evidence on the potential effects of maternal Cy exposure on offspring. A model of maternal Cy exposure was used to assess its neurobehavioral effects on young-adult offspring. We found that gestational Cy exposure affected pregnancy outcomes and fetal development, and that offspring showed impairments in anxiety as well as learning and memory, accompanied by impairments in hippocampal synaptic ultrastructure and synaptic plasticity. In addition, the IP3R-GRP75-VDAC1 apoptogenic pathway was also upregulated, and in vitro models showed that inhibition of this pathway alleviated neuronal apoptosis as well as synaptic plasticity damage. In conclusion, maternal Cy exposure during pregnancy can cause neurobehavioral abnormalities and synaptic damage in offspring, which may be related to neuronal apoptosis induced by activation of the IP3R-GRP75-VDAC1 pathway in the hippocampus of offspring. Our findings provide clues to understand the neurotoxicity mechanism of maternal Cy exposure to offspring during pregnancy.

Physiological testosterone attenuates profibrotic activities of rat cardiac fibroblasts through modulation of nitric oxide and calcium homeostasis.

Testosterone deficiency is associated with poor prognosis among patients with chronic heart failure (HF). Physiological testosterone improves the exercise capacity of patients with HF. In this study, we evaluated whether treatment with physiological testosterone contributes to anti-fibrogenesis by modifying calcium homeostasis in cardiac fibroblasts and we studied the underlying mechanisms. Nitric oxide (NO) analyses, calcium (Ca2+) fluorescence, and Western blotting were performed in primary isolated rat cardiac fibroblasts with or without (control cells) testosterone (10, 100, 1,000 nmol/L) treatment for 48 hours. Physiological testosterone (10 nmol/L) increased NO production and phosphorylation at the inhibitory site of the inositol trisphosphate (IP3) receptor, thereby reducing Ca2+ entry, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) expression, type I and type III pro-collagen production. Non-physiological testosterone-treated fibroblasts exhibited similar NO and collagen production capabilities as compared to control (testosterone deficient) fibroblasts. These effects were blocked by co-treatment with NO inhibitor (L-NG-nitro arginine methyl ester [L-NAME], 100 µmol/L). In the presence of the IP3 receptor inhibitor (2-aminoethyl diphenylborinate [2-APB], 50 µmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar phosphorylated CaMKII expression. When treated with 2-APB or CaMKII inhibitor (KN93, 10 µmol/L), testosterone-deficient and physiological testosterone-treated fibroblasts exhibited similar type I, and type III collagen production. In conclusion, physiological testosterone activates NO production, and attenuates the IP3 receptor/Ca2+ entry/CaMKII signaling pathway, thereby inhibiting the collagen production capability of cardiac fibroblasts.

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wild-type Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wild-type ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT), inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high-affinity receptor), and ITPR1-mediated cytoplasmic free calcium release in vitro. In addition, we show that gene transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT-mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

The role of Rho-kinase and calcium ions in constriction triggered by ET-1.

Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca2+ related to activation of IP3 receptors and to Rho-kinase activity.

Functional Tuning of Intrinsic Endothelial Ca2+ Dynamics in Swine Coronary Arteries.

RATIONALE: Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca(2+) transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca(2+) is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca(2+) signals in the coronary endothelium is poorly understood. OBJECTIVE: We aimed to determine whether a discrete spatial and temporal profile of Ca(2+) dynamics underlies endothelium-dependent relaxation of swine coronary arteries. METHODS AND RESULTS: Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca(2+) dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca(2+). In vessel rings, inhibition of specific Ca(2+)-dependent endothelial effectors, namely, small and intermediate conductance K(+) channels (K(Ca)3.1 and K(Ca)2.3) and endothelial nitric oxide synthase, produces additive tone, which is blunted by internal store depletion or inositol 1,4,5-trisphosphate receptor blockade. Stimulation of endothelial inositol 1,4,5-trisphosphate-dependent signaling with substance P causes idiosyncratic changes in dynamic Ca(2+) signal parameters (active sites, event frequency, amplitude, duration, and spatial spread). Overall, substance P-induced vasorelaxation corresponded poorly with whole-field endothelial Ca(2+) measurements but corresponded precisely with the concentration-dependent change in Ca(2+) dynamics (linearly translated composite of dynamic parameters). CONCLUSIONS: Our findings show that endothelium-dependent control of swine coronary artery tone is determined by spatial and temporal titration of inherent endothelial Ca(2+) dynamics that are not represented by tissue-level averaged Ca(2+) changes.

Can endothelial hemoglobin-α regulate nitric oxide vasodilatory signaling?

We used mathematical modeling to investigate nitric oxide (NO)-dependent vasodilatory signaling in the arteriolar wall. Detailed continuum cellular models of calcium (Ca2+) dynamics and membrane electrophysiology in smooth muscle and endothelial cells (EC) were coupled with models of NO signaling and biotransport in an arteriole. We used this theoretical approach to examine the role of endothelial hemoglobin-α (Hbα) as a modulator of NO-mediated myoendothelial feedback, as previously suggested in Straub et al. (Nature 491: 473-477, 2012). The model considers enriched expression of inositol 1,4,5-triphosphate receptors (IP3Rs), endothelial nitric oxide synthase (eNOS) enzyme, Ca2+-activated potassium (KCa) channels and Hbα in myoendothelial projections (MPs) between the two cell layers. The model suggests that NO-mediated myoendothelial feedback is plausible if a significant percentage of eNOS is localized within or near the myoendothelial projection. Model results show that the ability of Hbα to regulate the myoendothelial feedback is conditional to its colocalization with eNOS near MPs at concentrations in the high nanomolar range (>0.2 µM or 24,000 molecules). Simulations also show that the effect of Hbα observed in in vitro experimental studies may overestimate its contribution in vivo, in the presence of blood perfusion. Thus, additional experimentation is required to quantify the presence and spatial distribution of Hbα in the EC, as well as to test that the strong effect of Hbα on NO signaling seen in vitro, translates also into a physiologically relevant response in vivo.NEW & NOTEWORTHY Mathematical modeling shows that although regulation of nitric oxide signaling by hemoglobin-α (Hbα) is plausible, it is conditional to its presence in significant concentrations colocalized with endothelial nitric oxide synthase in myoendothelial projections. Additional experimentation is required to test that the strong effect of Hbα seen in vitro translates into a physiologically relevant response in vivo.

Mitochondrial Dysfunction and Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2.

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells.

Motor-Skill Learning Is Dependent on Astrocytic Activity.

Motor-skill learning induces changes in synaptic structure and function in the primary motor cortex through the involvement of a long-term potentiation- (LTP-) like mechanism. Although there is evidence that calcium-dependent release of gliotransmitters by astrocytes plays an important role in synaptic transmission and plasticity, the role of astrocytes in motor-skill learning is not known. To test the hypothesis that astrocytic activity is necessary for motor-skill learning, we perturbed astrocytic function using pharmacological and genetic approaches. We find that perturbation of astrocytes either by selectively attenuating IP3R2 mediated astrocyte Ca(2+) signaling or using an astrocyte specific metabolic inhibitor fluorocitrate (FC) results in impaired motor-skill learning of a forelimb reaching-task in mice. Moreover, the learning impairment caused by blocking astrocytic activity using FC was rescued by administration of the gliotransmitter D-serine. The learning impairments are likely caused by impaired LTP as FC blocked LTP in slices and prevented motor-skill training-induced increases in synaptic AMPA-type glutamate receptor in vivo. These results support the conclusion that normal astrocytic Ca(2+) signaling during a reaching task is necessary for motor-skill learning.

Intracellular Calcium Plays a Critical Role in the Microcystin-LR-Elicited Neurotoxicity Through PLC/IP3 Pathway.

Neurotoxicity of microcystin-leucine-arginine (MCLR) has been widely reported. However, the mechanism is not fully understood. Using primary hippocampal neurons, we tested the hypothesis that MCLR-triggered activation in intracellular free calcium concentration ([Ca(2+)](i)) induces the death of neurons. Microcystin-leucine-arginine inhibited cell viability at a range of 0.1 to 30 µmol/L and caused a dose-dependent increase in [Ca(2+)](i). This increase in [Ca(2+)](i) was observed in Ca(2+)-free media and blocked by an endoplasmic reticulum Ca(2+) pump inhibitor, suggesting intracellular Ca(2+) release. Moreover, pretreatment of hippocampal neurons with intracellular Ca(2+) chelator (O,O'-bis (2-aminophenyl) ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxy-methyl ester) and inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenyl borate) could block both the Ca(2+) mobilization and the neuronal death following MCLR exposure. In contrast, the ryanodine receptor inhibitor (dantrolene) did not ameliorate the effect of MCLR. In conclusion, MCLR disrupts [Ca(2+)](i) homeostasis in neurons by releasing Ca(2+) from intracellular stores, and this increase in [Ca(2+)](i) may be a key determinant in the mechanism underlying MCLR-induced neurotoxicity.

Urotensin II promotes vagal-mediated bradycardia by activating cardiac-projecting parasympathetic neurons of nucleus ambiguus.

Urotensin II (U-II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U-II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U-II and its receptor at this level. We report here that U-II produces an increase in cytosolic Ca(2+) concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor; and (ii) Ca(2+) influx through P/Q-type Ca(2+) channels. In addition, U-II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U-II into nucleus ambiguus elicits dose-dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U-II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

General anesthetic isoflurane modulates inositol 1,4,5-trisphosphate receptor calcium channel opening.

BACKGROUND: Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP3R) Ca-release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics. METHODS: Using single-channel patch clamp electrophysiology, the authors examined the gating of rat recombinant type 3 InsP3R (InsP3R-3) Ca-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist inositol 1,4,5-trisphosphate (InsP3). The authors also examined the effects of isoflurane on InsP3R-mediated Ca release from the endoplasmic reticulum and changes in intracellular Ca concentration ([Ca]i). RESULTS: Clinically relevant concentrations (approximately 1 minimal alveolar concentration) of the commonly used general anesthetic, isoflurane, activated InsP3R-3 channels with open probability similar to channels activated by 1 µM InsP3 (Po ≈ 0.2). This isoflurane modulation of InsP3R-3 Po depended biphasically on [Ca]i. Combination of isoflurane with subsaturating levels of InsP3 in patch pipettes resulted in at least two-fold augmentations of InsP3R-3 channel Po compared with InsP3 alone. These effects were not noted in the presence of saturating [InsP3]. Application of isoflurane to DT40 cells resulted in a 30% amplification of InsP3R-mediated [Ca]i oscillations, whereas InsP3-induced increase in [Ca]i and cleaved caspase-3 activity were enhanced by approximately 2.5-fold. CONCLUSION: These results suggest that the InsP3R may be a direct molecular target of isoflurane and plays a role in the mechanisms of anesthetic-mediated pharmacological or neurotoxic effects.

Diverse regulation of IP3 and ryanodine receptors by pentazocine through σ1-receptor in cardiomyocytes.

Although pentazocine binds to σ1-receptor (σ1R) with high affinity, the physiological relevance of its binding remains unclear. We first confirmed that σ1R stimulation with pentazocine rescues contractile dysfunction following pressure overload (PO)-induced cardiac hypertrophy ovariectomized (OVX) female rats. In in vivo studies, vehicle, pentazocine (0.5-1.0 mg/kg ip), and NE-100 (1.0 mg/kg po), a σ1R antagonist, were administered for 4 wk (once daily) starting from the onset of aortic banding after OVX. We also examined antihypertrophic effects of pentazocine (0.5-1 µM) in cultured cardiomyocytes exposed to angiotensin II. Pentazocine administration significantly inhibited PO-induced cardiac hypertrophy and rescued hypertrophy-induced impairment of cardiac dysfunctions such as left ventricular end-diastolic pressure, left ventricular developed pressure, and left ventricular contraction and relaxation (±dp/dt) rates. Coadministration of NE-100 with pentazocine eliminated pentazocine-induced amelioration of heart dysfunction. Interestingly, pentazocine administration inhibited PO-induced σ1R reduction and inositol-1,4,5-trisphosphate (IP3) receptor type 2 (IP3R2) upregulation in heart. Therefore, the reduced mitochondrial ATP production following PO was restored by pentazocine administration. Furthermore, we found that σ1R binds to the ryanodine receptor (RyR) in addition to IP3 receptor (IP3R) in cardiomyocytes. The σ1R/RyR complexes were decreased following OVX-PO and restored by pentazocine administration. We noticed that pentazocine inhibits the ryanodine-induced Ca(2+) release from sarcoplasmic reticulum (SR) in cultured cardiomyocytes. Taken together, the stimulation of σ1R by pentazocine rescues cardiac dysfunction by restoring IP3R-mediated mitochondrial ATP production and by suppressing RyR-mediated Ca(2+) leak from SR in cardiomyocytes.

Imatinib treatment inhibit IL-6, IL-8, NF-KB and AP-1 production and modulate intracellular calcium in CML patients.

Imatinib (IM) is considered the gold standard for chronic myeloid leukemia (CML) treatment, although resistance is emerging as a significant problem. The proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) play an important role in cell proliferation, survival, and resistance to glucocorticoid-mediated cell death. Several transcription factors such as NF-KB and AP-1 are activated in response to physiopathological increases and modulation of intracellular calcium levels. Our previous study demonstrated that lymphocytes from CML patients showed dysregulated calcium homeostasis and oxidative stress. Alteration in ionized calcium concentration in the cytosol has been implicated in the initiation of secretion, contraction, and cell proliferation. In this study, we hypothesized that IL-6, IL-8, NF-kB, AP-1, and intracellular calcium may be used as selective and prognostic factors to address the follow-up in CML patients treated with imatinib. Our results demonstrated a significant down-regulation in IL-6 and IL-8 release as well as NF-kB and AP-1 activation in lymphomonocytes from Imatinib-treated patients, compared to samples from untreated patients. In parallel, IM treatment, in vivo and in vitro, were able to modulate the intracellular calcium concentration of peripheral blood mononuclear cells of CML patients by acting at the level of InsP(3) receptor in the endoplasmic reticulum and at the level of the purinergic receptors on plasma membrane. The results of this study show that measurements of NF-kB, AP-1, IL-6, IL-8, and intracellular calcium in CML patients treated with Imatinib may give important information to the hematologist on diagnostic criteria and are highly predictive in patients with newly diagnosed CML.

Dopamine D1 receptors regulate type 1 inositol 1,4,5-trisphosphate receptor expression via both AP-1- and NFATc4-mediated transcriptional processes.

Although our recent report demonstrates the essential involvement of up-regulation of a regulator of intracellular Ca(2+) concentration, type 1 inositol 1,4,5-trisphosphate receptors (IP(3) Rs-1), mediated via dopamine D1-like receptor (D1DR) stimulation in the cocaine-induced psychological dependence, the exact mechanisms of regulation of IP(3) R-1 expression by D1DRs have not yet been clarified. This study attempted to clarify these mechanisms using mouse cerebral cortical neurons. An agonist for phosphatidylinositide-linked D1DRs, SKF83959, induced dose- and time-dependently IP(3) R-1 protein up-regulation following its mRNA increase without cAMP production. U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor), significantly inhibited the SKF83959-induced IP(3) R-1 up-regulation. Furthermore, immunohistochemical examinations showed that SKF83959 increased expression of both cFos and cJun in nucleus as well as enhanced translocation of both calcineurin and NFATc4 complex to nucleus from cytoplasm. In addition, SKF83959 directly recruited binding of both AP-1 and NFATc4 to IP(3) R-1 promoter region. These results indicate that D1DR activation induces IP(3) R-1 up-regulation via increased translocation of AP-1 as well as NFATc4 in Gαq protein-coupled calcium signaling transduction pathway.

Nanomolar ouabain increases NCX1 expression and enhances Ca2+ signaling in human arterial myocytes: a mechanism that links salt to increased vascular resistance?

The mechanisms by which NaCl raises blood pressure (BP) in hypertension are unresolved, but much evidence indicates that endogenous ouabain is involved. In rodents, arterial smooth muscle cell (ASMC) Na(+) pumps with an α(2)-catalytic subunit (ouabain EC(50) ≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na(+) pumps have an α(1)-subunit (ouabain EC(50) ≈ 5 µM). Human α(1)-Na(+) pumps, however, have high ouabain affinity (EC(50) ≈ 10-20 nM). We used immunoblotting, immunocytochemistry, and Ca(2+) imaging (fura-2) to examine the expression, distribution, and function of Na(+) pump α-subunit isoforms in human arteries and primary cultured human ASMCs (hASMCs). hASMCs express α(1)- and α(2)-Na(+) pumps. Further, α(2)-, but not α(1)-, pumps are confined to plasma membrane microdomains adjacent to sarcoplasmic reticulum (SR), where they colocalize with Na/Ca exchanger-1 (NCX1) and C-type transient receptor potential-6 (receptor-operated channels, ROCs). Prolonged inhibition (72 h) with 100 nM ouabain (blocks nearly all α(1)- and α(2)-pumps) was toxic to most cultured hASMCs. Treatment with 10 nM ouabain (72 h), however, increased NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and augmented ATP (10 µM)-induced SR Ca(2+) release in 0 Ca(2+), ouabain-free media, and Ca(2+) influx after external Ca(2+) restoration. The latter was likely mediated primarily by ROCs and store-operated Ca(2+) channels. These hASMC protein expression and Ca(2+) signaling changes are comparable with previous observations on myocytes isolated from arteries of many rat hypertension models. We conclude that the same structurally and functionally coupled mechanisms (α(2)-Na(+) pumps, NCX1, ROCs, and the SR) regulate Ca(2+) homeostasis and signaling in hASMCs and rodent ASMCs. These ouabain/endogenous ouabain-modulated mechanisms underlie the whole body autoregulation associated with increased vascular resistance and elevation of BP in human, salt-sensitive hypertension.

IP3-mediated octopamine-induced synaptic enhancement of crayfish LG neurons.

The biogenic amines, octopamine and serotonin, modulate the synaptic activity of the lateral giant interneuron (LG) circuitry of the crayfish escape behavior. Bath application of both octopamine and serotonin enhances the synaptic responses of LG to sensory stimulation. We have shown previously (Araki et al. J Neurophysiol 94:2644-2652, 2005) that a serotonin-induced enhancement of the LG response was mediated by an increase in cAMP levels following activation of adenylate cyclase; however, octopamine acts independently. Here, we clarify how octopamine enhances the LG response during sensory stimulation using physiological and pharmacological analyses. When phospholipase C inhibitor U-73122 was directly injected into the LG before biogenic amine application, it abolished the enhancing effect of octopamine on direct sensory input to the LG, but did not block indirect input via sensory interneurons or the effect of serotonin. Direct injection of IP(3), and its analogue adenophostin A, into the LG increased the synaptic response of the LG to sensory stimulation. Thus, IP(3) mediates octopamine-induced synaptic enhancement of the LG, but serotonin acts independently. These results indicate that both octopamine and serotonin enhance the synaptic responses of the LG to sensory stimulation, but that they activate two different signaling cascades in the LG.

Caveolae and propofol effects on airway smooth muscle.

BACKGROUND: The i.v. anaesthetic propofol produces bronchodilatation. Airway relaxation involves reduced intracellular Ca(2+) ([Ca(2+)](i)) in airway smooth muscle (ASM) and lipid rafts (caveolae), and constitutional caveolin proteins regulate [Ca(2+)](i). We postulated that propofol-induced bronchodilatation involves caveolar disruption. METHODS: Caveolar fractions of human ASM cells were tested for propofol content. [Ca(2+)](i) responses of ASM cells loaded with fura-2 were performed in the presence of 10 µM histamine with and without clinically relevant concentrations of propofol (10 and 30 µM and intralipid control). Effects on sarcoplasmic reticulum (SR) Ca(2+) release were evaluated in zero extracellular Ca(2+) using the blockers Xestospongin C and ryanodine. Store-operated Ca(2+) entry (SOCE) after SR depletion was evaluated using established techniques. The role of caveolin-1 in the effect of propofol was tested using small interference RNA (siRNA) suppression. Changes in intracellular signalling cascades relevant to [Ca(2+)](i) and force regulation were also evaluated. RESULTS: Propofol was present in ASM caveolar fractions in substantial concentrations. Exposure to 10 or 30 µM propofol form decreased [Ca(2+)](i) peak (but not plateau) responses to histamine by ~40%, an effect persistent in zero extracellular Ca(2+). Propofol effects were absent in caveolin-1 siRNA-transfected cells. Inhibition of ryanodine receptors prevented propofol effects on [Ca(2+)](i), while propofol blunted [Ca(2+)](i) responses to caffeine. Propofol reduced SOCE, an effect also prevented by caveolin-1 siRNA. Propofol effects were associated with decreased caveolin-1 expression and extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: These novel data suggest a role for caveolae (specifically caveolin-1) in propofol-induced bronchodilatation. Due to its lipid nature, propofol may transiently disrupt caveolar regulation, thus altering ASM [Ca(2+)](i).

Apoptosis induced clustering of IP(3)R1 in nuclei of non-differentiated PC12 cells.

Inositol 1,4,5-trisphosphate (IP(3)) receptors are emerging as key sites for regulation by pro- and anti-apoptotic factors. Induction of apoptosis for 3 h increased mRNA and protein levels of type 1 IP(3) receptors in non-differentiated (ND), but not in differentiated (D) PC12 cells. Inhibitors of the IP(3) R's calcium release-2-aminoethoxydiphenyl borate (2-APB) and xestospongin-completely prevented Bax and caspase-3 mRNA increase after treatment with the apoptosis inducer set (AIK), and this reinforces the importance of IP(3) R1 in the apoptosis of ND PC12 cells. Apoptosis induction not only increases the IP(3) R1 protein, but it also causes formation of IP(3) R1 clusters in the nucleus which most likely result from fusion of the nucleoplasmic reticulum and/or IP(3) R1 translocation to the nucleus. This is quite similar to the observations noted after overexpression of IP(3) R1 in PC12 cells. The amount of IP(3) induced calcium release was higher in control than in AIK-treated cells. From our results we propose that after the apoptosis induction the amount of intranuclear calcium decreased dramatically due to the increase of calcium permeability of the nuclear calcium store vesicles. Therefore, increase of the calcium permeability may result from IP(3) receptors translocation to nuclei that can boost the calcium transport through IP(3) receptors.

Trisk 32 regulates IP(3) receptors in rat skeletal myoblasts.

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation-contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP(3) receptors (IP(3)R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP(3)-mediated Ca(2+) release were assessed by measuring changes in [Ca(2+)](i) following the stimulation by bradykinin or vasopressin. The amplitude of the Ca(2+) transients evoked by 20 µM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca(2+). Similar observations were made when 0.1 µM vasopressin was used to initiate Ca(2+) release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca(2+) entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca(2+) transients; rather, they were due to the enhanced activity of IP(3)R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca(2+) release via IP(3)R.

Glycine release provoked by disturbed Na⁺, Na⁺ and Ca²âº homeostasis in cerebellar nerve endings: roles of Ca²âº channels, Na⁺/Ca²âº exchangers and GlyT2 transporter reversal.

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [³H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca²âº-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 µM), but not of 4-AP (1 mM), involved also external Ca²âº-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca²âº-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca²âº channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca²âº-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na⁺/Ca²âº exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca²âº originated through mitochondrial Na⁺/Ca²âº exchangers. Ca²âº-induced Ca²âº release (CICR) mediated by inositoltrisphosphate receptors (InsP3Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP3R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets.