In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca 2+ signals. In many cells, mitochondria act as Ca 2+ buffers by taking up and releasing Ca 2+ , but this simple buffering action by itself often cannot explain the organelle's effects on Ca 2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca 2+ channels in T lymphocytes as a mechanism of mitochondrial Ca 2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca 2+ stores causes sustained activation of the store-operated Ca 2+ current, I CRAC (CRAC, Ca 2+ release-activated Ca 2+ ). Inhibition of mitochondrial Ca 2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca 2+ -dependent inactivation of I CRAC . Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca 2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca 2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca 2+ ] i elevation, indicating a functional link between mitochondrial regulation of I CRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca 2+ channel activity and signal transmission from the plasma membrane to the nucleus.

We have monitored agonist-induced alpha 1B-adrenergic receptor (alpha 1BAR) redistribution by immunocytochemical procedures in concert with functional measurements of agonist-elicited [3H]inositol phosphate (InsP) production in human embryonal kidney 293 cells stably expressing alpha 1BAR cDNA (HEK293/alpha 1B). Anti-peptide antibodies directed against the carboxyl-terminal decapeptide of the alpha 1BAR were prepared and shown to react specifically with alpha 1BAR on immunoblots and in situ in HEK293/alpha 1B transfectants. Treatment of HEK293/alpha 1B cells with norepinephrine (10 microM) results in a rapid (5-15 min) and striking internalization of cell surface receptor as visualized by confocal immunofluorescence microscopy. Receptor redistribution is sustained in the presence of agonist, rapidly reversed upon agonist removal, and prevented by the alpha 1 antagonist prazosin. Receptor internalizes to endosomes, as shown by colocalization with transferrin receptor, an endosomal marker. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (50 nM) causes receptor endocytosis similar to agonist; agonist-induced internalization is blocked by the PKC inhibitor staurosporine (0.5 microM). In parallel experiments, agonist-induced [3H]InsP production is abolished by phorbol 12-myristate 13-acetate but potentiated by staurosporine. Inhibition of receptor internalization with hypertonic sucrose attenuates agonist-induced [3H]InsP formation; this effect is reversed by concomitant inhibition of PKC with staurosporine. These results suggest that PKC-dependent phosphorylation occurring as a consequence of alpha 1AR stimulation induces receptor desensitization and internalization. Internalized receptor is reactivated and continuously recycled to the cell surface during agonist exposure.

Controlled variation in intracellular calcium concentration is a key component of the immune response signaling pathway in lymphocytes. Store-operated calcium entry (SOCE) in these cells provides a prolonged increase in cytoplasmic Ca(2+) concentrations and ultimately leads to the production of pro-inflammatory cytokines. Molecules that inhibit SOCE could therefore be useful immunomodulating agents for the treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and other conditions. Although the presence of the SOCE signaling pathway in lymphocytes and other cells involved in the immune response has been known for many years, key proteins involved in SOCE were identified only recently. The identification of these proteins may further enable the identification of agents that inhibit SOCE without affecting other cellular processes. This contribution documents representative examples of the small-molecule inhibitors of SOCE that have been reported to date. Where possible, methods that were used to characterize the mechanism of action of the inhibitors are also described.

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The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq.

BACKGROUND Converging evidence has implicated endogenous neurotensin (NT) in the pathophysiology of brain processes relevant to schizophrenia. Prepulse inhibition of the startle reflex (PPI) is a measure of sensorimotor gating and considered to be of strong relevance to neuropsychiatric disorders associated with psychosis and cognitive dysfunction. Mice genetically engineered to not express NT display deficits in PPI that model the PPI deficits seen in schizophrenia patients. NT1 receptors have been most strongly implicated in mediating the psychosis relevant effects of NT such as attenuating PPI deficits. To investigate the role of NT1 receptors in the regulation of PPI, we measured baseline PPI in wildtype (WT) and NT1 knockout (KO) mice. We also tested the effects of amphetamine and dizocilpine, a dopamine agonist and NMDA antagonist, respectively, that reduce PPI as well as the NT1 selective receptor agonist, PD149163, known to increase PPI in rats. METHODS Baseline PPI and acoustic startle response were measured in WT and NT1 knockout KO mice. After baseline testing, mice were tested again after receiving intraperatoneal (IP) saline or one of three doses of amphetamine (1.0, 3.0 and 10.0 mg/kg), dizocilpine (0.3, 1.0 and 3.0 mg/kg) and PD149163 (0.5, 2.0 and 6.0 mg/kg) on separate test days. RESULTS Baseline PPI and acoustic startle response in NT1 KO mice were not significantly different from NT1 WT mice. WT and KO mice exhibited similar responses to the PPI-disrupting effects of dizocilpine and amphetamine. PD149163 significantly facilitated PPI (P < 0.004) and decreased the acoustic startle response (P < 0.001) in WT but not NT1 KO mice. CONCLUSIONS The data does not support the regulation of baseline PPI or the PPI disruptive effects of amphetamine or dizocilpine by endogenous NT acting at the NT1 receptor, although they support the antipsychotic potential of pharmacological activation of NT1 receptors by NT1 agonists.

Agonist occupied ␣ 1 -adrenoceptors (␣ 1 -ARs) engage several signaling pathways, including phosphatidylinositol hydrolysis, calcium mobilization, arachidonic acid release, mitogen-activated protein (MAP) kinase activation, and cAMP accumulation. The natural agonist norepinephrine (NE) activates with variable affinity and intrinsic efficacy all adrenoceptors, and in cells that coexpress ␣ 1 -and ␤-AR subtypes, such as cardiomyocytes, this leads to coactivation of multiple downstream pathways. This may result in pathway cross-talk with significant consequences to heart physiology and pathologic state. To dissect signaling components involved specifically in ␣ 1A -and ␤ 2 -AR signal interplay, we have developed a recombinant model system that mimics the levels of receptor expression observed in native cells. We followed intracellular Ca 2ϩ mobilization to monitor in real time the activation of both G q and G s pathways. We found that coactivation of ␣ 1A -and ␤ 2 -AR by the nonselective agonist NE or via a combination of the highly selective ␣ 1A -AR agonist A61603 and the ␤-selective agonist isoproterenol led to increases in Ca 2ϩ influx from the extracellular compartment relative to stimulation with A61603 alone, with no effect on the associated transient release of Ca 2ϩ from intracellular stores. This effect became more evident upon examination of an ␣ 1A -AR variant exhibiting a partial defect in coupling to G q , and we attribute it to potentiation of a non G q -pathway, uncovered by application of a combination of xestospongin C, an endoplasmic reticulum inositol 1,4,5-triphosphate receptor blocker, and 2-aminoethoxydiphenyl borate, a nonselective storeoperated Ca 2ϩ entry channel blocker. We also found that stimulation with A61603 of a second ␣ 1A -AR variant entirely unable to signal induced no Ca 2ϩ unless ␤ 2 -AR was concomitantly activated. These results may be accounted for by the presence of ␣ 1A /␤ 2 -AR heterodimers or alternatively by specific adrenoceptor signal cross-talk resulting in distinct pharmacological behavior. Finally, our findings provide a new conceptual framework to rationalize outcomes from clinical studies targeting ␣-and ␤-adrenoceptors.Given the diversity of physiological processes controlled by G protein-coupled receptors (GPCRs or 7-transmembrane receptors) and the large number of receptors from this family coexpressed in different tissues, it comes as no surprise that linear signal transduction models cannot accommodate the many observed pharmacological outcomes that follow receptor activation. It is generally understood that intracellular signal transduction pathways stemming from the activation of GPCRs interact significantly to add layers of complexity to their regulation, sometimes leading to novel signaling modes (Cordeaux and Hill, 2002). In addition, many receptors have been shown to couple to more than one G protein, and also initiate G protein-independent signaling upon stimulation (for review, see Gilchrist, 2007;Violin and Lefkowitz, 2007).Such GPCR signalin...