GRINL1A colocalizes with N-methyl D-aspartate receptor NR1 subunit and reduces N-methyl D-aspartate toxicity.

We hypothesized that proteins from the GRINL1A complex transcription unit called Gcom proteins modulate glutamatergic neurotransmission through interaction with the NR1 subunit of the N-methyl D-aspartate (NMDA) receptor. Cotransfection of hemagglutinin-tagged Gcom1 (GRINL1A combined transcript 1) and NR1 cDNAs into HEK293 cells revealed overlapping fluorescent signals in the plasma membrane. Coimmunoprecipitation studies demonstrated reciprocal coimmunoprecipitation from rat brain protein isolates, suggesting an interaction between GRINL1A proteins and the NMDA receptor. Anti-Gcom1 and anti-NR1 antibodies revealed colocalization of postsynaptic immunoreactivity in rat cortical and hippocampal neurons. Finally, anti-Gcom1 antibodies specifically inhibited NMDA excitotoxicity in rat cortical neurons, suggesting a functional interaction of Gcom and NR1 proteins. Our results are consistent with a facilatory role of GRINL1A proteins in glutamatergic signal transduction through interaction with the NMDA receptor.

beta-Adrenoceptor blockers protect against staurosporine-induced apoptosis in SH-SY5Y neuroblastoma cells.

The beta-adrenoceptor blockers exhibit a well-characterized anti-apoptotic property in the heart and kidney while less is known about the effect of this class of drugs on neuronal apoptosis. We studied the effects of three beta-adrenoceptor blockers propranolol (1-(isoproplyamino)-3-(naphthalene-1-yloxy)propan-2-ol), atenolol (2-[4-[2-hydroxy-3-(1-methylethylamino)propoxyl]phenyl]ehanamide), and ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), against staurosporine-induced apoptosis in SH-SY5Y human neuroblastoma cells. Staurosporine increased caspase 3-like activity, DNA fragmentation, PARP cleavage, and the number of TUNEL positive cells consistent with the induction of apoptosis. Propranolol and ICI 118551, but not atenolol, demonstrated a concentration-dependent inhibition of caspase 3-like activity. Propranolol and ICI 118551 directly inhibited the enzymatic activity of recombinant caspase 9 while atenolol did not; however, none of the beta-adrenoceptor blockers that were examined directly blocked caspases 2 or 3 activity. In isolated mitochondria, propranolol and ICI 118551 inhibited staurosporine-induced cytochrome c release while atenolol did not. We conclude that propranolol and ICI 118551 protect SH-SY5Y cells against staurosporine-induced apoptosis through a dual action on the mitochondria and on caspase 9 in a cell type and an apoptotic paradigm where the conventional inhibitors of mitochondrial permeability transition such as cyclosporin A and bongkrekic acid demonstrate no protection.

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells.

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.

Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis.

The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H2O2) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H2O2, but not to staurosporine. This selective protection against H2O2 was due to inhibition of caspase-3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H2O2-induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.

Cardiac mitochondrial connexin 43 regulates apoptosis.

Connexin 43 (Cx43) is thought to be present largely in the plasma membrane and its function solely to provide low resistance electrical connection between myocytes. A recent report suggested the presence of Cx43 in the mitochondria as well. We confirmed the presence of Cx43 in the mitochondria isolated from adult rat ventricles with the Cx43 immunoreactivity fractionating to the outer mitochondrial membrane. Mitochondrial Cx43 is mostly phosphorylated only detected by a phospho-specific antibody. Using a Ca2+ -sensitive electrode and Western blot, we showed that the gap junction inhibitors 18-beta-glycyrrhetinic acid (beta-GA), oleamide, and heptanol all induced concomitant release of Ca2+ and cytochrome C in isolated mitochondria whereas the inactive analog 18-beta-glycyrrhizic acid failed to do so. In low density neonatal myocyte culture with no appreciable cell-cell contacts, beta-GA induced apoptosis as assessed by TUNEL staining. Our results suggest a novel role of Cx43 as a regulator of mitochondrial physiology and myocyte apoptosis.

Kinetic properties of GABA rho1 homomeric receptors expressed in HEK293 cells.

The rho1 subunit of the ionotropic GABA receptors is thought to contribute to the formation of the GABA(C) receptors with pharmacological and physiological properties distinct from those of GABA(A) receptors. Previous characterization of this subunit expressed in the Xenopus oocytes revealed an ion channel with slow activation and deactivation and no desensitization, quite different from the properties of GABA(C) receptors observed in native cells. We expressed the human rho1 subunit in human embryonic kidney (HEK) 293 cells and quantitatively characterized the kinetic properties of these receptors using a rapid drug application device. The rho1 subunit expressed in HEK293 cells exhibited pharmacological and kinetic properties qualitatively identical to those described when rho1 was expressed in the oocytes. An apparent desensitizing current observed during a constant GABA application was determined to be secondary to an E(Cl) shift. Detailed kinetic analyses and parameter estimation for a five-state kinetic model revealed that the channel is best described by a set of rate constants with a notably faster GABA unbinding K(off) rate compared to the parameters proposed for the same subunit expressed in the oocytes. The same subunit expressed in hippocampal neurons showed activation and deactivation kinetics identical to the current characterized in HEK293 cells. The kinetic properties of rho1 subunit expressed in a nonoocyte model system may be better described quantitatively by the rate constants presented here.

A1 adenosine receptor knockout mice are protected against acute radiocontrast nephropathy in vivo.

The role of renal A1 adenosine receptors (A1AR) in the pathogenesis of radiocontrast nephropathy is controversial. We aimed to further elucidate the role of A1AR in the pathogenesis of radiocontrast nephropathy and determine whether renal proximal tubule A1AR contribute to the radiocontrast nephropathy. To induce radiocontrast nephropathy, A1AR wild-type (WT) or knockout (KO) mice were injected with a nonionic radiocontrast (iohexol, 1.5-3 g iodine/kg). Some A1WT mice were pretreated with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a selective A1AR antagonist) before iohexol injection. A1AR contribute to the pathogenesis of radiocontrast nephropathy in vivo as the A1WT mice developed significantly worse acute renal failure, more renal cortex vacuolization, and had lower survival 24 h after iohexol treatment compared with the A1KO mice. DPCPX pretreatment also protected the A1WT mice against radiocontrast-induced acute renal failure. No differences in renal cortical apoptosis or inflammation were observed between A1WT and A1KO mice. To determine whether the proximal tubular A1AR mediate the direct renal cytotoxicity of radiocontrast, we treated proximal tubules in culture with iohexol with or without 2-chloro-N6-cyclopentyladenosine (a selective A1AR agonist) or DPCPX pretreatment. We also subjected cultured proximal tubule cells overexpressing A1AR or lacking A1AR to radiocontrast injury. Iohexol caused a direct dose-dependent reduction in proximal tubule cell viability as well as proliferation. Neither the A1AR agonist nor the antagonist treatment affected proximal tubule viability or proliferation. Moreover, overexpression or lack of A1AR failed to impact the iohexol toxicity on proximal tubule cells. Therefore, we conclude that radiocontrast causes acute renal failure via mechanisms dependent on A1AR; however, renal proximal tubule A1AR do not contribute to the direct tubular toxicity of radiocontrast.

T-cadherin GPI-anchor is insufficient for apical targeting in MDCK cells.

T-cadherin is a 95kDa glycoprotein member of the cadherin family of adhesion molecules attached to the extracellular surface of the cell membrane through a glycosyl-phosphatidylinositol (GPI)-anchor. Whether a T-cadherin ectodomain apical targeting signal or the GPI-anchor itself targets this protein to the apical membrane is not known. Chimeras of the reporter EGFP and T-cadherin have demonstrated that a minimal construct consisting of the C-terminal 25 amino acids including the N690 (omega-site) of T-cadherin was sufficient to GPI-anchor the EGFP protein. However, efficient GPI-anchor with minimal secretion of the protein required an additional 5 residues (omega-1 to omega-5). The GPI-anchored chimeras fractionated to the Triton X-100 detergent insoluble fraction and were released to the cell culture supernatant by phosphoinositide-specific phospho-lipase C digestion. When expressed in MDCK cells, all GPI-anchored chimeras targeted to the basolateral membrane, while the T/N-chimera and the wild-type T-cadherin targeted to the apical membrane. Therefore, T-cadherin is an example of another rare GPI-anchored protein where the anchor itself is not sufficient for apical targeting in MDCK cells.

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides.

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.