Attenuation of Enkephalin Activity in Neuroblastoma × Glioma NG108—15 Hybrid Cells by Phospholipases

The role of membrane phospholipids in enkephalin receptor-mediated inhibition of adenylate cyclase (EC 4.6.1.1) activity in neuroblastoma X glioma NG108-15 hybrids was studied by selective hydrolysis of lipids with phospholipases. When NG108-15 cells were treated with phospholipase C from Clostridium welchii at 37 degrees C, an enzyme concentration--dependent decrease in adenylate cyclase activity was observed. The basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities were more sensitive to phospholipase C (EC 3.1.4.3) treatment than were the NaF-5'-guanylylimidodiphosphate (Gpp(NH)p)-sensitive adenylate cyclase activities. Further, Leu5-enkephalin inhibition of basal or PGE1-stimulated adenylate cyclase activity was attenuated by phospholipase C treatment, characterized by a decrease of enkephalin potency and of maximal inhibitory level. [3H]D-Ala2-Met5-enkephalinamide binding revealed a decrease in receptor affinity with no measurable reduction in number of binding sites after phospholipase C treatment. Although opiate receptor was still under the regulation of guanine nucleotide after phospholipase C treatment, adenylate cyclase activity was more sensitive to the stimulation of Gpp(NH)p. Thus, the reduction of opiate agonist affinity was not due to the uncoupling of opiate receptor from N-component. Further, treatment of NG108-15 hybrid cell membrane with phospholipase C at 24 degrees C produced analogous attenuation of enkephalin potency and efficacy without alteration in receptor binding. The reduction in enkephalin potency could be reversed by treating NG108-15 membrane with phosphatidylcholine, but not with phosphatidylserine, phosphatidylinositol, or cerebroside sulfate. The enkephalin activity in NG108-15 cells was not altered by treating the cells with phospholipase A2 o phospholipase C from Bacillus cereus. Hence, apparently, there was a specific lipid dependency in enkephalin inhibition of adenylate cyclase activity.     

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.     

Adaptive increase in adenylyl cyclase activity in NG108-15 and S49 cells induced by chronic treatment with inhibitory drugs is not due to a decrease in cyclic AMP concentrations.

NG108-15 neuroblastoma x glioma hybrid cells and S49 lymphoma cells exhibit an enhancement in adenylyl cyclase activity after chronic treatment with receptor agonists that acutely inhibit the enzyme. Using agonists that activate five distinct inhibitory receptors in NG108-15 cells, we have found that there is a correlation between the extent of acute inhibition of prostaglandin E1 (PGE1)-stimulated cAMP accumulation and efficacy for induction of enhanced PGE1 stimulation of cAMP accumulation after chronic treatment and withdrawal. Chronic treatment with dideoxyadenosine, which acutely inhibits adenylyl cyclase activity by a mechanism independent or cell surface receptors or pertussis toxin-sensitive G proteins, did not induce enhanced PGE1 stimulation of cAMP accumulation in NG108-15 cells or forskolin stimulation of cAMP accumulation in S49 cells. While control basal cAMP concentrations were acutely decreased by carbachol in NG108-15 cells and by somatostatin in S49 cells, when the cAMP concentrations were maintained above the control basal values with a phosphodiesterase inhibitor, chronic treatment with these inhibitory drugs nonetheless resulted in enhanced cAMP responses in both NG108-15 and S49 cells. These results provide evidence that the initial decrement in cAMP concentrations caused by inhibitory drug is not the requisite signal for inducing the subsequent sensitization of adenylyl cyclase in NG108-15 and S49 cells but that activation of a pertussis toxin-sensitive G protein is involved in the development of this important adaptation.     

Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.     

Modulation of sodium-sensitive GTPase by partial opiate agonists. An explanation for the dual requirement for Na+ and GTP in inhibitory regulation of adenylate cyclase

Opiates and opioid peptides inhibit adenylate cyclase and stimulate specific low Km GTPase activity in membranes from neuroblastoma x glioma NG108-15 hybrid cells. The effects of opiate agonists on both enzymes are mediated by high affinity stereospecific receptors and require Mg2+, GTP, and Na+. In the presence of Mg2+, Na+ inhibits basal GTPase activity; opiates stimulate GTP hydrolysis by antagonizing the Na+-induced inhibition. Activation of GTPase leads, in turn, to inactivation of GTP-stimulated adenylate cyclase activity. The intrinsic activities (or efficacies) of a series of opiates are identical for stimulation of GTPase and inhibition of adenylate cyclase. These results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.     

Enhancement of glutamate uptake mediates the neuroprotection exerted by activating group II or III metabotropic glutamate receptors on astrocytes

We investigated whether the activation of astroglial group II and III metabotropic glutamate receptors (mGluRs) could exert neuroprotective effects and whether the neuroprotection was related to glutamate uptake. Our results showed that the activation of astroglial group II or III mGluRs exerted neuroprotection against 1-methyl-4-phenylpyridinium (MPP+) astroglial conditioned medium-induced neurotoxicity in midbrain neuron cultures. Furthermore, MPP+ decreased glutamate uptake of primary astrocytes and C6 glioma cells, which was recovered by activating group II or III mGluRs. Specific group II or III mGluRs antagonists completely abolished the neuroprotective effects and the enhancement of glutamate uptake of their respective agonists. Our results showed that the primary cultured rat astrocytes and C6 glioma cells expressed receptor proteins for group II mGluR2/3, group III mGluR4, mGluR6 and mGluR7. C6 glioma cells expressed mRNA for group II mGluR3, group III mGluR4, mGluR6, mGluR7 and mGluR8. In conclusion, we confirmed that the activation of astroglial mGluRs exerted neuroprotection, and demonstrated that the mechanism underlying this protective role was at least partially related to the enhancement of glutamate uptake.     

Opiate-Dependent Changes in the Sensitivity of Adenylate Cyclase to Stimulatory Agonists and 5''-Guanylylimidodiphosphate Are Independent of G Protein Abundance and Eukaryotic ADP-Ribosyltransferase Activity in NG108-15 Cells

NG108-15 cells were exposed in culture to 1 microM [D-Ala2,D-Leu5]enkaphalin (DADLE) for 17 h. This treatment increased the maximum iloprost- and 5'-(N-ethylcarboxamido)adenosine-dependent activation of adenylate cyclase, as well as basal enzyme activity. In addition, there was an increase in the capacity of 5'-guanylylimidodiphosphate [Gpp(NH)p] to inhibit adenylate cyclase activity by direct interaction with the alpha-subunit of the Gi regulatory protein. A similar effect was observed if the cells were exposed to 10 microM carbachol. These treatments of NG108-15 cells did not alter the capacity of NaF to activate adenylate cyclase by direct interaction with Gs alpha. Exposure of NG108-15 cells to DADLE alone or DADLE plus carbachol had no effect on the capacity of pertussis toxin to ADP-ribosylate membrane proteins in these cells; neither was there any change in the activity of eukaryotic ADP-ribosyltransferase expressed in these cells. Under these conditions, the endogenous enzyme did not label any protein with a molecular mass similar to Gi alpha, 41 kDa. Treatment of the cells with DADLE or carbachol had no effect on the abundance of Gs alpha, Gi alpha, or G beta. The underlying mechanism for the changes in agonist-dependent stimulatory responses or Gpp(NH)p-dependent inhibition of adenylate cyclase remains obscure, but appears not to be mediated by eukaryotic ADP-ribosyltransferase activity or a change in the abundance of G proteins known to regulate adenylate cyclase.     

Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.     

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.     

Requirement for both choleragen and pertussis toxin to obtain maximal activation of adenylate cyclase in cultured cells

NG108-15 cells contain both the inhibitory and stimulatory guanyl nucleotide-binding regulatory proteins of the cyclase system. Choleragen activates cyclase directly by ADP-ribosylating the stimulatory guanyl nucleotide-binding protein; prostaglandin E1 does not further increase activity of cells treated with maximally effective concentrations of choleragen. Including pertussis toxin during incubation with this concentration of choleragen, however, further augments both cyclase activity and cAMP accumulation by intact cells. These observations suggest that the inhibitory guanyl nucleotide-binding protein exerts basal inhibition on catalytic activity which cannot be overcome by maximally effective concentrations of choleragen, stimulatory hormones, or both.     

Muscarinic receptor regulation of NG108-15 adenylate cyclase: requirement for Na+ and GTP

Cholinergic agonists inhibit the basal and PGE1-activated adenylate cyclase activity in membranes isolated from the mouse neuroblastoma x glioma hybrid cell NG108-15. Inhibition is observed with acetylcholine, acetyl-beta-methylcholine and carbachol and is blocked by two specific muscarinic antagonists, atropine and quinuclydinylbenzilate. Inhibition of basal and PGE1-activated activity is only partial. Carbachol-directed inhibition has an apparent Km of 6 microM in the presence or absence of PGE1. Both the guanine nucleotide GTP and the monovalent cation Na+ are required for this muscarinic inhibition of basal and PGE1-activated NG108-15 adenylate cyclase. The selectivity observed for monovalent cations (all chloride salts) in this process is Na+ congruent to Li+ greater than K+ greater than Choline+ with the ED50 for Na+ congruent 40 microM. Of the nucleotides tested, only IT (and not ATP, UTP or CTP) replaces GTP in this process. GTP at 10 microM represents a saturating nucleotide concentration. Opiate-directed inhibition of NG108-15 adenylate cyclase has recently been shown to exhibit a similar requirement for GTP and Na+ [Blume, A. J., Lichtshtein, D. and Boone, G. (1979) Proc. National Academy of Sciences, USA, in press]. The data presented here therefore support the hypothesis that the general transfer of inhibitory information from membrane receptors to adenylate cyclase involves both a Na+ and GTP-sensitive process.     

Positive and Negative Coupling of the Metabotropic Glutamate Receptors to a G Protein–activated K+ Channel,GIRK, in Xenopus Oocytes

Metabotropic glutamate receptors (mGluRs) control intracellular signaling cascades through activation of G proteins. The inwardly rectifying K⁺ channel, GIRK, is activated by the βγ subunits of G_i proteins and is widely expressed in the brain. We investigated whether an interaction between mGluRs and GIRK is possible, using Xenopus oocytes expressing mGluRs and a cardiac/brain subunit of GIRK, GIRK1, with or without another brain subunit, GIRK2. mGluRs known to inhibit adenylyl cyclase (types 2, 3, 4, 6, and 7) activated the GIRK channel. The strongest response was observed with mGluR2; it was inhibited by pertussis toxin (PTX). This is consistent with the activation of GIRK by G_i/G_o-coupled receptors. In contrast, mGluR1a and mGluR5 receptors known to activate phospholipase C, presumably via G proteins of the G_q class, inhibited the channel''s activity. The inhibition was preceded by an initial weak activation, which was more prominent at higher levels of mGluR1a expression. The inhibition of GIRK activity by mGluR1a was suppressed by a broad-specificity protein kinase inhibitor, staurosporine, and by a specific protein kinase C (PKC) inhibitor, bis-indolylmaleimide, but not by PTX, Ca²⁺ chelation, or calphostin C. Thus, mGluR1a inhibits the GIRK channel primarily via a pathway involving activation of a PTX-insensitive G protein and, eventually, of a subtype of PKC, possibly PKC-μ. In contrast, the initial activation of GIRK1 caused by mGluR1a was suppressed by PTX but not by the protein kinase inhibitors. Thus, this activation probably results from a promiscuous coupling of mGluR1a to a G_i/G_o protein. The observed modulations may be involved in the mGluRs'' effects on neuronal excitability in the brain. Inhibition of GIRK by phospholipase C–activating mGluRs bears upon the problem of specificity of G protein (GIRK interaction) helping to explain why receptors coupled to G_q are inefficient in activating GIRK.     

Multiple receptors linked to inhibition of adenylate cyclase accelerate Na+/H+ exchange in neuroblastoma x glioma cells via a mechanism other than decreased cAMP accumulation

alpha 2-Adrenergic receptors, a population of receptors linked to inhibition of adenylate cyclase, accelerate Na+/H+ exchange in NG108-15 neuroblastoma x glioma cells (Isom, L. L., Cragoe, E. J., Jr., and Limbird, L. E. (1987) J. Biol. Chem. 262, 6750-6757). We now report that two other receptor populations linked to inhibition of adenylate cyclase, muscarinic cholinergic and delta-opiate receptors, also alkalinize the interior of NG108-15 cells, as measured with the pH-sensitive fluorescent probe, 2,7-biscarboxyethyl-5(6)-carboxy-fluorescein. Manipulations that block Na+/H+ exchange, i.e. removal of extracellular Na+, reduction of extracellular pH to equal that of intracellular pH, and addition of 5-amino-substituted analogs of amiloride, all block alpha 2-adrenergic, delta-opiate, or muscarinic cholinergic receptor-induced alkalinization in a parallel fashion. These data suggest that all three populations of receptors alkalinize NG108-15 cells by acceleration of Na+/H+ exchange and do so via a shared or similar mechanism. Although these three receptor populations are linked to inhibition of adenylate cyclase, decreased production of cAMP does not appear to be the mechanism responsible for receptor-accelerated Na+/H+ exchange. Thus, ADP-ribosylation of intact NG108-15 cells with Bordetella pertussis islet-activating protein prevents attenuation of prostaglandin E1-stimulated cAMP accumulation by alpha 2-adrenergic, muscarinic, and delta-opiate agonists but has no measurable effect on the ability of these agonists to accelerate Na+/H+ exchange. Similarly, manipulations that block receptor-accelerated Na+/H+ exchange influence but do not block receptor-mediated attenuation of cAMP accumulation. Thus, the present data suggest that these two receptor-mediated biochemical events, acceleration of Na+/H+ exchange and attenuation of cAMP accumulation, occur through divergent mechanisms in NG108-15 cells.     

Dopamine-induced plasticity, phospholipase D (PLD) activity and cocaine-cue behavior depend on PLD-linked metabotropic glutamate receptors in amygdala

Cocaine-cue associations induce synaptic plasticity with long lasting molecular and cellular changes in the amygdala, a site crucial for cue-associated memory mechanisms. The underlying neuroadaptations can include marked alterations in signaling via dopamine (DA) receptors (DRs) and metabotropic glutamate (Glu) receptors (mGluRs). Previously, we reported that DR antagonists blocked forms of synaptic plasticity in amygdala slices of Sprague-Dawley rats withdrawn from repeated cocaine administration. In the present study, we investigated synaptic plasticity induced by exogenous DA and its dependence on mGluR signaling and a potential role for phospholipase D (PLD) as a downstream element linked to mGluR and DR signaling. Utilizing a modified conditioned place preference (CPP) paradigm as a functional behavioral measure, we studied the neurophysiological effects after two-weeks to the last cocaine conditioning. We recorded, electrophysiologically, a DR-induced synaptic potentiation in the basolateral to lateral capsula central amygdala (BLA-lcCeA) synaptic pathway that was blocked by antagonists of group I mGluRs, particularly, the PLD-linked mGluR. In addition, we observed 2-2.5 fold increase in PLD expression and 3.7-fold increase in basal PLD enzyme activity. The enhanced PLD activity could be further stimulated (9.3 fold) by a DA D1-like (D1/5R) receptor agonist, and decreased to control levels by mGluR1 and PLD-linked mGluR antagonists. Diminished CPP was observed by infusion of a PLD-linked mGluR antagonist, PCCG-13, in the amygdala 15 minutes prior to testing, two weeks after the last cocaine injection. These results imply a functional interaction between D1/5Rs, group I mGluRs via PLD in the amygdala synaptic plasticity associated with cocaine-cues.     

Nitric oxide inhibits Ca(2+) mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells

The present study was designed to determine whether the cADP-ribose-mediated Ca(2+) signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca(2+) mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca(2+) release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the alpha-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 microM) produced a rapid Ca(2+) release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca(2+) concentration ([Ca(2+)](i)). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca(2+)](i), but it had no effect on the U-46619-induced increase in [Ca(2+)](i). In the presence of 8-Br-cADPR (100 microM) and ryanodine (10 microM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1-100 microM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 microM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca(2+)](i) by inhibition of cADP-ribose-induced Ca(2+) mobilization.     

Cell-Free Desensitization of Opioid Inhibition of Adenylate Cyclase in Neuroblastoma × Glioma NG108–15 Hybrid Cell Membranes

When membranes from neuroblastoma X glioma NG108-15 hybrid cells were incubated in a cell-free system with opioid agonists, a time-, temperature-, and dose-dependent desensitization to opioid inhibition of adenylate cyclase activity was observed. The composition of the system during the incubation was manipulated to elucidate the biochemical mechanisms of desensitization. Receptor coupling appeared to be a prerequisite for desensitization, because both magnesium and sodium, which are necessary for coupling, were required for desensitization. Removal of ATP and addition of cyclic AMP or cyclic GMP had no effect on desensitization.     

Activation of Metabotropic Glutamate Receptors Inhibits Synapsin I Phosphorylation in Visceral Sensory Neurons

Activation of glutamate metabotropic receptors (mGluRs) in nodose ganglia neurons has previously been shown to inhibit voltage-gated Ca⁺⁺ currents and synaptic vesicle exocytosis. The present study describes the effects of mGluRs on depolarization-induced phosphorylation of the synaptic-vesicle-associated protein synapsin I. Depolarization of cultured nodose ganglia neurons with 60 mm KCl resulted in an increase in synapsin I phosphorylation. Application of mGluR agonists 1-aminocyclopentane-1s-3r-dicarboxylic acid (t-ACPD) and L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) either in combination or independently inhibited the depolarization induced phosphorylation of synapsin I. Application of the mGluR antagonist (RS)-α-Methyl-4-carboxyphenylglycine (MCPG) blocked t-ACPD-induced inhibition of synapsin phosphorylation but not the effects of L-AP4. In addition, application of either t-ACPD or L-AP4 in the absence of KCl induced depolarization had no effect on resting synapsin I phosphorylation. RT-PCR analysis of mGluR subtypes in these nodose ganglia neurons revealed that these cells only express group III mGluR subtypes 7 and 8. These results suggest that activation of mGluRs modulates depolarization-induced synapsin I phosphorylation via activation of mGluR7 and/or mGluR8 and that this process may be involved in mGluR inhibition of synaptic vesicle exocytosis in visceral sensory neurons of the nodose ganglia. Received 28 June 2000/Revised: 11 September 2000     

Activation of type 4 metabotropic glutamate receptor promotes cell apoptosis and inhibits proliferation in bladder cancer

Bladder cancer, the second most common genitourinary malignancy, severely endangers the human health. Rising evidence suggests that metabotropic glutamate receptors (mGluRs) are involve in tumor progression. In this study, we observed that metabotropic glutamate receptor 4 (mGluR4) was functionally expressed in normal and cancerous bladder cells and its expression was positively correlated with high bladder cancer grading. We further confirmed that the activation of mGluR4 by VU0155041, an mGluR4-specific agonist, decreased cyclic adenosine monophosphate (cAMP) concentration and cell viability, promoted apoptosis and inhibited proliferation in bladder cancer cells, whereas MSOP (group III mGluR antagonist) or mGluR4 knockdown eliminated the effects of mGluR4 activity. Western blotting revealed the decreased cyclin D1 expression, increased procaspase-8/9/3 cleavage, and unbalanced Bcl-2/Bax expression in bladder cancer cell lines after mGluR4 activation, and likewise MSOP and mGluR4 knockdown abrogated the actions of mGluR4 activity. In vivo study showed that mGluR4 activation significantly inhibited tumor growth of bladder cancer via suppressing proliferation and promoting apoptosis. Furthermore, upregulation of phosphatase and tensin homolog (PTEN) and inhibition of Akt phosphorylation were also observed after mGluR4 activation. Similar with VU0155041, the Akt-specific inhibitor markedly promoted apoptosis and inhibited proliferation. Nevertheless, the PTEN-specific inhibitor significantly abolished the mGluR4 activation-induced cell apoptosis and proliferative inhibition in bladder cancer cell lines. These results indicate that mGluR4 can regulate the switch between survival and death via the cAMP/PTEN/AKT signaling pathway in bladder cancer cells. Our findings suggest that mGluR4 has diagnostic and prognostic potential for bladder cancer, and the development of mGluR4 agonist may be a promising strategy for bladder cancer treatment.