Agonist-stimulated Inositol Phosphate Research Articles

Phospholipase C∈ Scaffolds to Muscle-specific A Kinase Anchoring Protein (mAKAPβ) and Integrates Multiple Hypertrophic Stimuli in Cardiac Myocytes

To define a role for phospholipase Cε (PLCε) signaling in cardiac myocyte hypertrophic growth, PLCε protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCε depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCε depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCε catalytic activity was required for hypertrophy development, yet PLCε depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCε was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCε-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCε complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCε integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy.

Pyk2 uncouples metabotropic glutamate receptor G protein signaling but facilitates ERK1/2 activation

Group I metabotropic glutamate receptors (mGluRs) are coupled via Gαq/11 to the activation of phospholipase Cβ, which hydrolyzes membrane phospholipids to form inositol 1,4,5 trisphosphate and diacylglycerol. This results in the release of Ca2+ from intracellular stores and the activation of protein kinase C. The activation of Group I mGluRs also results in ERK1/2 phosphorylation. We show here, that the proline-rich tyrosine kinase 2 (Pyk2) interacts with both mGluR1 and mGluR5 and is precipitated with both receptors from rat brain. Pyk2 also interacts with GST-fusion proteins corresponding to the second intracellular loop and the distal carboxyl-terminal tail domains of mGluR1a. Pyk2 colocalizes with mGluR1a at the plasma membrane in human embryonic kidney (HEK293) cells and with endogenous mGluR5 in cortical neurons. Pyk2 overexpression in HEK293 results in attenuated basal and agonist-stimulated inositol phosphate formation in mGluR1 expressing cells and involves a mechanism whereby Pyk2 displaces Gαq/11 from the receptor. The activation of endogenous mGluR1 in primary mouse cortical neuron stimulates ERK1/2 phosphorylation. Treatments that prevent Pyk2 phosphorylation in cortical neurons, and the overexpression of Pyk2 dominant-negative and catalytically inactive Pyk2 mutants in HEK293 cells, prevent ERK1/2 phosphorylation. The Pyk2 mediated activation of ERK1/2 phosphorylation is also Src-, calmodulin- and protein kinase C-dependent. Our data reveal that Pyk2 couples the activation mGluRs to the mitogen-activated protein kinase pathway even though it attenuates mGluR1-dependent G protein signaling.

Engineered Mutation of Some Important Amino Acids in Angiotensin II Type 1 (AT1) Receptor Increases the Binding Affinity of AT1-Receptor Antagonists

The present study was designed to examine the binding affinity and functional potency of selective angiotensin II type 1 (AT1)-receptor antagonists towards specific mutants of AT1 receptor using site-directed mutagenesis. We also compared our results with the wild-type AT1 receptor and investigated the possible reasons behind that. Both wild-type and mutant receptors were expressed in COS-7 cells and the binding affinities of the antagonists were determined by radioligand binding assay. Inhibition of agonist-stimulated inositol phosphate accumulation by the antagonists was also done. Substitution of asparagine235 of intracellular loop 3 of the AT1 receptor by arginine increased the binding affinity of the antagonists 5 – 34-fold, whereas the increase in the binding affinity of the antagonists in the phenylalanine239 mutant by arginine and tryptophan (F239R and F239W) were 3 – 19-fold and 2 – 15-fold higher, respectively, compared to the wild-type AT1 receptor. The results suggested that substitution by a positively charged or sterically hindered amino acid in the AT1 receptor allows it to interact with the acidic tetrazole moiety and carboxylate groups of the antagonists more strongly compared to the wild-type receptor. These findings may play an important role to change the binding affinity of the antagonists to an effective level for the pharmacological function of the drugs.

Calcineurin Inhibitor Protein (CAIN) Attenuates Group I Metabotropic Glutamate Receptor Endocytosis and Signaling

Group I metabotropic glutamate receptors (mGluRs) are coupled via phospholipase Cbeta to the hydrolysis of phosphoinositides and function to modulate neuronal excitability and synaptic transmission at glutamatergic synapses. The desensitization of Group I mGluR signaling is thought to be mediated primarily via second messenger-dependent protein kinases and G protein-coupled receptor kinases. We show here that both mGluR1 and mGluR5 interact with the calcineurin inhibitor protein (CAIN). CAIN is co-immunoprecipitated in a complex with Group I mGluRs from both HEK 293 cells and mouse cortical brain lysates. Purified CAIN and its C-terminal domain specifically interact with glutathione S-transferase fusion proteins corresponding to the second intracellular loop and the distal C-terminal tail domains of mGluR1. The interaction of CAIN with mGluR1 could also be blocked using a Tat-tagged peptide corresponding to the mGluR1 second intracellular loop domain. Overexpression of full-length CAIN attenuates the agonist-stimulated endocytosis of both mGluR1a and mGluR5a in HEK 293 cells, but expression of the CAIN C-terminal domain does not alter mGluR5a internalization. In contrast, overexpression of either full-length CAIN or the CAIN C-terminal domain impairs agonist-stimulated inositol phosphate formation in HEK 293 cells expressing mGluR1a. This CAIN-mediated antagonism of mGluR1a signaling appears to involve the disruption of receptor-Galpha(q/11) complexes. Taken together, these observations suggest that the association of CAIN with intracellular domains involved in mGluR/G protein coupling provides an additional mechanism by which Group I mGluR endocytosis and signaling are regulated.

Amino Acids of the Human α1d-Adrenergic Receptor Involved in Antagonist Binding

Computer simulations of the human α1d-adrenergic receptor (α1d-AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of α1d-AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [3H]prazosin and neither did it show agonist-stimulated inositol phosphates (IP) formation. On the other hand, the Glu237Ala and Ser258Ala mutant α1d-AR showed increased binding affinity for [3H]prazosin. Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor’s binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[3H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between α1d-AR and the radioligand [3H]prazosin as well as tamsulosin.

Identification of a Key Amino Acid of the Human 5-HT2B Serotonin Receptor Important for Sarpogrelate Binding

Based on radio-ligand binding and molecular modeling studies, sarpogrelate shows a moderate selectivity for 5-HT2B versus 5-HT2A receptors. To confirm the modeling data of sarpogrelate to 5-HT2B receptors predicting interaction of sarpogrelate towards Asp135 in helix 3 of 5-HT2B receptors, we constructed and characterized the mutation of this residue by site-directed mutagenesis. The Asp135Ala mutant did not exhibit any affinity for [3H]rauwolscine. Therefore, it was not possible to find sarpogrelate affinity to the mutant using [3H]rauwolscine. The mutation also abolished agonist-stimulated inositol phosphates formation. These results provide evidence that Asp135 is important for the interaction between 5-HT2B receptors and sarpogrelate.

Identification of Amino Acid Residues Important for Sarpogrelate Binding to the Human 5-Hydroxytryptamine2A Serotonin Receptor

The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)2A-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT2A receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [3H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [3H]ketanserin (Kd >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [3H]ketanserin. The mutation also abolished agonist-stimulated formation of [3H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [3H]ketanserin (Kd 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pKi value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pKb value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT2A receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.

The DRY Motif as a Molecular Switch of the Human Oxytocin Receptor

The human oxytocin receptor is known to exhibit promiscuous activity by coupling to both Galpha(q) and Galpha(i) G proteins to activate distinct signaling pathways. A single-amino acid substitution within the highly conserved E/DRY motif at the cytosolic extension of helix 3 [i.e., D136(3.49)N] increased the rate of both basal and agonist-stimulated inositol phosphate (IP(3)) accumulation of the receptor. Furthermore, like for a typical constitutively active receptor, the partial agonist arginine vasopressin behaved as a full agonist for the D136(3.49)N mutant. Subsequently, both oxytocin and arginine vasopressin showed an increased potency in stimulating IP3 accumulation as compared to the wild-type receptor. Very interestingly, our experiments provide strong evidence that the D136(3.49)N mutant inhibits receptor signaling via Galpha(i)-mediated pathways while increasing the activity through the Galpha(q)-mediated pathways. Molecular simulations of the free and OT-bound forms of wild-type OTR and of the D136(3.49)N constitutively active mutant suggest that the receptor portions close to the E/DRY and NPxxY motifs are particularly susceptible to undergoing structural modification in response to activating mutations and agonist binding. Furthermore, computational modeling suggests that the OT-bound form of wild-type OTR is able to explore more states than the OT-bound form of the D136(3.49)N constitutively active mutant, consistent with its G protein promiscuity. Taken together, these observations emphasize the important role of the E/DRY motif not only in receptor activation but also in the promiscuity of G protein coupling. Knowledge of the mechanism of selective G protein coupling could aid drug discovery efforts to identify signaling specific therapies.

Delta-opioid agonist-stimulated inositol phosphate formation in isolated, rabbit iris-ciliary bodies: role of G i/o proteins and Gβγ-subunits

Our laboratory has previously demonstrated the ability of kappa- and delta-opioid agonists to decrease aqueous flow rates and intraocular pressure of rabbits. The mechanisms by which these agents act in the ciliary body of the rabbit could involve inhibition of cAMP production, as well as increased generation of inositol phosphates (IPs). With regard to enhanced production of IPs, it has been suggested that their levels can be augmented by stimulation of phospholipase C via opioid receptors linked to either Gα-subunits derived from Gq proteins or Gβγ-subunits derived from G i/o-proteins. The aim of the current study is to investigate the role of pertussis toxin (PTX)-sensitive G-proteins and Gβγ-subunits in delta-opioid agonist-mediated changes in IP production in the isolated, rabbit iris-ciliary body (ICB). In one set of experiments, ICB segments were treated with the delta agonist, SNC80 (10 −12–10 −7 mol/l) alone. Other experiments were conducted utilizing SNC80 following pretreatment with either phosducin (Gβγ-subunit scavenger), PTX, or the delta antagonist, naltrindole. IP production was measured by ion exchange chromatography. Basal levels of IPs in the rabbit ICB were 58 287±2162, 15 218±969 and 2083±367 dpm/mg protein for IP 1, IP 2 and IP 3, respectively. The highly selective delta-opioid receptor agonist, SNC80, produced concentration-dependent increases in the levels of the IPs in the ICB, which were diminished in the presence of the delta antagonist, naltrindole, indicating the effect was mediated via activation of delta-opioid receptors. Pretreatment of tissues with PTX (75 ng/ml), completely abolished the concentration-dependent production of IPs generated by SNC80 (10 −11–10 −7 mol/l). In addition, pretreatment with phosducin (1 nmol/l) ablated the SNC80 (1 nmol/l)-induced increase in the formation of all three inositol phosphates. Results from this study indicate that the delta-opioid receptor-mediated increase in IP production is a PTX-sensitive G i/o response that involves participation of Gβγ-subunits. Thus, delta-opioid receptor activation by SNC80 in the ICB could be responsible, in part, for suppression of aqueous humor dynamics.

Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.

Influence of receptor number on functional responses elicited by agonists acting at the human adenosine A(1) receptor: evidence for signaling pathway-dependent changes in agonist potency and relative intrinsic activity.

Activation of A(1) adenosine receptors leads to the inhibition of cAMP accumulation and the stimulation of inositol phosphate accumulation via pertussis toxin-sensitive G-proteins. In this study we have investigated the signaling of the A(1) adenosine receptor in Chinese hamster ovary (CHO) cells, when expressed at approximately 203 fmol/mg (CHOA1L) and at approximately 3350 fmol/mg (CHOA1H). In CHOA1L cells, the agonists N(6)-cyclopentyladenosine (CPA), (R)-N(6)-(2-phenylisopropyl)adenosine, and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited cAMP production in a concentration-dependent manner. After pertussis toxin treatment, the agonist NECA produced a stimulation of cAMP production, whereas CPA and (R)-N(6)-(2-phenylisopropyl)adenosine were ineffective. In CHOAIH cells, however, all three agonists produced both an inhibition of adenylyl cyclase and a pertussis toxin-insensitive stimulation of adenylyl cyclase. All three agonists were more potent at inhibiting adenylyl cyclase in CHOA1H cells than in CHOA1L cells. In contrast, A(1) agonists (and particularly NECA) were less potent at stimulating inositol phosphate accumulation in CHOA1H cells than in CHOA1L cells. After pertussis toxin treatment, agonist-stimulated inositol phosphate accumulation was reduced in CHOA1H cells and abolished in CHOA1L cells. The relative intrinsic activity of NECA in stimulating inositol phosphate accumulation, compared to CPA (100%), was much greater in the presence of pertussis toxin (289.6%) than in the absence of pertussis toxin (155.2%). These data suggest that A(1) adenosine receptors can couple to both pertussis toxin-sensitive and -insensitive G-proteins in an expression level-dependent manner. These data also suggest that the ability of this receptor to activate different G-proteins is dependent on the agonist present.

Neurochemical effects of environmental chemicals: in vitro and in vivo correlations on second messenger pathways.

Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, toxic, and widely distributed environmental chemicals. There is now both epidemiological and experimental evidence that PCBs cause cognitive deficits; however, the underlying cellular or molecular mechanism(s) is not known. We have hypothesized that altered signal transduction/second messenger homeostasis by PCBs may be associated with these effects since second messengers in signal transduction pathways, such as calcium, inositol phosphates (IP), and protein kinase C (PKC), play key roles in neuronal development and their function. In vitro studies using cerebellar granule neurons and isolated organelle preparations indicate that ortho-PCBs increase intracellular free Ca2+ levels by inhibiting microsomal and mitochondrial Ca2+ buffering and the Ca2+ extrusion process. Ortho-PCBs also increase agonist-stimulated IP accumulation and cause PKC translocation at low micromolar concentrations where no cytotoxicity is observed. On the other hand, non-ortho-PCBs are not effective in altering these events. Further SAR studies indicate that congeners with chlorine substitutions favoring non-coplanarity are active in vitro, while congeners favoring coplanarity are relatively inactive. Subsequent in vivo studies have shown that repeated exposure to a PCB mixture, Aroclor 1254, increases PKC translocation and decreases Ca2+ buffering in the brain, similar to in vitro studies. These changes in vivo are associated with elevated levels of non-coplanar ortho-PCB congeners at levels equivalent to 40-50 microM in brain, the concentrations that significantly inhibited second messenger systems in neuronal cultures in vitro. Current research is focusing on PCB-induced alterations in second messenger systems following developmental exposure.

Differential regulation of human α1-adrenoceptor subtypes

We have compared the agonist-induced down-regulation of human alpha1A-, alpha1B- and alpha1D-adrenoceptors upon stable expression in rat-1 fibroblasts. During a 24-h incubation the agonist phenylephrine downregulated alpha1A- and alpha1 -adrenoceptors in a concentration-dependent manner. While maximum downregulation was similar for both subtypes, the threshold concentration for significant reductions was markedly higher for alpha1A- than for alpha(1B-adrenoceptors (10 microM vs. 100 nM). The downregulation of both subtypes by 100 microM phenylephrine was time-dependent, and significant reductions were observed already after 2-4 h. In contrast, incubation of alpha1D-adrenoceptor-expressing cells with phenylephrine increased receptor number in a time- and concentration-dependent manner. The downregulation of alpha1B-adrenoceptors by 100 microM phenylephrine for 24 h was accompanied by a matching reduction in mRNA abundance, but no such reduction was seen for alpha-adrenoceptors. These treatment conditions also caused a functional desensitization of agonist-stimulated inositol phosphate formation for alpha1A- and alpha1B- but not for alpha1D-adrenoceptors. Treatment with the phorbol ester phorbol-12-myristate-13-acetate did not change receptor density or mRNA abundance and did not cause functional desensitization. We conclude that human alpha1-adrenoceptor subtypes are differentially regulated by agonist treatment even if they are expressed in the same cell line.

A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor.

Construction of constitutively active mutants of the GnRH receptor, a member of the G-protein coupled receptor superfamily, would facilitate investigation of the mechanism of receptor activation. Point mutations were introduced in the human GnRH receptor in positions corresponding to those which caused constitutive activity in other G-protein coupled receptors. The effects of these mutations on ligand binding, receptor intracellular signaling and receptor expression were determined. Wild type and mutated receptor cDNAs were expressed in COS-1 cells. Basal and agonist-stimulated inositol phosphate production and ligand binding were determined. In addition, receptor mRNA levels, cell surface receptor stability and rate of internalization were measured. Although none of the mutant receptors exhibited constitutive activity, mutation of Phe-2 72 in transmembrane helix VI to Leu increased cell surface receptor numbers, with unchanged affinities for radiolabeled agonist, superagonist and antagonist peptides compared with wild type receptor. The cell surface receptor stability and rate of internalization were similar for wild type and F272L GnRH receptors. Thus a single amino acid mutation in transmembrane helix VI causes an increase in cell surface receptor numbers, which appears to result from an increased rate of receptor protein translation, processing or insertion into membranes.

Functional Microdomains in G-protein-coupled Receptors: THE CONSERVED ARGININE-CAGE MOTIF IN THE GONADOTROPIN-RELEASING HORMONE RECEPTOR

An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.

Molecular cloning, genomic characterization and expression of novel human α 1A-adrenoceptor isoforms

We have isolated and characterized from human prostate novel splice variants of the human α 1A-adrenoceptor, several of which generate truncated products and one isoform, α 1A-4, which has the identical splice site as the three previously described isoforms. Long-PCR on human genomic DNA showed that the α 1A-4 exon is located between those encoding the α 1A-1 and α 1A-3 variants. CHO-K1 cells stably expressing α 1A-4 showed ligand binding properties similar to those of the other functional isoforms as well as agonist-stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that α 1A-4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Disruption of Inositol Phosphate Accumulation in Cerebellar Granule Cells by Polychlorinated Biphenyls: A Consequence of Altered Ca2+Homeostasis

The present study examined the activation of protein kinase C (PKC) and disruption of Ca2+homeostasis as potential mechanisms underlying effects of the polychlorinated biphenyl (PCB) congener 2,2′-dichlorobiphenyl (DCB) on inositol phosphate (IP) signaling in cerebellar granule cells. DCB (100 μM) increased basal IP accumulation in cerebellar granule cells when the extracellular free Ca2+concentration ([Ca2+]e) was 0.75 mMbut not when [Ca2+]ewas 1 μM. Ionomycin (0.1 to 30 μM), a Ca2+ionophore, also increased basal IP accumulation and [Ca2+]iin a concentration-dependent manner in cerebellar granule cells in the absence of DCB; increases in basal IP accumulation induced by 100 μMDCB were not additive with ionomycin. Ionomycin also disrupted carbachol (CARB, 1 mM)-stimulated IP accumulation. A 30-min preincubation with 0.3 or 1.0 μMionomycin decreased CARB-stimulated IP accumulation, whereas simultaneous addition of 1.0 and 10 μMionomycin with CARB increased and decreased, respectively, IP accumulation. DCB caused concentration-dependent increases in intracellular free Ca2+concentration ([Ca2+]i) in cerebellar granule cells under experimental conditions identical to those used to measure IP accumulation. Following a one-half hour exposure to DMSO, 50 or 100 μMDCB, the [Ca2+]iwas 36, 103, and 453 nM, respectively. We examined whether direct or indirect activation of PKC underlies DCB-induced inhibition of agonist-stimulated IP accumulation. DCB (100 μM) did not alter PKC activity in cytosolic or membrane fractions of granule cell homogenates. In intact cells, 50 nMphorbol 12-myristate, 13-acetate (PMA) inhibited CARB-stimulated IP accumulation by 80%, an effect which was blocked completely by the PKC inhibitor bisindolylmaleimide (2 μM; BIM). However, inhibition of CARB-stimulated IP accumulation (90%) induced by 100 μMDCB was not relieved by BIM. These results suggest that (1) perturbations of Ca2+homeostasis may underlie DCB effects on IP accumulation, (2) at a time which corresponds to addition of agonists in IP accumulation assays, [Ca2+]iis elevated in cerebellar granule cells exposed to DCB, and (3) activation of PKC is not a mechanism by which DCB inhibits agonist-stimulated IP accumulation.

Can the mechanisms of aluminum neurotoxicity be integrated into a unified scheme?

Regardless of the host, the route of administration, or the speciation, aluminum is a potent neurotoxicant. In the young adult or developmentally mature host, the neuronal response to Al exposure can be dichotomized on morphological grounds. In one, intraneuronal neurofilamentous aggregates are formed, whereas in the other, significant neurochemical and neurophysiological perturbations are induced without neurofilamentous aggregate formation. Evidence is presented that the induction of neurofilamentous aggregates is a consequence of alterations in the posttranslational processing of neurofilament (NF), particularly with regard to phosphorylation state. Although Al has been reported to impact on gene expression, this does not appear to be critical to the induction of cytoskeletal pathology. In hosts responding to Al exposure without the induction of cytoskeletal pathology, impairments in glucose utilization, agonist-stimulated inositol phosphate accumulation, free radical-mediated cytotoxicity, lipid peroxidation, reduced cholinergic function, and altered protein phosphorylation have been described. The extent to which these neurochemical modifications correlate with the induction of a characteristic neurobehavioral state is unknown. In addition to these paradigms, Al is toxic in the immediate postnatal interval. Whether unique mechanisms of toxicity are involved during development remains to be determined. In this article, the mechanisms of Al neurotoxicity are reviewed and recommendations are put forth with regard to future research. Primary among these is the determination of the molecular site of Al toxicity, and whether this is based on Al substitution for divalent metals in a number of biological processes. Encompassed within this is the need to further understand the genesis of host- and developmental-specific responses.

Increased [3H]Phorbol Ester Binding in Rat Cerebellar Granule Cells and Inhibition of45Ca2+Sequestration in Rat Cerebellum by Polychlorinated Diphenyl Ether Congeners and Analogs: Structure–Activity Relationships

Our previous reports indicate thatortho-substituted non-coplanar polychlorinated biphenyl (PCB) congeners perturbed neuronal Ca2+-homeostasisin vitro,altered agonist-stimulated inositol phosphate accumulation, and caused protein kinase C (PKC) translocation. The structure–activity relationship (SAR) with 24 PCB congeners was consistent with a chlorination pattern that favored non-coplanarity while those with chlorination that favored coplanarity were less active. To test the hypothesis that coplanarity (or lack thereof) is a significant factor in the activity of PCBs, studies with related classes of chemicals such as the polychlorinated diphenyl ethers (PCDEs), in which coplanarity is more difficult to achieve regardless of degree and pattern of chlorination, were initiated. The selected PCDEs and their analogs are predicted to be active, since they are non-coplanar in nature. The effects of these chemicals were studied using the same measures for which PCBs had differential effects based on structural configuration. These measures include PKC translocation as determined by [3H]phorbol ester ([3H]PDBu) binding in cerebellar granule cells and45Ca2+sequestration as determined by45Ca2+uptake by microsomes and mitochondria isolated from adult rat cerebellum. All the PCDE congeners studied, increased [3H]PDBu binding in a concentration-dependent manner. The order of potency was 2,4,4′-trichlorodiphenyl ether > 4,4′-dichlorodiphenyl ether > diphenyl ether, 3,3′,4,4′-tetrachlorodiphenyl ether and, 2,2′,4,4′,5- and 2,3′,4,4′,5-pentachlorodiphenyl ethers. The structurally related diphenyl ether nitrofen and diphenyl ethaneso,p′-1,1,1-trichloro-2,2-bis[p-chlorophenyl]ethane (DDT) andp,p′-DDT increased [3H]PDBu binding to a similar extent (28–35% stimulation at 100 μM). All PCDE congeners and their analogs inhibited45Ca2+sequestration by microsomes and mitochondria. Of all the chemicals, unchlorinated diphenyl ether was the least active. These results are in agreement with previous SAR findings in which non-coplanar PCBs are active and support our hypothesis that the extent of coplanarity determined by a pattern of chlorination on certain aromatic hydrocarbons can weaken their potencyin vitro,although the extent of chlorination is also important.

Is the heterologous expression of metabotropic glutamate receptors (mGluRs) an appropriate method to study the mGluR function? Experience with human embryonic kidney 293 cells transfected with mGluR1

The cloning of metabotropic glutamate receptors (mgluRs) has initiated a new approach to the study of their function: the introduction of mGluR cDNA into cells that do not normally express mGluRs, thus allowing the heterologous receptor expression. We have transfected human embryonic kidney (HEK) 293 cells with the full length mGluRla cDNA and with its truncated variant which encodes the receptor termed mGluRlT (a receptor lacking the long intracellular domain and similar to the splice variant mGluR1c). Transient transfection of HEK-293 cells with mGluR1a, but not the mGluR1T cDNA, resulted in a significant increase in inositol phosphate (IP) formation in absence of any mGluR agonists. This effect was completely dependent on the presence of extracellular calcium, and unlike the agonist-stimulated IP formation it was insensitive to pertussis toxin. The prolonged activation of IP formation might affect the cell physiology. In an attempt to obtain stably transfected cells, we transfected about 1.5 × 10 6 HEK-293 cells with the plasmide conveying the full-length mGluR1a cDNA and the neomicin-resistance gene. Only 12 clones survived the antibiotic selection, and only one of these 12 clones continued to divide. The size of mRNA from the clone was smaller than the full-length mGluR1a mRNA. The shortened mRNA, revealed in the clone, apparently encoded a functional mGluR that was sensitive to glutamate, but unlike the mGluR1a, it did not respond to 1S,3R-ACPD (1S,3R-aminocyclopentane-1,3-dicarboxylic acid). A prudent use of the heterologous cell transfection technique is necessary in studying the xfunction and the pharmacology of mGluRs.