Metabotrophic Receptors Research Articles

Immunohistofluorescence Study of the Actions of Manganese on the Phospholipase C Mechanism of Dopamine D2‐Like Post‐Synaptic Receptors in Crassostrea virginica

Manganese (Mn) is a neurotoxin causing Manganism, a Parkinson's‐like disease. Mn neurotoxicity involves disruption of dopaminergic neurotransmission. The mechanism of how Mn produces its toxic effects is not fully resolved and reports postulate it to be more related to dopamine neuron dysfunction rather than degeneration. Lack of effective treatment for Mn toxicity is a major obstacle in clinical management of Manganism. Gill lateral cell (GLC) cilia of Crassostrea virginica are controlled by serotonergic‐dopaminergic innervations. Dopamine (DA) is the neurotransmitter causing cilio‐inhibition, serotonin cilio‐excitation. Previous work of our lab with C. virginica showed post‐synaptic DA receptors in GLC are D2 type (D2DR) G protein‐coupled metabotrophic receptors and that Mn disrupts the DA induced cilio‐inhibition of GLC cilia. These findings suggest the D2DR receptor is a potential site of action in Mn neurotoxicity. The D2DR signaling pathway involves inhibition of adenylyl cyclase and activation of phospholipase C (PLC). PLC has not been well studied in bivalves, nor has the effects of Mn on PLC been studied. PLC activation generates the second messengers inositol trisphosphate and diacylglycerol. In this study we hypothesize PLC can be visualized in oyster GLC by immunohistofluorescence, and if so we will determine if Mn effects its visualization. 1° antibodies to PLC and 2° FITC‐labeled antibodies were purchased from Abcam and Santa Cruz Biotechnology, respectively. Briefly, gills were dissected, snap frozen, cryostat sectioned at 10 microns, fixed with EDAC (N‐Ethyl‐N’‐(3‐dimethylaminopropyl) carbodiimide hydrochloride), treated with blockers, and incubated with 1°, then 2° antibodies. Sections were viewed on a Leica epilume fluorescence microscope with a Leica DFC400 camera, 50 watt mercury lamps and FITC excitation/emission filters. All sections were photographed with the same camera setting at 100, 200 and 400X. Phase contrast images also were photographed. We found gill sections showed bright green fluorescence in the cytoplasm and along cell membranes indicating the presence of PLC. In other experiments we treated gills for 1 or 48 hours with 500μM of Mn or zinc (Zn). Zn, which is known to cause allosteric inhibition of a number of enzymes, was used for comparisons. Fluorescence intensity of GLC was quantified using ImageJ from NIH. Results show fluorescence intensity in Mn treated GLC was 14 and 20% reduced compared to untreated cells for the 1 and 48 hour treatments, respectively. Gills treated with Zn did not show a difference. The study shows PLC is present in GLC of C. virginica and that Mn did cause a small, but significant reduction in PLC fluorescence intensity. This study provides new knowledge of the actions of Mn on the D2DR pathway in bivalve gill. Our future experiments will test if Mn negatively effects the physiological actions of PLC on GLC cilia activity.Support or Funding InformationSupported in part by grant 2R25GM06003 of the Bridge Program of NIGMS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Association of GRM7 Variants with Different Phenotype Patterns of Age-Related Hearing Impairment in an Elderly Male Han Chinese Population

Several single nucleotide polymorphisms (SNPs) of the Glutamate metabotrophic receptor 7 gene (GRM7) have recently been identified by the genome-wide association study (GWAS) as potentially playing a role in susceptibility to age-related hearing impairment (ARHI), however this has not been validated in the Han Chinese population. The aim of this study was to determine if these SNPs are also associated with ARHI in an elderly male Han Chinese population. In this case-control candidate genes association study, a total of 982 men with ARHI and 324 normal-hearing controls subjects were studied. Using K-means cluster analysis, four audiogram shape subtypes of ARHI were identified in the case group: ‘‘flat shape (FL)’’, ‘‘sloping shape (SL)’’, ‘‘2-4 kHz abrupt loss (AL) shape’’ and ‘‘8 kHz dip (8D) shape’’. Results suggested that the SNP rs11928865 (A>T) of GRM7 was significantly associated with ARHI after adjusting for non-genetic factors (p= 0.000472, OR= 1.599, 95%CI= 1.229~2.081). Furthermore, frequency of TT genotype (rs11928865) were significant higher in the SL subgroup and AL subgroup with compared to controls group (p= 9.41E-05, OR= 1.945, 95%CI= 1.393~2.715; p= 0.000109, OR= 1.915, 95%CI= 1.378~2.661 adjusted, respectively) after Bonferroni correction. However, there wasn’t significant difference in the frequency of the TT genotype between cases in the FL subgroup or the 8D subgroup with when compared with controls. Results of the current study suggest that, in an elderly male Han Chinese population, GRM7 SNP rs11928865 (TT) occurs more frequently in ARHI patients with SL and AL phenotype patterns.

Evidence that L-AP5 and D,L-AP4 can preferentially block cone signals in the rat retina

Several lines of evidence suggest that, as concentrations of two agonists of group III metabotropic glutamate receptors are increased, cone contributions to the b-wave are blocked before rod contributions. Application of L-AP5 (L-2-amino-5-phosphonobutyric acid) at concentrations of 50 microM and D,L-AP4 (D,L-2-amino-4-phosphonobutyric acid) at concentrations 2 microM had a greater effect in reducing the amplitude of the rat ERG b-wave at high light intensities than at low light intensities. The amplitude reduction occurs at flash intensities that saturate rod photoreceptor responses. When steady backgrounds are used to saturate rod photoreceptors, the b-wave responses show increased long-wavelength sensitivity. Responses on a rod saturating background are blocked by adding L-AP5 or AP4 at the above concentrations to the perfusate. Further evidence for metabotrophic receptors being involved comes from the observation that even when ionotropic glutamate receptors are pharmacologically blocked with MK801 and DNQX, AP4 selectively blocks cone contributions to the b-wave. Thus we suggest that the type III metabotrophic receptors on depolarizing cone bipolar cells or cone synaptic terminals are affected by concentrations of L-AP5 and D,L-AP4 that have minimal effects on rod bipolar cells or rod synaptic terminals.

Glutamatergic receptors regulate expression, phosphorylation and accumulation of neurofilaments in spinal cord neurons

Glutamatergic regulation of neurofilament expression, phosphorylation and accumulation in cultured spinal cord neurons was studied. At seven days in culture, 0.15% of the neurons were immunoreactive for non-phosphorylated neurofilaments, but essentially no cells immunoreactive for phosphorylated neurofilaments were seen. The number and size of the immunoreactive cells in culture corresponded well to those of rat and human spinal cord neurons in vivo. In spinal cord cultures, sublethal, long-lasting stimulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate or metabotrophic receptors, but not N-methyl- d-aspartate receptors, dose-dependently increased the number of non-phosphorylated neurofilament-immunoreactive cells, which was blocked by nifedipine, an antagonist of voltage-sensitive Ca 2+ channels. Stimulation of kainate or all non- N-methyl- d-aspartate receptors decreased the expression of medium-molecular-weight neurofilament messenger RNA. Blockade of AMPA/kainate receptors, but not of N-methyl- d-aspartate receptors, increased the amount of phosphorylated neurofilament protein and the number of phosphorylated neurofilament-immunoreactive cell bodies. The phosphorylated neurofilament-immunoreactive cell population was different from the non-phosphorylated neurofilament-immunoreactive neurons, which lost their axonal non-phosphorylated neurofilament immunoreactivity but showed intense cytoplasmic labeling in response to the blockade of AMPA/kainate receptors. Immunoreactivity for phosphoserine did not change upon glutamate receptor stimulation and blockade. The results show that activation of AMPA/kainate receptors decreases the expression of neurofilament messenger RNA and neurofilament phosphorylation in spinal cord neurons by a mechanism involving active voltage-sensitive Ca 2+ channels. Blockade of these receptors seems to disturb axonal neurofilament transport. Because AMPA/kainate receptors mediate chronic glutamatergic death of spinal motor neurons and these receptors have been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis, the observed alteration in neurofilament phosphorylation and distribution may contribute to the pathogenesis of chronic motor neuron diseases.

Possible role of nitric oxide in the development of L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

1. L-2-Chloropropionic acid (L-CPA) produces selective neuronal cell necrosis in rat cerebellum when administered orally at 750 mg kg-1 that is mediated in part through activation of N-methyl-D-aspartate (NMDA) receptors. Cerebellar granule cell death occurs between 30 and 36 h following L-CPA administration exhibiting a number of features in common with excitatory amino acid-induced cell death. We have used this in vivo model to examine the neurochemical processes following L-CPA-induced activation of NMDA receptors leading to neuronal cell death in the rat cerebellum. 2. The effects of a number of compounds which potently block nitric oxide synthase in vitro were examined on L-CPA-induced neurotoxicity 48 h following L-CPA dosing, to discover whether the neuronal cell death is mediated in part by excessive nitric oxide generation. Four inhibitors were studied, NG-nitro-L-arginine (L-NOARG), NG-nitro-L-arginine methyl ester (L-NAME), NG-iminoethyl-L-ornithine (L-NIO) and 3-bromo-7-nitroindazole (BrNI). 3. L-NAME (50 mg kg-1, i.p. twice daily) and BrIN (50 mg kg-1, i.p. twice daily) administration prevented the L-CPA-induced loss of granule cells which can reach up to 80-90% of the total cell number in rats treated with L-CPA alone. L-NOARG (50 mg kg-1, i.p. twice daily) and L-NIO administered at either 25 or 100 mg kg-1, twice daily did not produce any significant protection against L-CPA-induced neurotoxicity. 4. Both L-NAME and BrIN also prevented the L-CPA-induced increase in cerebellar water content and sodium concentrations. L-NIO when administered at the highest doses prevented the increase in cerebellar sodium concentration but not water content. L-NIO and L-NOARG were ineffective in preventing the L-CPA-induced increases in cerebellar water and sodium concentrations. 5. L-CPA-induced reductions in cerebellar aspartate and glutamate concentrations and increases in glutamine and GABA concentrations were prevented by L-NAME and BrIn, but not by L-NIO or L-NOARG. Also reductions in L-[3H]-glutamate binding to glutamate ionotrophic and metabotrophic receptors in the granule cell layer of rat cerebellum was prevented by L-NAME and BrIN, but not L-NIO or L-NOARG. 6. In conclusion, the neuroprotection offered by L-NAME and BrIN suggests that L-CPA-induced cerebellar granule cell necrosis is possibly mediated by or associated with excessive generation of nitric oxide. The inability of nitric oxide synthase inhibitors, L-NOARG and L-NIO to afford protection may result from their limited penetration into the brain (L-NIO) or rapid dissociation from the enzyme.

Cytokines in neurodegeneration and repair

Cytokines have diverse actions in the brain, some of which may facilitate either neurodegeneration or neuroprotection. The expression of cytokines, particularly interleukins-1 and -6 (IL-1, IL-6) and tumor necrosis factor α, is rapidly and markedly induced in response to experimentally induced or clinical neurodegeneration. We have demonstrated that central administration of the IL-1 receptor antagonist (IL-1ra) markedly inhibits neurodegeneration induced by focal cerebral ischaemia, local infusion of glutamate receptor agonists or traumatic brain injury in the rat. In contrast, IL-1ra offers no protection against degeneration of primary cortical neurones in culture caused by exposure to agonists of ionotrophic or metabotrophic receptors. In vivo, administration of IL-1β exacerbates ischaemic brain damage, whereas in cell culture, exogenous IL-1 is neuroprotective at concentrations in the nM range, an effect which appears to be mediated by release of endogenous nerve growth factor. Higher concentrations of IL-1 (μM range) are neurotoxic to neurones in culture and may mimic the involvement of IL-1 in neurodegeneration in vivo. Thus, excessive production of cytokines such as IL-1 appears to mediate experimentally induced neurodegeneration in vivo, while neuroprotective effects of low concentrations of the cytokine suggest a dual role for IL-1 in neuronal survival.