Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas.

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability. In the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 (SK2) and SK-MEL-5 (SK5), we show that glutamate is both necessary and sufficient to maintain cell viability, regardless of underlying genetic mutations. Addition of glutamate increased DNA synthesis, whereas removal of glutamate not only suppressed DNA synthesis but also promoted cell death in SK2 and SK5 melanoma cells. Using genetic and pharmacological inhibitors, we established that this effect of glutamate is mediated by the activation of mGlu1 receptors. The stimulatory potential of mGlu1 receptors was further confirmed in vivo in a melanoma cell xenograft model. In this model, subcutaneous injection of SK5 cells with short hairpin RNA-targeted downregulation of mGlu1 receptors resulted in a decrease in the rate of tumor growth relative to control. We also demonstrate for the first time that a selective mGlu1 receptor antagonist JNJ16259685 ((3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone) slows SK2 and SK5 melanoma tumor growth in vivo. Taken together, these data suggest that pharmacological inhibition of mGlu1 receptors may be a novel approach for the treatment of metastatic melanoma.

Axotomy and nerve growth factor regulate levels of neuronal nicotinic acetylcholine receptor alpha3 subunit protein in the rat superior cervical ganglion.

Neuronal nicotinic acetylcholine receptors (nAChRs) play a significant role in sympathetic transmission in the superior cervical ganglia (SCG), with most of the signal carried by a nAChR containing an alpha3 subunit. Work has shown that transection of the postganglionic nerves (axotomy) of the SCG results in a decrease in mRNA transcripts for alpha3, alpha5, alpha7 and beta4 and in protein expression of alpha7 and beta4. To evaluate effects of axotomy on alpha3 protein in the SCG, quantitative immunoblotting was used to demonstrate a dramatic decrease (> 80%) in the levels of this subunit 4 days after axotomy. Similarly, immunocytochemistry showed a marked decline in the number and the intensity of stained neurons for the alpha3 subunit as well as tyrosine hydroxylase. Ganglia explanted into culture for 4 days also showed a substantial decrease in alpha3 subunit protein. This decrease was partially prevented by the addition of nerve growth factor (NGF) to the culture medium at the time of explantation. Additionally, this decrease was reversed by the addition of NGF to the culture medium following 4 days in culture in the absence of NGF. These findings suggest that the loss of alpha3 subunit contributes to the reported decrease in ganglionic synaptic transmission that follows axotomy, and that NGF plays an important role in regulating the expression of alpha3-containing nAChRs in the SCG.

Enrichment of N-methyl-D-aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities.

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density-associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)-95, the NMDA receptor subunits, chapsyn-110, and the CaMKII alpha subunit. However, synaptophysin, SAP-102, and the GABA(A) receptor beta subunit exhibited lower enrichment levels compared to PSD-95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression.

A synthetic peptide corresponding to the C-terminus of the alpha 3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal alpha 3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the alpha 3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of alpha 3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the alpha 3 subunit in the SCG was also determined. The alpha 3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the alpha 3 subunit participate in important roles in specific regions of the rat brain and the SCG.

Evidence of a functional alpha7-neuronal nicotinic receptor subtype located on motoneurons of the dorsal motor nucleus of the vagus.

In vitro autoradiography using 125I-alpha-bungarotoxin (alpha-BGTx) and anti-alpha7 immunohistochemistry were performed on the dorsal motor nucleus of the vagus (DMV) of sham and chronically vagotomized rats to determine whether the alpha7-nicotinic acetylcholine receptor (nAChR) is located postsynaptically on DMV neurons whose axons contribute to the vagus nerve. Intense bilateral 125I-alpha-BGTx binding and anti-alpha7 immunostaining were observed in coronal brain sections containing the DMV of sham-vagotomized animals. Unilateral cervical vagotomy resulted in ipsilateral losses of 125I-alpha-BGTx binding and anti-alpha7 immunostaining from the DMV. Simultaneous staining of rat brainstem sections with anti-alpha7 and anti-choline acetyltransferase (ChAT) antibodies (to identify cholinergic DMV neurons that project into the vagus nerve) revealed that every DMV neuron that was stained for ChAT showed alpha7-staining as well. In vagotomized animals, no ChAT-positive neurons expressing alpha7-nAChRs remained in the ipsilateral DMV. We conclude that the alpha7-nAChR subtype is located postsynaptically on DMV neurons. To test whether the alpha7-nAChR is similar to the alpha7-homomeric nAChR, experiments were performed in anesthetized rats, and compounds were microinjected into the DMV while monitoring intragastric pressure (IGP). alpha-BGTx and strychnine antagonized nicotine-induced increases in IGP; no antagonism was observed with methyllycaconitine, a compound known to block the homomeric alpha7-nAChR subtype. Recovery from alpha-BGTx-induced antagonism of the nicotine response was observed. We conclude that there is a nAChR containing the alpha7-subunit in the DMV that is different from the homomeric alpha7-nAChR subtype.

Effects of post-mortem delay on subunits of ionotropic glutamate receptors in human brain.

The effect of post-mortem delay on the stability of the protein subunits that combine to form NMDA and AMPA type glutamate receptors has been assessed in samples of human brain tissue. While most of the subunits (i.e. GluR1, GluR2/3, GluR4, NR1) appear to be stable for up to 18 h post-mortem, the NR2A and NR2B subunits appear to be proteolyzed rapidly following death. These results are consistent with the concept that the proteolytic products of NR2A and NR2B, although at smaller molecular sizes than the full-length protein, are all identifiable on Western blots. Thus, a method is proposed that allows for the estimation of the levels of these labile proteins even in samples obtained up to 18 h post-mortem. Using this method we have estimated the levels of all AMPA and NMDA receptor subunits in selected (i.e. hippocampus, frontal and entorhinal cortex) brain tissue samples obtained from control patients and patients who have died with Alzheimer's disease. Modest decreases in NMDA receptor subunits NR1, NR2A, and NR2B were found in the hippocampus and in frontal cortex while little or no change in any of these subunits were documented in entorhinal cortex. Subunits for AMPA receptors (GluR1, GluR2/3, and GluR4) appeared to show a generalized decrease in all these tissues. As a surrogate marker for overall decreases due to generalized neuronal cell death, levels of neuron-specific enolase were measured in all tissues and were found to be nearly identical in control and Alzheimer's brains.

Changes in NMDA receptor subunit expression in response to contusive spinal cord injury.

Differential assembly of N-methyl-D-aspartate (NMDA) receptor subunits determines their functional characteristics. Using in situ hybridization, we found a selective increase of the subunits NR1 and NR2A mRNA at 24 h in ventral motor neurons (VMN) caudal to a standardized spinal cord contusion injury (SCI). Other neuronal cell populations and VMN rostral to the injury site appeared unaffected. Significant up-regulation of NR2A mRNA also was seen 1 month after SCI in thoracic and lumbar VMN. The selective effects on VMN caudal to the injury site suggest that the loss of descending innervation leads to increased NMDA receptor subunit expression in these cells after SCI, which may alter their responses to glutamate. In contrast, protein levels determined by western blot analysis show decreased levels of NR2A 1 month after SCI in whole thoracic segments of spinal cord that included the injury sites. No effects of injury were seen on subunit levels in cervical or lumbar segments. Taken together with our previous study showing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit down-regulation after injury, our data suggest that glutamate receptor composition is significantly altered after SCI. These changes need to be taken into account to properly understand the function of, and potential pharmacotherapy for, the chronically injured spinal cord.

Quantification of human H1 histamine receptor mRNA from peripheral blood.

STUDY OBJECTIVE: To develop a reverse transcription (RT)-polymerase chain reaction (PCR) technique to detect and quantify human histamine1 (H1) receptor mRNA in peripheral blood. METHODS: Primer pairs were based on the human H1 receptor nucleotide sequence. A competitive reference standard (CRS) was developed that used the same primers as wild-type mRNA but contained a 92-bp deletion. RT-PCR was performed with 5 microg of total RNA obtained from venous blood of six subjects that was added to known concentrations of CRS RNA. Linear regression comparing wild-type with CRS product was used to quantify wild-type mRNA. MEASUREMENTS AND MAIN RESULTS: Three subjects had detectable H1 mRNA, with a range of 31-435 pg. In three subjects PCR product was not detected, although the presence of RNA was confirmed. Redesigned primer pairs showed mRNA to H1 receptor in two of the remaining subjects, but it was undetectable in the third. CONCLUSION: RT-PCR can be used to detect and quantify human H1 receptor mRNA from peripheral blood.

Alterations in subunit expression, composition, and phosphorylation of striatal N-methyl-D-aspartate glutamate receptors in a rat 6-hydroxydopamine model of Parkinson's disease.

Recent evidence has linked striatal N-methyl-D-aspartate (NMDA) receptor function to the adverse effects of long-term dopaminergic treatment in Parkinson's disease. We have studied the abundance, composition, and phosphorylation of NMDA receptor subunits (NRs) in the rat 6-hydroxydopamine lesion model of parkinsonism. In lesioned striatum, the abundance of NR1 and NR2B in striatal membranes was decreased to 68 +/- 3.2 and 62 +/- 4.4%, respectively, relative to the unlesioned striata, whereas the abundance of NR2A was unchanged. Coimmunoprecipitation of NMDA receptors under nondenaturing conditions revealed that these changes reflected a selective depletion of receptors composed of NR1/NR2B, without alteration in receptors composed of NR1/NR2A. However, the abundance and composition of striatal NMDA receptors in extracts containing both cytoplasmic and membrane proteins were not altered in lesioned rats, suggesting that the changes in the membrane fraction resulted from intracellular redistribution of receptors. The phosphorylation of NR1 protein at serine 890 and serine 896, but not at serine 897, and the tyrosine phosphorylation of NR2B but not NR2A were decreased in the membrane fraction of the lesioned striatum. Chronic treatment of lesioned rats with L-dopa normalized the alterations in the abundance and subunit composition of the NMDA receptors in striatal membranes, and produced striking hyperphosphorylation, both of NR1 at serine residues, and NR2A and NR2B at tyrosine residues. These findings suggest that the adverse motor effects of chronic L-dopa therapy may result from alterations in regulatory phosphorylation sites on NMDA receptors.

Alterations in AMPA receptor subunit expression after experimental spinal cord contusion injury.

The AMPA-preferring subtype of ionotropic glutamate receptors (GluRs) is a hetero-oligomeric ion channel assembled from various combinations of four subunits: GluR1, GluR2, GluR3, and GluR4. Antagonists of these receptors can mitigate the effects of experimental spinal cord injury (SCI), indicating that these receptors play a significant role in pathophysiology after spinal trauma. We tested the hypothesis that SCI alters expression of AMPA receptors using a standardized thoracic weight-drop model of rat contusive spinal cord injury. AMPA receptor subunit expression was measured at 24 hr and at 1 month after SCI with quantitative Western blot analysis and in situ hybridization. GluR2 protein levels were preferentially reduced near the injury site 24 hr after SCI. This reduction persisted at 1 month. At a cellular level, a significant decrease in both GluR2 and GluR4 mRNA was found in spared ventral motor neurons adjacent to the injury site and distal to it, with other AMPA subunit mRNAs maintained at control levels. In contrast, only GluR1 mRNA was decreased in the sympathetic preganglionic neurons of the intermediolateral horn. These results suggest population-specific and long-lasting changes in neuronal AMPA receptor composition, which may alter response to glutamate after SCI. These alterations may contribute not only to acute neuropathological consequences of injury, but they may also be partially responsible for the altered functional state of preserved tissue seen chronically after SCI.

Biochemical studies of the structure and function of the N-methyl-D-aspartate subtype of glutamate receptors.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotoxicity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, and in situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.

Affinities of brompheniramine, chlorpheniramine, and terfenadine at the five human muscarinic cholinergic receptor subtypes.

Anticholinergic effects are presumed to be the mechanism for the efficacy of chlorpheniramine in symptomatic relief of the common cold. Terfenadine, a second-generation antihistamine, reportedly lacks anticholinergic side effects. We evaluated affinities of two commonly used over-the-counter antihistamines, brompheniramine and chlorpheniramine, as well as terfenadine in comparison with atropine at the five human muscarinic cholinergic receptor subtypes using CHO cells stably transfected with the individual subtypes. Atropine was more potent than all three drugs at m1-m5 (p<0.01). No significant difference was observed between chlorpheniramine and brompheniramine. Atropine, brompheniramine, and chlorpheniramine could not discriminate between m1-m5. Terfenadine demonstrated subtype selectivity at m3. In vitro comparisons in human muscarinic receptor subtypes could potentially be used to predict clinical anticholinergic effects of antihistamines and to target receptor-specific effects of such agents.

Developmental regulation of tyrosine phosphorylation of the NR2D NMDA glutamate receptor subunit in rat central nervous system.

A subunit-specific antibody against the N-methyl-D-aspartate (NMDA) receptor NR2D protein along with an antiphosphotyrosine antibody were employed to examine the developmental profile of the tyrosine phosphorylation of NR2D and its regulation by a protein phosphatase inhibitor in rat brain. NMDA receptor proteins from the thalamus at postnatal days 1, 7, 21, and 49 were solubilized under denaturing conditions and used in immunoprecipitations with these antibodies followed by quantitative immunoblot analysis of NR2D protein in the resulting immunopellets. The results indicate that the NR2D subunit is tyrosine phosphorylated in the brain. The quantified data examining the developmental profile of tyrosine phosphorylation of NR2D in the thalamus show that the level of tyrosine phosphorylation of NR2D protein increases five- to sixfold during development. In addition, the protein phosphatase inhibitor pervanadate (vanadyl hydroperoxide) was found to increase tyrosine phosphorylation of NR2D subunit threefold in brain slices, implying an active cycle of phosphorylation and dephosphorylation in situ. These studies demonstrate developmentally regulated tyrosine phosphorylation of NR2D protein in vivo, suggesting that tyrosine phosphorylation may be important for regulating the functions of this NMDA receptor subunit in the mammalian central nervous system.

Subunit composition of N-methyl-D-aspartate receptors in the central nervous system that contain the NR2D subunit.

The N-methyl-D-aspartate (NMDA) receptor is assembled using proteins from two gene families, NR1 and NR2. Although a few studies have examined the composition of NMDA receptors containing NR1, NR2A, and NR2B, the composition of native NMDA receptors that incorporate the NR2D subunit is not known. The goal of the current study was to examine the subunit composition of native NMDA receptors that contain the NR2D subunit in the rat central nervous system by immunoprecipitation of assembled NMDA receptors from rat brain tissues using specific antibodies against NR1, NR2A, NR2B, and NR2D subunits. NMDA receptors were solubilized using either nondenaturing (native) conditions, in which the subunits remain assembled in complexes, or denaturing conditions, in which the NMDA subunits are dissociated from one another. Each of the antibodies selectively and quantitatively immunoprecipitated only the corresponding subunit when the subunits were solubilized using denaturing conditions. In contrast, when NMDA receptors were solubilized under nondenaturing conditions, immunoprecipitation followed by quantitative immunoblot analysis of the resulting pellets show that the majority of the NR2D protein is associated with the NR1 subunit. In addition, the NR2D subunit forms a heteromeric assembly with NR1, as well as with NR2A and/or NR2B subunits, reflecting ternary complex formation. Finally, a binary complex composed of only NR1/NR2D subunits was found in the thalamus but not in the midbrain, where the complexes always contained either NR2A or NR2B, demonstrating that in the central nervous system, different subtypes of NR2D-containing NMDA receptors are present that vary in spatial expression, perhaps indicating distinct physiological and behavioral roles.

The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B).

A monoclonal antibody (R1JHL) against the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor has been developed that recognizes an epitope in the region of the amino-terminal amino acids 341-561 (a region common to all splice variants of NR1). This monoclonal antibody identifies a broad band at 115 kDa in immunoblots using membranes from NR1-transfected cells and from rat brain tissue. No cross-reactivity with any NR2 subunit is seen. With the goal to determine quantitatively the subunit composition of cortical NMDA receptors, we used the monoclonal antibody to NR1 and polyclonal antibodies against the NR2A and NR2B subunits to perform immunoprecipitations of receptor subunits from solubilized adult rat cortical membranes. Solubilization of the receptor subunits was accomplished under both nondenaturing (native) conditions, under which the subunits seem to remain associated with one another, and denaturing conditions, under which the subunits are associated from each other. Although each of these antibodies selectively immunoprecipitates only its corresponding (cognate) subunit when the subunits have been solubilized under denaturing conditions, each of the antibodies immunoprecipitates a sizable fraction of the other two NMDA receptor subunits when membranes are solubilized under nondenaturing conditions, indicating an interaction in situ. Using quantitative immunoblot analysis of the three subunits in both the pellets and supernatants from the immunoprecipitations, we found 1) the dominant NMDA receptor complex in adult rat cortex contains at least three subunits, NR1/NR2A/NR2B; 2) a smaller fraction of NMDA receptors are composed of only two subunits, NR1/NR2B or NR1/NR2A; 3) there are no complexes that contain NR2A/NR2B that do not contain NR1; 4) only a small fraction of each subunit is not associated with any other NMDA receptor subunit; 5) no coimmunoprecipitation of noncognate subunits occurs unless the subunits are assembled with each other in situ; and 6) there is no physical interaction between these NMDA receptor subunits and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluR2 or GluR3 subunits. These results suggest that functional studies with recombinant receptors composed of at least three subunits may be the most physiologically meaningful.

Regional and ontogenic expression of the NMDA receptor subunit NR2D protein in rat brain using a subunit-specific antibody.

A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an approximately 160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N-glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.

Ontogeny of NMDA R1 subunit protein expression in five regions of rat brain.

A polyclonal antiserum to a fusion protein corresponding to a region of the NMDA R1 (NR1) subunit (amino acids 656-811) was produced and affinity purified. A quantitative immunoblotting technique was developed using the fusion protein as a standard. By employing this method, ontogenic studies (day 2-42) of the density of NR1 protein were carried out in several regions of rat brain. The results showed that in all five of the brain regions examined [olfactory bulb (Ob), cortex (Cx), hippocampus (Hp), midbrain (Mb) and cerebellum (Cb)], levels of NR1 protein are low at birth and increase with similar patterns having a sharp rise within the first 3 weeks after birth. Levels increased 2.0 to 4.5-fold from postnatal day 2 to postnatal day 42. Although the general patterns of developmental expression are similar, large differences in the absolute amounts of NR1 protein among the five brain regions were observed. The maximal levels (pmol of fusion protein equivalent/mg +/- S.E.) of NR1 subunit attained during development in the five regions are: Hp 2.0 +/- 0.37 > Cx 1.4 +/- 0.11 > Ob 1.3 +/- 0.2 > Mb 1.0 +/- 0.10 > Cb 0.57 +/- 0.13. The temporal patterns of expression of NR1 protein are similar to results from studies examining the expression of NR1 mRNA. Furthermore, the absolute numbers obtained from our studies are close to those found using [(3)H]MK-801 binding suggesting that many of the NR1 subunits expressed in the brain exist in an active form.

Hippocampal muscarinic receptor function in spatial learning-impaired aged rats.

Efficiency of coupling of hippocampal muscarinic receptors to phosphoinositide (PI) turnover was investigated in behaviorally characterized young and aged Long-Evans rats using hippocampal minces and the method of partial receptor alkylation of Furchgott. Densities of the m1, m2, and m3 receptor proteins were determined using specific antibodies and immunoprecipitation. Spatial learning ability was quantified using a water maze. There were no differences in the levels of muscarinic receptor proteins between young and aged (27 months) rats or in rats with impaired spatial learning. The dissociation constant (KD) for the agonist oxotremorine-M and the KD/EC50 ratio, an indicator of receptor-effector coupling efficiency were similar in young and aged rats. However, the maximal PI turnover response to oxotremorine-M was decreased in impaired aged rats and this parameter was highly correlated with the spatial learning index (R = -0.825; p < 0.001). A reduction in effector stimulation in the absence of changes in receptor protein or coupling efficiency suggests that dysfunction in the hippocampal muscarinic receptor systems occurs at the level of phospholipase C or beyond.

Adenosine receptor activation potentiates phosphoinositide hydrolysis and arachidonic acid release in DDT1-MF2 cells: putative interrelations.

Studies were undertaken in an effort to discern possible mechanisms by which the A1 adenosine receptor agonist cyclopentyladenosine (CPA) enhances the norepinephrine-stimulated (NE-stimulated) hydrolysis of phosphoinositides in DDT1-MF2 cells. Measurements of arachidonic acid release revealed similar behaviours to those observed in measurements of phosphoinositide hydrolysis. In the presence of NE, both second messenger responses were potentiated by the addition of CPA, whereas in the absence of NE, CPA had little or no effect on either second messenger. The stimulation and potentiation of both second messenger responses were enhanced in the presence of extracellular calcium, and in each case these effects were persistent over time. For either second messenger system the stimulation by NE and the potentiation by CPA appeared to utilize separate mechanisms as evidenced by the fact that the potentiations by CPA were selectively antagonized by a cAMP analogue or by pertussis toxin, whereas the stimulations by NE were essentially unaffected by these agents. Inhibition of phospholipase A2 (PLA2) also blocked the potentiation of PLC by CPA, without affecting NE-stimulated phosphoinositide hydrolysis. Furthermore, in the presence of CPA, the exogenous administration of PLA2 was found to stimulate phosphoinositide hydrolysis in these cells. These data are consistent with a hypothesis whereby the apparent potentiation of NE-stimulated phosphoinositide hydrolysis by CPA is actually due to the stimulation by CPA of a second pathway of phospholipase C activity which is additive to that of NE. The activation of PLC and PLA2 by NE produces phospholipid products which may play a permissive role in the pathway coupling adenosine A1 receptors to these phospholipases. The formation of lysophosphatidic acid is suggested as one possible mediator of this permissive effect.

Characterization of NMDA receptor subunit-specific antibodies: distribution of NR2A and NR2B receptor subunits in rat brain and ontogenic profile in the cerebellum.

Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at approximately 175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, mid-brain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and mid-brain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [3H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth.(ABSTRACT TRUNCATED AT 250 WORDS)