ADP-Ribosylation of Membrane Proteins in Cholinergic Nerve Terminals

Abstract: Lysed Torpedo synaptosomes or washed synaptosomal membranes were incubated with [³²P]NAD⁺ and subjected to electrophoresis on SDS-polyacrylamide gels. More than eight membrane proteins were ADP-ribosylated. The most intensely labeled proteins were those of M_r= 62,000 and 82,000. Radiolabeling was more intense in synaptosomes than in other subcel-lular fractions. Cholera toxin caused ribosylation of additional synaptosomal proteins with M_r= 42,000 and (in some preparations) 49,000. Neither endogenous nor cholera toxin-catalyzed ADP-ribosylation required added guanyl nu-cleotides. Cholera toxin increased the adenylate cyclase activity of synaptosomal membranes, suggesting that the cholera toxin substrates are regulatory components of adenylate cyclase in these synaptosomes.     

[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions

Abstract: [³H]Nemonapride and [³H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [³H]nemonapride and [³H]spiperone give markedly different B _max values for preparations of D₂ dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D_2(short), D_2(long), and D₃] in different buffers. B _max values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [³H]spiperone and [³H]nemonapride do not differentiate between different forms or populations of D₂-like receptors.     

N-Methyl-d-Aspartate-Sensitive Glutamate Receptors Induce Calcium-Mediated Arachidonic Acid Release in Primary Cultures of Cerebellar Granule Cells

Abstract: In primary cultures of cerebellar granule cells, glutamate, aspartate, and N -methyl-d-aspartate (NMDA) induced a dose-dependent release of [³H]arachidonic acid ([³H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [³H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A₂ (PLA₂) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA₂ rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [³H]AA release was dependent on the presence of extracellular Ca²⁺ and was mimicked by the Ca²⁺ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [³H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA₂ that is mediated by Ca²⁺ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.     

Comparison of [3H]WIN 35,428 Binding, a Marker for Dopamine Transporter, in Embryonic Mesencephalic Neuronal Cultures with Striatal Membranes of Adult Rats

Abstract: In contrast to striatal membranes of adult rats, where high- ( K _D1= 34 n M ) and low- ( K _D2= 48,400 n M ) affinity binding sites for [³H]WIN 35,428 are present, in primary cultures of ventral mesencephalon neurons (CVMNs) only low-affinity binding sites were found ( K _D= 336,000 n M ). The binding of [³H]WIN 35,428 in CVMNs prepared from rat embryos was reversible, saturable, and located in cytosol. Although dopamine (DA) uptake blockers inhibited [³H]DA uptake at nanomolar concentrations in CVMNs, the displacement of [³H]WIN 35,428 binding in CVMNs by DA uptake inhibitors required 100-8,000 times higher concentrations than were needed to displace [³H]WIN 35,428 binding in striatal membranes. Piperazine derivatives, e.g., GBR-12909, GBR-12935, and rimcazole, inhibited [³H]WIN 35,428 binding in CVMNs more effectively than did cocaine, WIN 35,428, mazindol, nomifensine, or benztropin. A positive correlation ( r = 0.779; p < 0.001) was found between drug affinities for the striatal membrane sites labeled by [³H]WIN 35,428 and their abilities to inhibit DA uptake in CVMNs, whereas no correlation existed between the IC₅₀ values of drugs that inhibited [³H]WIN 35,428 binding and [³H]DA uptake in CVMNs. The cytosolic [³H]WIN 35,428 binding sites may be a piperazine acceptor and may not be involved in the regulation of the DA transporter.     

Enhanced sensitivity to cholera toxin in ADP-ribosylarginine hydrolase-deficient mice

Cholera toxin (CT) produced by Vibrio cholerae causes the devastating diarrhea of cholera by catalyzing the ADP-ribosylation of the alpha subunit of the intestinal Gs protein (Gsalpha), leading to characteristic water and electrolyte losses. Mammalian cells contain ADP-ribosyltransferases similar to CT and an ADP-ribosyl(arginine)protein hydrolase (ADPRH), which cleaves the ADP-ribose-(arginine)protein bond, regenerating native protein and completing an ADP-ribosylation cycle. We hypothesized that ADPRH might counteract intoxication by reversing the ADP-ribosylation of Gsalpha. Effects of intoxication on murine ADPRH-/- cells were greater than those on wild-type cells and were significantly reduced by overexpression of wild-type ADPRH in ADPRH-/- cells, as evidenced by both ADP-ribose-arginine content and Gsalpha modification. Similarly, intestinal loops in the ADPRH-/- mouse were more sensitive than their wild-type counterparts to toxin effects on fluid accumulation, Gsalpha modification, and ADP-ribosylarginine content. Thus, CT-catalyzed ADP-ribosylation of cell proteins can be counteracted by ADPRH, which could function as a modifier gene in disease. Further, our study demonstrates that enzymatic cross talk exists between bacterial toxin ADP-ribosyltransferases and host ADP-ribosylation cycles. In disease, toxin-catalyzed ADP-ribosylation overwhelms this potential host defense system, resulting in persistence of ADP-ribosylation and intoxication of the cell.     

Characterization of [3H]CP-96,501 as a Selective Radioligand for the Serotonin 5-HT1B Receptor: Binding Studies in Rat Brain Membranes

Abstract: 3-(1,2,5,6-Tetrahydro-4-pyridyl)-5- n -propoxyindole (CP-96,501) was found to be a more selective ligand at the serotonin 5-HT_1B receptor than the commonly used 5-HT_1B agonist, 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [³H]CP-96,501, was found to bind with a high affinity ( K _D, 0.21 n M ) to a single binding site ( n _H, 1.0). The receptor density of this site ( B _max, 72 fmol/mg of protein) matched that of the 5-HT_1B receptor determined with [³H]5-HT. Competition curves of 16 serotonergic compounds in [³H]CP-96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5-HT_1B receptor determined with [³H]5-HT or [¹²⁵I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [³H]CP-96,501 binding. In the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p], [³H]CP-96,501 binding was reduced, while the potency of CP-96,501 to displace [¹²⁵I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP-96,501. The results of this study suggest that [³H]CP-96,501 is a useful agonist radioligand for the 5-HT_1B receptor.     

Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

Effects of Monensin on Assembly of Po Protein into Peripheral Nerve Myelin

Abstract: The ionophore monensin has been used in a variety of systems to block secretion of glycoproteins or assembly of glycoproteins into membranes. We examined the effects of monensin on assembly of the Po glycoprotein into PNS myelin, and compared this agent with the glycosylation inhibitor tunicamycin in our system. Sciatic nerves from 9-day-old rat pups were sliced and incubated in vitro . Electron microscopy of the Schwann cells in slices incubated with monensin revealed extensive swelling of the Golgi complex. Incubation with 10⁻⁷ M monensin inhibited total protein synthesis by about 20% and fucose incorporation into protein about 35%. Following isolation of myelin, proteins were separated by sodium dodecyl sulfate gel electrophoresis. Monensin inhibited the appearance of Po in myelin, while causing its accumulation in a denser membrane fraction. In addition, a slightly faster-migrating species of P_o labeled with both [³H]fucose and [¹⁴C]glycine was observed in all fractions. Assembly of basic proteins into myelin was not affected. Preincubation with 10 μg/ml tunicamycin for 30 min prior to incubation with [³H]fucose and [¹⁴C]glycine for 2 h resulted in a 65% decrease in [³H]fucose incorporation into P_o, and the appearance of a new [¹⁴C]glycine-labeled peak that migrated in the region of the 23K protein reported by Smith and Sternberger. [³H]Fucose incorporation was inhibited earlier, and to a greater extent, than protein synthesis. Our results show that processing of the P_o glycoprotein is sensitive to both monensin and tunicamycin, and that monensin partially blocks assembly of P_o into myelin.     

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.     

Pharmacological and Regional Characterization of [3H]LY278584 Binding Sites in Human Brain

Abstract: Binding of [³H]LY278584, which has been previously shown to label 5-hydroxytryptamine₃ (5-HT₃) receptors in rat cortex, was studied in human brain. Saturation experiments revealed a homogeneous population of saturable binding sites in amygdala ( K _D= 3.08 ± 0.67 n M, B _max= 11.86 ± 1.87 fmol/mg of protein) as well as in hippocampus, caudate, and putamen. Specific binding was also high in nucleus accumbens and entorhinal cortex. Specific binding was negligible in neocortical areas. Kinetic studies conducted in human hippocampus revealed a K _on of 0.025 ± 0.009 n M ⁻¹ min⁻¹ and a K _off of 0.010 ± 0.002 min⁻¹. The kinetics of [³H]LY278584 binding were similar in the caudate. Pharmacological characterization of [³H]LY278584 specific binding in caudate and amygdala indicated the compound was binding to 5-HT₃ receptors. We conclude that 5-HT₃ receptors labeled by [³H]LY278584 are present in both limbic and striatal areas in human brain, suggesting that 5-HT₃ receptor antagonists may be able to influence the dopamine system in humans, similarly to their effects in rodent studies.     

Dopamine Agonist-Mediated Inhibition of Acetylcholine Release in Rat Striatum Is Modified by Thyroid Hormone Status

Abstract: K⁺-evoked acetyl[³H]choline ([³H]ACh) release was inhibited in a concentration-dependent manner by apomorphine and the D₂ agonist quinpirole in striatal slices prepared from euthyroid and hypothyroid rats. However, there was a significant increase in the maximum inhibition observed with both agonists in the hypothyroid compared with the euthyroid group, which paralleled the increased D₂ agonist sensitivity reported for stereotyped behavior. The D₂ antagonist raclopride decreased, and the D, antagonist SCH 23390 increased, the inhibition of [³H]ACh release by apomorphine, confirming an inhibitory role for D₂ receptors and an opposing role for D₁ receptors. Because there is no difference in D₁ or D₂ receptor concentration between the euthyroid and hypothyroid groups, it is suggested that thyroid hormone modulation of D₂ receptor sensitivity affects a receptor-mediated event. Following intrastriatal injection of pertussis toxin (PTX), apomorphine no longer inhibited [³H]ACh release. In fact, increased [³H]- ACh release was observed, an effect reduced by SCH 23390, providing evidence that D₁ receptors enhance [³H]- ACh release, and confirming that a PTX-sensitive G protein mediates the D₂ response. As it has been reported that thyroid hormones modulate G protein expression, this mechanism may underlie their effect on dopamine agonist- mediated inhibition of ACh.     

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein

Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.     

Adenine nucleotides directly stimulate pertussis toxin

Both cholera toxin and pertussis toxin catalyzed ADP-ribosylation of purified bovine brain tubulin. The effect of cholera toxin was evident in the absence or presence of nucleotides. In contrast, pertussis toxin required adenine nucleotides for its ADP-ribosylating activity. ATP, ATP gamma S, App(NH)p, deoxy-ATP, and ADP all supported pertussis toxin-catalyzed ADP-ribosylations in the absence or presence of EDTA, suggesting that nucleotide hydrolysis was not involved. Adenine nucleotides also promoted pertussis toxin-catalyzed ADP-ribosylation of heat-treated bovine serum albumin. This result suggests that adenine nucleotides directly affect pertussis toxin. ATP stimulation of pertussis toxin-catalyzed hydrolysis of NAD to ADP-ribose supports this hypothesis.     

Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.     

Interaction of Guanine Nucleotides with [3H]Kainate and 6-[3H]Cyano-7-Nitroquinoxaline-2,3-dione Binding in Goldfish Brain

Abstract— Recent reports have suggested that a major proportion of [³H]kainate binding in goldfish brain is to a novel form of G-protein-linked glutamate receptor. Here we confirm that guanine nucleotides decrease [³H]kainate binding in goldfish brain membranes, but that binding is also reduced to a similar extent under conditions where G-protein modulation should be minimised. Inclusion of GTPγS resulted in an approximately twofold decrease in the affinity of [³H]kainate binding and a 50% reduction in the apparent B _max values in both Mg²⁺/Na⁺ and Mg²⁺/Na⁺-free buffer when assayed at 0°c. The pharmacology of [³H]kainate binding is similar to that of well-characterised ionotropic kainate receptors but unlike that of known me-tabotropic glutamate receptors, with neither 1 S ,3 R -amino-1,3-cyclopentanedicarboxylic acid (1 S ,3 R -ACPD) nor ibo-tenic acid being effective competitors. The molecular mass of the [³H]kainate binding protein, as determined by radiation inactivation, was 40 kDa, similar to the subunit sizes of other lower vertebrate kainate binding proteins that are believed to comprise ligand-gated ion channels. Furthermore, GTP-γS also inhibited the binding of the non-NMDA receptor-selective antagonist 6-[³H]cyano-7-ni-troquinoxaline-2,3-dione. These data strongly suggest that the regulatory interaction between guanine nucleotides and [³H]kainate and 6-[³H]cyano-7-nitroquinoxaline-2,3-dione binding is complex and involves competition at the agonist/antagonist binding site in addition to any G-protein-mediated modulation.     

[3H]Xylamine Accumulation by Two Sympathetically Innervated Tissues

Abstract: The accumulation of [³H]xylamine ([ring-G-³H]- N -2'-chloroethyl- N -ethyl-2-methylbenzylamine; [³H]XYL) by rabbit thoracic aorta and rat vas deferens was studied in vitro . [³H]XYL uptake in both tissues saturated at the same concentrations and displayed the same time course as that for maximal inhibition of [³H]noradrenaline ([³H]NA) accumulation by XYL. Hydrolysis of [³H]XYL or coincubation with Na₂S₂O₃ reduced tissue accumulation of tritium by 80%. In aorta and vas deferens, about 45% and 65%, respectively, of the total [³H]XYL accumulation was blocked by 100 μmol/L desmethylimipramine (DMI), l -NA, or bretylium. In the absence of sodium ion, the uptake of [³H]XYL was reduced by approximately these same amounts. In both tissues nearly 70% of the [³H]XYL taken up was protein bound when measured as trichloroacetic acid-precipitated tritium. Of this radioactivity, the same proportion was sensitive to 100 μmol/L DMI or the absence of sodium ion as found for total tissue accumulation of [³H]XYL. Hydrolysis of [³H]XYL prior to incubation with vas deferens reduced the protein-bound tritium by more than 95%. The studies indicate that [³H]XYL interacts with NA uptake carrier in both rabbit aorta and rat vas deferens and that a substantial portion of the resulting protein-bound radioactivity is carrier-dependent.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

γ-Aminobutyric AcidA Receptor Function Is Desensitised in Rat Cultured Cerebellar Granule Cells Following Chronic Flunitrazepam Treatment

Abstract: This study examined γ-aminobutyric acid_A (GABA_A) receptor function in cultured rat cerebellar granule cells by using microphysiometry following chronic flunitrazepam exposure, and correlated the findings with the α1 and β2/3 subunit protein expression and [³H]muscimol binding after the same treatment paradigm. Flunitrazepam treatment reduced ( p < 0.05) the maximal GABA-stimulated increase in extracellular acidification rate ( E _max) (16.5 ± 1.2% and 11.3 ± 1.0%, 2-day control and treated cells, respectively; 17.4 ± 1.0% and 9.9 ± 0.7%, 7-day control and treated cells, respectively; best-fit E _max± SEM, n = 7), without affecting the GABA concentration required to elicit 50% of maximal response (EC₅₀) (1.2 ± 1.7 and 2.3 ± 1.8 µ M , 2-day control and treated cells, respectively; 1.7 ± 1.5 and 1.5 ± 1.5 µ M , 7-day control and treated cells, respectively; best-fit EC₅₀± SEM, n = 7). Flunitrazepam exposure also abolished the flunitrazepam potentiation of the GABA response, caused a transient reduction of the GABA_A receptor α1 and β2/3 subunit proteins over the initial 2 days, but did not alter [³H]muscimol binding compared with vehicle-treated cells. The results suggest that changes in GABA_A receptor subunit protein expression, rather than loss of [³H]muscimol binding sites, underlie the chronic flunitrazepam-mediated desensitisation of GABA_A receptor function.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.