Biomedical subjects

D M Chuang

Publications and source records attributed to D M Chuang.

At least 73 records · Page 4Linked to original sources

PubMed1993

Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells.

Cerebellar granule cells express m2- and m3-muscarinic acetylcholine receptors (mAChRs) and their corresponding mRNA with m3-mAChR being the predominant receptor subtype. After stimulation with the mAChR agonist, carbachol, m2- and m3-mAChR mRNA levels were decreased in a time- and concentration-dependent manner with the maximal down-regulation at 2 and 8 hr, respectively. Immunoprecipitation studies revealed that amounts of m2- and m3-mAChR protein also decreased at 8 and 24 hr, respectively. The carbachol-induced down-regulation of m3-mAChR mRNA was associated with a decrease in the transcription rate, but a substantial enhancement of the mRNA stability. Upon removal of carbachol after treatment for 8 hr, the levels of m3-mAChR mRNA and mAChR binding sites returned to their original values with a t1/2 of approximately 80 min and 6 hr, respectively. The carbachol-elicited loss of m2- and m3-mAChR mRNA was blocked by their corresponding receptor subtype-specific antagonists, AF-DX 116 (m2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (m3-selective), and was concurrent with an increase in c-fos mRNA levels. Exposure of granule cells to the nonselective mAChR antagonist, atropine, caused a time- and concentration-dependent increase in the level of both m2- and m3-mAChR mRNA and mAChR binding sites. At 24 hr, immunoprecipitable m3-mAChR protein was predominantly increased. The atropine-induced up-regulation of m3-mAChR mRNA was concurrent with a marked enhancement of the mRNA stability and its transcription rate. The elevated levels of m3-mAChR mRNA and binding sites declined to their untreated values after the removal of atropine. Treatment with AF-DX 116 and 4-DAMP also produced an increase in the level of m2- and m3-mAChR mRNA and their corresponding immunoprecipitable receptor protein. These results demonstrate that the mAChR agonist and antagonist induce a down- and up-regulation of mAChR expression, respectively, through receptor-mediated mechanisms in cerebellar granule cells. Moreover, at least for m3-mAChR mRNA, the agonist- and antagonist-induced effects are reversible and associated with corresponding changes in the transcription rate of this receptor mRNA species.

Animals↗

PubMed1993

Endothelin- and ATP-induced inhibition of adenylyl cyclase activity in C6 glioma cells: role of Gi and calcium.

Endothelin-1 (ET) and ATP mobilize Ca2+ in rat C6 glioma cells by stimulating phosphoinositide turnover. Both agents also inhibit adenylyl cyclase (AC) activity in C6 glioma cells. The goal of this study was to characterize the molecular mechanisms responsible for the inhibition of AC activity. The administration of either ET, ATP, A23187, or thapsigargin to cells simultaneously with isoproterenol for 5 min inhibited isoproterenol-stimulated cAMP synthesis by a maximum of 60%, 91%, 65%, and 68%, respectively. Pretreatment of cells with pertussis toxin (PTX) did not alter the inhibitory effects of A23187 or thapsigargin, whereas the inhibitory effects of ET or ATP were completely eliminated. Removal of extracellular Ca2+ and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester treatment failed to affect the inhibition caused by ET or ATP, whereas the inhibition caused by A23187 or thapsigargin was completely eliminated in Ca(2+)-free medium and was attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester treatment. The inhibition by both receptor agonists in the earlier phase (30 sec) of the AC reaction was, however, reduced by using either Ca(2+)-free medium or PTX pretreatment. The administration of 3-isobutyl-1-methylxanthine or Ro 20-1724 suggested that the inhibitory effects of A23187 and thapsigargin were partially due to Ca(2+)-dependent stimulation of PDE activity. Short term treatment with phorbol-12-myristate-13-acetate (PMA) had no effect on isoproterenol-stimulated AC activity. However, the inhibition of cAMP induced by ET or ATP, but not by A23187 or thapsigargin, was diminished by PMA, suggesting that the receptor signal via Gi was blocked by PMA treatment. The antagonistic effect of PMA was blocked by staurosporine. All four agents still inhibited AC activity in cells that had been treated with PMA for 24 hr to deplete protein kinase C. ET produced an additional decrease in AC activity in cells that had been treated with a maximally effective concentration of A23187 or thapsigargin. The ET- or ATP-induced decrease in cAMP levels showed homologous desensitization. These results demonstrate that ETZ receptors and ATP receptors in C6 glioma cells inhibit AC activity primarily by interaction with a PTX-sensitive G(i) and partially by elevation of [Ca(2+)]. Protein kinase C activation is not responsible for agonist-induced inhibition of AC but appears to uncouple the G(i)/AC system activated by ET or ATP.

Adenosine Triphosphate↗

PubMed1993

Tetrahydroaminoacridine is neurotrophic and promotes the expression of muscarinic receptor-coupled phosphoinositide turnover in differentiating cerebellar granule cells.

We have investigated whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug with potential antidementia activity, has a trophic action on differentiating cerebellar granule cells by using the method of [3H]inositol incorporation into inositol-containing phospholipid. Addition of THA (30-50 microM) prevented the extensive neuronal degeneration which occurred in the growth medium containing "low" K+ (15 mM). These effects were similar to the neuroprotective action caused by the presence of 100 microM N-methyl-D-aspartate (NMDA). Neurotrophic effects of THA and NMDA on cells grown in low K+ were also demonstrated by direct microscopic examination of cellular morphology. Measurement of phosphoinositide (PI) response in the rescued cells indicated that NMDA modestly promoted the PI response to carbachol and norepinephrine but markedly stimulated the activity induced by glutamate. In contrast, although THA had little or no influence on the maturation of the norepinephrine- and glutamate-induced PI response, it selectively enhanced the activity stimulated by carbachol. Furthermore, the THA treatment drastically increased the Vmax value of carbachol-induced PI turnover with no significant alteration in the EC50 value. Scatchard analysis of the binding of N-[3H]methylscopolamine to intact granule cells indicated a selective increase in the maximum binding value in cells grown in THA-supplementing medium. These observations suggest that THA seems to selectively up-regulate muscarinic cholinergic receptors.

4-Aminopyridine↗

PubMed1993

Paradoxical increase of 5-hydroxytryptamine2 receptors and 5-hydroxytryptamine2 receptor mRNA in cerebellar granule cells after persistent 5-hydroxytryptamine2 receptor stimulation.

Rat cerebellar granule cells express 5-hydroxytryptamine (5-HT)2 receptors that mediate phosphoinositide turnover by a pertussis toxin-sensitive mechanism. Prestimulation of these neurons with 10 microM 5-HT or (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane [(+/-)-DOI], a putative 5-HT2 receptor agonist, resulted in a time-dependent desensitization of the phosphoinositide response to 5-HT. The desensitization was detected within 30 min after prestimulation and reached a maximum (about 80%) decrement at 8 hr. However, [3H]ketanserin binding to 5-HT2 receptors in crude membranes or intact cerebellar granule cells was increased by treatment with 5-HT or DOI, in a time- and concentration-dependent manner. The increase occurred after the onset of desensitization and was fully manifest (about 160-190%) at 4 hr after stimulation. Although the Bmax and Kd were unchanged at 1 hr after 5-HT or DOI treatment, both parameters were significantly increased at 4 and 24 hr. The amount of 5-HT2 receptor mRNA detected by Northern blot hybridization using a 5-HT2 receptor-specific riboprobe was increased in parallel with the up-regulation of 5-HT2 receptor binding sites. Thus, an increase in 5-HT2 receptor mRNA was detected within 2 hr after 5-HT or DOI prestimulation, reached a maximum around 4 hr, and remained at a plateau for at least 24 hr. The levels of total RNA, m3 muscarinic acetylcholine receptor mRNA, and beta-actin mRNA were not significantly affected by these treatments. Our results demonstrated that 5-HT2 receptor binding sites and their mRNA undergo a paradoxical induction during persistent agonist stimulation.

Amphetamines↗

PubMed1993

Novel opioid binding sites associated with nuclei of NG108-15 neurohybrid cells.

Nuclear opioid binding sites have been discovered in NG108-15 neurohybrid cells. Marker enzyme analyses as well as electron and fluorescence microscopy studies attested to the high degree of purity of the nuclear preparations. Immunohistochemical studies on cryostat sections of NG108-15 cells with an antibody to the opioid receptor corroborated a nuclear localization. 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE), 3H-[D-Ala2,D-Leu5]enkephalin (3H-DADLE), and 3H-diprenorphine binding parameters, Kd and Bmax values, and heterologous competition binding and stereospecificity data satisfied criteria for the presence of delta-opioid sites in purified nuclear preparations. Neither mu-([D-Ala2,mephe4,gly-ol5] enkephalin), dihydromorphine, nor kappa-(U69593) specific binding was detectable in purified nuclear preparations. Rates of association and dissociation of 3H-[D-Ser2,L-Leu5]enkephalyl-Thr were comparable to values obtained previously for opioid receptors. Opioid binding was also shown in subnuclear preparations from NG108-15 cell cultures. Agonists, 3H-DADLE and 3H-DPDPE, bind with high affinity to nuclear membranes and with lower affinity to chromatin. In contrast, partial agonist 3H-diprenorphine high-affinity binding sites were predominant in chromatin, while low-affinity binding was found in the nuclear membrane. Accordingly, 5'-guanylylimidodiphosphate sensitivity of 3H-DADLE binding was detected in nuclear membranes but not in chromatin. Both agonist and partial agonist opioid binding to nuclear membrane and chromatin were abolished upon cycloheximide treatment of NG108-15 cells. Taken together, the results suggest that NG108-15 cells contain newly synthesized GTP binding regulatory protein (G-protein)-coupled delta-opioid receptors in nuclear membranes and uncoupled opioid binding sites in chromatin.

Binding Sites↗

PubMed1992

Fluoxetine modulates G protein alpha s, alpha q, and alpha 12 subunit mRNA expression in rat brain.

Signal-transducing G proteins are central to the coordination of receptor-effector communication. We have explored the effects of long-term fluoxetine administration of G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q and G alpha 12 mRNA expression in various rat brain regions using reverse transcriptase-polymerase chain reaction (RT-PCR)-mediated cross-species partial cDNA cloning. Northern blot analysis, and RNase protection assay techniques. Fluoxetine decreased G alpha s mRNA in midbrain, while mRNA expression of the novel G protein alpha subunits, G alpha q and G alpha 12, was increased in neostriatum and frontal cortex. We conclude that in addition to post-translational modification, regulation of G protein function by antidepressant drugs may occur at the level of gene expression.

Animals↗

PubMed1992

Carbamazepine-induced neurotoxicity and its prevention by NMDA in cultured cerebellar granule cells.

Long-term neurotoxicity of carbamazepine was studied in cultured cerebellar granule cells. Treatment of cells with carbamazepine for 3 days induced a dose-dependent neurotoxicity detected by a loss of [3H]ouabain binding to Na+,K(+)-ATPase, and [3H]N-methyl scopolamine binding to muscarinic cholinergic receptors as well as by direct morphologic examination. NMDA protected against carbamazepine-induced toxicity and this protection was blocked by 2-amino-5-phosphono-valerate (APV). The neurotoxicity induced by carbamazepine may be involved in the teratogenic and adverse effects of overdose associated with the treatment of manic-depressive illness and seizures.

2-Amino-5-phosphonovalerate↗

PubMed1992

Effects of chronic nicotine and haloperidol administration on muscarinic receptor-mediated phosphoinositide turnover in rat brain slices.

Muscarinic receptor-mediated phosphoinositide (PI) turnover in rat brain slices was assessed after chronic administration of nicotine (12 mg/kg/day) or haloperidol decanoate (1.5 mg/kg/day), either alone or in combination, for 6 weeks. Nicotine alone did not significantly alter carbachol-induced inositol monophosphate (IP1) accumulation in the frontal cortex, but did result in a significant increase in the hippocampus, and in a decrease in the striatum. Haloperidol alone attenuated carbachol-stimulated IP1 accumulation in all three brain regions. Chronic treatment with combined nicotine and haloperidol resulted in no significant change in carbachol-sensitive IP1 accumulation in either the frontal cortex or hippocampus but did result in a decrease in the striatum. The results suggest significant cross-talk between cholinergic and dopaminergic systems in affecting PI metabolism.

Animals↗

PubMed1992

Regulation of bradykinin-induced phosphoinositide turnover in cultured cerebellar astrocytes: possible role of protein kinase C.

Phosphoinositide hydrolysis was studied in primary cultures of rat cerebellar astrocytes prelabeled with [3H]myo-inositol. Among the agonists examined, the rank order of efficacies in causing phosphoinositide hydrolysis was bradykinin > endothelin-1 > ATP > norepinephrine. The bradykinin response was robust (24-fold increase) with EC50 value of 30 nM and saturating concentration of 1 microM. Preincubation of cells with pertussis toxin did not affect the activation of phosphoinositide turnover by bradykinin. Although short-term (within 90 min) treatment of cells with phorbol dibutyrate attenuated bradykinin-induced phosphoinositide breakdown, the inhibitory effect was lost after 3-6 h of phorbol dibutyrate treatment. Extended (24 h) preincubation resulted in a potentiation of bradykinin response. Homologous desensitization of bradykinin response was observed in cells prestimulated with bradykinin for up to 6 h. However, similar to the effect of phorbol dibutyrate, 24-h pretreatment with bradykinin selectively sensitized the response to bradykinin. Up-regulation of the bradykinin response was also observed in cells prestimulated with endothelin-1 or norepinephrine for 24 h, although these treatments resulted in only homologous desensitization to their own response. Our results suggest that cultured cerebellar astrocytes express bradykinin receptors coupled to phospholipase C and in these cells protein kinase C plays a more prominent role in the negative-feedback regulation of bradykinin-evoked phosphoinositide response.

Adenosine Triphosphate↗

PubMed1992

Potentiation by Ca2+ ionophores and inhibition by extracellular KCl of endothelin-induced phosphoinositide turnover in C6 glioma cells.

Interactions between endothelin-1 (ET)-induced phosphoinositide (PI) hydrolysis and agents that increase Ca2+ influx (i.e. A23187 and ionomycin) or induce depolarization (i.e. KCl) were investigated using C6 glioma. A23187 dose-dependently potentiated ET (30 nM)- and ATP (100 microM)-induced [3H]inositol phosphate (IP) accumulation. This potentiation was associated with an increase in the maximal stimulation elicited by both ET and ATP but their EC50 values were unchanged. This effect of A23187 occurred at concentrations that did not affect basal PI turnover; i.e. 10 nM-3 microM. Ionomycin within the range of 1 nM-1 microM also significantly enhanced ET-induced PI breakdown and this effect was associated with an increase of [Ca2+]i. KCl in a concentration-dependent manner (14.7-54.7 mM) markedly inhibited PI breakdown elicited by ET and ATP, but had much less inhibition on basal activity and no effect on A23187- and ionomycin-induced responses. In parallel, KCl added before or after ET, sharply attenuated the increase of ET-induced [Ca2+]i but did not affect basal level or ionomycin-induced [Ca2+]i response. Neither the potentiation by A23187 nor the inhibition by KCl of ET-induced PI turnover was observed in cultured cerebellar astrocytes. Our results suggest that the cell type-specific regulation by Ca2+ ionophores and KCl on ET-induced PI metabolism is closely related to perturbation of [Ca2+]i.

Adenosine Diphosphate↗

PubMed1992

Mechanisms of action of anticonvulsants in affective disorders: comparisons with lithium.

Until recently it appeared that lithium carbonate possessed a unique spectrum of clinical action in the acute and prophylactic treatment of manic and depressive episodes. It is now increasingly apparent that the anticonvulsants carbamazepine and valproate also share components of this spectrum of efficacy in the affective disorders but, in addition, are clinically effective in some lithium nonresponders. This clinical convergence can now drive a reexamination of the potential mechanisms of action of these compounds in the affective disorders. In spite of intensive study over several decades, the mechanism of action of lithium has remained elusive. A basic conundrum in the consideration of the actions of lithium has also been to explain how a simple ion could have such complex effects on multiple neurotransmitter systems and, in particular, have bimodal actions in the treatment of both manic and depressive phases of the illness. We suggest that a fundamental reconceptualization of both mania and depression as overactivated neural systems (either excitatory or inhibitory) could facilitate this conceptualization. Given the recent evidence linking lithium's effects to uncoupling receptor-mediated activity at the level of G-proteins or attenuating it at the level of second messenger systems mediated by adenylate cyclase or phosphoinositide turnover, these mechanisms become ideal candidates for considering how the drug could dampen overactivated systems potentially relevant to either depression or mania. The lag in onset of maximum therapeutic action of lithium, carbamazepine, and valproate further suggests that biologic effects associated with chronic compared with acute administration are the prime candidates for psychotropic effects. Comparison of the acute and chronic effects of carbamazepine with those of valproate is also offered to focus on the most likely receptor, second-messenger, and ion channel mechanisms involved in their anticonvulsant and psychotropic actions. It is hoped that better understanding of the comparative actions of lithium, carbamazepine, and valproate will allow better targeting of individual drugs for individual patients as well as, ultimately, the development of new and more selective treatments for the recurrent affective disorders.

Anticonvulsants↗

PubMed1992

Pharmacological characterization of endothelin-stimulated phosphoinositide breakdown and cytosolic free Ca2+ rise in rat C6 glioma cells.

Because increasing evidence indicates that glial cells are a target of endothelin, we have characterized endothelin-induced phosphoinositide (PI) turnover and Ca2+ homeostasis in C6 glioma cells. Endothelin-1 (ET) increased formation of 3H-inositol phosphate (IP) from PI and elicited an increase in cytosolic free Ca2+ ([Ca2+]i) in rat C6 glioma. In the presence of Li+, the increase in 3H-inositol trisphosphate formation was rapid, reaching its peak at 5 min after stimulation. ET also elicited a rapid and sustained increase in [Ca2+]i in a dose-dependent manner (1-100 nM). The rank orders of efficacy for ET-related peptides in increasing [Ca2+]i were ET = ET-2 greater than sarafotoxin greater than ET-3. Both ET-mediated stimulation of IP formation and [Ca2+]i increase were largely inhibited in the absence of external Ca2+ but unaffected by the depletion of external Na+ and the presence of dihydropyridine derivatives or verapamil. Inorganic Ca2+ channel blockers Cd2+, La3+, and Mn2+ at 1 mM inhibited both responses induced by ET. Cross-desensitization and nonadditivity were observed for both events among ET-related peptides tested, but not between ET and ATP. Pretreatment of cells with pertussis toxin (PTX) attenuated the PI response to ET, but had no effect on ET-elicited [Ca2+]i increase. ET-induced Ca2+ mobilization (measured in Ca(2+)-free medium) was only transient and was inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate. Moreover, the intracellular Ca2+ pools mobilized by ET and ATP appeared to overlap, as indicated by their partial heterologous desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1992

Antagonist-induced transient down-regulation of delta-opioid receptors in NG108-15 cells.

According to current concepts, agonists can effect the down-regulation of cell surface receptors, whereas antagonists can cause their up-regulation. We have discovered that the opioid antagonists naltrexone, naloxone, and ICI174864 induce a transient down-regulation of delta-opioid receptors before up-regulation, in NG108-15 cells. The possibility of an apparent loss of sites due to blockade by residual antagonist was ruled out by several lines of evidence. The reduction in delta receptors was time, temperature, and antagonist concentration dependent. This down-regulation could not be induced by either the highly mu-selective opioid antagonist cyclic D-Phe-Cys-Try-D-Trp-Arg-Thr-Pen-Thr-amide or the muscarinic antagonist atropine. In the same neurohybrid cells, the opioid agonist [D-Ala2,D-Leu5]enkephalin (0.1 microM, 60 min) effected a greater down-regulation of delta-opioid receptors. Similar qualitative changes in opioid binding of subcellular fractions were elicited with [D-Ala2,D-Leu5]enkephalin and naltrexone. However, the agonist was 2-fold more effective in reducing the heavy membrane population of receptors and 4-fold more potent in increasing the light membrane sites. Because heavy membranes are enriched in plasma membrane, whereas light membranes contain intracellular sites, these findings indicate that internalization occurs in both instances. Naltrexone and the delta-specific antagonists ICI174864 and naltrindole also diminished specific activities of two lysosomal enzymes, whereas opioid agonist-induced down-regulation was accompanied by an increase in their specific activities. Pretreatment of cell cultures with concanavalin A blocked both down-regulation and alterations in the lysosomal enzyme activities elicited by agonists and antagonists, suggesting that the latter is an opioid receptor-mediated process. The up-regulation of delta-opioid receptors by antagonists appears, then, to entail down-regulation that differs from that of agonists.

Amino Acid Sequence↗

PubMed1992

N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.

Animals↗

PubMed1991

Chronic haloperidol treatment attenuates receptor-mediated phosphoinositide turnover in rat brain slices.

The long-term effects of haloperidol on phosphoinositide turnover in rat brain slices were investigated. Continuous treatment with haloperidol decanoate (21 mg/kg I.M. biweekly for 6 weeks) significantly attenuated carbachol- and norepinephrine (NE)-induced inositol phosphate accumulation in rat frontal cortex and hippocampus. In the striatum, the haloperidol treatment also significantly decreased carbachol-stimulated inositol phosphate level but did not significantly affect NE-sensitive phosphoinositide turnover. These effects were not observed in rats treated with a single dose of haloperidol (1.5 mg/kg). Basel levels of inositol phosphate in these 3 brain regions did not change following continuous or single haloperidol doses.

Animals↗

PubMed1991

Expression and agonist-induced down-regulation of mRNAs of m2- and m3-muscarinic acetylcholine receptors in cultured cerebellar granule cells.

The regulation and expression of muscarinic acetylcholine receptor (mAChR) mRNA was studied in cultured cerebellar granule cells using Northern blot hybridization, mRNA species for m2- and m3-mAChRs but not m1- and m4-mAChRs were detected in these cells. The expression of mRNAs of both m2- and m3-mAChRs reached a maximum on the tenth day in culture but their expression patterns differed. Treatment of cerebellar granule cells after 8 days in culture with 100 microM carbachol led to differential down-regulation of the mRNA species of both mAChR subtypes present. Muscarinic receptor antagonists, atropine (1 microM) and pirenzepine (10 microM), prevented carbachol-induced m3-mAChR mRNA down-regulation observed at 8 h. However, exposure to either atropine or pirenzepine alone for 8 h led to a significant up-regulation of m3-mAChR mRNA. Thus, the mRNA species for both m2- and m3-mAChR subtypes are differentially expressed in culture and down-regulated by agonist stimulation. The loss of these mRNA species may play a role in the down-regulation of mAChR binding sites that occurs after desensitization.

Animals↗

PubMed1991

Endothelin- and sarafotoxin-induced phosphoinositide hydrolysis in cultured cerebellar granule cells: biochemical and pharmacological characterization.

Endothelin (ET)-1, -2, -3, big ET-1 and sarafotoxin S6b (S6b) dose-dependently increased phosphoinositide (PI) hydrolysis by 6- to 10-fold in cultured cerebellar granule cells prelabeled with [3H] myoinositol. The PI response elicited by ET-1 was dependent on the presence of extracellular Ca++, but was not reduced by organic (nisoldipine, nimodipine) or inorganic (Co++, Mn++) calcium channel blockers. Pretreatment of granule cells with tetrodotoxin or amiloride failed to affect the response to ET-1. Extracellular sodium depletion resulted in a marked increase in basal PI turnover; however, the net increase of PI turnover induced by ET-1 was unchanged. ET-induced PI breakdown could be partially inhibited by short or long term treatment with phorbol dibutyrate but was unaffected by pertussis toxin. ET- and S6b-induced PI turnover were dependent on the culturing time of granule cells, with the maximal response in a 4-day culture. The ET- and S6b-induced PI turnover appeared to be additive to that induced by carbachol, histamine, norepinephrine, serotonin, glutamate and maitotoxin. However, the responses induced by ET and S6b were nonadditive. Prestimulation of cells with ET or S6b for 30 sec to 24 hr resulted in dramatic loss of the ability of ET and S6b to stimulate PI hydrolysis, without affecting subsequent responsiveness induced by other stimuli, indicating homologous desensitization for ET- and S6b-induced responses. Moreover, our results further support the notion that ET and S6b act on the same population of receptors in cerebellar granule cells.

Animals↗

PubMed1991

Up-regulation of delta opioid receptors in neuroblastoma hybrid cells: evidence for differences in the mechanisms of action of sodium butyrate and naltrexone.

Opioid binding in subcellular fractions from neurohybrid cells was assessed using two models of up-regulation. Homologous up-regulation was achieved by treating NG108-15 cells with the opioid antagonist naltrexone. Na butyrate was added to NCB-20 cell cultures to affect heterologous up-regulation. In both paradigms light and heavy membranes were resolved by concanavalin A (con A) pretreatment of cells followed by density centrifugation. [3H][D-Ala2,D-Leu5]enkephalin (DADLE) and [3H]diprenorphine Bmax values for these fractions increased without changes in affinity. In contrast to 48 h of antagonist treatment, 5 min of exposure to naltrexone down-regulated heavy membrane delta sites. Under both conditions of up-regulation, inhibition of LM [3H]DADLE specific binding by 5'-guanylylimidodiphosphate was enhanced suggesting greater receptor coupling to guanine nucleotide binding regulatory proteins. Although attenuated by addition of cycloheximide, [3H]DADLE binding to total homogenates increased upon naltrexone treatment of NG108-15 cells. Heavy membrane Bmax values were also augmented in the presence of cycloheximide and naltrexone for 48 h. Activities of beta-glucuronidase and beta-hexoseaminidase were diminished in total homogenates and subcellular fractions from naltrexone-treated cells, suggesting an opioid-induced alteration in lysosomal enzyme trafficking. Comparable receptor down- and up-regulation and attenuation of lysosomal enzyme activity were elicited by the delta-selective opioid peptide antagonist (allyl)2 Tyr-Aib-Aib-Phe-Leu-OH. These results suggest that homologous up-regulation entails initial down-regulation and blockade of receptor degradation.

Binding Sites↗