Systematic account of animal poisonings in Germany, 2012-2015.

A systematic retrospective study on animal poisonings in Germany (wildlife excluded) between January 2012 and December 2015 was conducted. Data were collected on animal exposure calls to German poison centres, poisoning cases presenting to the University of Veterinary Medicine, Hannover Small Animal and Equine Clinics, cases involving off-label use of veterinary medicinal products reported to the Federal Office of Consumer Protection and Food Safety and toxicological submissions to the Institute of Pharmacology, Toxicology, and Pharmacy, Faculty of Veterinary Medicine, Ludwig-Maximilians-University, Munich. Descriptive statistics were used to characterise animal type, exposure reason, type and substance, year/month of exposure, case severity and outcome. An evaluation of the data and data sources was also carried out. Variation in poisoning patterns was seen. However, dogs and cats were the most frequently reported species and medicinal products, pesticides and plants were consistently implicated as top causes of poisoning. Advantages and disadvantages were associated with each data source; bias was found to be an important consideration when evaluating poisoning data. This study provided useful information on animal poisonings in Germany and highlights the need for standardised approaches for the collection, evaluation and integration of poisoning data from multiple sources.

[High incidence of jaundice in young calves in Southern Germany]. / Häufung von Ikterus bei jungen Kälbern in Süddeutschland.

OBJECTIVE: Between September, 2010, and August, 2011, a series of cases of jaundice of unknown origin in young calves was detected in a number of farms in Southern Germany. This paper describes the syndrome on the basis of 57 cases, and the approach taken to discover the cause. MATERIAL AND METHODS: The clinical course of the disease is described in 19 patients. Using a case definition (calves aged 1-3 weeks, total serum bilirubin > 20 µmol/l and/or serum glutamate dehydrogenase [GLDH] activity >50U/l and/or autopsy findings with striking liver pathology [jaundice, liver dystrophy, cirrhosis]), 36 farms were included in an epidemiological survey. In a feeding trial, two batches of a dietary supplement feed, previously used in diseased calves on farms, were fed at the dosage recommendations of the manufacturer to four clinically healthy calves over 5days. Four other calves served as controls. The calves were clinically monitored daily, and blood samples were investigated using clinical chemistry and haematology. RESULTS: Clinical examination revealed behavioural alterations (weakness, tonic-clonic seizures and bawling just before death), recumbency, jaundice and discolouration of faeces. In less severe cases without clinical signs, there was an increase in serum bilirubin concentration and/or GLDH activity. In the epidemiological survey of affected farms, the feeding of a diet supplement feed was registered in 54 of 57 cases. The feeding of two batches of that diet supplement feed to four clinically healthy calves resulted in a significant (p<0.05) increase in bilirubin and lactate concentrations, as well as the GLDH activity in serum, but without serious impairment of the general condition, whereas in control calves, no comparable changes were observed. CONCLUSION: The results of the epidemiological survey and the feeding trial suggest a causal involvement of a dietary supplement feed. The toxic principle is unknown. CLINICAL RELEVANCE: Knowledge of the clinical picture and the probable feed-related context is important to detect this disease early. The suspected dietary supplement feed has been taken off the market, but with other products similar problems may arise.

Suspected side effects of doxycycline use in dogs - a retrospective study of 386 cases.

This study investigated doxycycline-related side effects in a large population of dogs. Data from 386 dogs that had received doxycycline for the treatment of various infectious diseases were analysed retrospectively. Potential side effects that developed during treatment were documented, and correlations with signalment, dose, duration of treatment, frequency of application, doxycycline preparation and use of additional drugs were investigated. Vomiting was reported in 18.3 per cent of dogs, 7.0 per cent developed diarrhoea and 2.5 per cent developed anorexia. While being treated with doxycycline, 39.4 per cent of dogs showed an increase in alanine aminotransferase (ALT) activity and 36.4 per cent showed an increase in alkaline phosphatase (ALP) activity. There was a dose-related risk of an increase in ALP activity (P=0.011, odds ratio [OR]=1.27, 95 per cent confidence interval [CI] 1.06 to 1.53), and older dogs treated with doxycycline were more likely to develop an increase in ALT activity (P=0.038, OR=1.23, 95 per cent CI 1.01 to 1.50) and vomiting (P=0.017, OR=1.11, 95 per cent CI 1.02 to 1.21).

Segment-dependent expression of muscarinic acetylcholine receptors and G-protein coupling in the equine respiratory tract.

Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS binding identified a higher density of total muscarinic receptors (fmol/mg protein) in the trachea (720+/-59 nmol/L) than in bronchi (438+/-48 nmol/L) or lung (22 +/- 3 nmol/L). Competitive binding studies using [3H]NMS and the unlabelled subtype-selective antagonists pirenzepine and telenzepine (M1), methoctramine and himbacine (M2), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) (M3), tropicamide (M4) and mamba toxin (MT-3) (M4) indicated the presence of at least three muscarinic receptor subtypes in peripheral lung tissue (50:40:24-28%: M2>M3>M1), whereas in bronchus and trachea M2 subtypes (87-90%) predominated over M3 (14-22%), and M1 subtypes were lacking. No differences were found between tissues in high-affinity binding sites for carbachol in the absence (31-36%) or presence of guanosine 5'-triphosphate (GTP) (approximately 100%). Western blotting for G-protein alpha-subunits showed a much more robust expression of G(alphai1/2) in the trachea (with highest receptor density) than in the lung or bronchi, whereas G(alphas)-protein was dominantly expressed in bronchus. Concomitantly, carbachol inhibited isoproterenol- and GTP-stimulated adenylyl cyclase activity with increasing muscarinic receptor expression (trachea > bronchi > lung). We conclude that the expression and signalling pathways of muscarinic receptors in the equine respiratory tract are segment-dependent. These receptors might contribute to the pathogenesis of COPD in the horse and could provide potential drug targets for the therapeutic use of anticholinergics in this species.

Opioid tolerance/dependence in neuroblastoma x glioma (NG108-15) hybrid cells is associated with a reduction in spontaneous stimulatory receptor activity.

Chronic opioid regulation of stimulatory receptor activity was investigated in neuroblastoma x glioma (NG108-15) hybrid cells stably transfected to express the human beta(2)-adrenoceptor (beta(2)-AR). Expressed beta(2)-ARs are functionally coupled to G proteins and display ligand-independent signalling activity, as demonstrated by the ability of an inverse agonist to attenuate basal adenylyl cyclase (AC) activity. Despite the relative increase in basal AC activity due to the development of tolerance/dependence, chronic morphine treatment was found to completely abolish spontaneous beta(2)-AR activity by reducing basal receptor/G protein precoupling. A similar chronic opioid effect was observed in transiently transfected COS-7 cells. These results indicate that during the state of opioid tolerance/dependence basal levels of AC activity are no longer under the control of spontaneously active stimulatory receptors.

Adenylyl cyclase supersensitivity in opioid-withdrawn NG108-15 hybrid cells requires Gs but is not mediated by the Gsalpha subunit.

On the cellular level, opioid dependence is characterized by a significant elevation of adenylyl cyclase (AC) activity after drug withdrawal, a regulatory phenomenon termed "AC supersensitivity" or "cAMP overshoot." The present study examines the role of the stimulatory G protein (Gs) in the expression of naloxone precipitated opioid withdrawal in chronically morphine (10 microM; 3 days) treated neuroblastoma X glioma (NG108-15) hybrid cells. Determination of high-affinity [3H]forskolin binding to intact cells, which provides a direct parameter for the binding of the activated alpha-subunit of Gs (Gsalpha) to AC, revealed that the enhancement of AC activity after opioid withdrawal is not caused by an increased stimulation of effector activity by Gsalpha. Although not a direct function of Gs, the expression of AC supersensitivity required Gsalpha-mediated stimulation of AC, because 1) the enhancement of AC activity after opioid withdrawal was observed only in the presence of low, but not of high concentrations of forskolin, and 2) chemical inactivation of Gsalpha by low pH pretreatment abolished the induction of AC supersensitivity. Moreover, the regulatory mechanism underlying AC supersensitivity not only required the presence of activated Gsalpha per se, but functional intact stimulatory signal transduction pathways. Indeed, blockade of prostaglandin E1 receptor/Gs interaction in situ with a site-specific anti-Gsalpha antibody, as well as uncoupling of prostaglandin E1 receptor signaling by cholera toxin-catalyzed ADP-ribosylation of Gsalpha, prevented the expression of AC supersensitivity in membranes from opioid-withdrawn cells. These results suggest that the enhancement of AC activity in opioid-dependent cells, triggered by drug withdrawal, is not a direct Gsalpha effect, but involves a secondary regulatory event that requires costimulation of AC by acutely receptor-activated Gsalpha.

Regulation of stimulatory adenylyl cyclase signaling during forskolin-induced differentiation of mouse neuroblastoma x rat glioma (NG108-15) cells.

Chronic exposure of neuroblastoma x glioma (NG108-15) cells to substances that elevate intracellular cAMP levels results in morphological differentiation into a more neuronal-like phenotype. Here we report that forskolin-induced differentiation is accompanied by a biphasic regulation of stimulatory adenylyl cyclase (AC) signaling. While 1 day of forskolin exposure produces an initial increase in basal, [AIF4](-)-, and prostaglandin E1 (PGE1)-stimulated AC activities, stimulatory signal transduction is substantially reduced after complete differentiation of the cells (6 days). Western blot analysis revealed that these functional changes correlate well with changes in the quantity of G(s)alpha, the stimulatory component of AC. Additional forskolin-induced adaptations were found for PGE1 receptors, inhibitory G proteins and AC. These data demonstrate that neuronal differentiation of NG108-15 cells is associated with complex regulatory changes within the stimulatory PGE1 receptor system.

Chronic morphine treatment increases stimulatory beta-2 adrenoceptor signaling in A431 cells stably expressing the mu opioid receptor.

Chronic opioid regulation of stimulatory beta-2 adrenoceptor (beta-2 AR) signaling was investigated in human mammary epidermoid carcinoma A431 cells stably expressing the cloned rat mu opioid receptor. In the cell clone used (A431/mu 13; Bmax = 302.9 +/- 46 fmol/mg membrane protein), the addition of morphine acutely attenuated basal as well as (-)-isoproterenol-stimulated cAMP accumulation. Prolonged exposure of the cells to morphine (10 microM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/mu 13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal falls to precipitate a cAMP overshoot in this cell system. Sensitization of stimulatory AC signaling by chronic morphine develops in a time- and dose-dependent manner and is blocked by both naloxone and pertussis toxin. Investigation into the mechanism leading to up-regulation of AC revealed a 40% increase in the number of beta-2 ARs as assessed by [125I]-cyanopindolol binding experiments. No additional quantitative changes were found for stimulatory G proteins and the effector enzyme itself. Sensitization of AC appears to be mediated solely by the increase in beta-2 AR numbers, because (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol hydrochloride, which acts as an "inverse agonist" at the beta-2 AR, completely reversed elevated basal AC activities, and because the ratio between functional active beta-2 ARs and stimulatory G proteins remained unchanged. In conclusion, chronic exposure of clonal A431/ mu13 cells to morphine increases the capacity of stimulatory AC signaling by up-regulating beta-2 AR number. These results demonstrate participation of stimulatory receptor systems in the cellular mechanisms underlying opioid dependence.

Enhanced stimulatory adenylyl cyclase signaling during opioid dependence is associated with a reduction in palmitoylated Gs alpha.

Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 microM; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc- reconstitution assay. This finding is most likely due to an increased functional activity of Gs alpha because the addition of exogenous G beta gamma subunits, which per se stimulate AC in S49cyc- membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment incresed the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of Gs alpha. Indeed, metabolic labelling studies with [3H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated Gs alpha in the plasma membrane. Furthermore, high affinity [3H]forskolin binding experiments demonstrated that depalmitoylated Gs alpha is able to associated directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of Gs alpha provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of Gs alpha in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.

Stable expression and functional characterization of the cloned rat mu-opioid receptor in human epidermoid carcinoma (A431) cells.

Regulation of intracellular cAMP levels serves as a cellular model for chronic drug action. Since the adenylate cyclase effector system is under dual control of both stimulatory as well as inhibitory receptor systems, a permanent cell line was created in order to allow evaluation of acute and chronic opioid effects on stimulatory receptor function. For this purpose, the cloned rat mu-opioid receptor was stably expressed in human epidermoid carcinoma (A431) cells, which carries high levels of endogenous beta 2-adrenoceptors. Four out of 16 cell clones were found to express considerable amounts of [3H]diprenorphine binding sites and were further characterized. Scatchard analysis of saturation binding data revealed maximal binding capacities (Bmax) between 242.2 +/- 11 and 1,271.8 +/- 221 fmol/mg of membrane protein, whereas drug affinity was found similar among all cell clones tested (Kd = 1.4 +/- 0.2 nM). The expressed mu-receptors also mediated agonist inhibition of adenylate cyclase, indicating that these receptors are functionally coupled to intracellular signalling pathways. Long-term exposure of the cells to morphine (10 microM; 2 days) produced cellular correlates of chronic opioid action as displayed by both a decrease in the maximal degree of adenylate cyclase inhibition (tolerance) as well as an increase in overall effector activity (dependence). Thus, based on these parameters, mu-opioid receptor expressing A431 cells provide a promising tool to investigate cellular mechanisms of chronic drug action.

Chronic exposure of NG 108-15 cells to inhibitory acting drugs reduces stimulatory prostaglandin E1 receptor number.

Prolonged exposure of neuroblastoma x glioma (NG 108-15) hybrid cells to inhibitory acting drugs results in sensitization of adenylate cyclase. We now report that chronic activation (3 days) of either inhibitory delta-opioid receptors, alpha 2B-adrenoceptors, or muscarinic M4 receptors significantly decreases the number of stimulatory, adenylate cyclase-coupled prostaglandin E1 receptors. Pharmacological characterization further revealed that the loss of [3H]prostaglandin E1-binding sites most likely corresponds to a reduction of the number of high-affinity, G protein-coupled prostaglandin E1 receptors. The decline in functionally active prostaglandin E1 receptors developed in a time- and dose-dependent manner and could be prevented by pretreatment of the cells with pertussis toxin. Heterologous prostaglandin E1 receptor regulation was blocked by concomitant exposure of the cells to antagonists for inhibitory receptors and was rapidly reversed (t 1/2 < 30 min) upon termination of chronic inhibitory drug treatment. The decrease in high-affinity prostaglandin E1 receptors developed regardless of whether full or partial agonists were used for pretreatment. In addition, the concentrations of inhibitory drugs required to maximally affect prostaglandin E1 receptor number closely resembled those mediating maximal adenylate cyclase inhibition. The data demonstrate that chronic inhibitory drug treatment of NG 108-15 hybrid cells reduces the number of functionally active, excitatory prostaglandin E1 receptors. Thus, it is proposed that adaptations at the level of stimulatory receptor systems contribute to the regulatory mechanisms associated with drug dependence.

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins.

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.

Chronic activation of inhibitory delta-opioid receptors cross-regulates the stimulatory adenylate cyclase-coupled prostaglandin E1 receptor system in neuroblastoma x glioma (NG108-15) hybrid cells.

The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E1 (PGE1) receptor system in neuroblastoma x glioma (NG108-15) hybrid cells. Persistent activation of delta-opioid receptors by morphine (10 mumol/L; 3 days) substantially down-regulates the number of PGE1 binding sites by approximately 30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTP gamma S (100 mumol/L) further revealed that the remaining PGE1 binding sites are still capable of interacting functionally with their associated stimulatory G proteins, Gs. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the alpha subunit of Gs (Gs alpha) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc- reconstitution experiments, respectively. Evaluation of the functional interaction between PGE1 receptors and Gs by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of Gs alpha revealed a significant increase in the ability of PGE1 receptors to activate Gs alpha (3.3-fold increase in EC50; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 mumol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 mumol/L) had no further effect on sensitization in PGE1 receptor/Gs coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE1 receptor system represents a potential target of chronic delta-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.

The use of genomic DNA probes for in-gel hybridization.

Hybridization within agarose gels using oligonucleotide probes has been described in several publications; genomic DNA probes, however, have been used rarely and only with limited success. Here we present a simple and convenient procedure for in-gel hybridization using radiolabeled genomic DNA fragments. The protocol was improved by the use of formamide in the hybridization as well as in the washing step. This method was compared with the conventional Southern blotting technique and was shown to produce good results in restriction pattern analysis, as well as in chromosomal localization with the help of pulsed field gel electrophoresis.

Retinoic acid-induced differentiation of human neuroblastoma SH-SY5Y cells is associated with changes in the abundance of G proteins.

Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH-SY5Y cells: Gs alpha, Gi alpha 1, Gi alpha 2, Go alpha, Gz alpha, and G beta. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 mumol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of mu-opioid binding sites, the levels of the inhibitory G proteins Gi alpha 1 and Gi alpha 2 were found to be significantly increased. This coordinate up-regulation is accompanied by functional changes in mu-opioid receptor-stimulated low-Km GTPase, mu-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5'-(beta gamma-imido)triphosphate [Gpp(NH)p; 10 nmol/L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prostaglandin E1 (PGE1) receptors and Gs alpha, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE1 binding sites and PGE1-stimulated adenylate cyclase activity, but significantly reduced amounts of Gs alpha were found. This down-regulation is paralleled by a decrease in the stimulatory activity of Gs alpha as assessed in S49 cyc- reconstitution assays.(ABSTRACT TRUNCATED AT 250 WORDS)

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants.

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.

Alterations in the expression of G-proteins and regulation of adenylate cyclase in human neuroblastoma SH-SY5Y cells chronically exposed to low-efficacy mu-opioids.

Western-blot analysis of human neuroblastoma SH-SY5Y cells (mu- and delta-receptors) revealed the presence of the following G-protein subunits: Gi alpha 1, Gi alpha 2, Gs alpha, G(o) alpha, Gz alpha, and G beta, a pattern resembling that observed in central nervous tissue. Chronic treatment of differentiated [all-trans-retinoic acid (10 microM; 6 days)] SH-SY5Y cells with D(-)-morphine (10 microM; 3 days) significantly increased the abundance of all G-protein subunits identified. Co-incubation of morphine-exposed cells together with naloxone (10 microM; 3 days) or the mu-selective opioid antagonist CTOP (10 microM; 3 days), but not with the delta-selective antagonist ICI-174,864 (10 microM; 3 days), completely abolished this effect, suggesting that the increase in G-protein abundance is specifically mediated by mu-receptors. Moreover, the biologically inactive enantiomer L(+)-morphine (10 microM; 3 days) failed to produce a similar effect. G-protein up-regulation developed in a time- and dose-dependent manner and is most likely due to enhanced protein synthesis de novo, since concomitant treatment of the cells with cycloheximide (100 micrograms/ml; 3 days) prevented this effect. Chronic treatment with the low-efficacy mu-selective opioid peptide morphiceptin (10 microM; 3 days), but not with the highly potent mu-agonist DAGO (0.1 microM; 3 days) produced a comparable increase in G-protein abundance. Coincident with quantitative effects on G-protein levels in morphine-tolerant/dependent SH-SY5Y cells, we found elevated levels of basal, forskolin (1 microM)- and prostaglandin-E1 (1 microM)-stimulated adenylate cyclase activities. Reconstitution experiments using S49 cyc- lymphoma-cell membranes suggest that this increase is most likely due to elevated levels of functionally intact Gs. Chronic treatment with both morphine and DAGO induces high degrees of tolerance in this cell line. However, the intrinsic activity of G1 was unchanged, as assessed in functional studies with low-nanomolar concentrations of guanosine 5'-[beta gamma- imido]triphosphate. Our data demonstrate that chronic treatment of SH-SY5Y cells with low-efficacy mu-opioids increases G-protein abundance, a phenomenon which might contribute to the biochemical mechanisms underlying opioid tolerance/dependence.

Coupling of prostaglandin E1 receptors to the stimulatory GTP-binding protein Gs is enhanced in neuroblastoma x glioma (NG108-15) hybrid cells chronically exposed to an opioid.

This study investigates the functional state of the stimulatory GTP-binding protein GS in neuroblastoma x glioma NG108-15 hybrid cells chronically exposed to an opioid. For this purpose, a novel in situ reconstitution protocol was established using membranes selectively depleted of GS function by transient exposure to low pH and then reconstituted with purified exogenous stimulatory GTP-binding proteins. With prostaglandin E1 (PGE1) receptor-stimulated adenylate cyclase activity as an indicator, reconstituted membranes of cells previously rendered tolerant to the delta-opioid [D-Ala2,D-Leu5]enkephalin (DADLE) exhibited approximately 3-fold elevated cAMP generation upon stimulation with PGE1, compared with nontolerant reconstituted cell membranes. This effect developed dose-dependently with respect to the opioid concentration used for pretreatment of the cells and was blocked by concomitant exposure to naloxone. In contrast, receptor-independent activation of GS by the stable GTP analogue guanosine-5'-O-(3-thio)triphosphate did not reveal any difference in adenylate cyclase activity between reconstituted membranes of control and chronically DADLE-pretreated cells. Furthermore, the functional activity of endogenous GS displayed no difference between control and DADLE-tolerant cells, as assessed in S49 cyc- reconstitution assays using sodium cholate extracts derived from NG108-15 membranes. The data presented suggest that the increase in PGE1 receptor-mediated adenylate cyclase activity in opioid-tolerant/dependent NG108-15 hybrid cells most likely relates to enhanced coupling efficiency between the PGE1 binding site (receptor) and GS. Moreover, our results support the concept that supersensitivity to excitatory drugs reflects an adaptive mechanism of cells chronically exposed to an opioid.

Exonic polymorphism vs intronic simple repeat hypervariability in MHC-DRB genes.

Gene products encoded by the major histocompatibility complex often exhibit a high degree of polymorphism. In humans the HLA-DR polymorphism is due to more than 50 alleles with varying exon 2 sequences. Each group of DRB alleles contains a certain form of the basic simple repeat motif (gt)n(ga)m in intron 2. Identical alleles can be differentiated on the basis of the hypervariable repeat. In this study focused on cattle (Bos taurus) we identified different Bota-DRB alleles in a limited survey by amplification via polymerase chain reaction and sequencing. In addition DRB exon 2 sequences were also obtained from eight additional hoofed animal species (seven horned artiodactyls and one pig) revealing artiodactyl-specific polymorphic and nonpolymorphic substitutions. In the genus Bos the intronic simple repeat variability was compared with exonic DRB polymorphism. As in humans all Bota-DRB exons were always associated with specifically organized basic simple repeat structures. Yet the extent of simple repeat variability was lower in cattle compared to humans. Selective breeding in the process of domestication might be responsible for the diminished intronic hypervariability. Nevertheless, the hypermutable simple repeat sequences have been preserved in the same position and with the same principal structure for at least 70 x 10(6) years of evolution. Unexpectedly, the rate of intronic simple repeat and exonic changes appear quite similar.