Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

Molecular and cellular mechanisms of opiate action: Studies in the rat locus coeruleus

We have studied the molecular and cellular mechanisms underlying the acute and chronic effects of opiates on neurons of the rat locus coeruleus (LC). Acutely, opiates inhibit LC neurons by activating K⁺ channels and inhibiting a novel sodium-dependent inward current. Both of these actions are mediated via pertussis toxin-sensitive G-proteins, and regulation of the sodium current occurs through inhibition of the cyclic AMP pathway. In contrast to the acute effects of opiates, chronic treatment of rats with opiates increases levels of specific G-protein subunits, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the possibility that this upregulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these noradrenergic LC neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms by which opiates regulate the expression of these intracellular messenger proteins in the LC. These studies will lead to an improved understanding of the molecular and cellular basis of opiate addiction.     

Biochemical actions of chronic ethanol exposure in the mesolimbic dopamine system

In previous studies, we have demonstrated that chronic administration of morphine or cocaine produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing actions of these and other drugs of abuse. Since this neural pathway is also implicated in the reinforcing actions of ethanol, it was of interest to determine whether chronic ethanol exposure results in similar biochemical adaptations. Indeed, as seen for chronic morphine and cocaine treatments, we show here that chronic ethanol treatment increased levels of tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, and decreases levels of neurofilament protein immunoreactivity, in the VTA. Also like morphine and cocaine, ethanol increases levels of cyclic AMP-dependent protein kinase activity in the NAc. These actions of ethanol required long-term exposure to the drug, and were in most cases not seen in the substantia nigra or caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. Altered levels of tyrosine hydroxylase in catecholaminergic cells frequently reflect altered states of activation of the cells. Moreover, increasing evidence indicates that ethanol produces many of its acute effects on the brain by regulating NMDA glutamate and GAB_A receptors. We therefore examined the influence of chronic ethanol treatment on levels of expression of specific glutamate and GABA receptor subunits in the VTA. It was found that long-term, but not short-term, ethanol exposure increased levels of immunoreactivity of the NMDARl subunit, an obligatory component of NMDA glutamate receptors, and of the Glu Rl subunit, a component of many AMPA glutamate receptors; but at the same time, long-term ethanol exposure decreased immunoreactivity levels of the α1 subunit of the GABA_A receptor complex. These changes are consistent with an increased state of activation of VTA neurons inferred from the observed increase intyrosine hydroxylase (TH) expression. These results demonstrate that chronic ethanol exposure results in several biochemical adaptations in the mesolimbic dopamine system, which may underlie prominent changes in the structural and functional properties of this neural pathway related to alcohol abuse and alcoholism. © 1995 Wiley-Liss, Inc.     

Protein Kinases in the Rat Nucleus Accumbens are Involved in the Aversive Component of Opiate Withdrawal

The specific participation of protein kinases in the expression of the somatic signs of morphine withdrawal has been previously demonstrated, suggesting that changes in intracellular signalling systems are involved in opioid addiction. In the present study, the involvement of protein kinases in the aversive/dysphoric effects of morphine abstinence has been investigated in the nucleus accumbens, because of the critical role played by the mesolimbic system in the rewarding effects of opioids. Rats were chronically treated with morphine, twice a day for 5 days, with doses progressively increased from 5 to 30 mg/kg (i-p.). In addition, microinjections into the nucleus accumbens of the serine-threonine kinase inhibitors H7 or H8 (1 or 10 nmol per side) or saline once daily were also given, both in control and in morphine-treated animals. After these chronic treatments, withdrawal syndrome was induced by naloxone administration (0.1 mg/kg, s. c.), and the motivational component of morphine abstinence was studied using the place aversion paradigm. When administered at the highest dose (10 nmol), H7 and H8 strongly reduced the place aversion induced by naloxone in morphine dependent animals. Protein kinase inhibitors did not induce significant behavioural responses in non-dependent animals. Chronic morphine treatment induced a selective up-regulation of adenylate cyclase activity in the amygdala, without affecting other brain regions. The morphine-increased adenylate cyclase activity in amygdala was reversed by the chronic intra-accumbens microinjection of H7 or H8. These results suggest that serine-threonine kinases in the nucleus accumbens play an important role in the emotional/dysphoric properties which characterize opiate withdrawal.     

Abused drugs modulate RGS4 mRNA levels in rat brain: comparison between acute drug treatment and a drug challenge after chronic treatment

While different classes of abused drugs interact with distinct signaling substrates, it appears that all utilize receptors in the mesolimbic dopamine system to mediate their reinforcing effects. The regulator of G-protein signaling (RGS) proteins modulate G-protein coupled receptor (GPCR) signaling by increasing the rate of GTP hydrolysis of G proteins. This study was undertaken to determine whether morphine, cocaine, or amphetamine would modulate RGS4 mRNA levels in relevant brain regions. Acute administration of morphine and cocaine decreased levels of RGS4 mRNA in the reticulotegmental pontine nucleus (RtTg) and locus coeruleus (LC). Increases in RGS 4 mRNA levels were observed in the nucleus accumbens (NAc) and dorsal central gray (CGD). Acute drug challenge after chronic drug administration increased RGS4 mRNA in the CGD and decreased RGS4 levels in the red nucleus and RtTg. Interestingly, the LC exhibited biphasic modulation, with decreased RGS4 mRNA levels after acute administration and increased levels after chronic administration. These findings indicate that RGS4 mRNA levels are modulated in a similar manner by different drugs of abuse and imply that a common substrate could mediate some effects of abused drugs.     

Rat pineal Gsa, Gia and Goa: relative abundance and development.

The adult rat pineal gland contains relatively high concentrations of Gsa, low amounts of both Gia and Goa, and undetectable levels of GTa. During development the amounts of 45 kDa Gsa and of Gia remain constant. In contrast, 42 kDa Gsa and Goa are nearly absent at birth and increase in abundance markedly thereafter. GTa is undetectable at any age. It would appear that multiple mechanisms regulate the expression of G-proteins in the pineal gland.     

Repeated predictable or unpredictable stress: effects on cocaine-induced locomotion and cyclic AMP-dependent protein kinase activity

Stressful experiences appear to have a strong influence on susceptibility to drug taking behavior. Cross-sensitization between stress and drug-induced locomotor response has been found. Locomotor response to novelty or cocaine (10 mg/kg, i.p.), cyclic AMP-dependent protein kinase (PKA) activity in the nucleus accumbens and basal corticosterone levels were evaluated in male adult rats exposed to acute and chronic predictable or unpredictable stress. Rats exposed to a 14-day predictable stress showed increased locomotor response to novelty and to cocaine, whereas rats exposed to chronic unpredictable stress demonstrated increased cyclic AMP-dependent PKA activity in the nucleus accumbens. Both predictable and unpredictable stress increased basal corticosterone plasma levels. These experiments demonstrated that stress-induced early cocaine sensitization depends on the stress regime and is apparently dissociated from stress-induced changes in cyclic AMP-dependent PKA activity and corticosterone levels.     

Chronic intrathecal morphine administration produces homologous mu receptor/G-protein desensitization specifically in spinal cord

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.     

Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTPgammaS binding in rat brain

Chronic cocaine administration produces alterations in mu and kappa opioid receptor density as well as striatal and accumbens opioid-regulated adenylyl cyclase activity, suggesting a psychostimulant responsive interaction between opioidergic and dopaminergic systems. Stimulation of G-protein-coupled opioid receptors inhibits adenylyl cyclase production of cyclic AMP. The present study employed in situ [(35)S]GTPgammaS binding to measure opioid receptor-stimulated activation of G-proteins in response to acute and chronic cocaine exposure. Male Fischer rats received acute (1 or 3 days) or chronic (14 days) binge pattern cocaine administration. Three and 14 days of cocaine injections resulted in greater increases in the ability of the mu receptor agonist DAMGO to stimulate [(35)S]GTPgammaS binding in both the core and the shell of the nucleus accumbens, all regions of the caudate putamen and the cingulate cortex compared with saline-matched controls. The greatest increases in DAMGO-stimulated [(35)S]GTPgammaS binding were observed in the dorsal areas of the caudate putamen in animals that received 14 days of cocaine. No significant changes in delta (DPDPE), or kappa (dynorphin A(1-17)) receptor-stimulated [(35)S]GTPgammaS binding were found in any brain region in response to cocaine administration. These results demonstrate that binge pattern cocaine administration induce changes in mu but not delta or kappa opioid receptor-mediated G-protein activity. This study provides support for the hypothesis that the addictive properties of both psychostimulants and opiates may share common neurochemical signaling substrates.     

Acute "binge" cocaine increases mu-opioid receptor mRNA levels in areas of the rat mesolimbic mesocortical dopamine system

Autoradiography studies demonstrated that chronic "binge" cocaine administration increased mu-opioid receptor density in dopaminergically innervated rat brain regions, including the cingulate cortex, the nucleus accumbens, and the basolateral amygdala. The present study investigated the effects of a single day of binge-pattern cocaine administration (3 x 15 mg/kg, intraperitoneally [i.p.] at hourly intervals) on mu-opioid receptor mRNA levels in selected brain regions. Rats were sacrificed 30 min after the third injection and mRNA levels were measured by a quantitative solution hybridization RNase protection assay. Acute binge cocaine administration significantly increased mu-opioid receptor mRNA levels in the frontal cortex, nucleus accumbens, and amygdala, but not in the caudate-putamen, thalamus, hippocampus, and hypothalamus. As has been suggested for other G-protein coupled receptors, the rapid increase of MOR mRNA reported in this study might represent an adaptive response to compensate for a decrease in number of receptors following cocaine-induced opioid peptide release.     

Neurite outgrowth in dorsal root ganglia induced by islet neogenesis-associated protein peptide involves protein kinase A activation

Islet neogenesis-associated protein (INGAP) peptide is a candidate therapeutic for diabetes and corrects sensory dysfunction in experimental diabetes in mice. In this study, we investigated the mechanism of action by which INGAP peptide promotes neurite outgrowth in sensory neurons of the dorsal root ganglia. Treatment of dorsal root ganglia primary dispersed cultures with INGAP peptide led to the displacement of fluorescently labeled forskolin from adenylate cyclase, the cyclic AMP-generating enzyme that has been implicated in neuritogenesis. The addition of forskolin or dibutyryl cyclic AMP enhanced the effects of INGAP peptide on neurite outgrowth in dorsal root ganglia explant cultures. Furthermore, pharmacological inhibition of adenylate cyclase with SQ22,536 or of protein kinase A with H89 or KT5720 significantly reduced the neurite-promoting effects of INGAP peptide. These results suggest that INGAP peptide-induced neurite outgrowth in the dorsal root ganglia partially involves cyclic AMP-dependent activation of protein kinase A.     

Acute “binge” cocaine increases mu-opioid receptor mRNA levels in areas of the rat mesolimbic mesocortical dopamine system

Autoradiography studies demonstrated that chronic “binge” cocaine administration increased mu-opioid receptor density in dopaminergically innervated rat brain regions, including the cingulate cortex, the nucleus accumbens, and the basolateral amygdala. The present study investigated the effects of a single day of binge-pattern cocaine administration (3 × 15 mg/kg, intraperitoneally [i.p.] at hourly intervals) on mu-opioid receptor mRNA levels in selected brain regions. Rats were sacrificed 30 min after the third injection and mRNA levels were measured by a quantitative solution hybridization RNase protection assay. Acute binge cocaine administration significantly increased mu-opioid receptor mRNA levels in the frontal cortex, nucleus accumbens, and amygdala, but not in the caudate-putamen, thalamus, hippocampus, and hypothalamus. As has been suggested for other G-protein coupled receptors, the rapid increase of MOR mRNA reported in this study might represent an adaptive response to compensate for a decrease in number of receptors following cocaine-induced opioid peptide release.     

Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.     

Selective regulation of Gi alpha 1 expression and function in PC12 cells by cAMP.

Hormone and neutrotransmitter receptor systems regulate both the activity and expression of GTP-binding proteins (G-proteins). However, relatively little is known about the mechanism by which this regulation occurs. One G-protein subtype, Gi alpha 1, is expressed primarily in neuronal cells. Here, we demonstrate the selective regulation of Gi alpha 1 mRNA and protein levels by cAMP. Treatment of PC12 cells with forskolin increases Gi alpha protein levels. Similarly, incubation of PC12 cells with agents that increase intracellular levels of cAMP, including forskolin, dibutyryl-cAMP, and 8-bromo-cAMP, induce a two- to fourfold increase in Gi alpha 1 mRNA levels. Furthermore, the effect of increased intracellular cAMP is specific for Gi alpha 1 mRNA expression; the levels of mRNA encoding other G-protein subtypes remain unaltered. cAMP-stimulated Gi alpha 1 expression occurs within hours of treatment and is sustained for days. Increasing intracellular cAMP by activation of cell surface adenosine receptors also increases Gi alpha 1 mRNA levels. Treatment of PC12 cells with phorbol esters, NGF, or depolarizing concentrations of KCl did not increase Gi alpha 1 mRNA expression, demonstrating that Gi alpha 1 expression is specifically regulated by cAMP. Guanine nucleotide-mediated inhibition of adenylate cyclase activity was measured in order to determine if the change in Gi alpha protein expression was accompanied by a change in G-protein function. Adenylate cyclase activity in PC12 cells treated with an adenosine analog and therefore expressing higher levels of Gi alpha protein is more sensitive to inhibition by guanine nucleotides than in nontreated PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term upregulation of protein kinase A and adenylate cyclase levels in human smokers

Repeated injections of cocaine and morphine in laboratory rats cause a variety of molecular neuroadaptations in the cAMP signaling pathway in nucleus accumbens and ventral tegmental area. Here we report similar neuroadaptations in postmortem tissue from the brains of human smokers and former smokers. Activity levels of two major components of cAMP signaling, cAMP-dependent protein kinase A (PKA) and adenylate cyclase, were abnormally elevated in nucleus accumbens of smokers and in ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of the catalytic subunit of PKA were correspondingly higher in the ventral midbrain dopaminergic region of both smokers and former smokers. Protein levels of other candidate neuroadaptations, including glutamate receptor subunits, tyrosine hydroxylase, and other protein kinases, were within normal range. These findings extend our understanding of addiction-related neuroadaptations of cAMP signaling to tobacco smoking in human subjects and suggest that smoking-induced brain neuroadaptations can persist for significant periods in former smokers.     

Morphine and cocaine influence on CRF biosynthesis in the rat central nucleus of amygdala

The central nucleus of the amygdala is a CRF-containing limbic brain site which mediates both fear-like and avoidance behaviors; moreover it has been hypothesized that atypical stress responses may contribute to compulsive drug use. Therefore, we studied in rat amygdala the level of CRF mRNA by in situ hybrydization, and the level of the peptide using immunocytochemistry after acute and chronic administration of morphine and cocaine and after their withdrawal. Acute injection of morphine (20 mg/kg i.p.) increased CRF mRNA level, but did not change significantly CRF immunoreactivity in the central nucleus of the amygdala. Chronic morphine administration significantly increased the level of CRF mRNA 3, 24 and 48 h after the last dose. Both, acute and chronic cocaine administration increased CRF mRNA, but the peptide level was decreased only after acute cocaine administration. However, in the late withdrawal (48 h after the last dose of cocaine) both mRNA and the peptide levels tended to decrease.The above data suggest that amygdalar CRF system activity is potently activated after administration of morphine and cocaine, and that activation of this system observed at the time of withdrawal from morphine may be responsible for aversion and anxiety related to these states; therefore a CRF1 receptor may be a target for prospective pharmacotherapies of the withdrawal from abused drugs.     

Regulation of G proteins by chronic morphine in the rat locus coeruleus

A possible role for G proteins in contributing to the chronic actions of opiates was investigated in the rat locus coeruleus (LC). The LC is a relatively homogeneous brain region that appears to play an important role in mediating acute and chronic opiate action in animals, as well as in humans. It was found that chronic, but not acute, treatment of rats with morphine, under conditions known to induce states of opiate tolerance and dependence, produced an increase in the level of pertussis toxin-mediated ADP-ribosylation of G proteins in the LC. The morphine-induced increase in ADP-ribosylation occurred in both Gi and Go, and was observed over a 30-fold range of NAD concentrations used. Concomitant treatment of rats with the opiate receptor antagonist naltrexone blocked the ability of morphine to produce this effect. In contrast, chronic morphine had no effect on pertussis toxin-mediated ADP-ribosylation of Gi and Go in the other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. Chronic morphine also had no effect on cholera toxin-mediated ADP-ribosylation of Gs in the LC and these other brain regions. Preliminary immunoblot analysis revealed that increased ADP-ribosylation levels of the alpha subunit of Go in the LC were associated with equivalent increases in the immunoreactivity of this protein in this brain region. It is possible that the observed regulation of G-proteins by morphine in the LC represents part of the changes that underlie opiate addiction in these neurons.     

Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.     

Alterations in the activity of adenylate cyclase and high affinity GTPase in Alzheimer's disease

The aim of this study was to assess the effect that Alzheimer's disease has on the functional integrity of several signal transduction proteins. The relative levels of the G-protein α subunits G_sα-L, G_sα-S, G_iα-1, G_iα-2 and G_oα were measured by western blotting and found to be unchanged in membranes prepared from Alzheimer-diseased frontal cortex or hippocampus compared to control brains. However the activity of the G-protein associated enzyme, high affinity GTPase, was found to be reduced in the frontal cortex (reduced by 25%) and by a similar magnitude in the hippocampus (reduced by 27%) of Alzheimer subjects. The same membrane preparation were also assayed for the activity of adenylate cyclase. Basal enzyme activity was not significantly altered in Alzheimer diseased hippocampus, but was markedly reduced (by 45%) in the frontal cortex. The ability of fluoride and aluminum ions to stimulate adenylate cyclase was not significantly changed in either brain region. This suggests that G-proteins, especially G_s, are still able to interact with this enzyme. These results indicate that although the presence of Alzheimer's disease does not significantly alter G-protein levels, changes have taken place in the overall activity of these proteins. However this alteration does not affect their ability to stimulate adenylate cyclase activity.