Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia.

Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D(1) receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder.

Mecanismos de tolerancia a los opioides en el control del dolor / No disponible

Durante la última década hemos asistido al descubrimiento de una serie de proteínas de señalización de expresión exclusiva o mayoritaria en el sistema nervioso que se han revelado esenciales en el procesamiento delas señales iniciadas en los receptores opioides. Estos estudios nos han permitido avanzar de modo definitivo en el conocimiento de los mecanismos que a nivel neuronal son responsables de la aparición de tolerancia/desensibilización a los analgésicos opioides. Este tipo de investigación no es posible abordarlo en estudios clínicos por lo que cobran relevancia los modelos de experimentación animal, celular o molecular. Así, gracias a los avances de la investigación básica empezamos a comprender el porqué en la clínica del dolor la rotación de opioides o su combinación con analgésicos no opioides es beneficiosa para prolongar la eficacia anticonceptiva durante el mayor tiempo posible. Asimismo, podemos afirmar que hay razones objetivas para según que tipo de dolor necesitemos tratar elegir uno u otro opioide. En estas líneas hemos tratado de aproximar ambos aspectos, investigación básica y experiencia clínica, a fin de aportar información válida al profesional del tratamiento del dolor (AU)

During the last few years nervous system research has unveiled the crucial role of a series of regulatory proteins in opioid receptor signaling. Therefore, our understanding of the molecular mechanisms responsible for the development of tolerance to opioid-induced analgesia has greatly expanded. Obviously, research of this kind is not carried out in clinical studies, and has to be performed in animal models, cultured cells, and in vitro systems. Clinical approaches such as opioid rotation or the combination of opioids with other analgesic drugs bring about the benefit of pain control during extended periods of time. Thus, the advances achieved by the basic research help us understand the efficacy of these treatments in the control of human pain. Moreover, this knowledge also provides objective reasons for selecting opioids depending on the type of pain to be treated. In the present article we have tried to approximate the outcome of basic research to clinical experience. We believe that the information provided herein will be useful to those involved in treating pain suffering in humans (AU)

Glycosylated phosducin-like protein long regulates opioid receptor function in mouse brain.

Phosducin (Phd), a protein that in retina regulates rhodopsin desensitization by controlling the activity of Gt beta gamma-dependent G-protein-coupled receptor kinases (GRKs), is present in very low levels in the CNS of mammals. However, this tissue contains proteins of related sequence and function. This paper reports the presence of N-glycosylated phosducin-like protein long (PhLP(L)) in all structures of mouse CNS, mainly in synaptic plasma membranes and associated with G beta subunits and 14-3-3 proteins. To analyze the role PhLP(L) in opioid receptor desensitization, its expression was reduced by the use of antisense oligodeoxynucleotides (ODNs). The antinociception induced by morphine, [D-Ala(2), N-MePhe(4),Gly-ol(5)]-enkephalin (DAMGO), beta-endorphin, [D-Ala(2)]deltorphin II, [D-Pen(2,5)]-enkephalin (DPDPE) or clonidine in the tail-flick test was reduced in PhLP(L)-knock-down mice. A single intracerebroventricular (icv)-ED(80) analgesic dose of morphine gave rise to acute tolerance that lasted for 4 days, but which was prevented or reversed by icv-injection of myristoylated (myr(+)) G(i2)alpha subunits. PhLP(L) knock-down brought about a myr(+)-G(i2)alpha subunit-insensitive acute tolerance to morphine that was still present after 8 days. It also diminished the specific binding of (125)I-Tyr(27)-beta-endorphin-(1-31) (human) to mouse periaqueductal gray matter membranes. After being exposed to chronic morphine treatment, post-dependent mice required about 10 days for complete recovery of morphine antinociception. The impairment of PhLP(L) extended this period beyond 17 days. It is concluded that PhLP(L) knock-down facilitates desensitization and uncoupling of opioid receptors.

Agonists determine the pattern of G-protein activation in mu-opioid receptor-mediated supraspinal analgesia.

The opioids heroin, methadone, buprenorphine, and morphine produce supraspinal antinociception in CD-1 mice that is antagonized by Cys(2), Tyr(3), Orn(5), Pen(7)-amide but not by naltrindole or nor-binaltorphimine. The patterns of GTP-binding regulatory proteins (G-proteins) activation exhibited by these agonists at mu-opioid receptors were characterized. The expression of alpha-subunits of Gi-protein classes, Gi1, Gi2, Gi3, Go1, Go2 and Gz, and those of the Gq-protein family, Gq and G11, was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments demonstrated differences in the analgesic profiles of these opioids. Though the knock-down of G(i2)alpha or G(z)alpha subunits diminished the analgesic effects of the four opioids, impairment of G(i3)alpha did not modify the potency of morphine. In mice with reduced G(i1)alpha, G(o1)alpha or G(11)alpha levels, antinociception induced by heroin and methadone was diminished, but buprenorphine and morphine showed no change in their effects. Also, antinociception induced by heroin and buprenorphine, but neither morphine nor methadone, required intact G(o2)alpha or G(q)alpha levels. Thus, morphine, heroin, methadone, and buprenorphine showed different patterns of G-protein activation in evoking mu-opioid receptor-mediated supraspinal antinociception. Therefore, after binding identical receptors, each agonist determines the classes of GTP-binding regulatory transducer proteins to be activated.

Administration of myr(+)-G(i2)alpha subunits prevents acute tolerance (tachyphylaxis) to mu-opioid effects in mice.

The administration of efficacious doses of morphine or beta-endorphin causes acute tolerance (tachyphylaxis) to the effects of additional administrations of these opioids. Mice intracerebroventricularly (icv)-injected with biologically active myristoylated (myr(+))-G(i2)alpha subunits developed no tachyphylaxis to morphine antinociception in the tail-flick test. This treatment increased the potency of opioid-induced analgesia during the declining phase. Moreover, animals showing tachyphylaxis to opioid effects exhibited normal responses to the agonists after icv-administration of myr(+)-G(i2)alpha subunits. In morphine tolerant/dependent mice, an icv dose of 12 pmol/mouse myr(+)-G(i2)alpha subunits facilitated complete restoration of morphine antinociception in only 4 or 5 days instead of the 10 to 11 days required for post-dependent mice. This was observed when myr(+)-G alpha subunits were injected within the first 24 h of chronic morphine administration -- but not later when long-term tolerance takes place. These results suggest that during the course of an opioid effect a progressive reduction of receptor-regulated G-proteins occurs, and hence tachyphylaxis develops. Exogenous administration of myr(+)-G alpha subunits may be of therapeutic potential in improving agonist activity and accelerating the recovery of post-dependent receptors.

RGS9 proteins facilitate acute tolerance to mu-opioid effects.

This paper reports that regulators of G-protein signalling (RGS) proteins modulate the timing and amplitude of opioid signals by a push-pull mechanism. This is achieved without noticeable changes in the binding properties of opioids, e.g. beta-endorphin to mu-opioid receptors. The expression of RGS proteins was reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Knock down of RGS2 or RGS3 diminished morphine and beta-endorphin analgesia, whereas that of RGS9 or RGS12 enhanced this activity. In mice with impaired RGS9, but not impaired RGS2, the potency and, in particular, the duration of opioid antinociception increased. Further, the animals did not exhibit acute tolerance generated by a single and efficacious dose of morphine, nor did they develop tolerance after a daily i.c.v. injection of the opioid for 4 days. In a model of sustained morphine treatment, the impairment of RGS9 proteins facilitated increases in the response to the delivered opioid. This was only effective for 2--3 h after the subcutaneous implantation of an oily morphine pellet; later, tolerance developed. To reduce the impact of the chronic morphine acting on opioid receptors, other RGS proteins presumably substitute the GTPase-activating function of RGS9 on morphine-activated G-alpha-GTP subunits. The desensitization of mu-opioid receptors appears to be a cell membrane-limited process facilitated by RGS9's sequestering of agonist-segregated G alpha subunits.

Lack of dependence and rewarding effects of deltorphin II in mu-opioid receptor-deficient mice.

We have previously shown that the antinociceptive effects produced by the delta opioid-selective agonist deltorphin II are preserved in mu-opioid receptor (MOR)-deficient mice. We have now investigated rewarding effects and physical dependence produced by deltorphin II in these animals. Wild-type and MOR-deficient mice were implanted with a cannula into the third ventricle and deltorphin II was administered centrally. The rewarding effects induced by deltorphin II were then investigated using the place preference paradigm. Wild-type mice showed place preference for the compartment previously associated with deltorphin II and this effect was not observed in MOR-deficient mice. In a second experiment, mice received a chronic perfusion of deltorphin II over 6 days, via an Alzet minipump connected to the intraventricular cannula, and withdrawal was precipitated by naloxone administration. Wild-type animals showed a moderate but significant incidence of several somatic signs of withdrawal. This withdrawal response was suppressed in MOR-deficient mice. Analysis of the immunoreactivity levels of PKC-alpha, PKC-beta (I and II) and PKC-gamma isozymes in the cerebral cortex of mice infused chronically with deltorphin II showed a significant up-regulation of all these isozymes in the soluble fraction in wild-type but not in MOR-deficient mice. In conclusion, mu-opioid receptors, which are not involved in deltorphin II antinociception, appear to mediate the effects of chronic deltorphin II on rewarding responses, physical dependence and adaptive changes to PKC.

Activation of I(2)-imidazoline receptors enhances supraspinal morphine analgesia in mice: a model to detect agonist and antagonist activities at these receptors.

This work investigates the receptor acted upon by imidazoline compounds in the modulation of morphine analgesia. The effects of highly selective imidazoline ligands on the supraspinal antinociception induced by morphine in mice were determined. 2. Intracerebroventricular (i.c.v.) or subcutaneous (s.c.) administration of ligands selective for the I(2)-imidazoline receptor, 2-BFI, LSL 60101, LSL 61122 and aganodine, and the non selective ligand agmatine, increased morphine antinociception in a dose-dependent manner. Neither moxonidine, a mixed I(1)-imidazoline and alpha(2)-adrenoceptor agonist, RX821002, a potent alpha(2)-adrenoceptor antagonist that displays low affinity at I(2)-imidazoline receptors, nor the selective non-imidazoline alpha(2)-adrenoceptor antagonist RS-15385-197, modified the analgesic responses to morphine. 3. Administration of pertussis toxin (0.25 microg per mouse, i.c.v.) 6 days before the analgesic test blocked the ability of the I(2)-imidazoline ligands to potentiate morphine antinociception. 4. The increased effect of morphine induced by I(2)-imidazoline ligands (agonists) was completely reversed by idazoxan and BU 224. Identical results were obtained with IBI, which alkylates I(2)-imidazoline binding sites. Thus, both agonist and antagonist properties of imidazoline ligands at the I(2)-imidazoline receptors were observed. 5. Pre-treatment (30 min) with deprenyl, an irreversible inhibitor of monoamine oxidase B (IMAO-B), produced an increase of morphine antinociception. Clorgyline, an irreversible IMAO-A, given 30 min before morphine did not alter the effect of the opioid. At longer intervals (24 h) a single dose of either clorgyline or deprenyl reduced the density of I(2)-imidazoline receptors and prevented the I(2)-mediated potentiation of morphine analgesia. 6. These results demonstrate functional interaction between I(2)-imidazoline and opioid receptors. The involvement of G(i)-G(o) transducer proteins in this modulatory effect is also suggested.

In vivo modulation of G proteins and opioid receptor function by antisense oligodeoxynucleotides.

The work in our laboratory has been designed to characterize the transducer mechanisms coupled to neurotransmitter receptors in the plasma membrane. Particular attention has been paid to the physiological/pharmacological effects mediated by the opioid system. Antisense oligodeoxynucleotides have proved useful in correlating opioid receptor clones with those defined pharmacologically. The involvement of the cloned opioid receptors mu, delta, and kappa in analgesia has been determined by means of in vivo injection of ODNs directed to the receptor mRNAs. Using this strategy the classes of G-transducer proteins regulated by each type/subtype of opioid receptor in the promotion of antinociception have also been characterized. After displaying different patterns of binding to their receptors, opioids trigger a variety of intracellular signals. The physiological implications and therapeutic potential of these findings merit consideration.

Myr+-Gi2 alpha and Go alpha subunits restore the efficacy of opioids, clonidine and neurotensin giving rise to antinociception in G-protein knock-down mice.

In mice whose Gi/o-protein function had been impaired by antisense 'knock-down' or pertussis toxin treatment, i.c.v. injection of myr+-Gi/o alpha subunits restored the effectiveness of beta-endorphin, morphine, DPDPE, clonidine and neurotensin to produce antinociception. Myr+-G alpha subunits of the class of G-proteins actually impaired were more effective than unlike but related myr+-G alpha subunits. Selectivity was noted in that only exogenous myr+-G alpha subunits affected (enhanced) the activity of agonists in G alpha-deficient signalling systems. This treatment had little effect on agonist potency when the impairment resided at the receptor level. The potential of the opioids, clonidine and R-PIA to increase G alpha-related in vitro hydrolysis of GTP was also re-established after injecting myr+-Gi2 alpha subunits into Gi2-knocked-down mice. Myr+-Gi2 alpha subunits pre-incubated with GTPgammaS or GDPbetaS before i.c.v. injection did not improve the activity of agonists in vivo (antinociception) or in vitro (regulation of low Km GTPase). After impairing the function of PKCbeta1 by antisense treatment or with the inhibitor H7, the effect of myr+-G alpha subunits on agonist potency was prevented. Electron microscope analysis showed the entry of gold-conjugated myr+-G alpha subunits into neural cells. These particles were found in the cytoplasm, associated with the plasma membranes of different neuronal processes and also in synaptic junctions. In cultured neurons and astrocytes myr+-Gi2 alpha-associated fluorescence was internalised in a dose-dependent manner and distributed in the plasma membrane and cytosol, as well as in nuclei of dividing astrocytes. Thus, G alpha subunits in CSF enter into neurons and functionally couple to the receptor-triggered signalling cascade. As G-proteins have been implicated in the pathophysiology of several neural disorders, this finding may be valuable in the therapy of such dysfunctions.

Use of selective antagonists and antisense oligonucleotides to evaluate the mechanisms of BUBU antinociception.

Evidence suggests that the antinociceptive effects of selective delta-opioid receptor agonists may involve an activation of the mu-receptor in some experimental conditions. The aim of this study was to clarify the receptors involved in the antinociceptive responses of the selective and systemically active delta-opioid receptor agonist Tyr-D-Ser-(O-tert-butyl)-Gly-Phe-Leu-Thr-(O-tert-butyl) (BUBU). The antinociception induced by systemic (i.v.) or central (i.c.v.) administration of BUBU was measured in the hot plate (jumping and paw lick latencies) and tail immersion tests in mice. In both tests, the responses were more intense when BUBU was administered by central route. The pre-treatment with the mu-opioid receptor antagonist cyprodime blocked the effects induced by central BUBU in the hot plate and tail immersion tests. The delta-opioid receptor antagonist naltrindole had no effect on BUBU-induced antinociception in the hot plate but decreased BUBU responses in the tail immersion test. Further evidence for this dual receptor action of BUBU was demonstrated by using antisense oligodeoxynucleotides. Thus, a reduction in central BUBU-induced antinociception was observed in the tail immersion test after the administration of antisense probes that selectively blocked the expression of mu- or delta-opioid receptors. These findings clearly indicate using a dual pharmacological and molecular approach that BUBU mediates its antinociceptive effects via activation of both mu- and delta-opioid receptors.

Endomorphin-1 and endomorphin-2 show differences in their activation of mu opioid receptor-regulated G proteins in supraspinal antinociception in mice.

Endomorphin-1 and endomorphin-2 are tetrapeptides of the brain whose binding profiles and analgesic activities indicate that they are endogenous ligands at micro opioid receptors. To analyze the classes of G transducer proteins activated by these opioids in the production of supraspinal antinociception, the expression of alpha subunits of the G(i) protein class, G(i1), G(i2), G(i3), G(o1), G(o2), and G(z), and those of the G(q) protein family, G(q) and G(11), was reduced by administration of antisense oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. The ODN treatments promoted differences in the analgesic effects displayed by morphine, [D-Ala(2),N-MePhe(4), Gly-ol(5)]enkephalin (DAMGO), and the novel opioids endomorphin-1 and endomorphin-2. The impairment of G(i1)alpha and G(i3)alpha function led to a weaker analgesic response to the endomorphins and to the alpha(2)-adrenoceptor agonist clonidine, whereas the effects of morphine and DAMGO were not affected. An antisense probe targeting G(i2)alpha blocked the antinociceptive effects of endomorphin-2, morphine, DAMGO, and clonidine but was without effect on the activity of endomorphin-1. Mice receiving the ODN to G(z)alpha subunits showed impaired response to all agonists. The knockdown of either G(o1)alpha, G(o2)alpha, G(q)alpha, or G(11)alpha had little or no influence on the antinociception induced by any of the opioids in the study. Thus, agonists exhibit differences in activating the variety of GTP-binding proteins regulated by mu opioid receptors.

Antisense oligodeoxynucleotide targeting distinct exons of the cloned mu-opioid receptor distinguish between endomorphin-1 and morphine supraspinal antinociception in mice.

Antisense oligodeoxynucleotides (ODN) were used to investigate the supraspinal antinociceptive effects of endomorphin-1, an endogenous peptide whose analgesic profile suggests that it is a ligand at the mu-opioid receptor. To selectively restrict the expression of this receptor, five ODN targeting distinct exons of the gene sequence were injected subchronically by the intracerebroventricular route (i.c.v.) into mice. The antinociception induced by endomorphin-1 was greatly reduced in animals receiving the ODN directed to nucleotides 677-697, which code for a sequence located on the second extracellular loop of the mu receptor. ODN-mu(un), one of the two antisense ODN directed to exon 1, also impaired endomorphin-1 antinociception. ODN targeting exons 2 and 4 were totally inactive. In contrast, all five ODN blocked the antinociception induced by morphine and beta-casomorphin. The analgesic potency of endomorphin-1, morphine, and beta-casomorphin remained unaltered by administration of an ODN to nucleotides 29-46 of the murine delta-opioid receptor gene sequence of a random-sequence ODN. This suggest the existence of diverse molecular forms for the mu-opioid receptor that mediate the antinociceptive effects of endomorphin-1 and morphine/beta-casomorphin.

Antibodies and antisense oligodeoxynucleotides to mu-opioid receptors, selectively block the effects of mu-opioid agonists on intestinal transit and permeability in mice.

1. We have studied the effects of mu and delta opioids on intestinal function (permeability, PER; gastrointestinal transit, GIT), and their antagonism after the intracerebroventricular (i.c.v.) administration of specific antibodies (ABs) or antisense oligodeoxynucleotides (ODN) to mu-receptors (OR). Central versus peripheral site/s of action of subcutaneous (s.c.) mu-opioids, were also assessed. 2. Male Swiss CD-1 mice were used. GIT was measured with charcoal and PER by the passage of 51Cr-EDTA from blood to lumen. 3. Morphine and fentanyl (i.c.v. and s.c.) inhibited GIT and PER in a dose-related manner; they were more potent by i.c.v. route, both on GIT and PER (70 and 17 times for morphine and fentanyl). They also had a greater effect on GIT than PER (4.3 and 1.6 times). DPDPE had a lower potency than mu-agonists in all experiments, and no dose-response could be obtained after s.c. administration on GIT. 4. Pretreatment with i.c.v. ABs (24 h) or antisense ODN (5 days), decreased the effects (GIT and PER) of i.c.v. morphine and fentanyl, while those of DPDPE remained unchanged. The ABs did not alter the peripheral effects of mu-opioids. 5. The results show that (i.c.v. or s.c.) mu opioids produce dose-related inhibitions of PER and GIT, being more potent by the i.c.v. route. Delta-opioids had a greater effect on PER than GIT, while the opposite occurred for mu-agonists. Pretreatment with ABs or ODN to mu-OR, blocked the central effects of mu (but not delta) agonists on GIT and PER.

Stimulation of mu- and delta-opioid receptors enhances phosphoinositide metabolism in mouse spinal cord: evidence for subtypes of delta-receptors.

The accumulation of inositol phosphates (IPs) induced by agonist-activated opioid receptors was analysed in mouse spinal cord slices pre-labelled with myo-[3H]inositol. Agonists showing selectivity to mu-opioid receptors, morphine and [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO), promoted concentration-dependent increases in the formation of IPs. The activation of delta-opioid receptors by the selective agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II produced similar increases in phosphoinositide (PI) metabolism. Pre-treatment of the slices with pertussis toxin (PTX) blocked the effect of opioid agonists on IP production. The involvement of Gi/Go-protein (guanine nucleotide-binding protein) classes in this opioid effect is therefore suggested. The activity of the opioid agonists was reduced by the opioid antagonists naltrexone and naloxone. The antagonist at delta1-receptors, 7-benzylidenenaltrexone (BNTX), exhibited greater potency than the antagonists at delta2-receptors, naltriben methanesulphonate (NTB) or naltrindrole 5'-isothiocyanate (NT II), in reducing the activating effect of DPDPE on phosphoinositide metabolism. Conversely, NTB and NT II were more potent antagonists of the activity of [D-Ala2]deltorphin II than BNTX. This work demonstrates the coupling of spinal mu- and delta-opioid receptors to phospholipase C and the generation of IPs. It also provides biochemical evidence for pharmacological subtypes of delta-opioid receptors in the activation of this signalling pathway.

Transport of CSF antibodies to Galpha subunits across neural membranes requires binding to the target protein and protein kinase C activity.

In the light of functional studies, it has been suggested that antibodies directed to alpha subunits of G-proteins delivered into cerebrospinal fluid (CSF) reached and blocked the function of neural transducer proteins. Current understanding indicates that IgGs do not move freely across plasma membranes. Therefore, to characterize the uptake of these antibodies by neural cells, anti-Gi2alpha IgGs were labeled with 125I, fluorescein or with gold particles. The expression of Galpha subunits was also reduced by blocking their mRNA with antisense oligodeoxynucleotides (ODN). Following intracerebroventricular (icv) injection of gold-conjugated anti-Gi2alpha IgGs, electrondense particles entered and became distributed in the cytoplasm and plasma membranes of neural cells. Scattered particles were also found in dendrites and nuclei. Unlabeled IgGs diminished cerebral signals of fluorescein-labeled anti-Galpha IgGs, indicating that this uptake can be saturated. Cerebral radiostaining promoted by in vivo anti-Gi2alpha 125I-IgGs was almost absent in Gi2alpha knocked-down mice, but not after decreasing the quantity of Gzalpha subunits. The immunosignals of CSF anti-Galpha 125I-IgGs, as well as the impairment of opioid-evoked antinociception, were increased by agonist-induced activation of G protein-coupled receptors. The impairing effect of the antibodies on opioid-evoked antinociception was prevented by agents blocking the cellular uptake of proteins, i.e., cytochalasin B, BSA, DMSO, H7, and by down regulation of protein kinase Cbeta1 (PKCbeta1). In mice treated with an ODN to PKCbeta1 mRNA, 125I-IgGs to Gi2alpha subunits remained bound to periventricular structures and did not spread to deeper areas of the CNS. These results indicate that IgGs delivered into the CSF show a saturable binding to Galpha subunits that translocate to the external side of the neural membrane before being internalized by a PKCbeta1-dependent mechanism.

Influence of Gz and Gi2 transducer proteins in the affinity of opioid agonists to mu receptors.

The affinity displayed by different opioids to mu receptors (ORs) was determined in mouse brain membranes incubated with antibodies directed to Galpha subunits of the guanine nucleotide-binding proteins Gi2 and Gz. Assays were conducted with 10 pm 125I-Tyr27-beta-endorphin in the presence of 300 nm N, N-diallyl-Tyr-(alpha-aminoisobutyric acid)2-Phe-Leu-OH (ICI-174 864), which prevented the binding of the iodinated neuropeptide to delta-ORs. Gpp(NH)p or the preincubation of mouse brain membranes with IgGs to Gi2alpha or Gzalpha subunits, promoted reductions in the affinity exhibited by the labelled probe. The potencies of beta-endorphin, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) and [D-Pen2,5]enkephalin (DPDPE) were reduced after impairing the coupling of mu-ORs to Gi2 or Gz proteins. Morphine showed a loss of affinity towards the mu-OR after preincubation of membranes with IgGs to Gzalpha subunits. However, it retained its potency after treatment with the anti-Gi2alpha IgGs. Conversely, [D-Ala2, D-Leu5]enkephalin (DADLE) and [D-Ser2, Leu5] enkephalin-Thr6 (DSLET) showed decreased affinity to mu-ORs after treatment with anti-Gi2alpha IgGs, with no noticeable change following the use of IgGs to Gzalpha subunits. The affinity exhibited by the opioid antagonists naloxone, naltrexone, naloxonazine and [Cys2,Tyr3,Orn5, Pen7 amide]somatostatin analogue (CTOP) remained unchanged after either treatment. Therefore, the affinity exhibited by opioid agonists of mu-ORs, but not antagonists, depends on the nature of the G-protein coupled to these receptors.

delta Opioid receptor subtypes activate inositol-signaling pathways in the production of antinociception.

To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists.

The delta2-opioid receptor subtype stimulates phosphoinositide metabolism in mouse periaqueductal gray matter.

The delta(delta)-opioid agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II increased the formation of inositol phosphates (IPs) in mice periaqueductal gray matter (PAG) slices pre-labeled with myo-[3H]inositol. Both delta-agonists caused an increase in IP accumulation in a dose-dependent manner (1-100 microM) and which was pertussis toxin (0.5 microg/mouse, i.c.v.) sensitive. This effect was blocked by the delta-antagonist ICI-174.864 (10 microM). The presence of subtypes of the delta-opioid receptor (delta1 and delta2) in PAG has been suggested by pharmacological studies. In this brain structure, naltrindrole 5'-isothiocyanate (5'-NTII), but not 7-benzylidenenaltrexone (BNTX), antagonized the effects of DPDPE and [D-Ala2]deltorphin II, suggesting the involvement of a population of delta receptors sensitive to the delta2-antagonist NT II on this effect. To further investigate the participation of delta-receptor subtypes in the stimulation of IPs formation, mice were injected with antisense oligodeoxynucleotides (ODNs) directed to nucleotides 7-26 or 2946 of the cloned delta-receptor mRNA, and PAG slices from these animals were used in in vitro assays. The results demonstrate that the reported increase of phosphoinositide (PI) hydrolysis depends on the agonist activation of the delta2-opioid receptor subtype in the PAG.

Local changes in GTP-binding protein immunoreactivities in human epileptogenic neocortex.

The relative levels of guanine nucleotide-binding protein alpha-subunits Gi1alpha, Gi2alpha, Gi3alpha, Go(alpha), Gs(alpha), and Gx/z(alpha) were measured in neocortex removed at surgery from patients with intractable temporal lobe epilepsy. Immunoreactivity was quantified using specific polyclonal antisera against the Galpha-subunits according to the Laurell "rocket" immunoelectrophoresis technique. We compared the G protein contents of spiking (active) and nonspiking (nonactive) cortical regions, based on intraoperative electrocorticography, within the same and different patients. There were no clear trends for lower or higher levels of G-protein subtypes to be found in the samples of protein extracts from nonspiking regions as compared to spiking regions. However, comparison of paired samples of spiking and nonspiking cortex within the same patient demonstrated that levels of certain G-protein subtypes were either increased or decreased in all patients. This indicates that cortical regions with enhanced neuronal activity may produce microzonal alterations in the levels of G proteins. Moreover, our results suggest that high levels of Gi1alpha and low levels of the other G-protein subtypes appear to be associated with a greater susceptibility to maintaining spiking activity.