Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate

Activation of the serotonin_1A receptor decreases CA₁ population spike amplitude and inhibits forskolin-stimulated adenylate cyclase in rat hippocampus. Pretreatment of rats with pertussis toxin blocked both responses. Because the electrophysiological and biochemical responses to serotonin were correlated after pertussis toxin treatment, we conclude that both responses are mediated by a common regulatory protein, presumably G_i.     

Influences of pertussis toxin, guanine nucleotides and forskolin on adenylate cyclase in striatal membranes of infant, adult and senescent rats

To identify age-related changes in the dopamine (DA) receptors-GTP-binding protein-adenylate cyclase system, the following experiments were performed at 7 days (infant), 70 days (adult) and 2 years (senescent) in striatal membranes of rats: (1) effects of GTP in the presence and absence of pertussis toxin (islet-activating protein, IAP) on adenylate cyclase in the presence of forskolin alone, (2) the same in the presence of forskolin plus DA and (3) the corresponding effect of guanyl-5'-yl-beta, alpha-imido-diphosphate (GppNHp). GTP caused biphasic effects: the activation at 1 microM and the inhibition at 100 microM on forskolin-stimulated cyclase activity at 70 days. The inhibition was suppressed with IAP pretreatment. In infant membranes, 100 microM GTP inhibited the activity in the absence and presence of 100 microM DA and IAP induced a reversal from the inhibition to the stimulation only in the presence of DA. In senescent animals, neither GTP nor IAP affected the activity. GppNHp at 1, 10 and 100 microM only activated and did not inhibit forskolin-stimulated cyclase at each stage. GppNHp-caused stimulation was no more affected by IAP. IAP ADP-ribosylated 41,000 dalton proteins at each stage and the specific activity of ADP-ribosylation was not changed during development and aging. It is suggested that inhibitory GTP-binding protein (Gi) with molecular size of 41,000 dalton is involved in DA receptor-adenylate cyclase system whose function is low at infant stage and markedly decreased at senescent stage in rat striatal membranes.     

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.     

Modification of morphine-induced locomotor activity by pertussis toxin: biochemical and behavioral studies in mice

The effect of pertussis toxin (PTX) on the locomotor-enhancing action of systemic and intracerebroventricular (i.c.v.) morphine was investigated in mice. Mice were i.c.v. injected with either PTX (0.25 and 0.5 μg) or saline as a control. The s.c. (5–20 mg/kg) and i.c.v. (7–30 nmol) administration of morphine produced a dose-related locomotor-enhancing action in control mice. The peak effect of morphine (30 nmol, i.c.v.)-induced hyperlocomotion was observed 90 min after the morphine injection. At the same time, morphine significantly increased dopamine (DA) metabolism in the limbic forebrain (nucleus accumbens and olfactory tubercle). Similarly, the selective μ-opioid receptor agonist[d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin (DAGO, 4 nmol, i.c.v.) also significantly increased locomotor activity and DA metabolism in the limbic forebrain. Both morphine- and DAGO-induced hyperlocomotion and elevation of DA turnover were antagonized by pretreatment with the μ antagonist β-funaltrexamine (β-FNA). These results suggest that the locomotor-enhancing action of morphine results from the activation of central μ-opioid receptors, and that the activation of the mesolimbic DA system may be involved in the expression of morphine-induced hyperlocomotion in mice. Furthermore, pretreatment with PTX (0.5 μg, i.c.v., 6 days prior to the testing) significantly reduced hyperlocomotion and elevation of DA turnover in the limbic forebrain which had been induced by administrations of morphine (30 nmol, i.c.v.) and DAGO (4 nmol, i.c.v.). These findings suggest that the central PTX-sensitive GTP-binding protein (G-protein) mechanism may play an important role in opioids-induced locomotor-enhancing action. Furthermore, the activation of mesolimbic DA transmission by μ-opioid agonists may also be mediated by a PTX-sensitive G-protein mechanism in mice.     

Presynaptic inhibition by neuropeptide Y and baclofen in hippocampus: insensitivity to pertussis toxin treatment

Neuropeptide Y (NPY) presynaptically inhibits excitatory transmission in area CA1 of rat hippocampus. As postsynaptic NPY receptors in certain other tissues have been shown to be coupled to G-proteins, we have tested the hypothesis that the hippocampal NPY effects are also mediated by G-proteins. Pretreatment of rats with pertussis toxin (PTX) was ineffective in blocking NPY's presynaptic inhibitory actions in area CA1 of the hippocampal slice. The presynaptic inhibitory action of baclofen was also unaffected by PTX pretreatment. However, in these same PTX-pretreated slices, the postsynaptic hyperpolarizing actions of baclofen and 5-hydroxytryptamine were blocked. We suggest that pre- and postsynaptic receptors possess different coupling mechanisms to their effectors.     

Intrahippocampal injection of pertussis toxin blocks adenosine suppression of synaptic responses

Adenosine exerts prominent inhibitory effects on synaptic transmission via a presynaptic action. Using the hippocampal slice preparation, we have found in electrophysiological experiments that this action of adenosine is blocked by intrahippocampal injections of pertussis toxin. In biochemical studies, we have confirmed that this treatment affects the GTP-binding proteins, G_i and G_o, in this preparation. These results indicate that both pre- and postsynaptic actions of adenosine involve pertussis toxin-sensitive GTP-binding proteins.     

Intra-accumbens pertussis toxin sensitizes rats to the locomotor activating effects of a single cocaine challenge

Drugs of abuse share common neurochemical signaling substrates, many of which are components of the cAMP cascade. Interestingly, a number of these substrates have been linked to drug-influenced behaviors. This study sought to understand the role of one signaling substrate, inhibitory G-proteins, in a drug-induced phenomenon known as behavioral sensitization. Specifically, we used pertussis toxin (PTX) as a tool to investigate the relationship between cocaine-induced alterations in cAMP signaling and behavior. Vehicle (1 micro l/side) or PTX (0.15 or 0.25 micro g/1 micro l/side) was bilaterally infused into the nucleus accumbens of rats. Locomotor activity was assessed on days 7, 14 and 21 post-infusion. Intra-accumbal PTX produced a dose-dependent increase in locomotor activity. On day 21 following behavioral monitoring for 1 h, rats were acutely challenged with cocaine (15 mg/kg, i.p.) and behavioral data were accumulated for an additional 2 h. Intra-accumbal PTX sensitized rats to the locomotor-activating effects of a single cocaine challenge which was dose-dependent. After behavioral testing, brains were removed and processed for in vitro receptor autoradiography using the D(1) receptor ligand [3H] SCH 23390. No changes in D(1) dopamine receptor binding were observed. These findings suggest a role for inhibitory proteins (G(i)/G(o)) within the nucleus acumbens in locomotor activity and also cocaine-induced behavioral sensitization.     

Role of receptor regulation in opioid tolerance mechanisms

The molecular basis of opioid tolerance/dependence has long eluded researchers, but recent advances in receptor regulation have suggested a useful conceptual approach to the problem. In NG108-15 neuroblastoma x glioma hybrid (NG) cells, opioid agonists inhibit adenylate cyclase in a dose-dependent, naloxone-antagonizable fashion. Chronic treatment with opioid agonists results in a series of molecular processes that, in a tolerance-like fashion, counteract this inhibition. These processes include desensitization and down-regulation of receptors and an increase in adenylate cyclase activity. Opioid inhibition of adenylate cyclase and opioid receptor down-regulation also have been observed in the brain. However, most studies have found that the receptors coupled to adenylate cyclase are not of the mu type, which are thought to be the primary mediators of opioid analgesia. Down-regulation has been observed for both mu and delta opioid receptors in the brain. However, in most cases, the time course of down-regulation is not correlated with that for tolerance development, and chronic morphine treatment does not result in down-regulation. Thus, opioid receptors in the brain, like those in NG cells, are subject to dynamic regulation by agonists, which probably has an important role in their function. However, it remains to be established that opioid receptor regulation is the basis of opioid tolerance and dependence.     

Differential modulatory roles of cholera toxin and pertussis toxin in the regulation of pain responses induced by excitatory amino acids administered intrathecally in mice

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in excitatory amino acids induced pain response. Intrathecal (i.t.) injection of glutamate (20 microg), N-methyl-D-aspartic acid (NMDA; 60 ng), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 13 ng), and kainic acid (12 ng) showed pain response. Pretreatment with CTX (0.05 and 0.5 microg, i.t.) attenuated pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t. in a dose-dependent manner. On the other hand, i.t. pretreatment with PTX further increased the pain response induced by glutamate, NMDA, AMPA and kainic acid administered i.t., especially at the dose of 0.5 microg. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play opposite roles in modulating the pain response induced by spinally administered. Furthermore, CTX- and PTX-sensitive G-proteins appear to modulate pain response induced by stimuli of both NMDA and non-NMDA glutamate receptors.     

Accelerated maternal responding following intra-VTA pertussis toxin treatment

Prior studies have supported a role for mesolimbic dopaminergic mechanisms in the regulation of maternal behavior. Accordingly, the ventral tegmental area (VTA) and its dopaminergic projections to the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in both the onset and maintenance of normal maternal behavior. To date, studies of direct manipulation of VTA neurochemistry at the onset of maternal behavior have been limited. The current study was undertaken to directly test the hypothesis that enhancement of dopaminergic transmission in the mesolimbic dopamine system can stimulate maternal activity using a pup-induced virgin model. Nulliparous female rats were stereotaxically infused with pertussis toxin (PTX 0, 0.1, or 0.3 μg/hemisphere) into the VTA to chronically stimulate the activity of dopaminergic projection neurons. After 3 days of recovery, maternal responding to donor pups was tested daily, and latency (in days) to full maternal behavior was recorded. Intra-VTA PTX treatment produced a robust dose-dependent decrease in maternal behavior latency, and a long-lasting increase in locomotor activity. These effects were associated with significantly decreased dopamine D1 receptor mRNA expression in the NAc. No effects of PTX treatment on mesolimbic dopamine utilization or mPFC receptor expression were observed. The findings indicate that chronic neural activation in the VTA accelerates the onset of maternal behavior in virgin female rats via modification of the NAc dopamine D1 receptor.     

Enhancement of histamine-induced vascular leakage by pertussis toxin in SJL/J mice but not BALB/c mice

Pertussis toxin (PTX) from Bordetella pertussis is known to enhance inflammatory responses which involve histamine and serotonin, including cell-mediated delayed-type hypersensitivity reactions. In this study we examined the effects of PTX on histamine-modulated microvascular responses. The actions of histamine on arteriole diameter and post-capillary leaky site formation in the cremaster muscle were measured intra-vitally in two inbred strains of mice (viz. BALB/c and SLJ). In SJL mice the rate and extent of histamine-induced leaky site formation were greatly enhanced (from 8.3 to 21.0 leaky sites per 0.1 cm²) by pre-exposure to PTX. In sharp contrast, PTX did not alter histamine-induced leaky site formation in BALB/c mice. Histamine-mediated dilation in arterioles in both strains of mice were not enhanced by PTX. PTX may enhance the development of inflammatory responses by enhancing histamine-induced leaky site formation of the microvasculature.     

Independence of, and interactions between, cannabinoid and opioid signal transduction pathways in N18TG2 cells

N18TG2 neuroblastoma cells co-express δ-opioid and CB1-cannabinoid receptors. Both receptors are negatively coupled to adenylyl cyclase through pertussis toxin-sensitive GTP-binding proteins. In the present study, we confirmed the independent activity of opioid and cannabinoid agonists, and investigated chronic interactions between the two signal transduction pathways in these cells. Opioid and cannabinoid agonists stimulated [ ]guanosine-5′-O-(3-thiotriphosphate) binding to N18TG2 membranes. When the opioid agonist etorphine and the cannabinoid agonist desacetyllevonantradol (DALN) were applied together, the stimulation was similar to the arithmetic sum of the two separate effects. This additivity existed even after partial ablation of the G-proteins reservoir with a low concentration of pertussis toxin, indicating that opioid and cannabinoid receptors activate different pools of G-proteins in N18TG2 cells. Chronic treatment of the cells with either opioid or cannabinoid agonists induced desensitization to the respective drug. In addition, asymmetric cross-desensitization was found: while long-term exposure to DALN induced homologous desensitization, and did not reduce the effect of etorphine, long-term exposure to etorphine attenuated the cannabinoid activation of G-proteins. Chronic exposure to either DALN or etorphine not only induced desensitization, but also elevated the basal activity of G-proteins in the exposed cells. The combination of the two drugs did not yield an additive activation, suggesting that chronic exposure of N18TG2 cultures to cannabinoid and opioid agonists modified a common responding element within the cells. This work presents the N18TG2 neuroblastoma as a suitable experimental model to study the molecular mechanism(s) underlying chronic interactions between opioid and cannabinoid drugs.     

Effects of pertussis toxin on behavioral responses during different withdrawal periods from chronic cocaine treatment

1. The role of Gi-proteins on cataleptic responses induced by SCH23390 and haloperidol in chronic cocaine-treated mice was examined by intracerebroventricullor (i.c. v.) and intravenous (i. v.) injections of pertussis toxin (PTX), which catalyzes adenosine diphosphate (ADP)-ribosylation of Gi-proteins. 2. In animals pretreated chronically with cocaine (10 mg/kg, s.c. on alternating days for 21 days), haloperidol (0.1 mg/kg i.p.) exerted an enhanced cataleptic response, but SCH23390 (0.1 mg/kg i.p.) produced an attenuated response at day 1, which converted to a supernormal response, when it was administered 20 days after the last cocaine injection. 3. The attenuated SCH23390 cataleptic response (D1 receptor supersensitivity induced one day after chronic cocaine treatment), was reversed one day after a single dose of PTX, which by itself had no effect, whereas the enhanced haloperidol catalepsy was further enhanced with same dose of toxin. 4. On the other hand, the enhanced SCH23390- and haloperidol-induced cataleptic responses seen during longer withdrawal period (20 days) were potentiated 20 days after a single coadministration of PTX. The stimulatory effects of PTX on the enhanced SCH23390-induced cataleptic response (D1 receptor subsensitivity induced during long-term withdrawal periods from chronic cocaine treatment), may be due to an indirect inhibition of D1 receptors (a synergistic effect) via blockade of postsynaptic dopamine D2 receptors. 5. The postsynaptic D1 receptor supersensitivity and D2 receptor subsensitivity induced one day after chronic cocaine treatment may involve greater Gi-protein ADP-ribosylation in the presynaptic cell body (VTA) than that in the postsynaptic cell body. On the other hand, the subsensitivity of postsynaptic dopamine D1 and D2 receptors (the enhanced SCH23390- and haloperidol-induced cataleptic responses) seen during longer withdrawal periods may mainly involve Gi-protein ADP ribosylation in the postsynaptic cell body, and which may be mediated by a PTX-sensitive muscarinic M2 and/orGABAB receptor activation.     

Involvement of metabotropic glutamate receptors in Gi- and Gs-dependent modulation of adenylate cyclase activity induced by a novel cognition enhancer NS-105 in rat brain

The effect of a novel cognition enhancer [(+)-5-oxo-

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-prolinepiperidinamide monohydrate] (NS-105) on cAMP formation was investigated in both slices and membranes of the rat cerebral cortex. NS-105 (10⁻⁸–10⁻⁶ M) inhibited forskolin-stimulated cAMP formation in membranes, however, the compound significantly enhanced the cAMP formation in pertussis toxin-pre-treated membranes, an action that was abolished by cholera toxin. In contrast, in digitonin-permeabilized membranes, NS-105 had no influence on Mn²⁺-stimulated cAMP formation. Both of the inhibitory and facilitatory actions of NS-105 on cAMP formation were mimicked by a metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and an adrenergic α₂ agonist UK-14,304, and blocked by a mGluR antagonist 2-amino-3-phosphonopropanoate but not by an α₂ antagonist yohimbine. In cortical slices, NS-105 (10⁻⁸–10⁻⁷ M) inhibited forskolin-stimulated cAMP accumulation but enhanced isoproterenol-stimulated cAMP accumulation, as did by a GABA_B agonist (−)baclofen. On the other hand, (−)baclofen, while it significantly inhibited cAMP accumulation in slices, did no longer inhibit cAMP accumulation, when treated with NS-105 (10⁻⁸–10⁻⁵ M). Similarly, (−)baclofen-induced inhibition of the cAMP accumulation was reversed by 1S,3R-ACPD and UK-14,304. NS-105 (10⁻⁶) increased [³⁵S]GTPγS binding in the intact but not digitonin-permeabilized cortical membranes, as produced by UK-14,304, although the compound (10⁻⁹–10⁻³ M) had no influence on various neurotransmitter receptor bindings, including α₂ receptors. These results suggest that NS-105 modulates adenylate cyclase activity by stimulating mGluRs which might coupled to both G_i/G_o and G_s. © 1997 Elsevier Science B.V. All rights reserved.     

Modification of morphine-induced analgesia and toxicity by pertussis toxin

The present study evaluates the effect of pertussis toxin (PTX) on morphine-induced analgesia and lethality. Mice were injected with 0.2 microgram PTX intracerebroventricularly (i.c.v.) and 0.2 micrograms PTX intrathecally (i.t.) or saline. Mice were tested for morphine-induced analgesia (tail flick) and lethality 16 days later; mice were also examined for pentobarbital-induced mortality. Morphine analgesic potency was decreased by approximately 4-fold in PTX-treated mice compared to controls. Conversely, the lethal potency of morphine was increased by 10-fold in PTX-treated mice compared to controls. PTX treatment did not alter the lethal potency of pentobarbital. Morphine-induced analgesia and lethality were dose-dependently antagonized by naloxone in both PTX and saline-treated groups. The results of this study suggest that morphine analgesia is mediated through PTX-sensitive G proteins. On the other hand, morphine-induced lethality appears to be limited by PTX-sensitive factor(s) since PTX treatment enhanced morphine's lethal potency. The increase in lethal potency of morphine may be due to unmasking of an excitatory opioid receptor mediated effect by PTX.     

Effects of pertussis toxin on caudate neuron electrophysiology: studies with dopamine D1 and D2 agonists

The selective D₁ and D₂-agonists SKF-38393 and N-0437 respectively, were tested in caudate pretreated with PT. A paired recording paradigm was used where the contralateral untreated caudate served as a control. Micropipettes were used to locally apply SKF-38393 and N-O437 onto neurons in both control and PT-pretreated caudate. A significant attenuation of the responses to the D₂ agonist were observed after PT administration. Only 1 out 12 cells tested on the PT side demonstrated any response to locally applied N-O437, whereas 90% of the neurons responded to the drug on the control side. Neurons from both the PT-pretreated and control caudates responded to locally applied SKF-38393. In addition to the specific D₁ and D₂ receptor agonists, DA and the indirect dopamine agonist PCP were tested for changes in responsiveness. Dopamine was equally efficacious at both control and PT-pretreated caudate neurons, which suggest that dopamine locally applied from the micropipette can interact with the unperturbed D₁ receptors in the PT-pretreated caudate. On the other hand, the response to PCP was significantly attenuated after PT administration, which suggest that endogenously released DA preferentially interacts with the D₂ receptor subtype. Taken together these data suggest an important role for the D₂ receptor in the physiology of dopamine responsiveness in the caudate nucleus.     

Interaction of the α-2 adrenergic- and opioid receptor with the cGMP system in the mouse cerebellum

The α-2 adrenergic agonist dexmedetomidine (Dex), 3–300 μg/kg, i.p., decreased cerebellar cGMP in a dose-dependent manner. Fentanyl (F), an opioid agonist, increased cerebellar cGMP at 0.3 mg/kg, s.c., and decreased it at doses ≥1 mg/kg. The inhibitory effect was receptor specific, that of Dex being blocked by the α-2 adrenergic antagonist yohimbine, 5 mg/kg, i.p.; that of F by the opioid antagonist naloxone, 5 mg/kg, i.p. In contrast the stimulatory effect of F was blocked by both naloxone and yohimbine. Yohimbine also enhanced the inhibitory effect of F. In mice pretreated with pertussis toxin, 2 μg/mouse, given i.c.v. 72 h before the agonists, the decrease in cGMP induced by Dex or F was not affected, while the stimulatory effect of F was reversed to an inhibitory effect. When inhibiting doses of F and Dex were administered together, the cGMP response was smaller than the sum of the individual responses. Dex attenuated in a dose-dependent manner the decrease in cGMP induced by F, and unmasked or enhanced the stimulatory effect of F. These results show that the α-2 adrenergic- and opioid-receptors are coupled to the cGMP effector system and suggest that the two pathways converge at a common post-receptor site in the cascade of events transducing the receptor signal to cGMP regulation.     

Upregulation of Adenosine Al Receptors and Forskolin Binding Sites Following Chronic Treatment with Caffeine or Carbamazepine: A Quantitative Autoradiographic Study

The effects of feeding a diet enriched in caffeine or carbamazepine (CBZ) were investigated in rats in a quantitative autoradiographic study of adenosine A1 receptors (labeled by [3H]cyclohexyladenosine, [3H]CHA) and adenylate cyclase (labeled by [3H]forskolin). Although regional distribution of [3H]CHA and [3H]forskolin binding sites differed in some areas, chronic CBZ as well as chronic caffeine upregulated both of them. The changes in receptor densities occurred in the same brain microregions, suggesting that caffeine and CBZ act as antagonists at similar subpopulations of adenosine A1 receptors and [3H]forskolin binding sites. Therefore, a selective interaction of these two drugs with distinct adenosine A1 receptors (and adenylate cyclase) probably does not explain the differential effects of caffeine and CBZ on neuronal activity.     

Opioids potentiate transmitter release from SK-N-SH human neuroblastoma cells by modulating N-type calcium channels

Opioids induce dual (inhibitory and excitatory) regulation of depolarization-evoked [³H]dopamine release in SK-N-SH cells through either μ or δ receptors. The potentiation of dopamine release by opioid agonists is mediated by N-type voltage-dependent calcium channels and does not involve G_i/G_o proteins. Removal of the excitatory opioid effect by blockade with ω-conotoxin, an N-channel antagonist, reveals the inhibitiory effect of opioids on release, thus suggesting that both modulatory effects of opioids are exerted in parallel.