Inhibition and superactivation of the calcium-stimulated isoforms of adenylyl cyclase

It was shown previously that chronic exposure to opiate agonists increases adenylyl cyclase (AC) activity, a phenomenon termed AC superactivation (or supersensitization). More recently, we showed that acute Gi/o- coupled receptor activation inhibits the activity of several AC isozymes, including Ca2+/calmodulin-stimulated AC-I and -VIII, whereas chronic receptor activation induces their superactivation. Here, we report that both acute Mu-opioid receptor-induced inhibition and chronic induced superactivation of AC-I and -VIII are pertussis toxin sensitive. In addition, we show that proteins that interfere with the activity of Gbetagamma subunits (Gbetagamma scavengers) strongly attenuate the acute inhibition of AC-I and -VIII and the superactivation of AC-I, and abolish the superactivation of AC-VIII. Based on these results, we suggest that Gbetagamma is involved in the acute inhibition and chronic agonist-induced superactivation of AC types I and VIII.     

Changes in the cAMP-related signal transduction mechanism in postmortem human brains of heroin addicts

Summary. Immunoreactivities of adenylyl cyclase (AC) type I (AC-I), and basal, forskolin- and Mn²⁺-stimulated AC activities with or without calcium and calmodulin (Ca²⁺/CaM) were estimated in temporal cortex (TC)-and nucleus accumbens (NAc) membranes from brains of heroin addicts and controls. Immunoreactivity of AC-I was significantly decreased in TC from brains of heroin addicts, but that did not change in NAc. Ca²⁺/CaM-sensitive AC activity was significantly lower in TC from brains of heroin addicts, but that activity in NAc did not show significant difference compared with the control. Some previous reports demonstrated that Ca²⁺/CaM-sensitive AC activity in membranes from postmortem human brain reflected the function of AC-I. Therefore, the downregulation of AC-I in TC plays an important role in the molecular mechanism of chronic opiate addiction in human brain. Received August 1, 2000; accepted August 28, 2000     

Deletion of the N-terminus of a K+ channel brings about short-term modulation by cAMP and β1-adrenergic receptor activation

On deletion of the N-terminus of RCK1 K⁺ channel, acute modulation of the channel by cAMP-elevating treatments is revealed. This modulation is studied inXenopus oocytes using two-electrode voltage-clamp, site-directed mutagenesis, and SDS-PAGE analyses. Treatments by Sp-8-Br-cAMPS, a membrane-permeant cAMP analog, and by isoproterenol, a β1-adrenergic receptor (β1R) agonist, both increased the current amplitudes with no effect on the voltage dependency of activation. The effect of isoproterenol was blocked by coexpression of either {ie269-1} or {ie269-2} proteins. The channel protein is phosphorylated on the Sp-8-Br-cAMPS treatment at Ser446; however, a phosphorylation-deficient variant in which this site has been altered is still modulated by Sp-8-Br-cAMPS and isoproterenol. Expression of the full-length channel with Kvβ1.1 auxiliary subunit renders the channel at the same modulation as that of the truncated one. Taken together, the RCK1 channel can be acutely modulated by cAMP and β1R activation possibly through protein kinase A (PKA) activation, but not through direct channel phosphorylation; the involvement of the N-terminus in this modulation is discussed.     

Ca2+/CaM-sensitive adenylyl cyclase activity is decreased in the Alzheimer's brain: Possible relation to type I adenylyl cyclase

Summary Immunoreactivities of four subtypes of adenylyl cyclase (AC) (types I, II, IV and V/VI), and basal, forskolin- and Mn²⁺-stimulated AC activities with or without calcium and calmodulin (Ca²⁺/CaM) were estimated in parietal cortex membranes from cases with dementia of the Alzheimer type (DAT) and age-matched controls. Immunoreactivities of AC-I and AC-II were significantly decreased, but those of AC-IV and AC-V/VI did not change in DAT brains. There was a significant correlation of AC-I immunoreactivity with Ca²⁺/CaM-sensitive AC activity, but not with the Ca²⁺/CaM-insensitive activity. Ca²⁺/CaM-sensitive AC activity was significantly lower in DAT than in the control, indicating that impairment of Ca²⁺/CaM-sensitive AC-I is clearly involved in the pathophysiology of DAT.     

Mu opioid receptor mutant, T394A, abolishes opioid-mediated adenylyl cyclase superactivation

This study was to characterize the effects of a point-mutant at C-terminal of mu opioid receptor (MOR), namely MOR T394A, in chronic opioid-induced cellular responses. After 18 h of exposure to [D-Ala, N-Me-Phe, Gly-ol] enkephalin (DAMGO), adenylyl cyclase (AC) superactivation, a hallmark for the cellular adaptive response after chronic opioid stimulation, was observed in the cells expressing wild-type receptor, but was totally abolished in the cells expressing MOR T394A. Receptor phosphorylation was also attenuated in cells with MOR T394A after prolonged preexposure to agonist. Furthermore, MAP kinase kinase-1 (MKK1) overexpression was able to rescue AC superactivation in cells with MOR T394A, but showed no effect in the wild-type MOR-expressing cells. These results indicated that the amino acid T394 at C-terminus of MOR played a critical role in chronic agonist-induced AC superactivation and receptor phosphorylation.     

Inhibition of AC-II activity following chronic agonist exposure is modulated by phosphorylation

Chronic exposure to opiate agonists (followed by agonist withdrawal) leads to a large increase in the activity of adenylyl cyclase (AC) isozymes I, V, VI, and VIII, a phenomenon defined as AC superactivation (or supersensitization). On the other hand, AC isozymes belonging to the AC-II family (AC-II, AC-IV, and AC-VII) show decreased activity, referred to as superinhibition. Using COS-7 cells transiently transfected with μ-opioid receptor and AC-II, we show here that inhibition of PKC and tyrosine kinase activities synergistically reduced the level of AC-II superinhibition. Moreover, inhibitor of Raf-1 kinase also led to a decrease in AC-II superinhibition. These data suggest that Raf-1, activated by PKC and tyrosine kinase, has a role in the regulation of AC-II superinhibition.     

Modulation of 5-HT1A receptor mediated response by fluoxetine in rat brain

Summary. Radioligand binding studies were done to investigate the effect of chronic administration of fluoxetine on 5-HT₁ receptor mediated response to adenylate cyclase (AC) in rat brain. Our studies revealed a significant decrease in the densities of 5-HT₁ and 5-HT_1A receptor sites in cortex and hippocampus of rat brain after chronic administration of fluoxetine (10 mg/Kg body wt.). However there was no significant change in the affinity of [³H]5-HT and [³H]DPAT for 5-HT₁ and 5-HT_1A receptor sites, respectively. However, in striatum, along with a significant (75%) downregulation of 5-HT₁ sites, the affinity of [³H]5-HT to these sites was increased, as revealed by decrease in Kd (0.50 ± 0.08 nM). Displacement studies showed that fluoxetine has higher affinity for 5-HT_1A receptors with a Ki value of 14.0 ± 2.8 nM, than 5-HT₁ sites. No significant change was observed in basal AC activity in any region after fluoxetine exposure. However, in cortex of experimental rats the 5-HT stimulated AC activity was significantly increased (16.03 ± 0.97 pmoles/mg protein; p < 0.01), when compared to 5-HT stimulated AC activity (12.98 ± 0.78 pmoles/mg protein) in control rats. The increase in 5-HT stimulated AC activity in cortex may be due to the significant downregulation of 5-HT_1A sites in cortex after fluoxetine exposure as these sites are negatively coupled to AC. The observed significant decrease in 5-HT₁ sites with concomitant increase in 5-HT stimulated AC activity, after fluoxetine treatment, suggests that fluoxetine, which has high affinity for these sites, acts by modulating the 5-HT_1A receptor mediated response in brain. Accepted August 25, 1999     

Involvement of an altered 5-HT -{6} receptor function in behavioral symptoms of Alzheimer's disease

We studied the hypothesis that disturbances in 5-HT_{6} receptor function in the temporal cortex may contribute to clinical symptoms of Alzheimer's disease (AD). 5-HT_{6} density and 5-HT levels were significantly decreased in a cohort of AD patients prospectively assessed for cognitive/behavioral symptoms. cAMP formation after stimulation with the selective 5-HT_{6} receptor agonist E-6801 was significantly lower (p<0.01) in AD (170.02 +/- 27.53 pmol/mg prot.) compared to controls (823.33 +/-196.67). In addition, the ratio cAMP formation after stimulation with E-6801/5-HT_{6} receptor density was significantly lower (p< 0.01) in AD (6.67 +/- 0.83) compared to controls (16.67 +/- 3.33). Splitting these results by sex, 5-HT_{6} receptor activation was significantly lower (p< 0.01) in AD females compared to males (121.67 +/- 30.02 vs. 231.67 +/- 34.17 pmol/mg prot). 5-HT_{6} density and 5-HT levels were significantly correlated (p < or = 0.01) in both controls and AD patients, although in AD, this correlation was lost in females. Psychosis factor was the best predictor of reduced 5-HT levels or adenylate cyclase activity after E-6801 stimulation, the former result being due to females. It may be suggested that psychotic symptoms may be related to a dysregulation of 5-HT_{6} activation by 5-HT in the temporal cortex. These results are discussed in terms of purported influence of sex and therapeutical approaches to psychosis in AD.     

The immunoreactivity and activity of adenylate cyclase type I are changed in the hippocampal CA1 region after transient forebrain ischemia in gerbils

Adenylate cyclase (AC) has a specific sensitivity to Ca²⁺/calmodulin. AC-I, one of the mediator of learning and memory, plays an important role in signal transduction underlying learning and memory function. In the present study, we found ischemia-related changes of AC-I in the hippocampal CA1 region, but not in the CA2/3 region, after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, AC-I immunoreactive neurons were detected in pyramidal and non-pyramidal cells in the hippocampus proper. AC-I immunoreactivity was significantly increased at 3 h in the CA1 region after ischemic insult. Thereafter, AC-I immunoreactivity was gradually decreased. Four days after ischemic insult, AC-I-immunoreactive CA1 pyramidal cells in the stratum pyramidale were very few due to delayed neuronal death. The results of Western blot analysis showed that changes of AC-I protein contents were similar to immunohistochemical data after ischemic insult. Gpp(NH)p-dependent AC-I activity in hippocampal CA1 region was not changed in all groups, while Ca²⁺/calmodulin-dependent AC-I activity in hippocampal CA1 region was significantly decreased 24 h after ischemia–reperfusion. These results suggest that the decrease of AC-I activity may be associated with impairment of neurodevelopment and neuroplasticity including learning and memory although the AC-I immunoreactivity was maintained 24 h postischemic group compared to that of the sham-operated group.     

Prolonged Morphine Treatment Alters Expression and Plasma Membrane Distribution of β-Adrenergic Receptors and Some Other Components of Their Signaling System in Rat Cerebral Cortex

β-Adrenergic signaling plays an important role in regulating diverse brain functions and alterations in this signaling have been observed in different neuropathological conditions. In this study, we investigated the effect of a 10-day treatment with high doses of morphine (10 mg/kg per day) on major components and functional state of the β-adrenergic receptor (β-AR) signaling system in the rat cerebral cortex. β-ARs were characterized by radioligand binding assays and amounts of various G protein subunits, adenylyl cyclase (AC) isoforms, G protein-coupled receptor kinases (GRKs), and β-arrestin were examined by Western blot analysis. AC activity was determined as a measure of functionality of the signaling system. We also assessed the partitioning of selected signaling proteins between the lipid raft and non-raft fractions prepared from cerebrocortical plasma membranes. Morphine treatment resulted in a significant upregulation of β-ARs, GRK3, and some AC isoforms (AC-I, -II, and -III). There was no change in quantity of G proteins and some other signaling molecules (AC-IV, AC-V/VI, GRK2, GRK5, GRK6, and β-arrestin) compared with controls. Interestingly, morphine exposure caused a partial redistribution of β-ARs, G_sα, G_oα, and GRK2 between lipid rafts and bulk plasma membranes. Spatial localization of other signaling molecules within the plasma membrane was not changed. Basal as well as fluoride- and forskolin-stimulated AC activities were not significantly different in membrane preparations from control and morphine-treated animals. However, AC activity stimulated by the beta-AR agonist isoprenaline was markedly increased. This is the first study to demonstrate lipid raft association of key components of the cortical β-AR system and its sensitivity to morphine.     

Clinical correlates of {18F}fluorodopa uptake in five grafted Parkinsonian patients

To assess the efficacy of fetal mesencephalic grafts in Parkinson's disease, it is important to know if the grafted cells survive and are functional. Positron emission tomography (PET) and {¹⁸F}fluorodopa ({¹⁸F}dopa) have been used to demonstrate the survival of the grafted cells, but the relationship of {¹⁸F}dopa uptake changes in the grafted striatum to motor function remains unclear. We investigated this question with 16 serial PET scan in 5 severe parkinsonian patients unilaterally grafted in whom we found a significant and progressive increase of the {¹⁸F}dopa uptake in the grafted putamen. The number of patients was too small to assess the sensitivity of {¹⁸F}dopa PET scans in individual patients. Yet, by analyzing the 16 serial PET scans we found a correlation between the {¹⁸F}dopa uptake (K_i) in the grafted putamen and the percentage of daily time spent “on,” suggesting that K_i changes have a functional meaning. In addition, the K_i values were correlated with the contralateral finger dexterity to the same extent in both the grafted and nongrafted putamen. These results indicate that {¹⁸F}dopa uptake reflects the motor function of the opposite side of the body, similarly in the grafted and ungrafted putamen, at least in terms of these tasks. Finally, extrapolating from these correlations offers the suggestion that clinical optimal results of the graft could be achieved if the graft brings the K_i values in the putamen to about two standard deviations of mean control values.     

Mice lacking adenylyl cyclase‐5 cope badly with repeated restraint stress

Physiological responses to acute stress proceed with the activation of the hypothalamus‐pituitary‐adrenal gland (HPA) system. Many brain regions are known to modulate the HPA axis activation in stress responses, but the detailed neural circuits and signaling system in the upstream of the HPA axis have to be explored further. Type 5 adenylyl cyclase (AC5) is highly concentrated in the dorsal striatum and nucleus accumbens, which are implicated in reward and stress‐related behavior. AC5^–/– mice exposed to daily 2‐hr restraint stress for only 3–5 days showed poor stress‐coping responses, including severe body weight loss, poor coat condition, respiratory difficulties, and freezing behavior. Plasma corticosterone levels during 2‐hr stress sessions increased in AC5^–/– mice compared with those of AC5^+/+ mice. However, neither the corticosterone receptor antagonist RU486 nor the CRH receptor antagonist NBI27914 blocked their poor stress coping, whereas the administration of the GABA_A receptor allosteric modulator diazepam or the D1 dopamine receptor antagonist SCH23390 prior to restraint stress sessions changed their stress‐coping response to the stressed AC5^+/+ mouse level. Stress‐triggered c‐Fos expression was completely blunted in the dorsal striatum of AC5^–/–. These results suggest that the AC5‐associated signal system and neural network are involved in the regulation of anxiety and stress‐coping response. © 2009 Wiley‐Liss, Inc.     

Adenylyl cyclase activity in Alzheimer's disease brain: stimulatory and inhibitory signal transduction pathways are differently affected

Adenylyl cyclase (AC) activity was studied in post mortem hippocampus and cerebellum from eight patients with Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and seven non-demented control patients. AC was stimulated via stimulatory guanine nucleotide binding proteins (Gs) using guanosine triphosphate (GTP) and GppNHp (both 10⁻⁴M) or directly with either forskolin (10⁻⁴M) or Mn²⁺ (10⁻²M). Inhibition of AC via A₁-receptors was performed with N⁶-cyclohexyladenosine (CHA) under basal conditions and in the presence of forskolin (10⁻⁵M). In both brain regions AC activity was significantly reduced in AD/SDAT when compared to controls. Under basal conditions and after stimulation via Gs mean reduction in hippocampus and cerebellum was 47.7% and 58.2%, respectively. The reduction was less pronounced after direct activation of the AC, amounting to 21.8% in hippocampus and 28.1% in cerebellum. CHA inhibited basal and forskolin-stimulated AC concentration-dependently by about 20% (basal) and 30% (forskolin). Inhibition by CHA was similar in hippocampus and cerebellum and tended to be more pronounced in AD/SDAT than in controls. Since the reduction of AC activity in AD/SDAT is greater after stimulation via Gs than after direct activation of the catalytic subunit, we suggest that both Gs and the catalytic subunit seem to be impaired. The fact that CHA-mediated inhibition of AC is not significantly different in AD/SDAT and controls, indicates that in contrast to Gs-, inhibitory G-proteins (Gi) coupling to AC remains intact in Alzheimer's disease.     

Somatostatin depresses the excitability of subicular bursting cells: Roles of inward rectifier K+ channels,KCNQ channels and Epac

The hippocampus is a crucial component for cognitive and emotional processing. The subiculum provides much of the output for this structure but the modulation and function of this region is surprisingly under‐studied. The neuromodulator somatostatin (SST) interacts with five subtypes of SST receptors (sst₁ to sst₅) and each of these SST receptor subtypes is coupled to Gi proteins resulting in inhibition of adenylyl cyclase (AC) and decreased level of intracellular cAMP. SST modulates many physiological functions including cognition, emotion, autonomic responses and locomotion. Whereas SST has been shown to depress neuronal excitability in the subiculum, the underlying cellular and molecular mechanisms have not yet been determined. Here, we show that SST hyperpolarized two classes of subicular neurons with a calculated EC₅₀ of 0.1 μM. Application of SST (1 μM) induced outward holding currents by primarily activating K⁺ channels including the G‐protein‐activated inwardly‐rectifying potassium channels (GIRK) and KCNQ (M) channels, although inhibition of cation channels in some cells may also be implicated. SST‐elicited hyperpolarization was mediated by activation of sst₂ receptors and required the function of G proteins. The SST‐induced hyperpolarization resulted from decreased activity of AC and reduced levels of cAMP but did not require the activity of either PKA or PKC. Inhibition of Epac2, a guanine nucleotide exchange factor, partially blocked SST‐mediated hyperpolarization of subicular neurons. Furthermore, application of SST resulted in a robust depression of subicular action potential firing and the SST‐induced hyperpolarization was responsible for its inhibitory action on LTP at the CA1‐subicilum synapses. Our results provide a novel cellular and molecular mechanism that may explain the roles of SST in modulation of subicular function and be relevant to SST‐related physiological functions.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

Chronic regulation of colonic epithelial secretory function by activation of G protein‐coupled receptors

Background Enteric neurotransmitters that act at G protein‐coupled receptors (GPCRs) are well known to acutely promote epithelial Cl^? and fluid secretion. Here we examined if acute GPCR activation might have more long‐term consequences for epithelial secretory function. Methods Cl^? secretion was measured as changes in short‐circuit current across voltage‐clamped T₈₄ colonic epithelial cells. Protein expression was measured by western blotting and intracellular Ca²⁺ levels by Fura‐2 fluorescence. Key Results While acute (15 min) treatment of T₈₄ cells with a cholinergic G_qPCR agonist, carbachol (CCh), rapidly stimulated Cl^? secretion, subsequent CCh‐induced responses were attenuated in a biphasic manner. The first phase was transient and resolved within 6 h but this was followed by a chronic phase of attenuated responsiveness that was sustained up to 48 h. CCh‐pretreatment did not chronically alter responses to another G_qPCR agonist, histamine, or to thapsigargin or forskolin which elevate intracellular Ca²⁺ and cAMP, respectively. This chronically acting antisecretory mechanism is not shared by neurotransmitters that activate G_sPCRs. Conditioned medium from CCh‐pretreated cells mimicked its chronic antisecretory actions, suggesting involvement of an epithelial‐derived soluble factor but further experimentation ruled out the involvement of epidermal growth factor receptor ligands. Acute CCh exposure did not chronically alter surface expression of muscarinic M₃ receptors but inhibited intracellular Ca²⁺ mobilization upon subsequent agonist challenge. Conclusions & Inferences These data reveal a novel, chronically acting, antisecretory mechanism that downregulates epithelial secretory capacity upon repeated G_qPCR agonist exposure. This mechanism involves release of a soluble factor that uncouples receptor activation from downstream prosecretory signals.     

Glycine increases the number of somatostatin receptors and somatostatin-mediated inhibition of the adenylate cyclase system in the rat hippocampus

The glycine and somatostatin (SS) neurotransmission systems in the brain have been implicated in the function of sensory, motor, and nociceptive pathways. To investigate a possible relationship between these two components, we studied the influence of glycine on the binding of ¹²⁵I-Tyr¹¹-SS to its receptors and on SS-like immunoreactivity (SSLI) levels in the rat hippocampus and frontoparietal cortex. An intracerebroventricular (i.c.v.) dose of 16 or 160 nmol of glycine induced an increase in the total number of specific SS receptors in the hippocampus but not in the frontoparietal cortex at 15 min following injection, with no changes in the affinity constant. This effect seems to be mediated by inhibitory strychnine-sensitive glycine receptors since pretreatment with the antagonist strychnine (80 μg/100 g body weight, intravenously) abolished this response. No significant changes in SSLI content were detected in either brain region of glycine- and strychnine plus glycine-treated rats as compared to control values. Since SS receptors are coupled via guanine nucleotide-binding G proteins to the adenylyl cyclase (AC) system, we also examined the inhibitory effects of SS and the guanine nucleotide Gpp(NH)p on AC activity in hippocampal membranes of control, glycine- and strychnine plus glycine-treated rats since the increase in SS receptors was observed only in this brain area. No significant differences were observed for the forskolin (FK)-stimulated AC enzyme activities in hippocampal membranes from all the experimental groups studied. In the hippocampus of the glycine-(160 nmol) treated group, however, basal AC activity was significantly lower, and the capacity of SS to inhibit FK-stimulated AC activity was increased as compared to the control group. Pretreatment with strychnine prevented the increase in SS-mediated inhibition of AC activity. The functional activity of the inhibitory guanine nucleotide-binding protein G₁, as determined by the inhibitory effect of the stable GTP analogue Gpp(NH)p on FK-stimulated AC activity, was significantly higher in hippocampal membranes of glycine- (160 nmol) treated rats as compared to controls. This suggests that the increased inhibition of AC activity by SS in the glycine-treated group may be due to the increase in G₁ activity and/or the increase in the number of SS receptors observed. Alternatively, the greater G₁ activity may be responsible for the increased binding of ¹²⁵I-Tyr¹¹-SS to its receptors observed after glycine administration. Altogether, these data suggest that the hippocampal somatostatinergic system can be regulated by strychnine-sensitive glycine receptors in the rat. © 1996 Wiley-Liss, Inc.     

Somatostatin receptor-GTP binding regulatory protein-adenylyl cyclase system in hippocampal membranes of strychnine-treated rats

Wistar rats were injected with either a non-convulsive dose (37.5 μg/100 g body weight (b.wt.), intravenously (i.v.)) or a convulsive dose (50 or 80 μg/100 g b.wt., i.v.) of strychnine. Binding of¹²⁵I-Tyr¹¹-somatostatin (¹²⁵I-Tyr¹¹-SS) to its specific receptors was measured in hippocampal membranes 15 min after strychnine injection at these three doses. The non-convulsive dose of strychnine did not affect binding of SS in the hippocampus whereas both convulsive doses decreased the number of specific SS receptors without influencing their apparent affinity. Somatostatin-like immunoreactivity (SSLI), SS-modulated adenylel cyclase (AC) activity and the inhibitory guanine-nucleotide binding regulatory protein were also measured in rats treated with 80 μg/100 g b.wt. of strychnine. SSLI content remained stable. No significant differences were seen for the basal and forskolin (FK)-stimulated AC enzyme activities in the hippocampus of strychnine-treated rats when compared to the control group. The capacity of SS to inhibit basal and FK-stimulated AC activity in the hippocampus was significantly lower in the strychnine group than in the control group. The ability of the stable GTP analogue 5′-guanylylimidodiphosphate [Gpp(NH)p] to inhibit FK-stimulated AC activity was also decreased in hippocampal membranes from strychnine-treated rats. These results suggest that the attenuated inhibition of AC by SS in hippocampal membranes from strychnine-treated rats may be caused by decreases in both G_i activity and in the number of SS receptors. Alternatively, an uncoupling of SS receptors from G_i and/or a decrease in the level of functional G_i may result in both a decrease in apparentB_max for SS binding and in SS-mediated inhibition of AC. Since recent studies of other authors support the view that SS is predominantly an inhibitory transmitter in the hippocampus, it is possible that the decreased inhibition of AC activity by SS as well as the decrease in the number of SS receptors found in the hippocampus of strychnine-treated rats may be a mechanism involved in the development of increased seizure susceptibility.     

The glycine site of the NMDA receptor contributes to neurokinin1 receptor agonist facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons

We have investigated the role of the glycine recognition site of the N-methyl-d-aspartate receptor (the Gly_NMDA site) in the facilitation of NMDA receptor agonist-evoked activity in rat dorsal horn neurons that is brought about by neurokinin ₁ (NK₁) receptor agonist and the contribution of protein kinase C (PKC) activation to this phenomenon. Ionophoresis of the selective NMDA receptor agonist 1-aminocyclobutane-ci's-1,3-dicarboxylic acid (ACBD) produced a sustained increase in the firing rate of single laminae III-V neurons recorded extracellularly using multibarrelled glass electrodes. The highly selective NK₁ receptor agonist acetyl-[Arg⁶, Sar⁹, Met(O₂)¹¹]-SP_6–11 (Sar⁹-SP) greatly facilitated this response, but under the present conditions had no effect when applied alone or with a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptor agonist) at the same current. In the presence of the Gly_NMDA site antagonists 2-carboxy-4,6-dichloro-(1H)-indole-3-propanoic acid (MDL 29951), 7-chloro-3-(cyclopropylcarbonyl)-4-hydroxy-2(1H)-quinoline (L701,252), 5,7-dinitroquinaxoline-2,3-dione (MNQX) or 7-chlorothiokynurenic acid (7-CTK), or the PKC inhibitors, chelery-thrine or GF109203X, the Sar⁹-SP-induced facilitation of ACBD-evoked activity was prevented, generally restoring activity to a level similar to that in the presence of ACBD alone, whilst an AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo (f) quinoxaline-2,3-dione (NBQX) did not inhibit the facilitation. At the same ionophoretic currents these compounds had no effect on ACBD-evoked activity in the absence of Sar⁹-SP but were inhibitory at significantly greater currents. To further substantiate the importance of the Gly_NMDA site in the interaction, the effects of NMDA receptor antagonists selective for alternative recognition sites on the NMDA receptor were investigated. MK-801, a non-competitive NMDA receptor antagonist and arcaine, a competitive inhibitor at the polyamine site, were applied to the facilitated activity seen in the presence of Sar⁹-SP and ACBD, and to ACBD-evoked activity alone. Unlike the Gly_NMDA site antagonists and PKC inhibitors, these compounds reduced both facilitated and ACBD-evoked activity at similar currents. Furthermore, like the NK₁ receptor agonist, a selective Gly_NMDA site agonist 1-aminocyclopropane carboxylic acid (ACPC) caused facilitation of ACBD-evoked activity which was also blocked by currents of L701,252 that did not alter activity evoked by ACBD alone. These data suggest that activation of the Gly_NMDA site (perhaps as a consequence of glycine release or modification of its influence by intracellular signalling cascades) is an essential component of the means by which NK₁ receptor activation results in facilitated responsiveness of dorsal horn neurons towards NMDA receptor agonists.