A neuropeptide FF agonist blocks the acquisition of conditioned place preference to morphine in C57Bl/6J mice

Neuropeptide FF behaves as an opioid-modulating peptide that seems to be involved in morphine tolerance and physical dependence. Nevertheless, the effects of neuropeptide FF agonists on the rewarding properties of morphine remain unknown. C57BL6 mice were conditioned in an unbiased balanced paradigm of conditioned place preference to study the effect of i.c.v. injections of 1DMe (D-Tyr1(NMe)Phe3]NPFF), a stable agonist of the neuropeptide FF system, on the acquisition of place conditioning by morphine or alcohol (ethanol). Morphine (10 mg/kg, i.p.) or ethanol (2 g/kg, i.p.) induced a significant place preference. Injection of 1DMe (1-20 nmol), given 10 min before the i.p. injection of the reinforcing drug during conditioning, inhibited the rewarding effect of morphine but had no effect on the rewarding effect of ethanol. However, a single injection of 1DMe given just before place preference testing was unable to inhibit the rewarding effects of morphine. By itself, 1DMe was inactive but an aversive effect of this agonist could be evidenced if the experimental procedure was biased. These results suggest that neuropeptide FF, injected during conditioning, should influence the development of rewarding effects of morphine and reinforce the hypothesis of strong inhibitory interactions between neuropeptide FF and opioids.     

Regionally and functionally distinct serotonin3 receptors control in vivo dopamine outflow in the rat nucleus accumbens

Central serotonin(3) (5-HT(3)) receptors control the mesoaccumbens dopamine (DA) pathway. This control is thought to be conditional and might involve regionally distinct subpopulations of 5-HT(3) receptors. Here, using in vivo microdialysis in rats, we assessed the relative contribution of nucleus accumbens (Nacc) 5-HT(3) receptors to the overall influence exerted by 5-HT(3) receptors on accumbal DA release induced by different drugs or treatments. In freely moving rats, pre-treatment with 5-HT(3) antagonists (0.1 mg/kg ondansetron and/or 0.03 mg/kg MDL 72222, s.c.) reduced DA efflux enhanced by morphine (1-10 mg/kg, s.c.) and haloperidol (0.01 mg/kg, s.c.), but not amphetamine (1-2.5 mg/kg, i.p.) or cocaine (10-20 mg/kg, i.p.), the latter two drugs do not trigger depolarization-stimulated DA exocytosis. Intra-Nacc administration of ondansetron (1 microm) in freely moving rats reduced the DA effects elicited by 10 mg/kg morphine, but not 1 mg/kg morphine or haloperidol. The 5-HT(1A) agonist 8-OH-DPAT (0.1 mg/kg, s.c.), known to decrease central 5-HT tone, reduced 10 but not 1 mg/kg morphine-stimulated DA outflow in freely moving rats. In halothane-anaesthetized rats, intra-Nacc ondansetron (1 microm) application reduced dorsal raphe nucleus electrical stimulation (20Hz)-induced DA outflow. Our results show that regionally distinct populations of 5-HT(3) receptors control the depolarization-dependent exocytosis of DA and suggest that the involvement of Nacc 5-HT(3) receptors occurs only when central DA and 5-HT tones are concomitantly increased.     

Role of the delta-opioid receptor in (1DMe)NPYF mediated antinociception

A selective delta-opioid antagonist, naltrindole, was used to study the role of the delta-opioid receptor in the antinociceptive actions of a synthetic NPFF analog, (1DMe)NPYF. I.t. (1DMe)NPYF (5 nmol) produced antinociception in the tail flick test and (1DMe)NPYF (0.5 nmol) potentiated the antinociceptive effect of i.t. morphine 7.8 nmol. (1DMe)NPYF (5 nmol) had an antihyperalgesic effect in carrageenan inflammation and it significantly reduced mechanical allodynia in the spinal nerve ligation model. All these effects were prevented or significantly reduced by pretreatment with naltrindole (28 nmol) (P < 0.01-0.001). These data suggest that activation of spinal delta-opioid receptors plays an important role in mediating the spinal antinociceptive effects of (1DMe)NPYF.     

Cholera and pertussis toxins inhibit differently hypothermic and anti-opioid effects of neuropeptide FF

In mice pretreated intracerebroventricularly (i.c.v.) with pertussis or cholera toxins, effects of neuropeptide FF (NPFF), on hypothermia and morphine-induced analgesia, were assessed. NPFF and a potent NPFF agonist, 1DMe (0.005-22 nmol) injected into the lateral ventricle decreased morphine analgesia and produced naloxone (2.5 mg x kg(-1), s.c.)-resistant hypothermia after administration into the third ventricle. Cholera toxin (CTX 1 microg, i.c.v.) pretreatment (24 or 96 h before) inhibited the effect of 1DMe on body temperature, but failed to reverse its anti-opioid activity in the tail-flick test. CTX reduced hypothermia induced by a high dose of morphine (8 nmol, i.c.v.) but not the analgesic effect due to 3 nmol morphine. Pertussis toxin (PTX) pretreatment inhibited both morphine-hypothermia and -analgesia but did not modify hypothermia induced by 1DMe. The present results suggest that NPFF-induced hypothermia depends on the stimulation of Gs (but not Gi) proteins. In contrast, anti-opioid effects resulting from NPFF-receptor stimulation do not involve a cholera toxin-sensitive transducer protein.     

Involvement of spinal kappa opioid receptors in the antagonistic effect of dynorphins on morphine antinociception.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.     

Hemodynamic responses of conscious rats following intrathecal injections of prodynorphin-derived opioids: independence of action of intrathecal arginine vasopressin

Experiments were conducted (i) to determine the hemodynamic (blood pressure and heart rate) responses of conscious rats following intrathecal (IT) administration of endogenous prodynorphin-derived opioids into the lower thoracic space, (ii) to identify the receptors involved in mediating their cardiovascular responses, and (iii) to reveal any possible hemodynamic interactions with the neuropeptide arginine vasopressin. Male Sprague-Dawley rats were surgically prepared with femoral arterial and venous catheters as well as a spinal catheter (into lower thoracic region, T9-T12). After recovery, hemodynamic responses were observed in conscious rats for 5-10 min after IT injections of artificial cerebrospinal fluid (CSF) solution, prodynorphin-derived opioids (dynorphin A, dynorphin B, dynorphin A (1-13), dynorphin A (1-10), alpha- and beta-neoendorphin, leucine enkephalin (LE), methionine enkephalin (ME), arginine vasopressin (AVP), or norepinephrine (NE)). IT injections of AVP (10 or 20 pmol), dynorphin A (1-13), or dynorphin A (10-20 nmol) caused pressor effects associated with a prolonged and significant bradycardia. Equimolar (20 nmol) concentrations of LE, ME, alpha- and beta-neoendorphin, and dynorphin A (1-10) caused no significant blood pressure or heart rate changes. Combined IT injections of dynorphin A (1-13) and AVP caused apparent additive pressor effects when compared with the same dose of either peptide given alone. IT infusion of the specific AVP-V1 antagonist d(CH2)5Tyr(Me)AVP before subsequent IT AVP, dynorphin A (1-13), or NE administration inhibited only the subsequent pressor responses to AVP. The kappa-opioid antagonist (Mr2266) infused IT blocked the pressor actions of subsequent dynorphin A administration and not AVP or NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurokinin A, calcitonin gene-related peptide, and dynorphin A (1-8) in spinal dorsal horn contribute to descending inhibition evoked by nociceptive afferent pathways: an immunocytochemical study

Immunocytochemical technique was used to compare the contents of neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dynorphin A (1-8) (DynA) on two sides of the lumbar dorsal horn of rats in which the unilateral thoracic dorsalateral funiculus (DLF) was transected while formalin (0.2 ml, 0.5%) was injected equally into two hindpaws. The results showed that all the NKA-like, CGRP-like, and DynA (1-8)-like immunoreactivities were significantly lower in the superficial laminae of the dorsal horn on the side ipsilateral to the lesioned DLF than that on the side with intact DLF. This implies that peripheral noxious inputs activate the supraspinal descending inhibitory systems which in turn modulate the transmission of noxious message at the spinal level by changing the release of related neuropeptides.     

Spinal Cord Dynorphin Precursor Intermediates Decline During Late Gestation

Abstract: This laboratory has previously reported that the maternal opioid analgesia associated with pregnancy and parturition is mediated, at least in part, by a maternal spinal cord dynorphin/κ opioid system. This analgesia is accompanied by an increase in dynorphin peptides (1–17 and 1–8) in the lumbar spinal cord. Levels of trypsin-generated arginine⁶-leucine-enkephalin (Leu-Enk-Arg)-immunoreactive determinants were also determined and used to reflect the content of dynorphin precursor intermediates. In spinal tissue, the amount of dynorphin A (1–17) contained in the form of precursor is, at a minimum, 10-fold higher than the content of mature dynorphin A (1–17) or dynorphin (1–8). During gestational day 22, the content of dynorphin precursor is reduced significantly (∼50%). The decline in the magnitude of dynorphin precursor intermediates in the spinal cord of pregnant rats vastly exceeds the magnitude of increase in the content of dynorphin peptides (1–17 and 1–8). This difference can best be explained by postulating a corresponding increase in the rate of release of spinal cord dynorphin (1–17). It is suggested that enhanced processing of dynorphin precursor intermediates represents the initial biochemical level of adaptation of spinal dynorphin neurons to increased demands of pregnancy.     

Physical association between neuropeptide FF and micro-opioid receptors as a possible molecular basis for anti-opioid activity

Neuropeptide FF (NPFF) modulates the opioid system by exerting functional anti-opioid activity on neurons, the mechanism of which is unknown. By using a model of SH-SY5Y cells, we recently postulated that anti-opioid activity likely takes place upstream from the signaling cascade, suggesting that NPFF receptors could block opioid receptors by physical interaction. In the present study, fluorescence techniques were used to monitor the physical association and the dynamic of NPFF2 and micro-opioid (MOP) receptors tagged with variants of the green fluorescent protein. Importantly, cyan fluorescent protein-tagged NPFF2 receptors retained their capacity to antagonize opioid receptors. Fluorescence resonance energy transfer (FRET) and coimmunoprecipitation studies indicate that NPFF and MOP receptors are close enough to generate a basal FRET signal. The opioid agonist Tyr-D-Ala-Gly-NMe-Phe-Gly-ol disrupts by 20-30% this FRET signal, mainly because it concomitantly induces 40% internalization of receptors. In contrast, the NPFF analog 1DMe significantly increases by 10-15% the basal FRET signal, suggesting an association between both receptors. In addition, 1DMe reduces, by half, MOP receptor internalization, indicating that, besides a functional blockade of opioid receptors, the NPFF analog also inhibits their internalization. Finally, as a first report showing the modulation of the mobility of a G-protein-coupled receptor by another one, fluorescence recovery after photobleaching analysis reveals that 1DMe modifies the lateral diffusion of MOP receptors in the cell membrane, changing them from a confined to a freely diffusing state. By promoting NPFF-MOP receptor heteromerization, 1DMe could disrupt the domain organization of MOP receptors in the membrane, resulting in a reduction of opioid response.     

Methionine5-enkephalin and opiate binding sites in the neurohypophysis of the bird, Gallus domesticus.

Uncertainties with respect to the cellular localization, binding characteristics and function of Met-enkephalin in the neurohypophysis of mammalian species prompted us to examine the neurohypophysis of a non-mammalian species for opioid material and opioid binding sites. In extracts of the neurohypophysis of the domestic fowl we found immunoassayable Met-enkephalin, but could not detect dynorphin(1-8)-like material. Met-enkephalin immunoreactivity was co-localized with mesotocin in the same nerve endings. Stereospecific opiate binding was specifically located in neurosecretosomes (isolated neurosecretory terminals) of the mesotocin type, as shown by autoradiography. Enkephalins therefore may modulate mesotocin release in an autocrine manner. The neurohypophysis of this common bird appears to be a favorable model for studies of enkephalin function in the absence of dynorphin.     

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.     

Ultrastructural demonstration of synaptic connections between calcitonin gene-related peptide immunoreactive axons and dynorphin A(1–8) immunoreactive dorsal horn neurons in a rat model of peripheral inflammation and hyperalgesia

Synaptic contact between dynorphin A(1–8)-like immunoreactive lamina V spinal neurons and calcitonin gene-related peptide-like immunoreactive axon terminals was demonstrated using the immuno-electron microscopic mirror technique in a rat model of peripheral inflammation and hyperalgesia. Adjacent tissue sections were immunocytochemically labeled for either dynorphin A(1–8) or calcitonin gene-related peptide and examined at the electron microscopic level for the presence of synaptic contacts. The results suggest that some opioid neurons which exhibit a dynamic increase in dynorphin peptide associated with peripheral inflammation and hyperalgesia receive direct monosynaptic input from presumptive nociceptive primary afferents.     

Opposing interplay between Neuropeptide FF and nitric oxide in antinociception and hypothermia

This study examined the ability of the anti-opioid Neuropeptide FF (NPFF) to modify the endogenous activity of nitric oxide (NO). Antinociceptive and hypothermic effects of 1DMe (D.Tyr-Leu-(n.Me)Phe-Gln-Pro-Gln-Arg-Phe-NH(2)), an NPFF agonist, and of L-NAME (N(omega)nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, were investigated in mice. Intraperitoneal (i.p.) injection of L-NAME induced, in the hot plate test, a dose-dependent antinociception not reversed by naloxone, an opioid antagonist, but inhibited by L-Arg, the NO synthesis precursor. Intracerebroventricular (i.c.v.) injections of 1DMe inhibit the antinociceptive activity of L-NAME in a dose-dependent manner. On the contrary, L-NAME markedly potentiated hypothermia induced by 1DMe injected in the third ventricle. These data show that Neuropeptide FF receptors exert a dual effect on endogenous NO functions and could modulate pain transmission independently of opioids.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

Caffeine inhibition of rat carotid body chemoreceptors is mediated by A2A and A2B adenosine receptors

Caffeine, an unspecific antagonist of adenosine receptors, is commonly used to treat the apnea of prematurity. We have defined the effects of caffeine on the carotid body (CB) chemoreceptors, the main peripheral controllers of breathing, and identified the adenosine receptors involved. Caffeine inhibited basal (IC50, 210 microm) and low intensity (PO2 approximately 66 mm Hg/30 mm K+) stimulation-induced release of catecholamines from chemoreceptor cells in intact preparations of rat CB in vitro. Opposite to caffeine, 5'-(N-ethylcarboxamido)adenosine (NECA; an A2 agonist) augmented basal and low-intensity hypoxia-induced release. 2-p-(2-Carboxyethyl)phenethyl-amino-5'-N-ethylcaboxamido-adenosine hydrochloride (CGS21680), 2-hexynyl-NECA (HE-NECA) and SCH58621 (A2A receptors agents) neither affected catecholamine release nor altered the caffeine effects. The 8-cycle-1,3-dipropylxanthine (DPCPX; an A1/A2B antagonist) and 8-(4-{[(4-cyanophenyl)carbamoylmethyl]-oxy}phenyl)-1,3-di(n-propyl)xanthine (MRS1754; an A2B antagonist) mimicking of caffeine indicated that caffeine effects are mediated by A2B receptors. Immunocytochemical A2B receptors were located in tyrosine hydroxylase positive chemoreceptor cells. Caffeine reduced by 52% the chemosensory discharges elicited by hypoxia in the carotid sinus nerve. Inhibition had two components with pharmacological analysis indicating that A2A and A2B receptors mediate, respectively, the low (17 x 10(-9) m) and high (160 x 10(-6) m) IC50 effects. It is concluded that endogenous adenosine, via presynaptic A2B and postsynaptic A2A receptors, can exert excitatory effects on the overall output of the rat CB chemoreceptors.     

A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

Fluorescence polarization assays for high-throughput screening of neuropeptide FF receptors

We have developed the first fluorescence polarization assays of human neuropeptide FF2 receptors in 384-well microtiter plates. Assays are completed in a single well with no transfer, separation, or wash steps. The performance is suitable for high-throughput drug screening applications with regard to speed of analysis, magnitude of displaceable signal, precision, and sensitivity of various reagents. The rank order of potency of agonists and antagonists agrees well relative to the published radiometric filtration assays: DMe NPFF > NPFF > frog PP (Rana temporaria pancreatic polypeptide) > PQRFamide > BIBP 3226. The effect of highly colored compounds is very small on the polarization signal up to micromolar concentrations. The method serves as a simple and fast alternative to radioligand binding assays of antiobesity drug candidates related to NPFF receptors.     

Comparative spinal analgesic action of dynorphin1-8, dynorphin1-13, and a kappa-receptor agonist U50,488

The effect of intrathecal injections of dynorphin1-8 (DYN1-8), dynorphin1-13 (DYN1-13), and a putative kappa agonist, U50,488 was tested in the rat tail-flick test. DYN1-8 and DYN1-13 (5, 10, 20 micrograms) produced a dose-related biphasic antinociceptive response consisting of an initial and a delayed response. Injection of U50,488 (20, 40 60 micrograms) produced a monophasic response. The antinociceptive effect of DYN1-8 (5, 10, 20 micrograms) and DYN1-13 (20 micrograms), was present 24 h postintrathecal injection. Pretreatment with systemic naloxone (2 mg/kg s.c.) attenuated the delayed response, but not the initial response induced by DYN1-8 and DYN1-13. The initial response was attenuated by pretreatment with intrathecal naloxone at a dose of 0.5 and 2.0 micrograms. The antinociceptive effect of U50,488 (20, 60 micrograms) was not affected by pretreatment with 2.0 micrograms intrathecal naloxone, but was significantly reduced by 4 micrograms of the antagonist. Both DYN1-8 and DYN1-13 (5 micrograms) augmented the antinociceptive effect of intrathecally administered morphine (5, 10 micrograms). Intrathecal injection of DYN1-8 (5, 10, 20 micrograms), DYN1-13 (5 micrograms), and morphine (10 micrograms) reduced the spontaneous output of urine measured at 2 and 24 h postintrathecal injection. A similar injection of U50,488 (20 micrograms) had no significant action on the urinary output. The results show that long and short dynorphin fragments have a comparable activity and the spinal antinociceptive actions of dynorphin are sensitive to low doses of intrathecal naloxone. The activity profile of spinally administered dynorphins differs from that of the kappa agonist U50,488.     

Release of a dynorphin A (1-8) degrading enzyme from the spinal cord by intraventricular morphine in the rat

Intraventricular injection of morphine sulfate, 40 micrograms, released an enzyme from the spinal cord into the perfusate which degraded dynorphin A (1-8) and, to a lesser extent, dynorphin A (1-13) in urethane anesthetized rats. The enzyme did not degrade dynorphin A (1-17), Met-enkephalin, Leu-enkephalin, substance P and neurotensin. This dynorphin A (1-8) degrading enzyme was inhibited by aprotinin, thiorphan, and, to a lesser extent, by bacitracin but was not inhibited by bestatin. A kinetic study of the interaction between dynorphin A (1-8) and aprotinin with the enzyme indicated that it is competitive in nature. The pharmacological significance of the findings is still unknown.     

N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures

N-Methyl D-aspartate (NMDA) receptor activation of extracellular-signal regulated kinase (ERK) was examined in primary cortical cultures. Tetrodotoxin, NMDA receptor antagonists, or reduced extracellular calcium (0.1 mm) greatly decreased basal levels of phospho-ERK2, indicating that activity-dependent activation of NMDA receptors maintained a high level of basal ERK2 activation. This activity-dependent activation of phospho-ERK2 was blocked by pertussis toxin and inhibition of calcium/calmodulin-dependent kinase II and phosphatidylinositol 3-kinase but not by inhibition of protein kinase C or cAMP-dependent protein kinase. Addition of a calcium ionophore or 100 microm NMDA decreased phospho-ERK2 in the presence of 1 mm extracellular calcium but enhanced phospho-ERK2 in 0.1 mm extracellular calcium. The reduction in basal phospho-ERK2 by 100 microm NMDA was also reflected as a decrease in phospho-cAMP response element-binding protein. Inhibition of tyrosine phosphatases and serine/threonine phosphatases protein phosphatase 1 (PP1), PP2A, and PP2B did not prevent the inhibitory effect of NMDA. In the presence of tetrodotoxin, NMDA produced a bell-shaped dose-response curve with stimulation of phospho-ERK2 at 10, 25, and 50 microm NMDA and reduced stimulation at 100 microm NMDA. NMDA (50 microm) stimulation of phospho-ERK2 was completely blocked by pertussis toxin and inhibitors of phosphatidylinositol 3-kinase and was partially blocked by a calcium/calmodulin-dependent kinase II inhibitor. These results suggests that NMDA receptors can bidirectionally control ERK signaling.