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NEUROPHARMACOLOGY

Somatostatin-Induced Activation and Up-Regulation of N-Methyl-D-aspartate Receptor Function: Mediation through Calmodulin-Dependent Protein Kinase II, Phospholipase C, Protein Kinase C, and Tyrosine Kinase in Hippocampal Noradrenergic Nerve Endings

Pharmacology and Toxicology Section, Department of Experimental Medicine (A.P., M.F., F.L., M.R.), Center of Excellence for Biomedical Research (A.P., M.R.), and Department of Endocrinological and Metabolic Sciences (M.A.), University of Genoa, Genoa, Italy

Received for publication October 21, 2004
Accepted December 15, 2004.

	Abstract

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Somatostatin receptors and glutamate N-methyl-D-aspartate (NMDA) receptors coexist on hippocampal noradrenergic axon terminals. Activation of somatostatin receptors was previously found to positively influence the function of NMDA receptors regulating norepinephrine release. The somatostatin receptors involved were pharmacologically characterized as sst5 type in experiments in Mg²⁺-free solutions. Here, we first confirm the pharmacology of these receptors using selective sst5 ligands in Mg²⁺-containing solutions. Moreover, we show by Western blot that the sst5 protein exists on purified hippocampal synaptosomal membranes. We then investigated the pathways connecting the two receptors using as a functional response the release of norepinephrine from rat hippocampal synaptosomes in superfusion. The release of norepinephrine evoked by somatostatin-14 plus NMDA/glycine was partly prevented by the protein kinase C inhibitor GF109203X [dihydrochloride3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione] and by the nonreceptor tyrosine kinase (Src) inhibitors PP2 [3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine] and lavendustin A; it was largely and almost totally abolished by the phospholipase C inhibitor U73122 [GenBank] [1-(6-[([17]-3-methoxyextra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione] and by the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 [N-(2-[N-[4-chlorocinnamyl]-N-methyl-amino-methyl]phenyl)-N-(2-hydroxyethyl)-4-methoxy-benzene-sulfonamide-phosphate salt], respectively; and it was unaffected by the protein kinase A inhibitor H89 [N-(2-[p-bromocinnamylamino]ethyl)5-isoquinolinesulfonamide hydrochloride]. The norepinephrine release evoked by somatostatin-14/NMDA/glycine was inhibited when anti-phosphotyrosine antibodies had been entrapped into synaptosomes. Entrapping the recombinant activated tyrosine kinase pp60^c-Src strongly potentiated the release of norepinephrine elicited by NMDA/glycine in Mg²⁺-free medium but failed to permit NMDA receptor activation in presence of external Mg²⁺ ions. The results suggest the involvement of CaMKII in the sst5 receptor-mediated activation of NMDA receptors in presence of Mg²⁺ and of the PLC/PKC/Src pathway in the up-regulation of the ongoing NMDA receptor activity.

Interactions between NMDA receptors and G protein-coupled receptors (GPCRs) coexpressed on membranes have been reported by several laboratories (Lu et al., 1999; Pittaluga et al., 2000; Lan et al., 2001; Heidinger et al., 2002; Kotecha et al., 2003). In particular, activation of GPCRs can affect NMDA receptor function, representing a major mechanism of glutamate transmission modulation. The cross talks between coexisting GPCRs and NMDA receptors involve intracellular kinase pathways that may differ among the different receptor-receptor interactions.

Depending on the subunits targeted and the kinases involved, phosphorylation can affect either negatively or positively the NMDA receptor function (see Kotecha and MacDonald, 2002). NR1 and NR2 subunits can undergo phosphorylation by several kinases including protein kinases A (PKA; Leonard and Hell, 1997), protein kinases C (PKC; Zheng et al., 1999; Liao et al., 2001), cytosolic tyrosine kinases of the Src family (Yu et al., 1997; Lu et al., 1999; Salter and Kalia, 2004), and Ca²⁺/calmodulin-dependent kinase II (CaMKII; Leonard et al., 1999; Soderling et al., 2001). Although phosphorylation of NR1 subunits generally limits NMDA receptor function (Zukin and Bennett, 1995), phosphorylation of NR2 subunits enhances NMDA receptor-mediated effects (see MacDonald et al., 2001).

NMDA receptors exist on central nervous system noradrenergic nerve endings, where they mediate exocytotic release of norepinephrine (NE; Jones et al., 1987; Fink et al., 1992; Raiteri et al., 1992). In rat hippocampus, these receptors, which contain NR2B subunits (Pittaluga et al., 2001), colocalize with somatostatin (SRIF) receptors, which are positively coupled to phosphoinositide breakdown and, based on a pharmacological study with selective ligands, belong to the sst5 subtype (Pittaluga et al., 2000). Activation of SRIF receptors fails to affect NE release but seems to permit activation and up-regulation of NMDA receptors in presence of physiological concentrations of extracellular Mg²⁺ and without depolarization (Pittaluga et al., 2000).

The mechanisms underlying the positive effects of SRIF on NMDA receptor function are not known. We previously suggested the involvement of inositoltrisphosphate (IP₃) and PKC (Pittaluga et al., 2000). The aims of the present work were to investigate the phosphorylative pathway(s) involved in the SRIF-NMDA receptor-receptor interaction using synaptosomes in superfusion, a technique particularly suitable for identifying receptors that coexist on the same nerve terminal and understanding their cross talks (see Raiteri and Raiteri, 2000); and to distinguish between processes leading to activation of release-enhancing NMDA receptors and up-regulation of receptors already in the open channel state. The results suggest the involvement of CaMKII in the sst5 receptor-mediated activation and of PKC and Src in the sst5 receptor-mediated up-regulation of NMDA receptor function.

	Materials and Methods

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Animals and Brain Tissue Preparation. Adult male rats (Sprague-Dawley, 200–250 g) were housed at constant temperature (22 ± 1°C) and relative humidity (50%) under a regular light-dark schedule (light 7:00 AM to 7:00 PM). Food and water were freely available. The experimental procedures were approved by the Ethical Committee of the Pharmacology and Toxicology Section, Department of Experimental Medicine, in accordance with the European legislation (European Communities Council Directive of November 24 1986, 86/609/EEC).

The animals were killed by decapitation, and the hippocampi were rapidly removed at 0 to 4°C. Crude synaptosomes were prepared according to Raiteri et al. (1992). Briefly, the ventral-medial part of the hippocampus was homogenized in 40 volumes of 0.32 M sucrose, buffered at pH 7.4 with phosphate (final concentration, 0.01 M). The homogenate was centrifuged at 1000g for 5 min to remove nuclei and cellular debris, and crude synaptosomes were isolated from the supernatant by centrifugation at 12,000g for 20 min. In some experiments, the tissue was homogenized in buffered sucrose containing 225 units/ml Src (pp60^c-Src) or 20 µg of antibody anti-phosphotyrosine to entrap these agents into subsequently isolated synaptosomes (Raiteri et al., 2000).

Release Experiments. The synaptosomal pellets were resuspended in physiological medium having the following composition: 125 mM NaCl, 3 mM KCl, 1.2 mM MgSO₄, 1.2 mM CaCl₂, 1 mM NaH₂PO₄, 22 mM NaHCO₃, and 10 mM glucose (aeration with 95% O₂ and 5% CO₂), pH 7.2 to 7.4. Synaptosomes were incubated for 15 min at 37°C with [³H]NE (final concentration, 30 nM) in the presence of 0.1 µM 6-nitroquipazine to avoid false labeling of serotonergic terminals.

Identical portions of the synaptosomal suspension were layered on microporous filters at the bottom of parallel superfusion chambers thermostated at 37°C (Raiteri and Raiteri, 2000). Synaptosomes were superfused at 0.5 ml/min with standard physiological solution aerated with 95% O₂ and 5% CO₂ at 37°C. Synaptosomes were first equilibrated during 36 min of superfusion; subsequently, nine consecutive 1-min fractions (t = 36–45 min) were collected. Synaptosomes were exposed to agonists at the end of the third fraction collected (t = 39 min) until the end of the superfusion, whereas antagonists were added 8 min before agonists. When indicated, the superfusion medium was replaced, at t = 20 min, with a medium from which Mg²⁺ ions were omitted. Fractions collected and superfused synaptosomes were counted for radioactivity.

Western Blot of sst5 Protein. To obtain a cellular preparation particularly enriched in isolated nerve endings, synaptosomes were prepared by homogenizing hippocampi in 40 volumes of 0.32 M sucrose buffered at pH 7.4 with Tris (final concentration, 0.01 M) and then purified by Percoll gradient. Briefly, the homogenate was centrifuged at 1000g for 5 min, to remove nuclei and debris, and synaptosomal fraction was purified by Percoll-sucrose density (2–20% v/v) gradient centrifugation for 5 min at about 33,500g. The 10 to 20% Percoll interface was removed, washed to eliminate Percoll, and synaptosomes were isolated from the supernatant by centrifugation at 12,000g for 20 min. Synaptosomes were then lysed in 1 ml of ice-cold water and pellets isolated by centrifugation at 7000g. The pellets were solubilized in a lysis buffer containing 20 mM HEPES (pH 7.4), 5 mM EDTA, 3 mM EGTA, 150 mM NaCl, and 4 mg/ml dodecyl-B-D-maltoside for 1h at 4°C and then ultracentrifuged at 100,000g for 1 h at 4°C. The supernatant containing solubilized synaptic membrane proteins was subjected to standardized colorimetric analysis to evaluate the protein content. Membrane protein (200 µg) was denatured and fractionated under reducing conditions on 10% SDS-PAGE, then transferred electrophoretically to Hybond C-Extra nitrocellulose membranes (Amersham Biosciences Inc., Piscataway, NJ). After transfer, nonspecific binding sites were blocked by Tris-buffered saline-Tween (t-TBS; 0.02 M Tris, 0.137 M NaCl, and 0.1% Tween 20) containing 5% nonfat dried milk. After three washes with t-TBS, membranes were incubated for 16 h at 4°C with a 1:150 dilution of goat anti-rat SST5r polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in t-TBS containing 3% bovine serum albumin. Membranes were washed three times with t-TBS, then incubated for 2 h at 22°C with a 1:5000 dilution of the antigoat horseradish peroxidase-linked IgG. After three washes with t-TBS, immunoreactive bands were detected by the chemiluminescence detection system enhanced chemiluminescence Western blot analysis system (Amersham Biosciences Inc.). The immunoreactive bands were visualized by autoradiography after 0.5 min of exposure to Hyperfilm MP (Amersham Biosciences Inc.).

Calculations and Statistics. The radioactivity released into each superfusate sample was expressed as a percentage of the total synaptosomal tritium content at the start of the fraction collected (fractional efflux). When the time course of the effect is reported, drug effects are expressed as percentage of effect and evaluated as the ratio between the percentage of tritium released into each fraction after the first and that in the first fraction collected. This ratio was compared with the corresponding ratio obtained under control conditions (no drug added).

Analysis of variance was performed by analysis of variance followed by Dunnett's test or Newman-Keuls multiple comparisons test or Student's t test as appropriate. Data were considered significant for p < 0.05 at least. Appropriate controls with antagonists and inhibitors were always run in parallel.

Chemicals. 1-[7,8-³H]Norepinephrine (specific activity 39 Ci/mmol) RPN-800 prestained molecular mass marker, chemiluminescence detection system enhanced chemiluminescence Western blot analysis system, and Hyperfilm MP were from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Hybond C-Extra nitrocellulose membranes were from Amersham Biosciences Inc. NMDA and PP2 were from Tocris Cookson Inc. (Bristol, UK). SRIF-14 was from Peninsula Laboratories (Belmont, CA). Glycine, GF109203X, U73122 [GenBank] , lavendustin A [5-amino-[(N-2,5-dihydroxybenzyl)-N'-2-hydroxybenzyl]salicylic acid], H89, and KN93 were purchased from Sigma/RBI (Natick, MA). Src (pp60^c-Src) and mouse anti-phosphotyrosine (clone 4G10) were from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated anti goat secondary antibody was purchased from Chemicon International (Temecula, CA). Goat anti-rat sst5R polyclonal antibody was from Santa Cruz Biotechnology, Inc. MK801 (dizocilpine) was from Merck Sharp and Dohme (Hoddesdon, UK), whereas 6-nitroquipazine maleate was from Duphar (Amsterdam, The Netherlands). BIM-23056 and L362,855 were gifts from Drs. P. P. A. Humphrey (Glaxo Institute of Applied Pharmacology, Cambridge, UK) and D. Hoyer (Neuroscience Research NOVARTIS Institute for Biomedical Research, Basel, Switzerland).

	Results

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The Function of NMDA Receptors in the Presence of Mg²⁺ Ions Is Facilitated by the Action of Somatostatin sst5 Receptors Present in the Hippocampus. As previously shown (Pittaluga et al., 2000), exposure of hippocampal synaptosomes to SRIF-14 plus NMDA/glycine evokes release of preloaded [³H]NE in the presence of a physiological concentration of Mg²⁺ ions (1.2 mM). When added alone, neither SRIF-14 (1 nM) nor NMDA (100 µM)/glycine (1 µM) can affect the release of [³H]NE. The release induced by SRIF-14 plus NMDA/glycine depends on NMDA receptor activation since MK801, a selective NMDA channel blocker inactive on its own, almost totally prevented the releasing effect (1 µM MK-801 = 2.65 ± 3.01%, 1 nM SRIF-14 + 100 µM NMDA + 1 µM glycine = 73.08 ± 6.56%, + 1 µM MK-801 = 15.60 ± 7.89%, p < 0.05 at least; results expressed as percentage of increase over basal release).

In the work by Pittaluga et al. (2000), the positive effect of SRIF-14 on the NMDA-evoked release of NE was related to activation of SRIF receptors exhibiting sst5 pharmacology. Since that pharmacological characterization was carried out in Mg²⁺-free solutions, it was important to ascertain if sst5 receptor ligands behaved similarly in solutions containing a physiological concentration of Mg²⁺ ions. As shown in Fig. 1, BIM-23056, a selective sst5 receptor antagonist, totally prevented the release of [³H]NE induced by SRIF-14/NMDA/glycine, suggesting that only sst5 receptors participate in the somatostatin-NMDA receptor-receptor interaction. The figure also shows that the sst5 partial agonist L362,855 mimicked in part SRIF-14.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Top, effect of SRIF-14 on the NMDA/glycine-evoked release of [3H]NE, in the presence of 1.2 mM Mg2+, is mimicked by the partial sst5 agonist L362,855 and prevented by the selective sst5 antagonist BIM-23056. Results are expressed as percentages of increase over basal release. Data are means ± S.E.M. of three experiments run in triplicate. *, p < 0.05 versus control. Bottom, representative Western blot of sst5 receptor proteins located in purified rat hippocampal synaptosomal membranes. Each lane corresponds to a synaptosomal membrane suspension prepared from a different animal. The presence of sst5 protein was detected by Western blot technique using selective antibodies raised against sst5 receptor protein.

The pharmacological characterization of the receptors involved as sst5 type may seem surprising. Based on morphological results, the presence of receptors of the sst5 type in some central nervous system regions is in fact controversial (Fehlmann et al., 2000; Rocheville et al., 2000a; Schulz et al., 2000; Kang et al., 2003). We therefore analyzed proteins from purified synaptosomal membranes for the presence of the sst5 receptor protein. As shown in Fig. 1, Western blot analysis with anti-sst5 antibodies recognized a component with an apparent mass of 60 to 70 kDa, suggesting that sst5 protein is expressed in nerve ending membranes. The prominent band with the apparent mass of 60 to 70 kDa should correspond to the monomeric form of the sst5 receptor (Rocheville et al., 2000b).

Somatostatin-NMDA Receptor-Receptor Interaction: Involvement of PLC and PKC but Not PKA. Table 1 shows that U73122 [GenBank] (0.1 µM), a selective inhibitor of PLC function, largely prevented the release evoked by SRIF-14/NMDA/glycine indicating the involvement of PLC present in noradrenergic nerve terminals. It is known that activated PLC promotes phosphoinositide breakdown followed by IP₃ and diacylglycerol production and possible consequent activation of intraterminal PKCs. Accordingly, GF109203X, a selective PKC blocker, inhibited in part the release of [³H]NE provoked by SRIF-14/NMDA/glycine in Mg²⁺-containing medium (Table 1).

Little is known about the relations between PKA activity and NMDA receptor function. PKA-targeted sites were proposed to reside within the NR1 subunit, although PKA was shown to influence also the functional activity of NMDA receptors by modifying NR2 subunits (Leonard and Hell, 1997). These events, however, have been mainly related to inhibitory effects on NMDA receptor function. Under our experimental conditions, the PKA selective inhibitor H89, added at 0.5 µM, failed to affect the release of [³H]NE induced by SRIF-14/NMDA/glycine (Table 1), suggesting that PKA-mediated phosphorylation of the NMDA receptor is unlikely to modulate NE release. Under very similar experimental conditions, H89 had been found to inhibit the up-regulation of NMDA receptors provoked by nicotine receptor activation in noradrenergic axon terminals of the hippocampus (Risso et al., 2004). At the concentrations applied, the enzyme blockers used did not modify, on their own, the spontaneous release of tritium (see legend to Table 1).

Involvement of Src. It is known that PKC can phosphorylate NMDA NR2 subunits directly, on serine and threonine, as well as indirectly, on tyrosine, by activating cytosolic tyrosine kinases of the Src family (Yu et al., 1997; Lu et al., 1999; MacDonald et al., 2001; Salter and Kalia, 2004). The possibility that the SRIF-NMDA receptor-receptor interaction involves the Src signaling was evaluated by studying the effects of the selective Src inhibitors PP2 and lavendustin A. As shown in Fig. 2, addition to the superfusion medium of PP2 (1 µM) or lavendustin A (5 µM) inhibited in part the [³H]NE release induced by SRIF-14/NMDA/glycine from hippocampal synaptosomes. The spontaneous release of tritium was not affected by the kinase inhibitors used (see legend to Fig. 2A).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Effects of kinase inhibitors on the released [3H]NE induced by SRIF-14 + NMDA + glycine from rat hippocampal synaptosomes. A, effects of Src inhibitors. Open square, control (no drug added); gray square, SRIF-14 (1 nM) + NMDA (100 µM) + glycine (1 µM); black diamond, +PP2 (1 µM); gray diamond, +lavendustin A (5 µM). PP2 and lavendustin A failed on their own to affect the basal release of [3H]NE (1 µM PP2 = 6.34 ± 5.87, 5 µM lavendustin A = –2.51 ± 3.67). B, effect of the CaMKII inhibitor KN93. Open square, control (no drug added); gray square, SRIF-14 (1 nM) + NMDA (100 µM) + glycine (1 µM); black square, +KN93 (1 µM). KN93 failed on its own to affect the basal release of [3H]NE (1 µM KN93 = 4.87 ± 5.32). Results are expressed as percentages of increase over basal release. Data are means ± S.E.M. of at least four experiments run in triplicate. *, p < 0.05 versus control; #, p < 0.05 versus SRIF-14 + NMDA + glycine.

Since the effects of PKC and Src inhibitors (used at the maximally effective concentration; data not shown) were only partial, we tested a combination of the two inhibitors. As reported in Table 2, addition of GF109203X together with PP2 did not produce additive inhibitory effect on the SRIF-14/NMDA/glycine-evoked release of [³H]NE.

Involvement of CaMKII. NMDA receptor subunits have been identified as CaMKII anchoring proteins in postsynaptic densities. Upon kinase autophosphorylation, CaMKII can bind the cytosolic C-terminal region of NR2B subunits and affect the function of the NMDA-associated ionic channel (Leonard et al., 1999; Soderling et al., 2001). Therefore, we investigated the possibility that the SRIF receptor-NMDA receptor interaction that occurs presynaptically involves CaMKII-mediated phosphorylative processes by analyzing the effect of the selective CaMKII inhibitor KN93 on the release of [³H]NE induced by SRIF-14/NMDA/glycine. As shown in Fig. 2B, KN93, added at 1 µM, almost totally inhibited the evoked [³H]NE release. The spontaneous release of tritium was not affected by 1 µM KN93 (see legend to Fig. 2).

Synaptosomal Entrapping of Anti-Phosphotyrosine Antibodies Prevents the SRIF-14/NMDA/Glycine-Evoked Release of [³H]NE. The finding that kinases of the Src family participate in the interaction between SRIF and NMDA receptors implies that the cross talk between the two receptors includes phosphorylation of tyrosine residues. To evaluate the involvement of a tyrosine phosphorylative pathway, antibodies raised against phosphotyrosines were entrapped into synaptosomes. We had previously shown that synaptosomes isolated after homogenization of brain tissue in the presence of anti-syntaxin or anti-SNAP25 antibodies exhibited decreased transmitter release when exposed to depolarizing stimuli, indicating that the above antibodies had been entrapped into nerve endings (Raiteri et al., 2000). As shown in Fig. 3, entrapping anti-phosphotyrosine antibodies into hippocampal synaptosomes decreased by about 50% the release of [³H]NE elicited by SRIF-14/NMDA/glycine. Entrapping anti-phosphotyrosine antibodies did not modify the spontaneous release of [³H]NE (control synaptosomes = 0.71 ± 0.02%, entrapped synaptosomes = 0.66 ± 0.05%, results expressed as percentage of tritium released into the first fraction collected).

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effect of entrapped anti-phosphotyrosine antibodies on the [3H]NE release induced by SRIF-14 + NMDA + glycine from rat hippocampal synaptosomes. Synaptosomes with or without entrapped antiphosphotyrosine antibodies were exposed to SRIF-14 (1 nM) + NMDA (100 µM) + glycine (1 µM). Empty bar, control synaptosomes; black bar, antibody-staffed synaptosomes. Results are expressed as percentage of increase over basal release. Data are means ± S.E.M. of three experiments run in triplicate. *, p < 0.05 versus control.

Src Mediates Up-Regulation, but Not Activation, of NMDA Receptors. The finding that Src inhibitors prevented only in part (50%) the NE release elicited by SRIF-14/NMDA/glycine (Fig. 2A) may suggest that Src does not mediate the permissive role played by SRIF on the activation of NMDA receptors in the presence of Mg²⁺ ions. To shed light on this aspect, we entrapped into synaptosomes the recombinant tyrosine kinase pp60^c-Src, an activated form of Src (Yu et al., 1997; Lu et al., 1999), and studied the effects of NMDA/glycine on the release of [³H]NE in the presence or absence of external Mg²⁺ ions.

Table 3 shows that entrapped pp60^c-Src failed to permit NMDA receptor activation since no releasing effect could be observed when synaptosomes staffed with pp60^c-Src were exposed to NMDA/glycine in presence of a physiological concentration (1.2 mM) of Mg²⁺ ions. On the contrary, pp60^c-Src strongly potentiated the ongoing NMDA-induced release, as shown by the results obtained when pp60^c-Src-staffed synaptosomes were exposed to NMDA/glycine in the absence of external Mg²⁺ ions. Entrapping pp60^c-Src did not modify the spontaneous release of [³H]NE (control synaptosomes = 0.56 ± 0.05%, entrapped synaptosomes = 0.64 ± 0.07%, results expressed as percentage of tritium released into the first fraction collected).

	Discussion

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The main findings of the present study are that activation of sst5 receptors present on hippocampal noradrenergic terminals positively affects the function of coexisting NMDA receptors through pathways involving CaMKII, PLC, PKC, and Src; somatostatin seems to trigger NMDA receptor activation through CaMKII; and the PLC/PKC/Src pathway up-regulates NMDA receptor function once the receptor is gated to an open state.

Noradrenergic axon terminals in the hippocampus possess NMDA receptors mediating exocytotic-like NE release (Jones et al., 1987; Fink et al., 1992; Raiteri et al., 1992). In the absence of depolarizing stimuli, activation of these receptors by NMDA/glycine only occurs when external medium is Mg²⁺ free. However, if SRIF-14, inactive on its own on NE release, is added together with NMDA/glycine, release of NE occurs also in medium containing physiological concentrations of Mg²⁺. Due to the characteristics of the technique used to monitor release (a monolayer of synaptosomes up-down superfused in conditions minimizing indirect effects; see Raiteri and Raiteri, 2000), sst5 receptors and NMDA receptors are likely to coexist and interact in noradrenergic axon terminals (Pittaluga et al., 2000).

The release of NE elicited by SRIF-14/NMDA/glycine, in the presence of external Mg²⁺, was completely prevented by the sst5 receptor antagonist BIM-23056 and by the NMDA receptor antagonist MK-801. Furthermore, it was largely reduced when PLC was inhibited by U73122 [GenBank] . It was reported that SRIF receptors of the sst5 type can couple to PLC and to enhancement of phosphoinositide metabolism (Wilkinson et al., 1997), with consequent production of IP₃ and diacylglycerol. Thus, it seems that PLC activation by SRIF acting at sst5 receptors plays an important role in the function of NMDA receptors in the presence of extraterminal Mg²⁺. The mechanism is probably indirect and mediated by downstream signaling molecules of PLC, in particular PKC. Accordingly, the release of NE provoked by SRIF/NMDA/glycine was in part reduced by selective PKC inhibitors (Table 1; see also Pittaluga et al., 2000). PKC can phosphorylate NR1 or NR2 subunits leading to inhibition or potentiation of NMDA receptor responses, respectively (MacDonald et al., 2001; Kotecha and MacDonald, 2002). The finding that PKC blockade inhibited the evoked NE release is compatible with the view that PKC participates in the phosphorylation of NR2 subunits of the NMDA receptor. A number of PKA phosphorylation sites exist on NMDA receptors (Leonard and Hell, 1997). Here, we found that inhibition of PKA by H89 had no effect on the release of NE evoked by SRIF-14/NMDA/glycine, excluding the involvement of the enzyme.

Our results suggest that Src-mediated tyrosine phosphorylation plays a role. This is supported by the inhibition of the evoked NE release observed in the presence of two Src inhibitors as well as by the finding that anti-phosphotyrosine antibodies prevented the effect of SRIF-14/NMDA/glycine.

The release-enhancing NMDA receptor present on hippocampal noradrenergic terminals is an NR2B-containing receptor (Pittaluga et al., 2001). NMDA receptor subunit NR2B is the major tyrosine phosphorylated protein in the postsynaptic density (Moon et al., 1994). Src is associated with NMDA receptors, and phosphorylation by Src up-regulates NMDA receptor currents (Yu et al., 1997; Lu et al., 1999). Several studies have addressed the signaling mechanisms controlling Src family kinase activation during glutamatergic transmission. One likely signal is the cell adhesion kinase /proline-rich tyrosine kinase 2 (CAK/Pyk2; Huang et al., 2001), which can be stimulated by PKC; in turn, CAK binds and activates Src family kinases (Dikic et al., 1996). Thus, the PLC/PKC/CAK/Src pathway may well couple sst5 receptor activation and NMDA receptor function in hippocampal noradrenergic neurons. This cascade has been proposed to mediate up-regulation of NMDA currents by various GPCRs (Lu et al., 1999; Heidinger et al., 2002; Kotecha et al., 2003; Salter and Kalia, 2004).

The sst5-NMDA receptor-receptor interaction seems to strictly depend on CaMKII activity since KN93 completely abolished the evoked NE release. Increased cytosolic Ca²⁺ availability from various sources, including stimulation of IP₃ receptors consequent to PLC activation, could trigger rapid CaMKII autophosphorylation and translocation to NR2B subunits of the NMDA receptor (Leonard et al., 1999; see Soderling et al., 2001). The interaction between NR2B and CaMKII was reported to lock the enzyme in an active conformation (Bayer et al., 2001); according to the authors, binding of CaMKII to NR2B and enzyme autophosphorylation can function synergistically, thus constituting a feed-forward pathway able to positively affect NMDA-mediated transmission.

Antagonists at sst5 and NMDA receptors completely abolished the SRIF-14/NMDA/glycine-evoked NE release. Inhibition of CaMKII also totally blocked the evoked release of the catecholamine. In contrast, PKC and Src inhibitors prevented only in part the SRIF-14/NMDA/glycine effect. The finding that PKC and Src inhibitors, when combined, did not elicit additive effect is consistent with PKC and Src working in series.

At this point, it seems important to recall that NMDA and glycine, added in the presence of Mg²⁺ ions and without depolarization, are unable to elicit NE release, whereas release occurs when SRIF-14 is added with the NMDA receptor coagonists. Therefore, one could distinguish two aspects of the SRIF action: a permissive role on the activation of the NMDA receptors, followed by up-regulation of the receptor ongoing activity.

To shed light on the question, we entrapped into synaptosomes pp60^c-Src, a recombinant Src in the activated form (Yu et al., 1997; Lu et al., 1999), having assumed that Src comes last in the PLC/PKC/CAK/Src sequence. Should Src permit NMDA receptor activation, NE release would be observed when pp60^c-Src-staffed synaptosomes are exposed to NMDA/glycine in the presence of Mg²⁺. However, if Src only potentiates the ongoing NMDA activity, NMDA/glycine would release NE from pp60^c-Src-staffed synaptosomes only in Mg²⁺-free medium. Entrapped pp60^c-Src failed to permit NMDA receptor activation in Mg²⁺-containing medium but potentiated the ongoing activity of NMDA, suggesting that the PKC/CAK/Src cascade mediates up-regulation of the function of NMDA receptors already activated. This idea does not exclude the participation of other agents, considering the impressive number of proteins constituting the NMDA receptor complex (Husi et al., 2000).

How sst5 receptors mediate activation of NMDA receptors on noradrenergic neurons remains to be established. Multiple mechanisms can be envisaged. Based on our results, a likely possibility is that phosphorylation of NR2B subunits by CaMKII leads to removal of Mg²⁺ from the NMDA receptor channel and to activation of the receptor in absence of depolarization. Another possibility stems from the increasing evidence that GPCRs can interact with NMDA receptors by physically associating with their subunits (Liu et al., 2000; Fiorentini et al., 2003; Salter, 2003). SRIF receptors of the sst2 type were found to physically associate with a scaffolding protein which can also bind to NR subunits (Peineau et al., 2003). One may speculate that also sst5 can perform a similar interaction, resulting in NMDA receptor activation.

Kotecha et al. (2003) have found that, in cultured hippocampal neurons, coactivation of mGluR5 and NMDA receptors up-regulate NMDA receptor currents through the PKC/CAK/Src cascade. Interestingly, NMDA channels must be gated to an open state during stimulation of mGluR5 for the up-regulation to occur; in fact, no potentiation could be observed in presence of Mg²⁺. Differently, coapplication of SRIF-14 and NMDA/glycine elicited NE release in presence of external Mg²⁺, compatible with the idea that somatostatin initially permits NMDA receptor activation in the presence of physiological concentrations of Mg²⁺ and without depolarization and subsequently mediates up-regulation of receptor function through the PKC/CAK/Src pathway. Together with previous reports (see Kotecha and MacDonald, 2002 and refs. therein), the present results confirm the multiplicity of the pathways that can be implicated in GPCR-NMDA receptor interactions, indicating the importance of their careful characterization.

To our knowledge, a clear distinction between activation and up-regulation of NMDA receptor function by GPCRs has not been previously considered. The reason may be that, in the experimental systems generally used, one can measure a basal channel activity that may be decreased or, more frequently, augmented by GPCR activation. In our system, the endogenous agonists released are immediately removed by the medium up-down superfusing the synaptosomal thin layer, so that NMDA and SRIF receptors remain virtually ligand-free and functionally silent. Addition of NMDA/glycine cannot elicit any NE release if Mg²⁺ is present, but release occurs if SRIF and NMDA/glycine are added concomitantly. Thus, our technique permits to identify conditions leading to NMDA receptor activation or to potentiation of the receptors already activated in absence of Mg²⁺.

Considering the involvement of NMDA receptors, somatostatin, and norepinephrine in memory and learning (Olias et al., 2004 and refs. therein), the effects of SRIF on the NMDA-evoked release of NE in the hippocampus deserves further investigation, also in view of the development of selective somatostatin receptor agonists (for instance, see Rohrer et al., 1998) to be employed in conditions of cognitive impairments.

	Acknowledgements

 
We thank Maura Agate for excellent assistance in preparing the manuscript.

	Footnotes

 
This work was supported by grants from Istituto Superiore di Sanitá (Programma Nazionale di Ricerca sull'AIDS: Progetto "Patologia, clinica e terapia dell'AIDS") and from Italian Ministero dell'Istruzione, dell'Università e della Ricerca.

doi:10.1124/jpet.104.079590.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; GPCR, G protein-coupled receptor; PKA, protein kinase A; PKC, protein kinase C; Src, tyrosine kinase; CaMKII, Ca²⁺/calmodulin-dependent protein kinase II; NE, norepinephrine; SRIF, somatostatin; IP₃, inositoltrisphosphate; t-TBS, Tris-buffered saline-Tween; PP2, 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-D]pyrimidin-4-amine; GF109203X, dihydrochloride3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione; U73122 [GenBank] , 1-(6-[([17]-3-methoxyextra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione; H89, N-(2-[p-bromocinnamylamino]ethyl)5-isoquinolinesulfonamide hydrochloride; KN93, N-(2-[N-[4-chlorocinnamyl]-N-methyl-amino-methyl]-phenyl)-N-(2-hydroxyethyl)-4-methoxy-benzene-sulfonamide-phosphate salt; MK801, dizocilpine; BIM-23056, D-Phe-Phe-Tyr-D-TRP-Lys-Val-Phe-D-Nal-NH₂; L362,855, c[Aha-Phe-Trp-D-Trp-Lys-Thr-Phe]; PLC, phospholipase C; CAK/Pyk2, cell-adhesion kinase /proline-rich tyrosine kinase 2.

Address correspondence to: Dr. Anna Pittaluga, Dipartimento di Medicina Sperimentale, Sezione Farmacologia e Tossicologia, Viale Cembrano 4, 16148 Genova, Italy. E-mail: pittalug{at}pharmatox.unige.it

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