Beverley Nicol

Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K+‐stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 ± 14% and was naloxone (10 μm)‐insensitive excluding an activation of μ, δ and κ opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.

Comparison of the effects of [Phe1Ψ(CH2‐NH)Gly2]Nociceptin (1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors

Nociceptin(NC) is the endogenous ligand for the opioid receptor like‐1 receptor (NC‐receptor). [Phe1ΨC(CH2‐NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea‐pig and mouse tissues. In this study, we investigated the effects of a range of NC C‐terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]‐Tyr14‐NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms.

Effects of intravenous anesthetic agents on glutamate release: a role for GABAA receptor-mediated inhibition.

Background: Many anesthetic agents are known to enhance the &agr;1&bgr;2&ggr;2S&ggr;-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation. Methods: Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37°C, samples for glutamate assay were obtained at 2-min intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1–100 &mgr;M) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1–300 &mgr;M), propofol (10 &mgr;M), or ketamine (30 &mgr;M) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1). Results: Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 ± 0.33 (mean ± SD, n = 96). Ketamine and thiopental produced a concentration-dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 &mgr;M, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 &mgr;M. Bicuculline also reversed the effects of propofol but not those of ketamine. Conclusions: The authors’ data indicate that thiopental, propofol, and ketamine inhibit K+-evoked glutamate release from rat cerebrocortical slices. The inhibition produced by thiopental and propofol is mediated by activation of GABAA receptors, revealing a subtle interplay between GABA-releasing (GABAergic) and glutamatergic transmission in anesthetic action.A antagonist; mechanisms of anesthesia; neurotransmitter release; rat cerebrocortical slices.)

Nocistatin reverses nociceptin inhibition of glutamate release from rat brain slices

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.

μ- and κ-opioids inhibit K evoked glutamate release from rat cerebrocortical slices

Abstract We have examined the effects of a range of opioid receptor subtype selective agonists on K + evoked glutamate release from perfused rat cerebrocortical slices. Dual application (S 1 and S 2 ) of K + (46 mM) evoked dual monophasic glutamate release profiles. When areas under the release curves were calculated an S 2 /S 1 ratio for control slices of 1.07 ± 0.08 ( n = 75) was obtained, this was reduced by 80% with EGTA (0.1 mM) treatment confirming the presence of a Ca 2+ regulated release process. Morphine produced a dose-dependent inhibition of the S 2 /S 1 ratio. At 1 μM this amounted to 78 ± 12% (mean ± SEM; n = 6). ( d -Ala 2 ,MePhe 4 ,gly(ol) 5 )enkephalin (DAMGO; 60 ± 12%, n = 6 at 1 μM), and spiradoline (53 ± 14% at 1 and 71 ± 11% at 100 μM, both n = 6) also inhibited glutamate release in a cyprodime (10 μM) and norbinaltorphimine (10 μM) reversible manner. ( d -Pen 2,5 )enkephalin (DPDPE; 1 μM) was ineffective. All agents tested did not affect basal glutamate release. Collectively these data implicate a role for μ and κ opioids in the control of evoked glutamate release and their potential for neuroprotective therapy.

Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.

Nociceptin/orphanin FQ inhibits glutamate release from rat cerebellar and brain stem slices

We have previously demonstrated that nociceptin/orphanin FQ (N/OFQ), inhibits K(+) depolarisation-evoked glutamate release from rat cerebrocortical slices. In this study we have examined the effects of N/OFQ on glutamate release from rat cerebellar and brain stem slices as there are regional differences in nociceptin/orphanin FQ receptor (NOP) expression. Slices were depolarised with two pulses of 46 mM K(+) (S(1) and S(2)) with N/OFQ added after S(1). Glutamate (non-radioactive) was measured using a fluorescence-based assay. N/OFQ effects were assessed by measuring area under S(1) and S(2) release curves and calculation of S(2)/S(1) ratios. In cerebellar slices K(+) evoked S(2)/S(1) ratio was 1.17+/-0.10 (n=28). This was reduced in a concentration dependent (EC(50) 22 nM; E(max) 46%) and naloxone (10 microM) insensitive manner by N/OFQ. In the brain stem K(+) evoked glutamate release was considerably reduced compared to cerebellum. In several preparations K(+) failed to evoke a significant release. In those that did K(+) evoked S(2)/S(1) ratio was 1.03+/-0.07 (n=13). A total of 100 nM N/OFQ reduced this by 38+/-12% and this response was naloxone insensitive. Due to this small response and its variability we could not construct a full concentration response curve. In conclusion we have demonstrated a functional NOP in rat cerebellum and brain stem that inhibits the release of glutamate.

Rapid communicationNocistatin reverses nociceptin inhibition of glutamate release from rat brain slices

We have examined the effects of the recently described heptadecapeptide nocistatin on K+-evoked glutamate release from rat cerebrocortical slices in vitro. In vivo, nocistatin reverses the action of nociceptin. Nocistatin (100 nM, n = 7) did not inhibit K+-evoked glutamate release alone. Nociceptin (100 nM) inhibited glutamate release by 51.7 +/- 8.3% (P < 0.05, n = 6) and this was fully reversed by nocistatin (100 nM). Nocistatin also appears to be an antagonist of nociceptin action in vitro.