Modulation of cyclic AMP formation by putative metabotropic receptor agonists.

1. The effects of various metabotropic glutamate receptor agonists on [3H]-cyclic AMP accumulation and phosphoinositide hydrolysis were investigated in guinea-pig cerebral cortical slices prelabelled with [3H]-adenine or [3H]-inositol. 2. 1-Aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD), L-2-amino-4-phosphonobutanoate (L-AP4) and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited concentration-dependent inhibitions of forskolin-stimulated [3H]-cyclic AMP accumulation, with IC50 values of 2.1 +/- 0.3, 71 +/- 17 and 0.2 +/- 0.1 microM respectively. 3. 1S,3R-ACPD and L-CCG-I increased the cyclic AMP responses to histamine H2 receptor stimulation with EC50 values of 7 +/- 2 microM and 19 +/- 2 microM respectively. 1S,3R-ACPD (EC50 values 17 +/- 2 microM) and L-CCG-I (EC50 value 15 +/- 3 microM) potentiated the cyclic AMP responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM). This potentiating effect of L-CCG-I was reduced in the presence of a protein kinase C inhibitor, and also in the absence of extracellular calcium. In contrast, L-AP4 inhibited the NECA response in a concentration-dependent manner, with an IC50 value of 120 +/- 20 microM. 4. L-AP4 (at concentrations up to 1 mM) failed to stimulate phosphoinositide hydrolysis in guinea-pig cerebral cortical slices, but both 1S,3R-ACPD (EC50 value 35 +/- 6 microM) and L-CCG-I (approximately 160 microM) elicited concentration-dependent stimulations of phosphoinositide turnover. 5. These results confirm the existence of at least two distinct subtypes of metabotropic receptor in guinea-pig cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of the metabotropic glutamate receptor inhibiting D-[3H]-aspartate output in rat striatum.

1. The effects of several agonists of the metabotropic glutamate receptor (mGluR) were studied in adult rat striatal slices by measuring (i) KCl (30 mM)-induced output of previously taken up D-[3H]-aspartate (Asp), (ii) forskolin (30 microM)-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation and (iii) phophoinositide (PI) hydrolysis. 2. K(+)-induced efflux of D-[3H]-Asp was inhibited by the following mGluR agonists: (1S,3S,4S)-(carboxycyclopropyl)glycine (L-CCG-I), (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and quisqualic acid (Quis). 2-Amino-4-phosphonobutyrate (L-AP4) was inactive up to 300 microM. The maximal inhibition of D-[3H]-Asp output was 60 +/- 8%. The EC50s of mGluR agonists were: 0.5 microM for L-CCG-I, 100 microM for 1S,3R-ACPD and 100 microM for Quis. 3. Forskolin-induced cyclic AMP accumulation was also inhibited by mGluR agonists. The maximal inhibition was 50 +/- 4% and was obtained at a concentration of 10 microM for L-CCG-I and 100 microM for 1S,3R-ACPD. The EC50s for this inhibition were: 0.9 microM for L-CCG-I and 20 microM for 1S,3R-ACPD. Quis (300 microM) inhibited cyclic AMP accumulation by approximately 20%. L-AP4 slightly potentiated cyclic AMP accumulation. 4. PI hydrolysis was stimulated by mGluR agonists. The most potent compound was Quis (100 microM), which increased inositol phosphate formation up to 2.2 fold over control values. Its EC50 was 15 microM. L-CCG-I and 1S,3R-ACPD increased inositol phosphate formation by approximately 1.8 fold and their EC50 values were 30 and 25 microM, respectively. L-AP4 did not affect PI hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterisation of 5-HT receptors positively coupled to adenylyl cyclase in the rat hippocampus

The pharmacological properties of 5-hydroxytryptamine (5-HT) receptors positively coupled to adenylyl cyclase in the rat hippocampus were investigated using selective agonists and antagonists. 5-HT (0.008–125 μM) stimulated cyclic AMP formation in homogenates of rat hippocampus in a concentration-dependent manner. The maximal increase in cyclic AMP formation occurred at 1 μM (141 ± 6%) and the half-maximal effect (EC₅₀) at 50 ± 22 nM. Cyclic AMP accumulation induced by 1 μM 5-HT was partly inhibited by the selective 5-HT_1A receptor antagonist WAY 100,635 (1 μM), the selective 5-HT₄ receptor antagonist SB 203,186 (1 μM), and the 5-HT_2A/C/ 5-HT₇ receptor antagonist mesulergine (25 μM). WAY 100,635, SB 203,186 and mesulergine inhibited the effect of 5-HT (1 μM) by 47%, 33% and 49%, respectively. The combination of WAY 100,635 (1 μM) with SB 203,186 (1 μM) or mesulergine (25 μM) resulted in stronger inhibition than with each antagonist alone, and the combination of all three antagonists produced almost total blockade (95%) of 5-HT-induced cyclic AMP accumulation. 5-Carboxamidotryptamine (5-CT; 0.008–125 μM), a 5-HT₁/5-HT₇ receptor agonist, and SDZ 216–454 (0.008– 125 μM), a selective 5-HT₄ receptor agonist, concentration-dependently stimulated cyclic AMP formation, but the maximal effect of each agonist was smaller than that of 5-HT alone. SDZ 216–454 (5 μM) and 5-CT (5 μM) in combination stimulated cyclic AMP formation in an additive manner. 8-OH-PIPAT and 8-OH-DPAT, two selective 5-HT_1A agonists, produced a small but significant increase in cyclic AMP formation at concentrations above 0.04 μM and 10 μM, respectively. These findings suggest that at least three 5-HT receptor subtypes, i.e. 5-HT_1A, 5-HT₇ and 5-HT₄ receptors, are involved in mediating 5-HT-induced cyclic AMP formation in rat hippocampus. Received: 28 October 1998 / Accepted: 16 March 1999     

Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea-pig cerebellum.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2.L-Glutamate, 1-aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD) and RS-3,5-dihydroxyphenylglycine (DHPG) evoked concentration-dependent increases in the accumulation of [3H]-inositol phosphates with pEC50 values of 2.98 +/- 0.02, 4.45 +/- 0.06 and 4.47 +/- 0.07, respectively. Maximal responses to these agents were 43 +/- 8, 52 +/- 12 and 84 +/- 11% of the response to 1 mM histamine, respectively. 3. The phosphoinositide response to 1S,3R-ACPD was antagonized in the presence of (+)-alpha-methyl-4-carboxyphenylglycine, with a calculated pKi value of 3.55 +/- 0.03. 4. Forskolin-stimulated accumulation of [3H]-cyclic AMP was not significantly altered in the presence of 10 microM DCG-IV or 300 microM 1S,3R-ACPD. Similarly, 300 microM 1S,3R-ACPD failed to alter isoprenaline-(1 microM) or 2-chloroadenosine (2-CA, 30 microM)-stimulated accumulation of [3H]-cyclic AMP. 5. Forskolin-stimulated accumulation of [3H]-cyclic AMP was concentration-dependently inhibited in the presence of L-glutamate and L-serine-O-phosphate (L-SOP) with pIC50 values of 2.91 +/- 0.17 and 2.86 +/- 0.04 with maximal inhibitions of 47 +/- 2 and 92 +/- 3%, respectively. L-2-Amino-4-phosphonobuty-rate (L-AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71 +/- 7% at 3 mM. 6. 2-CA-evoked accumulation of [3H]-cyclic AMP was also inhibited by L-glutamate and L-SOP with pIC50 values of 2.71 +/- 0.03 and 2.72 +/- 0.08 and maximal inhibitions of 51 +/- 5 and 99 +/- 0%, respectively. L-AP4 inhibited the 2-CA response without saturating, evoking an inhibition of 68 +/- 1% at 3 mM. 7. Isoprenaline-evoked accumulation of [3H]-cyclic AMP was inhibited by L-glutamate and L-SOP with pIC50 values of 3.21 +/- 0.01 and 2.96 +/- 0.08 and maximal inhibitions of 88 +/- 2 and 93 +/- 3%, respectively. 8. These results suggest that the guinea-pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.     

α2-Adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins

The aim of this study was to use a ³H-adenine pre-labelling technique to characterise the effect of ₂-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10^–7–10^–4 M) stimulated ³H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated ³H-cyclic AMP accumulation approximately 7–8 fold. The response reached a peak after 5 min. In the presence of rolipram (10^–5 M), basal ³H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 ± 57 dpm; rolipram: 3088 ± 229, n \2 = 3) and forskolin (3 × 10^–5 M) stimulated ³H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 × 10^–5 M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10^–9–10^–4 M) and UK14304 (10^–9–10^–4 M) inhibited forskolin-stimulated ³H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC₅₀ values of 7.61 ± 0.37 (n \s = 4) and 7.76 ± 0.23 (n \s = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10^–4 M) nor UK14304 (10^–4 M) altered basal ³H-cyclic AMP levels. Phenylephrine (10^–4 M) had no effect on basal ³H-cyclic AMP levels and produced a 25.4 ± 7.1% inhibition of the forskolin-stimulated response, an effect that was reversed by 10^–6 M rauwolscine. Rauwolscine (10^–9–10^–6 M) produced a concentration-dependent reversal of the inhibitory effect of UK14304 10^–6 M on forskolin-stimulated ³H-cyclic AMP accumulation with a mean pK _i of 8.35 ± 0.39 (n = 3), but had no effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. Similarly, prazosin (3 × 10^–8–3 × 10^–5 M) or imiloxan (3 × 10^–8––3 × 10^–5 M) produced a concentration-dependent reversal of the UK14304 (10^–7 M)-induced inhibition of forskolin-stimulated ³H-cyclic AMP accumulation, with mean pK _i values of 6.32 ± 0.22 (n = 4) and 6.01 ± 0.30 (n = 3), respectively; neither drug had any effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. This suggests that the receptor is of the _2A-adrenoceptor subtype. It can be seen from these studies that it is possible to measure changes in cyclic AMP accumulation in porcine vascular smooth muscle using a pre-labelling technique, and it has been possible to demonstrate the presence of functional ₂-adrenoceptors, stimulation of which results in inhibition of forskolin-stimulated cyclic AMP formation.     

5-Hydroxytryptamine 5-HT1B receptors inhibiting cyclic AMP accumulation in rat renal mesangial cells

A clonal cell line derived from rat renal mesangial cells was shown to express endogenous 5-hydroxytryptamine (serotonin, 5-HT) receptors that mediate inhibition of cyclic AMP accumulation. These receptors were characterized as being of the 5-HT _1B receptor subtype. 5-HT ₁ receptor agonists inhibited forskolin-stimulated cyclic AMP accumulation in rat renal mesangial cells (60–70% maximal inhibition) with the following rank order of potency (mean pEC ₅₀ values±SEM, n=3): ergotamine (9.58±0.51)>RU 24969 (8.67±0.23)=5-CT (8.42±0.06)=CP 93129 (8.15±0.27)>5-HT (7.75±0.11) >sumatriptan (6.29±0.30)>8-OH-DPAT (4.32±0.15). 5-HT ₂ and 5-HT ₄ receptor agonists were without effect. 5-HT-induced inhibition of cyclic AMP accumulation was abolished by a pre-treatment of the cells with pertussis toxin. (–)Propranolol was a partial agonist (27% maximal inhibition, pEC ₅₀ 7.19±0.24, n=3); when used as an antagonist at 1?µM, it shifted the concentration- response curve of 5-HT to the right (pK _B 7.22±0.35, n=3). Methiothepin was a competitive antagonist of 5-HT (pA ₂ 8.04±0.10, Schild slope 0.87±0.21, n=3). Rauwolscine (10?µM) had no antagonist activity. There was a significant correlation ( r=0.98, P=0.0001) between the cyclic AMP data obtained in rat mesangial cells and 5-HT _1B binding data reported in rat brain cortex. The same pattern of responses was observed in early passages of primary cultures of rat mesangial cells. This study shows that rat mesangial cells can be used as a convenient source of functional 5-HT _1B receptors. It also constitutes further evidence for the widespread distribution of 5-HT _1B receptors outside the brain.     

Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.     

Effect of selective phosphodiesterase inhibitors on synaptic transmission in the guinea-pig ileum

The effect of selective phosphodiesterase (PDE) inhibitors was studied on neural transmission within the enteric nervous system employing a two-compartment bath (containing the oral and the anal end of a segment of guinea-pig ileum, respectively). Ascending excitatory enteric nerve pathways were activated by electrical field stimulation (10 Hz for 2 s, 45 mA, 0.5 pulse duration) in the anal compartment and the resulting contraction of the intestinal circular muscle in the oral compartment was recorded. The partitioned bath enables PDE inhibitors and other drugs to be applied to enteric nerve pathways (in the anal compartment) without interfering with the recording of the smooth muscle contraction in the oral compartment. The PDE 4 inhibitors rolipram (0.01–10 μM) and Ro-20-1724 (0.01–10 μM) significantly (P<0.01) inhibited (10–91% and 9–83%, respectively) the nerve-mediated contractions. When both rolipram and Ro-20-1724 were tested after phentolamine (1 μM) or yohimbine (0.1 μM), they were significantly (P<0.01) less effective. By contrast prazosin (1 μM) was ineffective. Vinpocetine (50 μM), milrinone (30 μM) and zaprinast (100 μM), which inhibit PDE 1, 3 and 5, respectively, did not modify the nerve-mediated contractions. 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cyclic AMP) or N⁶,2’-O-dibutyryladenosine 3’,5’ cyclic monophosphate (dibutyryl cyclic AMP), two analogues of cyclic AMP, at lower concentrations (0.1–1 μM) significantly (P<0.01) inhibited (15–73% and 5–49%, respectively) the nerve-mediated contractions, while at higher concentrations (10–100 μM) they caused a significant (P<0.01) potentiating (48–68% and 77–78%, respectively) effect. These results indicate that inhibition of PDE 4 (but not PDE 1, PDE 3 or PDE 5) produces a depression of neural transmission within the enteric nervous system, possibly by releasing noradrenaline acting at α₂-adrenoceptors on enteric neurons. Received: 21 November 1997 / Accepted: 11 March 1998     

Ligand binding profile of the rat metabotropic glutamate receptor mGluR3 expressed in a transfected cell line

A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [³H]glutamate as radioligand. The rank order of affinity was l-glutamate>(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD)>L(+)-2-amino-3-phosphonopropionic acid (L-AP3)>quisqualic acid>L(+)-2-amino-4-phosphonobutyric acid (L-AP4)>ibotenic acid. The active enantiomers of several phenylglycines displayed K_i values of 300 to 400 M. The nonactive enantiomers and the standard ionotropic glutamate receptor ligands N-methyl-d-aspartic acid (NMDA), (R,S)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid only weakly displaced [³H]glutamate. In this cell line, l-glutamate and (2S,3S,4S)--(Carboxycyclopropyl)-glycine (L-CCG-I) reduced cAMP levels in a dose-dependent manner. The sensitivity of this system and its easy applicability make it feasible to envisage ligand binding assays on cell lines expressing cloned receptors as useful screening tools to discover and characterize new and specific agonists and antagonists.     

Evidence that antipsychotic drugs are inverse agonists at D2 dopamine receptors

1. The effects of a number of D₂-like dopamine receptor antagonists have been determined on forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary (CHO) cells expressing the human D_2short dopamine receptor (CHO-D2S cells).
2. Dopamine inhibited the effect of forskolin (as expected for a D₂ receptor). However, all of the antagonists tested, apart from UH232 and (−)-butaclamol, were able to increase cyclic AMP accumulation above the forskolin control level. (+)-Butaclamol elicited a similar stimulation of forskolin-stimulated cyclic AMP accumulation in a CHO cell line expressing human D_2long dopamine receptors whereas it exhibited no stimulating effect on forskolin-stimulated cyclic AMP accumulation in untransfected CHO-K1 cells.
3. There was a strong correlation between the EC₅₀ values of these compounds for potentiation of cyclic AMP accumulation and their K_i values from radioligand binding experiments in CHO-D2S cells.
4. The effects of both (+)-butaclamol and dopamine in CHO-D2S cells were inhibited by pre-treatment with pertussis toxin indicating a role for Gi/Go proteins.
5. UH232 did not significantly affect forskolin-stimulated cyclic AMP accumulation but this substance was able to inhibit the effects of both dopamine and (+)-butaclamol in a concentration-dependent manner. Thus the effects of (+)-butaclamol on forskolin-stimulated cyclic AMP accumulation are mediated directly via the D₂ receptor rather than by reversal of the effects of an endogenous agonist.
6. These data suggest that the D₂ dopamine receptor antagonists tested here, many of which are used clinically as antipsychotic drugs, are in fact inverse agonists at human D₂ dopamine receptors.

     

Blockade of GABAB receptors facilitates muscarinic agonist-induced epileptiform activity in immature rat piriform cortex in vitro

The effects of the selective GABA_B receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) on muscarinic (mAChR) and metabotropic glutamate (mGluR) responsiveness were studied in slices of piriform cortex from both immature (P16–P22) and adult (≥P40) rats, using a conventional intracellular recording technique. In both adult and immature slices, CGP 52432 (1 μM) had no effect on neuronal membrane properties, whereas it selectively abolished the late inhibitory postsynaptic potential (IPSP) evoked by local electrical stimulation of association fibre terminals. Age-related changes in mAChR (but not mGluR) responsiveness were also detected. In adult neurones, bath-application of the mAChR agonist oxotremorine-M (OXO-M; 10 μM), or the selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 10 μM) evoked similar membrane depolarization and inhibition of evoked excitatory postsynaptic potentials (EPSPs). However, while 1S,3R-ACPD and OXO-M produced indistinguishable slow excitatory effects in immature slices, during superfusion with OXO-M, neurones exhibited spontaneous paroxysmal depolarizing shifts (PDSs) that were suppressed in the presence of atropine (1 μM) or the selective GABA_B receptor agonist β-parachlorophenyl-γ-aminobutyric acid [(–)baclofen; 10 μM]. Also, application of OXO-M resulted in a pronounced prolongation (rather than a decrease) of electrically evoked postsynaptic potentials (PSPs) which now exhibited recurrent superimposed spike discharges. In adult slices, in the continuous presence of CGP 52432 (1 μM; 20 min pre-incubation), a subsequent exposure to 10 μM OXO-M or 1S,3R-ACPD failed to induce any spontaneous epileptiform activity, and evoked PSPs were consistently suppressed. In contrast, in immature slices, after incubation in CGP 52432 (1 μM; 20 min), a subsequent application of a low dose of OXO-M (2.5 μM), which was inactive per se, was able to produce spontaneous PDSs and a prolongation of evoked PSPs. We conclude that a reduction in GABA_B-mediated synaptic inhibition in immature slices (in co-operation with other factors) may contribute to the facilitation of excitatory neurotransmission and therefore play a role in the generation of mAChR-induced epileptiform activity. Received: 16 March 1998 / Accepted: 11 May 1998     

In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys<Superscript>7,10</Superscript>]N/OFQ(1–13)NH<Subscript>2</Subscript>

Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G_i-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys^7,10]N/OFQ(1–13)NH₂ (c[Cys^7,10]). c[Cys^7,10], the template N/OFQ(1–13)NH₂ and N/OFQ displaced the binding of [³H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO_hNOP) with pK _i values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ stimulated the binding of guanosine triphosphate gamma [³⁵S] to CHO_hNOP cells with pEC₅₀/E _max (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC₅₀ values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys^7,10] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys^7,10], N/OFQ(1–13)NH₂ and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1–13)NH₂ produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys^7,10]. In summary, c[Cys^7,10] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.     

Pharmacology and mode of action of bradykinin on mouse-isolated trachea

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10^–7M) a relaxation with bradykinin (3·10^–9–3·10^–7) was found. The maximal response amounted 69.7±4.1% (n=15) with a pD₂ value of 7.2±0.21. The selective bradykinin B₂ receptor antagonist HOE 140 (10^–10–10^–8M) antagonized the bradykinin-induced relaxation, while the bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) had no influence. The selective bradykinin B₁ receptor agonist des-Arg⁹-bradykinin (10^–6M) caused a small relaxation (8.4±2.5%, n=6), which could be antagonized completely by the selective bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) while addition of the selective bradykinin B₂ receptor antagonist HOE 140 (10^–8M) was without effect. In the presence of indomethacin (10^–6M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK₁ receptor antagonist RP 67851 (10^–6M) or the presence of the nitric oxide synthase inhibitor L-NAME (3·10^–4M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B₂ receptors. This response is probably mediated by prostaglandines. Received: 14 November 1996 / Accepted: 5 March 1997     

Structure-activity relationships for a series of phenylglycine derivatives acting at metabotropic glutamate receptors (mGluRs).

1. The actions of a series of twelve phenylglycine derivatives at metabotropic glutamate receptors (mGluRs) linked to both phosphoinositide hydrolysis (PI) and cyclic AMP were investigated. 2. PI hydrolysis was determined by the accumulation of [3H]-inositol-monophosphate ([3H]-IP1) in neonatal ral cortical slices prelabelled with [3H]-myo-inositol. The non-selective mGluR agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid ((1S,3R)-ACPD) produced a concentration-dependent increase in [3H]-IP1 (EC50 approximately 20 microM). This agonist was subsequently used to investigate potential antagonist activity of the phenylglycine derivatives. Of the compounds tested (+)-alpha-methyl-4-carboxyphenylglycine (M4CPG) and (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) were the most active with KP values of 0.184 +/- 0.04 mM and 0.367 +/- 0.2 mM respectively. 3. Activity at adenylyl cylase-coupled mGluRs was investigated by determining the accumulation of [3H]-cyclic AMP in adult rat cortical slices. [3H]-cyclic AMP accumulation, elicited by 30 microM forskolin, was inhibited by (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-1) and L-2-amino-4-phosphonobutanoate (L-AP4) with respective EC50 values of 0.3 microM and 10 microM. Neither agonist was able to inhibit completely forskolin stimulated cyclic AMP accumulation; this is evidence that not all adenylyl cyclase is susceptible to modulation by mGluRs. Phenylglycine derivatives were examined for their ability to antagonize the inhibition of [3H]-cyclic AMP accumulation by L-CCG-1 or L-AP4 at their EC50 concentrations. 4. A rank order of potency of the phenylglycine derivatives as antagonists of L-AP4 and L-CCG-1 was obtained. The most effective compound. (RS)-alpha-methyl-3-carboxymethylphenylglycine (M3CMPG) had IC50 values in the order of 1 microM against L-AP4 and 0.4 microM against L-CCG-1. 5. The results from this study indicate that phenylglycine-derived compounds can discriminate between groups of metabotropic glutamate receptors and may also display some selective activity between subtypes within groups. Future work based on these findings may lead to the development of more selective and potent compounds as important pharmacological tools.     

Metabotropic glutamate receptor modulation of cAMP accumulation in the neonatal rat hippocampus

The pharmacology and cellular mechanism by which metabotropic glutamate receptor (mGluR) activation modulates cAMP formation was studied in cross-chopped hippocampal slices from neonatal (7 day old) rats. The selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), and other non-selective mGluR agonists produced concentration-related stimulation of basal cAMP formation in this tissue. The relative agonist potency order was 1S,3R- ACPD = quisqualate > ibotenate 1R,3S-ACPD. 1S,3R-ACPD stimulated cAMP accumulation was antagonized in a stereoselective manner by -2-amino-3-phosphonopropionate ( -AP3), but not by higher chain homologues such as -2-amino-4-phosphonobutyrate ( -AP4) and 2-amino-5-phosphonopentanoate (AP5). 1S,3R-ACPD-enhanced cAMP formation was greatly inhibited by incubation with adenosine deaminase. In the adult rat hippocampus, 1S,3R-ACPD did not appreciably increase basal cAMP, but inhibited forskolin-stimulated cAMP formation, and this effect was observed with or without adenosine deaminase. In the presence of the adenosine receptor antagonist and cAMP phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), 1S,3R-ACPD did not enhance cAMP formation in the neonatal hippocampus, but inhibited forskolin-stimulated cAMP (like in the adult tissue). These results demonstrate that mGluRs that increase cAMP in the neonatal hippocampus have a unique pharmacology when compared to mGluRs that decrease cAMP accumulation and increase phosphoinositide hydrolysis. 1S,3R-ACPD stimulation of cAMP in the neonatal rat hippocampal slice involves potentiation of responses to endogenous adenosine. Negatively coupled cAMP linked mGluRs are also present in the neonatal tissue, but are masked by the predominence of the positively coupled mGluR cAMP response.     

L-2-amino-4-phosphonobutyrate (L-AP4) is an agonist at the type IV metabotropic glutamate receptor which is negatively coupled to adenylate cyclase: European Journal of Pharmacology — Molecular Pharmacology Section, 227 (1992) 361–362

Glutamate and L-AP4 inhibited forskolin-stimulated cyclic AMP (cAMP) production in baby hamster kidney (BHK) cells transfected with the type IV metabotropic receptor (mGluR4). In situ hybridization revealed a high level of mRNA for the mGluR4 in the entorhinal cortex, but not in the dentate gyrus. These data demonstrate that mGluR4 receptors are negatively coupled to the cAMP cascade, and suggest that the mGluR4 receptor may be the previously described presynaptic L-AP4 receptor.     

Blockade of voltage-sensitive sodium channels by NS-7, a novel neuroprotective compound, in the rat brain

The effects of 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7), a novel neuroprotective compound, on the voltage-sensitive sodium channels (VSSC) were examined in the rat brain and cardiac myocytes. NS-7 inhibited [³H]batrachotoxinin A 20α-benzoate (BTX) binding (neurotoxin receptor site 2) in brain membranes with a Ki value of 1 μM , while the compound was less effective in the cardiac myocytes (Ki = 13 μM). Aconitine, on the other hand, inhibited [³H]BTX binding to brain membranes and cardiac myocytes with the same potency. In contrast, NS-7 had no affinity for [³H]saxitoxin binding in brain (neurotoxin receptor site 1). In superfused slices of the rat cerebral cortex, NS-7 inhibited the veratridine (5 μM)-evoked glutamate release in a concentration-dependent manner, the IC₅₀ value of which was 7.7 μM, whereas the compound showed a weak and not significant suppression of KCl-evoked glutamate release. The tissue concentrations of NS-7 in the rat cerebral cortex and heart were 89 and 28 nmole/g tissue, respectively, 5 min after its intravenous injection (8 mg/kg). Furthermore, in the cerebral cortex, NS-7 distributed preferentially to the membrane-enriched synaptosomal fraction. Since neurotoxin receptor site 2 is located in the transmembrane region of the VSSC moiety, the channel function may be substantially inhibited by a peripheral administration of NS-7. These results suggest that the blockade of neurotoxin receptor site 2 of VSSC in the brain contributes to the neuroprotective action of NS-7. Received: 29 October 1996 / Accepted: 20 January 1997     

Cisplatin-induced toxicity in immortalized renal cell lines established from transgenic mice harboring temperature sensitive SV40 large T-antigen gene

 We established renal cell lines from definite nephron segments which were microdissected from kidneys of transgenic C57BL/6 mice, harboring the large T-antigen gene of temperature-sensitive mutant simian virus 40, pSVtsA58(ori-). Cell culture was under a humidified atmosphere of 5% CO₂ in air, on collagen-coated dishes, and in RITC80-7 medium with 5% fetal bovine serum, 10 μg/ml transferrin, 1 μg/ml insulin, 10 ng/ml recombinant human EGF, penicillin and streptomycin. Cell line which kept contact inhibition character was established from each segment. Cells derived from distal tubule, cortical and outer medullary collecting duct possessed their cyclic AMP response to arginine-vasopressin, like their original nephron segment. On the other hand, cells derived from terminal proximal tubules (S3 segment) formed a cobblestone-like confluent monolayer, and did not respond to arginine-vasopressin like their fresh segments. Since cisplatin, a well-known nephrotoxic substance, damages proximal tubules (especially S3) rather than collecting ducts, we assayed cell number, protein content, and ATP content of cultured S3 cells at various times after addition of 0.2 mM cisplatin. Decrease of cell number, total protein content and total ATP content of culture cells occurred after 10 h incubation with 0.2 mM cisplatin. The 50% lethal dose (LD₅₀) of cisplatin in S3 cells was 4×10^{–  5} M after 20 h incubation and 8.5×10^{–  6} M after 40 h incubation. Outer medullary collecting duct (OMCD) cells were damaged 30% maximally after 20 h incubation with cisplatin, and LD₅₀ in them became 2.5×10^{–  5} M after 40 h incubation. We could show that the LD₅₀ of cisplatin in the OMCD cell line was three times higher than that in the S3 cell line. Thus, these cell lines are the first in the kidney to definite the segmental origin and to maintain some differentiated unique functions. They are valuable for studies on intrarenal site-specific actions and possible mechanisms of action of pharmacological and toxic substances. Received: 3 May 1995 / Accepted 4 September 1995     

L-2-amino-4-phosphonobutyrate (L-AP4) is an agonist at the type IV metabotropic glutamate receptor which is negatively coupled to adenylate cyclase.

Glutamate and L-AP4 inhibited forskolin-stimulated cyclic AMP (cAMP) production in baby hamster kidney (BHK) cells transfected with the type IV metabotropic receptor (mGluR4). In situ hybridization revealed a high level of mRNA for the mGluR4 in the entorhinal cortex, but not in the dentate gyrus. These data demonstrate that mGluR4 receptors are negatively coupled to the cAMP cascade, and suggest that the mGluR4 receptor may be the previously described presynaptic L-AP4 receptor.     

Molecular mechanisms involved in the asymmetric interaction between cannabinoid and opioid systems

The aim of this work was to study the mechanism of cross-modulation between cannabinoid and opioid systems for analgesia during acute and chronic exposure. Acute coadministration of ineffectual subanalgesic doses of the synthetic cannabinoid CP-55,940 (0.2 mg/kg i.p.) and morphine (2.5 mg/kg i.p.) resulted in significant antinociception. In chronic studies, a low dose of CP-55,940 (0.2 mg/kg, i.p.) that per se did not induce analgesia in naive animals produced a significant degree of antinociception in rats made tolerant to morphine, whereas in rats made tolerant to CP-55,940, morphine challenge did not produce any analgesic response. To identify the mechanism of these asymmetric interactions during chronic treatment, we investigated the functional activity of cannabinoid and μ opioid receptors and their effects on the cyclic AMP (cAMP) cascade. Autoradiographic-binding studies indicated a slight but significant reduction in cannabinoid receptor levels in the hippocampus and cerebellum of morphine-tolerant rats, whereas CP-55,940-stimulated [³⁵S]GTPγS binding showed a significant decrease in receptor/G protein coupling in the limbic area. In CP-55,940 exposed rats, μ opioid receptor binding was significantly raised in the lateral thalamus and periaqueductal gray (PAG), with an increase in DAMGO-stimulated [³⁵S]GTPγS binding in the nucleus accumbens. Finally, we tested the cAMP system's responsiveness to the cannabinoid and opioid in the striatum and dorsal mesencephalon. In vivo chronic morphine did not affect CP-55,940's ability to inhibit forskolin-stimulated cAMP production in vitro and actually induced sensitization in striatal membranes. In contrast, in vivo chronic CP-55,940 desensitized DAMGO's efficacy in inhibiting forskolin-stimulated cAMP production in vitro. The alterations to the cAMP system seem to mirror the behavioral responses, indicating that the two systems may interact at the postreceptor level. This might open up new therapeutic opportunities for relief of chronic pain through cannabinoid–opioid coadministration.