Development of a homogeneous MAP kinase reporter gene screen for the identification of agonists and antagonists at the CXCR1 chemokine receptor.

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Galpha(i/o) or Galpha(q) families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sap1a following MAP kinase activation. In contrast to other reporter gene assays for Galpha(i/o)-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Galpha(i)-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC(50) = 7.0 +/- 0.1) > GCP-2 (pEC(50) = 6.3 +/- 0.1) > NAP-2 (pEC(50) < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Galpha(i/o)-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

Novel non-indolic melatonin receptor agonists differentially entrain endogenous melatonin rhythm and increase its amplitude.

In this study we have examined the ability of melatonin and four synthetic melatonin receptor agonists to entrain endogenous melatonin secretion in rats, free running in constant darkness. The circadian melatonin profile was measured by trans-pineal microdialysis, which not only reveals the time of onset and end of production (phase), but also the amplitude of the rhythm. Exogenous melatonin given at the onset of subjective darkness (clock time 12 h) was effective to entrain endogenous melatonin production. Only one agonist, 2-chloroacetamido-8-methoxytetralin (AH-017), mimicked this action. Two other agonists, 4-methoxy-2-(methylene propylamide)indan (GG-012) and N-[2-[2,3,7,8-tetrahydro-1H-furo(2, 3-g)indol-1-yl]ethyl]acetamide (GR196429), induced a phase-delay under free running conditions, possibly by increasing tau (tau) period. One agonist, 2-acetamido-8-methoxytetralin (AH-001) did not show any phase effect on the free running rhythm. Unexpectedly, all melatonin receptor agonists increased the amplitude of melatonin secretion. The amount of the increase varied from just below the level of significance (AH-001) to an approximately 2-fold increase (GG-012 and GR196429). This is in clear contrast to entrainment with melatonin, which significantly decreased the amplitude. It is hypothesized that entrainment and effects on amplitude of melatonin secretion are mediated by different mechanisms which can be differentially modulated using specific ligands.

Signalling by CXC-chemokine receptors 1 and 2 expressed in CHO cells: a comparison of calcium mobilization, inhibition of adenylyl cyclase and stimulation of GTPgammaS binding induced by IL-8 and GROalpha.

The effect of interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha) on [35S]-guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding, forskolin-stimulated cyclic AMP accumulation and cytosolic calcium concentration were determined in recombinant CHO cells expressing HA-tagged CXC-chemokine receptors 1 and 2 (CXCR1 and CXCR2). Radioligand binding assays confirmed that the binding profiles of the recombinant receptors were similar to those of the native proteins. IL-8 displaced [125I]-IL-8 binding to CXCR1 and CXCR2 with pKi values of 8.89+/-0.05 and 9.27+/-0.03, respectively. GROalpha, a selective CXCR2 ligand, had a pKi value of 9.66+/-0.39 at CXCR2 but a pKi>8 at CXCR1. Calcium mobilization experiments were also consistent with previous reports on native receptors. Activation of both receptors resulted in stimulation of [35S]GTPgammaS binding and inhibition of adenylyl cyclase. A comparison of the functional data at CXCRI showed that a similar potency order (IL-8> >GROalpha) was obtained in all three assays. However, at CXCR2 whilst the potency orders for calcium mobilization and inhibition of adenylyl cyclase were similar (IL-8 > or = GROalpha), the order was reversed for stimulation of [35S]GTPgammaS binding (GROalpha > IL-8). All of the functional responses at both receptors were inhibited by pertussis toxin (PTX), suggesting coupling to a Gi/Go protein. However, the calcium mobilization induced by IL-8 at CXCR1 was not fully inhibited by PTX, suggesting an interaction with a G-protein of the Gq family. Our results with pertussis toxin also suggested that, in the [35S]GTPgammaS binding assay, CXCR1 displays some constitutive activity. Thus, we have characterized the binding and several functional responses at HA-tagged CXCRs 1 and 2 and have shown that their pharmacology agrees well with that of the native receptors. We also have preliminary evidence that CXCR1 displays constitutive activity in our cell line and that CXCR2 may traffic between different PTX sensitive G-proteins.

Pharmacological characterisation of melatonin mt1 receptor-mediated stimulation of [35S]-GTPgammaS binding.

The activation of G-proteins by melatonin mt1 receptors was studied by measuring [35S]-guanosine-5'-(3-thiotriphosphate) ([35S]-GTPgammaS) binding to membranes prepared from Chinese hamster ovary (CHO) cells stably expressing human mt1 receptors. Melatonin stimulated [35S]-GTPgammaS binding in a concentration-dependent manner (pEC50, 8.77+/-0.02). The optimal (212+/-4%) increase over basal levels of binding (basal = 100%) was observed following incubation of membranes (12.5 microg protein/well) for 120 min at 30 degrees with [35S]-GTPgammaS (0.1 nM), in the presence of GDP (10 microM), NaCl (100 mM), and MgCl2 (10 mM). Melatonin analogues stimulated [35S]-GTPgammaS binding with a rank order (2-iodomelatonin > melatonin = S20098 > GR196429 > 6-chloromelatonin = 6-hydroxymelatonin >> N-acetylserotonin > or = GR135531 = mt1 luzindole = 5-HT = 0), which was identical to their affinities for the high affinity state of the receptor (correlation coefficient 0.94). All agonists evoked similar maximum increases in [35S]-GTPgammaS binding. EC50 values were 14- to 63-fold lower than binding affinities. The melatonin receptor antagonist luzindole (0.1-10 microM) evoked a parallel rightward shift in the melatonin concentration-response curve, with a pKB of 7.19+/-0.13, which is similar to its affinity in radioligand binding studies for human mt1 receptors. Stimulation of [35S]-GTPgammaS binding was abolished by pretreatment of cells with pertussis toxin (18 hr, 100 ng/mL) prior to preparation of membranes. Melatonin was without effect in CHO cells which lacked the mt1 receptor. Thus, melatonin and melatonin analogues stimulate [35S]-GTPgammaS binding with a profile which is consistent with binding to mt1 receptors causing activation of Gi/Go G-proteins.

GR196429: a nonindolic agonist at high-affinity melatonin receptors.

N-[2-[2,3,7,8-tetrahydro-1H-furo(2,3-g)indol-1-yl]ethyl]acetamide (GR196429) is a novel, nonindolic melatonin receptor agonist. GR196429 had high affinity for human mt1 (pKi 9.9) and MT2 (pKi 9.8) receptors expressed in Chinese hamster ovary cells and for 2-[125I]-iodomelatonin binding sites in human cerebellum, guinea pig superior colliculus and hypothalamus and chicken retina and tectum (pKi 8.8-9.5). GR196429 was inactive at a wide range of other hormone and neurotransmitter receptors. In Chinese hamster ovary cells expressing human mt1 or MT2 receptors, both melatonin and GR196429 dose-dependently inhibited forskolin-stimulated cAMP accumulation. In rabbit isolated retina, GR196429 inhibited calcium-dependent [3H]-dopamine release with potency (IC50 30 pM) and maximum effect (76 +/- 5% at 1 nM) similar to those of melatonin. The response was antagonized by the melatonin receptor antagonist luzindole (1 microM). In slices of rat brain suprachiasmatic nucleus, perfusion (1 h) with GR196429 at zeitgeber time 10 phase advanced the circadian peak in neuronal activity measured on the following day, with a maximum phase advance of 2.7 +/- 0.3 h at 10 pM and an EC50 of 0.6 pM, results that indicated a melatonin-like action on the phase of the circadian clock. CNS penetration and duration of receptor occupancy was determined in an ex vivo radioligand binding assay. In membranes of guinea pig superior colliculus prepared 30 min after administration of GR196429 (s.c.), 2-[125I]-iodomelatonin binding was inhibited with an ED50 of 0.04 mg/kg. After a dose of 1 mg/kg, binding was significantly inhibited for at least 3 h. Thus GR196429 is a potent and selective agonist at high-affinity melatonin receptors, which modulates circadian rhythms in an in vitro model of the circadian clock and which readily penetrates the CNS.

Temperature and agonist dependency of tachykinin NK1 receptor antagonist potencies in rat isolated superior cervical ganglion.

Using rat isolated superior cervical ganglion we have further characterised tachykinin NK1 receptors and investigated the possible existence of tachykinin NK1 receptor subtypes. At 37 degrees C, tachykinin NK1 receptor antagonists GR82334 ([D-Pro9[spiro-gamma- lactam]Leu10,Trp11]physalaemin-1(1-11)), CP-99,994 ((+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine) and (+/-)-RP67580 (7,7-diphenyl-2[1-imino-2(2-methoxy- phenyl)-ethyl]perhydroisoindol-4-one (3aR,7aR)) antagonised more potently depolarisation responses evoked by GR73632 (delta Ava]L-Pro9,N-MeLeu10]SP-(7-11)), septide ([pGlu6,Pro9]SP-(6-11)) and neurokinin A than those evoked by substance P, substance P O-methyl ester and [Sar9,Met(O2)11]substance P. GR73632 and substance P O-methyl ester evoked depolarisation responses of similar magnitude, unaffected by addition of tetrodotoxin, but which cross-desensitised. At 22 degrees C, the ability of GR82334 and (+/-)-RP67580 to inhibit substance P O-methyl ester-evoked but not GR73632-evoked responses was enhanced greatly. These results suggest a single population of tachykinin NK1 receptors in this preparation. The agonist and temperature dependency of tachykinin NK1 receptor antagonist potency in rat isolated superior cervical ganglion may reflect different conformational changes in the tachykinin NK1 receptor induced by partial or full sequence substance P analogues.

The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

Modulation of the rat suprachiasmatic circadian clock by melatonin in vitro.

The pineal hormone melatonin regulates daily and seasonal rhythms, at least in part through an action on the mammalian biological clock in the suprachiasmatic nuclei (SCN). Melatonin was tested in vitro (10(-15)-10(-6) M; ZT9.5-10.5) for its effect on the circadian peak in neuronal firing rate in the rat SCN slice. It produced a concentration-related phase advance (maximum advance = 3 +/- 0.3 h at 10(-9) M, n = 3; minimum effective concentration = 10(-13) M; EC50 = 1.2 x 10(-12) M). The melatonin receptor antagonist luzindole (10(-5) M) blocked the phase-advance produced by melatonin (10(-9) M), whilst having no effect on its own. These data show that the effect of melatonin on the SCN clock, measured via the circadian rhythm of neuronal firing rate in the nuclei, is consistent with a concentration-dependent action via a high affinity melatonin receptor.

New spiropiperidines as potent and selective non-peptide tachykinin NK2 receptor antagonists.

The synthesis of a series of 2-(5-fluoro-1H-indol-3-yl)ethyl spiropiperidines is described together with their tachykinin NK2 receptor affinities measured in a rat colon binding assay. Equivalent NK2 receptor binding affinity was observed for the spirooxazolidinone 3-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-1-oxa-3,8-diazaspiro[4.5] decan-2-one (3a), the imidazolidinone 3-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-1,3,8-triazaspiro[4.5 ] decan-2-one (3s), and the pyrrolidinone 2-benzyl-8-[2-(5-fluoro-1H-indol-3-yl)ethyl]-2,8-diazaspiro[4.5]decan -3 - one (3t). Substitution in the phenyl ring of compound 3a produced no significant enhancement in NK2 binding affinity. Replacement of the phenyl ring in 3a with other aromatic rings resulted in a significant loss in binding affinity. Compound 3a was shown to be a potent NK2 receptor antagonist in guinea pig trachea where it also demonstrated 1000-fold selectivity for NK2 receptors over NK1. In the anesthetized guinea pig, compound 3a administered by the intravenous or oral route displayed potent and long-lasting antagonist activity against NK2 receptor agonist induced bronchoconstriction.

The anxiolytic-like activity of GR159897, a non-peptide NK2 receptor antagonist, in rodent and primate models of anxiety.

The non-peptide NK2 receptor antagonist, GR159897, was evaluated in two putative models of anxiety, the mouse light-dark box and the marmoset human intruder response test. Effects were compared to the structurally dissimilar NK2 antagonist, (+/-) SR48968 and the benzodiazepines, diazepam and chlordiazepoxide. GR159897 (0.0005-50 micrograms/kg SC) caused significant and dose-dependent increases in the amount of time mice spent in the more aversive light compartment of the light-dark box, with no effect on locomotor activity. (+/-)SR48968 (0.0005-0.5 microgram/kg SC) and diazepam (1-1.75 mg/kg SC), also increased time spent in the light compartment, without effect on locomotor activity. In the marmoset human intruder response test, GR159897 (0.2-50 micrograms/kg SC) significantly increased the amount of time marmosets spent at the front of the cage during confrontation with a human observer ("threat"). Similar effects were produced by (+/-)SR48968 (10-50 micrograms/kg SC) and chlordiazepoxide (0.3-3.0 mg/kg SC). These results provide further evidence, in both rodent and primate species, for the ability of NK2 antagonists to restore behaviours which have been suppressed by novel aversive environments. Such effects indicate that NK2 antagonists may have anxiolytic activity.

GR159897, a potent non-peptide antagonist at tachykinin NK2 receptors.

GR159897 ((R)-1-[2-(5-fluoro-1H-indol-3-yl)ethyl]-4-methoxy-4- [(phenylsulfinyl)methyl]piperidine) is a novel, highly potent and selective non-peptide antagonist at tachykinin NK2 receptors. GR159897 inhibited binding of the NK2 receptor antagonist radioligand [3H]cyclohexylcarbonyl-Gly-Ala-(D)Trp-Phe-NMe2 ([3H]GR100679) to human ileum NK2 receptors transfected into Chinese hamster ovary cells (pKi 9.5) and to rat colon membranes (pKi 10.0). GR159897 was a competitive antagonist of contractions induced by the NK2 receptor agonist [Lys3,Gly8-R-gamma-lactam-Leu9]neurokinin A-(3-10) (GR64349) in guinea-pig trachea (pA2 8.7), and had negligible activity at human NK1 receptors transfected into Chinese hamster ovary cells (pKi 5.3), NK1 receptors in guinea-pig trachea (pKB < 5) or NK3 receptors in guinea-pig cerebral cortex (pKi < 5). In vivo, in the anaesthetised guinea-pig, GR159897 (0.12 mg.kg-1 i.v.) potently antagonised bronchoconstriction induced by GR64349 (dose-ratio = 28), with a long duration of action (3 h). GR159897 should be a useful tool for studying the physiological and pathophysiological role of tachykinin NK2 receptor activation.

GR138676, a novel peptidic tachykinin antagonist which is potent at NK3 receptors.

GR138676, a conformationally constrained analogue of neurokinin B, is a novel, potent NK3 receptor antagonist. GR138676 was a competitive antagonist of neurokinin B-dependent arachidonic acid mobilization from prelabelled Chinese hamster ovary cells stably transfected with a human NK3 receptor gene (pKB 8.3) and of contractions induced by senktide in rat portal vein (pKB 8.2). However, GR138676 was also a competitive antagonist of the increase in intracellular calcium evoked by the selective NK1 agonist, GR73632, in the human astrocytoma U373MG cell-line (pKB 8.3). GR138676 had little activity at NK2 receptors, inhibiting binding of the NK2 antagonist radioligand [3H]-GR100679 to Chinese hamster ovary cells transfected with the human ileum NK2 receptor with a pKi of 6.0. In summary, despite its activity at NK1 receptors, GR138676 will be a useful tool for characterizing NK3 receptors as well as defining the physiological and pathophysiological function of this receptor subtype.

Synthesis and characterization of selective fluorescent ligands for the neurokinin NK2 receptor.

Several fluorescent probes for the NK2 receptor were designed, synthesized, and pharmacologically characterized. These fluorescent ligands are analogues of the selective NK2 heptapeptide antagonist N-alpha-benzoyl-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (1, GR94800). They were obtained by substitution of 2,n-diaminoalkyl amino acid (n = 3-6) for Ala1 and the subsequent coupling of the fluorophore NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) or fluoresceinthiocarbamyl to the N-omega amino group. The fluorescent derivatives retained high binding affinities for the NK2 receptor in transfected CHO cells. In contrast, fluorescent derivatives made by replacing the N-alpha-benzoyl group of 1 by NBD or fluorescein were considerably less active. The effect on ligand potency of varying the length of the spacer arm between the peptide moiety and the fluorescent group was also studied. The most potent fluorescent antagonists were N-alpha-benzoyl-Dab(gamma-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (5B), pKi = 8.87 for NK2; N-alpha-benzoyl-Orn (delta-NBD)-Ala-D-Trp-Phe- D-Pro-Pro-Nle-NH2 (4B), pKi = 8.84; and N-alpha-benzoyl-Lys(epsilon-NBD)-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH2 (3B), pKi = 8.83. These three compounds were highly selective for NK2 over NK3 and NK1 receptors. We show that these fluorescent ligands are useful tools for the detection of NK2 receptor expression by flow cytometry. Additionally, these fluorescent probes should prove valuable for fluorescence microscopy and study of ligand-receptor interaction by spectrofluorimetry.

Anxiolytic activity of tachykinin NK2 receptor antagonists in the mouse light-dark box.

The tachykinin NK2 receptor antagonists, GR100679 (0.02-200 micrograms/kg s.c.) and (+/-)-SR489698 (0.05-5.0 micrograms/kg s.c.), dose-dependently increased the time which mice spent in the light side of the light-dark box. There was no evidence of sedation or other over behaviours. The amplitudes of these effects were similar to that evoked by diazepam (1.75 mg/kg s.c.). These results indicate a disinhibitory action of NK2 antagonists on suppressed behaviours in a novel aversive environment. This suggests an involvement of NK2 receptors in anxiety-related behaviours.

Ontogeny and characterization of [125I]Bolton Hunter-eledoisin binding sites in rat spinal cord by quantitative autoradiography.

The distribution and characteristics of [125I]Bolton Hunter-eledoisin binding sites in rat lumbar spinal cord were studied during postnatal development by in vitro receptor autoradiography. At three, six and 10 days of age, specific [125I]eledoisin binding was distributed throughout the dorsal and ventral horns of the spinal cord. In contrast, from day 24 onwards, specific binding of [125I]eledoisin was confined to superficial layers of the dorsal horn, with negligible amounts of specific binding in the ventral horn. [125I]Eledoisin binding to neonatal (three day) and adult (eight to 12 weeks) spinal cord sections was characterized using tachykinin agonists. In both dorsal and ventral horns of neonatal spinal cord, the rank order of potency of agonists indicated that the majority (64%) of specific [125I]eledoisin binding was to neurokinin-3 binding sites. The identity of the non-neurokinin-3 sites labelled by [125I]eledoisin remains to be determined. In adult rat spinal cord, [125I]eledoisin appeared to bind exclusively to neurokinin-3 binding sites. These results suggest that major changes take place in the localization of neurokinin-3 receptors during postnatal ontogeny of the rat spinal cord. These changes may reflect an important role for tachykinins in neuronal plasticity of the developing spinal cord.

Investigation into species variants in tachykinin NK1 receptors by use of the non-peptide antagonist, CP-96,345.

The affinity of the non-peptide antagonist CP-96,345 for tachykinin NK1 receptors has been estimated in a range of species by use of both radioligand binding and functional assays. CP-96,345 was 30-120 fold less active at NK1 receptors in rat and mouse than in the other species examined, including man. These results demonstrate the existence of species variations in NK1 receptors.

Receptor-selective, peptidase-resistant agonists at neurokinin NK-1 and NK-2 receptors: new tools for investigating neurokinin function.

The pharmacological profiles of two novel neurokinin agonists have been investigated. delta Ava[L-Pro9,N-MeLeu10]SP(7-11) (GR73632) and [Lys3,Gly8-R-gamma-lactam-Leu9] NKA(3-10) (GR64349) are potent and selective agonists at NK-1 and NK-2 receptors respectively. In the guinea-pig isolated trachea preparation, contractions induced by these agonists were largely unaffected by inclusion of peptidase inhibitors in the bathing medium, indicating that these agonists are resistant to metabolism by peptidases. In the anaesthetised guinea-pig, both agonists were more potent bronchoconstrictor agents than either NKA or the SP analogue, SP methylester. In the anaesthetised rat, the NK-1 agonist, GR73632 was more potent than SP, NKA or NKB at causing the histamine-independent extravasation of plasma proteins into the skin after intradermal administration. The NK-2 agonist, GR64349 and the NK-3 agonist, senktide were without significant effect in this model. These agonists are useful tools for characterizing neurokinin receptor-mediated actions both in vitro and in vivo.

Serotonin and migraine.

Migraine has long been considered as a "vascular headache" but clearly neurological mechanisms are involved. The pathophysiology appears to somehow involve serotonin, both peripherally and centrally, but its involvement may be just epiphenomenal. Adding to the enigma it is apparent that many of the presently available drugs for the treatment of migraine interact in one way or another with serotonin receptors. However, they tend to have a number of other unrelated actions and they are only of limited clinical value. Interestingly a promising new drug for the treatment of the acute attack, sumatriptan, has a very selective action as an agonist at a specific 5-HT1-like receptor sub-type, mediating vasoconstriction, which is localized on cranial blood vessels. Its action may, or may not, be independent of any involvement of serotonin in the genesis of migraine. Hopefully though, current attempts to determine sumatriptan's mechanism of action will shed further light on the pathology of migraine itself and the putative involvement of serotonin.

Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathway--I. Behavioural, neurochemical and histological characterization of the lesion.

1-Methyl-4-phenylpyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been chronically infused (10 micrograms/24 h for 7 days) via osmotic minipumps into the left median forebrain bundle of the rat in order to determine whether it can induce permanent damage to the nigrostriatal dopamine system. Its effects were assessed over a period of 6 months post lesion. Four to 5 days following minipump implantation, all MPP+-treated animals displayed spontaneous ipsilateral postural bias indicating a marked imbalance in striatal dopamine and degeneration of the ipsilateral nigrostriatal dopamine pathway. After 3-5 weeks, MPP+-infused animals showed dose-related ipsilateral and contralateral circling in response to methamphetamine (1-5 mg/kg i.p.) and apomorphine (0.05-0.25 mg/kg s.c.) respectively. In vivo, using bilateral monitoring of striatal dopamine in MPP+-infused animals at 2 and 4 months by push-pull perfusion, both basal and methamphetamine- (2.5 mg/kg i.p.) stimulated release of dopamine was undetectable in the ipsilateral striatum, indicating a complete loss of dopamine terminals. In contrast, in the contralateral striatum of these animals and in striata of saline-infused animals, there were 4-5-fold increases in dopamine release in response to methamphetamine. Six months after lesion, animals infused with MPP+ continue to exhibit robust rotational behaviour in response to methamphetamine and apomorphine. In the ipsilateral striatum of the MPP+-infused animals the tissue concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were all undetectable; however, the levels of noradrenaline, serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not significantly different from control values. In contrast to the striatum, MPP+ had no significant effect on the levels of dopamine and its metabolites in the ipsilateral nucleus accumbens; in addition, the levels of noradrenaline and serotonin and its metabolite were comparable to control levels. Histological examination revealed a marked loss of cells and severe gliosis in the substantia nigra pars compacta of MPP+-infused animals. The present results provide evidence that direct infusion of MPP+ into the medial forebrain bundle of the rat can lead to a complete loss of dopamine neurons in the pars compacta of the substantia nigra with ensuing behavioural, neurochemical and biochemical changes characteristic of the lesion.(ABSTRACT TRUNCATED AT 400 WORDS)