Exogenous melatonin entrains rhythm and reduces amplitude of endogenous melatonin: an in vivo microdialysis study.

The circadian rhythm of melatonin production was studied using on-line, in vivo microdialysis in the rat pineal gland. With this technique it was possible to record a pronounced melatonin rhythm with very high time resolution. Three phase-markers of the rhythm were calculated from the data, indicating increase (IT50), decrease (DT50) and amplitude of the rhythm. Comparing these phase markers led to several conclusions. Entrainment of the rhythm under constant darkness was performed with melatonin administration at different circadian stages [circadian time (CT) 8 and CT12] and for different periods of time (2 weeks and 4 weeks). Also, entrainment was established by applying 15 min light pulses at CT0. Entrainment of IT50 with melatonin partially uncoupled it from DT50. Four weeks entrainment in constant darkness (DD) caused a phase-delay in DT50 of 2.2 hr. Entrainment of IT50 with light at CT0 for 2 weeks in DD caused a phase-advance in DT50 of 1.3 hr. The entrainment with melatonin was restricted to a narrow window for melatonin to be applied, since injections at CT8 did not result in entrainment. Exogenous melatonin reduced the amplitude of the rhythm of endogenous melatonin. This effect was not circadian time dependent, since administration at CT8 for 2 weeks and at CT12 for 4 weeks resulted in a highly significant decrease. Light did not seem to have an effect on the amplitude. The data presented here provide us with new information about the nature of entrainment by melatonin. Since the present development of melatonergic agents for clinical use focuses on the entrainment capacity, effects of these compounds on amplitude of circadian rhythms needs to be addressed. In vivo microdialysis seems to be a good technique for that.

GR127935: a potent and selective 5-HT1D receptor antagonist.

GR127935 is the most potent 5-HT1D receptor antagonist yet described, possessing nanomolar affinity at human 5-HT1D receptors. Sumatriptan-induced contractions of the dog isolated basilar artery and saphenous vein are antagonised by GR127935 in an insurmountable manner indicative of its slow dissociation from the 5-HT1D receptor. 5-HT1D receptor-mediated hypothermia and rotational behaviour in guinea-pigs are antagonised potently, and with long duration, by GR127935, administered by a variety of routes. GR127935 also blocks central 5-HT1D autoreceptors in vitro and in vivo. GR127935 has much lower affinity at other 5-HT, and non-5-HT, receptors. In functional studies, GR127935 fails to affect 5-HT2 receptor-mediated 'wet dog shakes' in guinea-pigs and 5-HT1A receptor-mediated inhibition of 5-HT release in rat dorsal raphé nucleus. The compound has a good safety profile in all species tested. It is concluded that GR127935 is a useful pharmacological tool to characterise 5-HT1D receptor function.

Involvement of 5 -HT1D receptors in cortical extracellular 5-HT release in guinea-pigs on exposure to the elevated plus maze.

Previous studies have shown that guinea-pigs handled daily from birth exhibit on exposure to the elevated plus maze similar behaviour to rats and increased cortical extracellular 5-HT determined by in vivo microdialysis. The present study investigates the effects of a non-selective 5-HT(1) agonist 5-carboxamidotryptamine (5-CT) and the 5-HT(1D) antagonist GR 127935 on behaviour and the release of cortical extracellular 5-HT both in a familiar environment and on exposure to the elevated plus maze. In the familiar environment of the home cage GR 127935 (0.3mg/kg i.p.) had no effect on extracellular 5-HT. The non-selective agonist 5-CT (0.1 mg/kg i.p) produced a prolonged decrease (-25%) in cortical 5-HT release, an effect noT antagonized by GR 127935 (0.3mg/kg). Under aversive conditions, exposure to the elevated plus maze, the release of extracellular 5-HT increased (155% of basal release), an effect abolished by 5-CT. Pre-treatment with the selective 5.HT(1D) antagonist GR 127935 antagonized the effect of 5-CT on the aversion-induced increase in extracellular 5-HT on exposure to the elevated plus maze, but did not change the effects of 5-CT on basal 5-HT release. The results suggest that GR 127935 is an effective antagonist at the 5 -HT(1D) terminal autoreceptor in vivo under conditions of increased 5- HT function. Furthermore, the results indicate that the 5-HT( 1D) receptor in the frontal cortex is functionally active under aversive conditions.

Effects of the 5-HT1D receptor antagonist GR127935 on extracellular levels of 5-HT in the guinea-pig frontal cortex as measured by microdialysis.

The involvement of 5-HT1D receptors in the regulation of 5-HT release in the guinea-pig brain was examined using the novel 5-HT1D receptor blocking drug GR127935. Levels of 5-HT were measured in frontal cortex of anaesthetized guinea-pigs using microdialysis. The infusion of GR127935 (100 nM) through the dialysis probe into frontal cortex caused a significant increase (61 +/- 8%) in cortical extracellular levels of 5-HT. The increase was transient (approximately 40 min) even in the continuous presence of GR127935. The transient increase was abolished by tetrodotoxin (1 microM). The 5-HT1 receptor agonist GR46611 (10 mg/kg s.c.) caused a significant and sustained (> 100 min) reduction in extracellular levels of 5-HT (65 +/- 5%). This response was abolished in animals pre-treated with GR127935, 0.05 mg/kg i.p. Paradoxically, systemic administration of higher doses of GR127935 (0.1-1 mg/kg i.p.) in naive anaesthetized guinea-pigs caused significant and sustained (> 120 min) decreases (> 65%) in cortical levels of 5-HT. The increase in extracellular 5-HT seen following infusion of GR127935 into frontal cortex may be due to GR127935 blocking 5-HT terminal autoreceptors causing a subsequent increase in the outflow of 5-HT from pre-synaptic terminals. This conclusion is supported by the ability of GR127935 to block the decrease in 5-HT induced by the 5-HT1 receptor agonist GR46611.(ABSTRACT TRUNCATED AT 250 WORDS)

5-HT1D as well as 5-HT1A autoreceptors modulate 5-HT release in the guinea-pig dorsal raphé nucleus.

5-Hydroxytryptamine (5-HT) release was measured by fast cyclic voltammetry in guinea-pig dorsal raphé nucleus slices. Release was reproducibly evoked by a single 0.1 msec pulse of electrical stimulation. The 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT) produced a concentration-related inhibition of the stimulated 5-HT release, with 50% inhibition at 47 nM. This inhibition was competitively antagonized by N-tert-butyl 3-4-(2-methoxypheny)piperazin-1-yl-2- phenylpropanamide dihydrochloride [(+/-)WAY 100135], a selective 5-HT1A receptor antagonist (pA2 = 7.9). The 5-HT1D receptor agonist sumatriptan also produced a concentration-related inhibition of 5-HT release, with 50% inhibition at 40 nM. The effect of sumatriptan on 5-HT release was antagonized by the 5-HT1D receptor antagonist 2'-methyl-4'-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxyli c acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR127935) (pA2 = 8.7). Both (+/-)WAY 100135 and GR127935 increased the 5-HT release evoked by a train of 5 pulses at 1 Hz, suggesting that they were antagonizing the feedback of endogenously released 5-HT onto its autoreceptors. These findings demonstrate for the first time the presence of functional 5-HT1D as well as 5-HT1A autoreceptors in the guinea-pig dorsal raphé nucleus.

Stimulation of central 5-HT1D receptors causes hypothermia in the guinea-pig.

The 5-HT(1) receptor agonist GR46611 (3-30 mg/kg s.c.) caused a dose-related decrease in rectal temperature in the adult guinea-pig. A lower dose (20 µg) administered directly into the lateral cerebral ventricle also caused a hypothermic response, suggesting that this effect is centrally mediated. GR46611-induced (10 mg/kg s.c.) hypothermia was not attenuated by WAY100135 (3-10 mg/kg s.c.), ritanserin (0.3-1 mg/kg s.c.), spiperone (0.1-0.3 mg/kg s.c.) and ondansetron (0.1-1 mg/kg s.c.), suggesting that 5-HT(1A), 5-HT(2A), 5-HT( 2C) and 5-HT(3) receptors are unlikely to be involved in this response. In contrast, the poorly selective 5-HT receptor antagonist, metergoline (1-10 mg/kg s.c.), and the potent 5-HT(1D) receptor antagonist, GR127935 (0.1-1 mg/kg p.o.), antagonized the effects of GR46611. The present data suggest that antagonism of GR46611-induced hypothermia may be useful for assessing the potency and duration of action of centrally-acting 5-HT( 1D) receptor antagonists in the guinea-pig.

The pharmacology of fluparoxan: a selective alpha 2-adrenoceptor antagonist.

1. This paper describes the pharmacology of the novel alpha 2-adrenoceptor antagonist fluparoxan (GR 50360) which is currently being studied clinically as a potential anti-depressant. Idazoxan and yohimbine were included in many studies for comparison. 2. In the rat isolated, field-stimulated vas deferens and the guinea-pig isolated, field-stimulated ileum preparations, fluparoxan was a reversible competitive antagonist of the inhibitory responses to the alpha 2-adrenoceptor agonist UK-14304 with pKB values of 7.87 and 7.89 respectively. In the rat isolated anococcygeus muscle, fluparoxan was a much weaker competitive antagonist of the contractile response to the alpha 1-adrenoceptor agonist phenylephrine with a pKB of 4.45 giving an alpha 2: alpha 1-adrenoceptor selectivity ratio of greater than 2500. 3. In the conscious mouse, fluparoxan (0.2-3.0 mg kg-1) was effective by the oral route and of similar potency to idazoxan in preventing clonidine-induced hypothermia and antinociception. In the rat, UK-14304-induced hypothermia (ED50 = 1.4 mg kg-1, p.o. or 0.5 mg kg-1, i.v.) and rotarod impairment (ED50 = 1.1 mg kg-1 p.o. or 1.3 mg kg-1, i.v.) were antagonized by fluparoxan. Fluparoxan, 0.67-6 mg kg-1, p.o., also prevented UK-14304-induced sedation and bradycardia in the dog. 4. In specificity studies fluparoxan had low or no affinity for a wide range of neurotransmitter receptor sites at concentrations up to at least 1 x 10(-5) M. It displayed weak affinity for 5-HT1A (pIC50 = 5.9) and 5-HT1B (pKi = 5.5) binding sites in rat brain. 5. We conclude that fluparoxan is a highly selective and potent alpha 2-adrenoceptor antagonist. The density of rat brain [3H]-dihydroalprenolol binding sites was reduced by 26% when fluparoxan was administered chronically for 6 days at a dose of 12 mg kg- 1 orally twice daily. The down-regulation of beta-adrenoceptors by fluparoxan is consistent with its antidepressant potential.

Evidence that the unilateral activation of 5-HT1D receptors in the substantia nigra of the guinea-pig elicits contralateral rotation.

1. The effects of various 5-hydroxytryptamine (5-HT) receptor agonists were examined following unilateral infusion into the substantia nigra (SN) of the guinea-pig. 2. The 5-HT1 receptor agonists, 5-carboxamidotryptamine (5-CT) (2-25 micrograms), sumatriptan (10-25 micrograms) and RU24969 (25 micrograms) all induced a marked contralateral rotation. In contrast, the selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT, 10-25 micrograms) produced only a very small response, whilst the selective 5-HT1C/5-HT2 receptor agonist (+-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride ((+/-)-DOI) (25 micrograms) and the 5-HT3 receptor agonist, 2-methyl 5-HT (2-Me5-HT, 25 micrograms) were without effect. 3. The contralateral rotation induced by 5-CT (10 micrograms) was attenuated following pretreatment with the non-selective 5-HT1/5-HT2 receptor antagonists methiothepin (1 mg kg-1, s.c.) and metergoline (5-10 mg kg-1, s.c.) but not the 5-HT1C/5-HT2 antagonist ritanserin (1 mg kg-1, s.c.) or the 5-HT3 antagonist, ondansetron (0.5 mg kg-1, s.c.). An involvement of dopaminergic systems in the rotational response to 5-CT was implied by the antagonism of 5-CT-induced rotation by haloperidol (0.3 mg kg-1, s.c.). 4. At doses lower than those required to produce contralateral rotation, 5-CT (0.08-0.4 micrograms) and sumatriptan (2 micrograms) induced a small, but nonetheless consistent, ipsilateral rotation. 5. The data with agonists and antagonists taken together suggest that 5-CT-induced contralateral rotation may be mediated by 5-HTID receptor activation but definitive classification of the receptor will not be possible until selective 5-HTID-antagonists become available. This may therefore represent the first model to study this receptor subtype in vivo.

Inhibition of DOI-induced wet dog shakes in the guinea-pig by 5-HT2 receptor antagonists.

The preferential 5-HT( 2)/5-HT(1C) receptor agonist DOI (0.1-4 mg/kg s.c.) caused an increase in locomotor activity, grooming and 'wet-dog' shakes (WDS) in the adult guinea-pig. The DOI-induced WDS behaviour was potently inhibited by several antagonists that have high affinity for the 5-HT(2) binding site. The WDS response is likely to be centrally-mediated since the effects of peripherally administered DOI were poorly antagonized by the peripherally-acting 5-HT(2) receptor antagonist BW501C67. Although these studies do not exclude an effect of DOI at 5-HT(1C) receptors, the high potency of ketanserin and spiperone in attenuating the effects of DOI would suggest an effect at the 5-HT(2) receptor. The present data suggest that antagonism of the directly-acting agonist DOI may be useful for assessing the selectivity and duration of action of centrally-acting 5-HT(2) receptor antagonists in the guinea-pig.

Lack of antinociceptive activity of sumatriptan in rodents.

The present study attempts to determine whether the novel anti-migraine drug sumatriptan has antinociceptive activity in rodents. Sumatriptan had little or no antinociceptive activity against a range of noxious stimuli and we therefore conclude that it is unlikely that the beneficial effects of the drug in treating migraine are due to a non-specific analgesic action.

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.

Evaluation of the receptor selectivities of opioid drugs by investigating the block of their effect on urine output by beta-funaltrexamine.

The effects of a series of opioid drugs on urine output in the water-loaded rat were studied and also the block of those effects by the irreversible opioid receptor antagonist, beta-funaltrexamine (beta-FNA). Fentanyl, d-propoxyphene, profadol, bromadoline, buprenorphine and nalbuphine produced only a decrease in urine output, which was antagonised by pretreatment with beta-FNA, 40 mg/kg s.c., 24 hr beforehand. These drugs were thus characterized as selective mu receptor agonists. U-50,488, tifluadom, Mr2034, proxorphan, ethylketocyclazocine and butorphanol all produced an initial decrease in urine output, which was antagonized by beta-FNA, and therefore probably mu receptor mediated, followed by a beta-FNA insensitive diuretic effect, which was probably kappa receptor mediated. For U-50,488, tifluadom, Mr2034 and proxorphan the threshold dose for increasing urine output was lower than that for decreasing it, suggesting that these four compounds are kappa-selective agonists. For ethylketocyclazocine and butorphanol, the threshold doses for producing both effects were similar, suggesting that these two drugs are non-selective agonists. SKF 10,047 produced a diuretic effect at low dose-levels, which may be kappa receptor mediated, and a beta-FNA insensitive decrease in urine output at higher dose-levels, which may suggest a sigma receptor mediated effect.

Reversal by beta-funaltrexamine of the antinociceptive effect of opioid agonists in the rat.

The effect of the irreversible opioid receptor antagonist, beta-funaltrexamine (beta-FNA), on antinociception produced by mu- and kappa-receptor agonists was studied in the rat. beta-FNA, 20 to 80 mg kg-1, s.c., given 24 h before testing, produced a dose-related antagonism of the effects of morphine in the paw pressure, hotplate and tail-flick tests. Following the 80 mg kg-1 dose, the degree of antagonism of morphine was stable for up to 48 h after dosing, but was reduced by 5 days and had disappeared by 8 days. In the paw pressure test, beta-FNA, 40 mg kg-1, s.c., antagonized the effects of fentanyl, buprenorphine, tifluadom, ethylketocyclazocine and proxorphan; it was without effect against the highly selective kappa-agonist, U-50,488. In light of these results, the possible opioid receptor selectivities of both the agonists and beta-FNA are reassessed.

Alpha-adrenoceptor-mediated antinociception and sedation in the rat and dog.

The present study compared the potency of a range of alpha-adrenoceptor agonists in producing antinociception and sedation in the rat and dog. In the rat, the selective alpha 2-adrenoceptor agonists, guanabenz, UK 14304 and guanfacine, were more potent as sedative agents than as antinociceptive agents. For compounds which have similar activities at both alpha 1 and alpha 2-adrenoceptors, such as clonidine, alinidine, oxymetazoline and naphazoline, there was little separation between effective doses for antinociception and sedation. In marked contrast, the selective alpha 1-adrenoceptor agonists, ST 587 and methoxamine, were more potent as antinociceptive agents than as sedatives. Similarly, ICI 106,270 and CP 18,534, two agonists with a greater alpha 1 to alpha 2-adrenoceptor ratio than clonidine, were also more potent in antinociceptive tests than in sedative tests. In the conscious dog, clonidine was 8-10 times more potent than ICI 106,270 and CP 18,534 at increasing nociceptive thresholds to mild electrical stimulation of the toothpulp. At equi-antinociceptive doses, the ranked order of potency for inducing sedation was clonidine greater than or equal to ICI 106,270 greater than CP 18,534. Dose-related bradycardia was also induced by each of the three alpha-adrenoceptor agonists at antinociceptive doses. These data suggest that antinociceptive activity can probably be mediated by either alpha 1 or alpha 2-adrenoceptors, whereas sedation appears to be mediated solely by the alpha 2-subtype. Thus, it may be possible to separate the antinociceptive and sedative effects of sympathomimetic agents, but it is unlikely that these agents would be completely devoid of cardiovascular effects.

Antagonism of alpha-adrenoceptor agonist-induced antinociception in the rat.

The present study investigated the effects of WB4101, a selective alpha 1-adrenoceptor blocking agent, and idazoxan, a selective alpha 2-adrenoceptor blocking agent, on antinociception and sedation in the rat mediated by adrenoceptors. Selective alpha 1-adrenoceptor agonists, e.g. ST587 and methoxamine induced antinociception but only elicited slight sedation; their antinociceptive effects were antagonized by WB4101 but not by idazoxan. In contrast, the marked sedative and antinociceptive effects induced by the selective alpha 2-adrenoceptor agonist, UK 14,304, were attenuated by idazoxan, but were little affected by WB4101. The mixed alpha 1/alpha 2-adrenoceptor agonists, clonidine and ICI 106,270, had differing profiles with respect to their antagonist interactions; the antinociceptive and sedative effects induced by clonidine were antagonized by idazoxan, whereas the antinociceptive effects of ICI 106,270 were antagonized by WB4101. The slight sedative effects induced by ICI 106,270 were not attenuated by either WB4101 or idazoxan; therefore, these studies give no insight into the mechanism of action for sedation induced by ICI 106,270. These data suggest that antinociception may be mediated by either alpha 1 or alpha 2-adrenoceptors; whereas sedation is predominantly alpha 2-adrenoceptor mediated.

Effect of beta-funaltrexamine on opioid side-effects produced by morphine and U-50, 488H.

Pretreatment of rats with the irreversible mu-opioid receptor antagonist, beta-funaltrexamine (beta-FNA), 20-40 mg kg-1 s.c., produced a dose-related antagonism of the reduction in respiratory rate, gastrointestinal (GI) propulsion, rotarod reaction latencies and body temperature produced by morphine administration 24 h later, suggesting that these effects are mediated via mu-opioid receptors. The kappa-receptor agonist, U-50,488H, was without effect on respiratory rate at the doses tested, but produced hypothermia, sedation and low maximum inhibition of GI propulsion. These effects of U-50,488H were not blocked by beta-FNA suggesting that they are mediated via kappa-receptors.

Effects of opiates on urine output in the water-loaded rat and reversal by beta-funaltrexamine.

The effects of several mu and kappa opioid receptor agonists on urinary excretion were examined in the water-loaded rat. Mu agonists induced anti-diuresis whereas kappa agonists caused diuresis. Furthermore, high efficacy kappa agonists eg. U50, 488H and tifluadom, produced marked diuretic effects, whereas kappa partial agonists eg. nalorphine, produced low maximum diuresis. Compounds having activity at both mu and kappa receptors eg. ethylketocyclazocine(EKC) and MR2034, produced a biphasic effect, an initial anti-diuretic effect followed by a more sustained diuretic effect. The irreversible mu receptor antagonist, beta-funaltrexamine (beta-FNA) antagonized the anti-diuretic effects elicited by fentanyl, pentazocine and buprenorphine and the initial anti-diuretic effect induced by EKC but did not inhibit the diuretic effects induced by U50, 488H and EKC. Thus, the use of beta-FNA in the water-loaded rat provides us with a valuable in vivo test which differentiates both mixed and selective opioid receptor agonists.

The effects of a series of capsaicin analogues on nociception and body temperature in the rat.

A series of capsaicin analogues have been synthesized in an attempt to try to separate the antinociceptive properties of capsaicin from its hypothermic effects. In capsaicin analogues with an unbranched alkyl side-chain of varying length, maximum antinociceptive and hypothermic activity was achieved with a chain length of 8-9 C atoms. Decreasing the chain length decreased both parameters equally. Substitution in the aromatic ring abolished activity. Several compounds related to homovanilloyl dodecylamide, an analogue of capsaicin in which the acylamide linkage is reversed, produced some antinociceptive activity without concomitant hypothermia, but the maximum antinociceptive effect achieved was very small. In the present series of capsaicin analogues good antinociception without hypothermia was not found.

Antinociceptive profile of dynorphin in the rat.

Morphine, bremazocine, dynorphin(1-17) and two of its fragments (1-13) and (1-8) all increased nociceptive pressure thresholds in the rat after intracerebroventricular administration. Morphine also increased nociceptive heat thresholds in the hotplate test at similar dose-levels. In contrast, bremazocine and the dynorphins were totally inactive against the noxious heat stimulus. This profile of antinociceptive activity is typical of kappa receptor agonists.