Microanatomy of the male and female reproductive tracts in the long-tailed butterfly ray Gymnura poecilura, an elasmobranch with unusual characteristics.

The anatomy of the male and female reproductive systems was investigated in the long-tailed butterfly ray Gymnura poecilura using gross observation and light microscopy. The testes are highly asymmetrical, to the extent that only the left testis is functional and the right testis is completely absent. Both of the male genital ducts are present and symmetrical, although spermatozoa only occur in the left duct. The genital ducts are straight and unconvoluted, with regular incomplete internal partitions throughout. Females do not possess a right ovary, nor do the oviducal glands exhibit distinct club and papillary zones, and the baffle zone lacks baffle plates. In all sections of the gland, the tubules display different secretory activities depending on the proximity to the gland lumen. The gland produces a thin egg membrane that encases each egg individually, while the endometrium is formed into trophonemata.

Synthesis and muscarinic activity of quinuclidinyl- and (1-azanorbornyl)pyrazine derivatives.

The synthesis and cortical muscarinic activity of a novel series of pyrazine-based agonists is described. Quinuclidine and azanorbornane derivatives were prepared either by reaction of lithiated pyrazines with azabicyclic ketones, followed by chlorination and reduction, or by reaction of the lithium enolate of the azabicyclic ester with 2-chloropyrazines followed by ester hydrolysis and decarboxylation. Substitution at all three positions of the heteroaromatic ring has been explored. Optimal muscarinic agonist activity was observed for unsubstituted pyrazines in the azanorbornane series. The exo-1-azanorbornane 18a is one of the most efficacious and potent centrally active muscarinic agonists known. Studies on the 3-substituted derivatives have provided evidence of the preferred conformation of these ligands for optimal muscarinic activity. Substitution at C6 gave ligands with increased affinity and reduced efficacy. Moving the position of the diazine ring nitrogens to give pyrimidine and pyridazine derivatives resulted in a significant loss of muscarinic activity.

Synthesis and serotonergic activity of 5-(oxadiazolyl)tryptamines: potent agonists for 5-HT1D receptors.

The synthesis and 5-HT1D receptor activity of a novel series of 5-(oxadiazolyl)tryptamines is described. Modifications of the oxadiazole 3-substituent, length of the linking chain (n), and the amine substituents are explored and reveal a large binding pocket in the 5-HT1D receptor domain. Oxadiazole substituents such as benzyl are accommodated without loss of agonist potency or efficacy. The incorporation of polar functionality on a phenyl or benzyl spacer group results in a 10-fold increase in affinity and functional potency. Optimal 5-HT1D activity is observed when the heterocycle is conjugated with the indole and the benzyl sulfonamides 20t and 20u represent some of the most potent 5-HT1D agonists known. Replacement of O for S in the heterocycle leads to a further increase in potency. Deletion of oxadiazole N-2 does not reduce activity, suggesting the requirement for only one H-bond acceptor in this location. The selectivity of these compounds for 5-HT1D receptors over other serotonergic receptors is discussed. Sulfonamide 20t shows > or = 1000-fold selectivity for 5-HT1D over 5-HT2, 5-HT1C, and 5-HT3 receptors and 10-fold selectivity with respect to 5-HT1A receptors. The functional activity of this series of compounds is studied and demonstrates high 5-HT1D receptor potency and efficacy comparable to that of 5-HT.

Synthesis and serotonergic activity of N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl]ethylamine and analogues: potent agonists for 5-HT1D receptors.

The synthesis and the 5-HT receptor activity of a novel series of N,N-dimethyltryptamines substituted in the 5-position with an imidazole, triazole, or tetrazole ring are described. The objective of this work was to identify potent and selective 5-HT1D receptor agonists with high oral bioavailability and low central nervous system penetration. Compounds have been prepared in which the azole ring is attached through either nitrogen or carbon to the indole. Conjugated and methylene-bridged derivatives have been studied (n = 0 or 1). Substitution of the azole ring has been explored either alpha or beta to the point of attachment to indole. In a series of N-linked azoles (X = N), simple unsubstituted compounds have high affinity and selectivity for 5-HT1D receptors. It is proposed that for good affinity and selectivity a hydrogen bond acceptor interaction with the 5-HT1D receptor, through a beta-nitrogen in the azole ring, is required. In a series of C-linked triazoles and tetrazoles (X = C), optimal affinity and selectivity for the 5-HT1D receptor was observed when the azole ring is substituted at the 1-position with a methyl or ethyl group. This study has led to the discovery of the 1,2,4-triazole 10a (MK-462) as a potent and selective 5-HT1D receptor agonist which has high oral bioavailability and rapid oral absorption. The in vitro activity and the preliminary pharmacokinetics of compounds in this series are presented.

Synthesis and serotonergic activity of 3-[2-(pyrrolidin-1-yl)ethyl]indoles: potent agonists for the h5-HT1D receptor with high selectivity over the h5-HT1B receptor.

The design, synthesis, and biological evaluation of a novel series of 3-[2-(pyrrolidin-1-yl)ethyl]indoles with excellent selectivity for h5-HT1D (formerly 5-HT1Dalpha) receptors over h5-HT1B (formerly 5-HT1Dbeta) receptors are described. Clinically effective antimigraine drugs such as Sumatriptan show little selectivity between h5-HT1D and h5-HT1B receptors. The differential expression of h5-HT1D and h5-HT1B receptors in neural and vascular tissue prompted an investigation of whether a compound selective for the h5-HT1D subtype would have the same clinical efficacy but with reduced side effects. The pyrrolidine 3b was initially identified as having 9-fold selectivity for h5-HT1D over h5-HT1B receptors. Substitution of the pyrrolidine ring of 3b with methylbenzylamine groups gave compounds with nanomolar affinity for the h5-HT1D receptor and 100-fold selectivity with respect to h5-HT1B receptors. Modification of the indole 5-substituent led to the oxazolidinones 24a,b with up to 163-fold selectivity for the h5-HT1D subtype and improved selectivity over other serotonin receptors. The compounds were shown to be full agonists by measurement of agonist-induced [35S]GTPgammaS binding in CHO cells expressed with h5-HT receptors. This study suggests that the h5-HT1D and h5-HT1B receptors can be differentiated by appropriate substitution of the ligand in the region which binds to the aspartate residue and reveals a large binding pocket in the h5-HT1D receptor domain which is absent for the h5-HT1B receptor. The compounds described herein will be important tools to delineate the role of h5-HT1D receptors in migraine.

3-(Piperazinylpropyl)indoles: selective, orally bioavailable h5-HT1D receptor agonists as potential antimigraine agents.

Clinically effective antimigraine drugs such as Sumatriptan have similar affinity at h5-HT1D and h5-HT1B receptors. In the search for a h5-HT1D-selective agonist as an antimigraine agent, a novel series of 3-(propylpiperazinyl)indoles have been synthesized and evaluated at h5-HT1D and h5-HT1B receptors. This class of compounds has provided subnanomolar, fully efficacious h5-HT1D agonists with up to 200-fold selectivity for the h5-HT1D receptor over the h5-HT1B receptor. Unlike other h5-HT1D-selective series, several propylpiperazines demonstrate good oral bioavailability. The optimum compound was 1-(3-[5-(1,2, 4-triazol-4-yl)-1H-indol-3-yl]propyl)-4-(2-(3-fluorophenyl)ethyl)p ipe razine (7f) which has excellent selectivity for h5-HT1D receptors over other 5-HT receptor subtypes and good oral bioavailability in three species. Compound 7f has been selected for further investigation as a potential development candidate in the treatment of migraine.

The roles of spinal adenosine receptors in the control of acute and more persistent nociceptive responses of dorsal horn neurones in the anaesthetized rat.

1. We describe here the effects of intrathecal selective adenosine receptor agonists on acute and more persistent evoked responses of dorsal horn nociceptive neurones recorded in intact rats anaesthetized with halothane. 2. The effects of the A1 receptor agonist, N6-cyclopentyladenosine and the non-selective agonist 2-chloroadenosine as well as the A2a receptor agonist, 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride were gauged on the C-, A delta-, A beta-fibre, post-discharge and wind-up responses produced by peripheral tanscutaneous stimulation. The antagonists, theophylline and 8(p-sulphophenyl) theophylline were also tested alone and to reverse the agonist effects. 3. Subcutaneous formalin (5%) was used to produce a more prolonged nociceptive response initiated by peripheral inflammation. 4. Both N6-cyclopentyladenosine and 2-chloroadenosine produced inhibitions of the C-fibre evoked responses, wind-up and post-discharge of the neurones with no significant effects on the A beta responses. By contrast, the A delta evoked responses were facilitated over the same time course and dose-range as the inhibitions. N6-cyclopentyladenosine was more potent and effective than 2-chloroadenosine. In marked contrast to these agonists, the A2a agonist produced only weak non-specific inhibitions. Theophylline and 8(p-sulphophenyl) theophylline alone had no effect on the acute responses but prevented or reversed inhibitory effects of N6-cyclopentyladenosine. 5. The formalin response was markedly inhibited by spinal N6-cyclopentyladenosine with both the acute first phase and more prolonged second phase being dose-dependently inhibited. N6-cyclopentyladenosine was considerably more potent on the formalin response than on the other neuronal measures. 6 The results suggest a role of adenosine Al receptors in the modulation of both acute and inflammatory nociception in the spinal cord.

L-694,247: a potent 5-HT1D receptor agonist.

1. The 5-hydroxytryptamine (5-HT) receptor binding selectivity profile of a novel, potent 5-HT1D receptor agonist, L-694,247 (2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl ]- 1H-indole-3-yl]ethylamine) was assessed and compared with that of the 5-HT1-like receptor agonist, sumatriptan. 2. L-694,247 had an affinity (pIC50) of 10.03 at the 5-HT1D binding site and 9.08 at the 5-HT1B binding site (sumatriptan: pIC50 values 8.22 and 5.94 respectively). L-694,247 retained good selectivity with respect to the 5-HT1A binding site (pIC50 = 8.64), the 5-HT1C binding site (6.42), the 5-HT2 binding site (6.50) and the 5-HT1E binding site (5.66). The pIC50 values for sumatriptan at these radioligand binding sites were 6.14, 5.0, < 5.0 and 5.64 respectively. Both L-694,247 and sumatriptan were essentially inactive at the 5-HT3 recognition site. 3. L-694,247, like sumatriptan, displayed a similar efficacy to 5-HT in inhibiting forskolin-stimulated adenylyl cyclase in guinea-pig substantia nigra although L-694,247 (pEC50 = 9.1) was more potent than sumatriptan (6.2) in this 5-HT1D receptor mediated functional response. L-694,247 (pEC50 = 9.4) was also more potent than sumatriptan (6.5) in a second 5-HT1D receptor mediated functional response, the inhibition of K(+)-evoked [3H]-5-HT release from guinea-pig frontal cortex slices. 4. The excellent agreement observed for L-694,247 between the 5-HTlD radioligand binding affinity and the functional potency confirm that the two functional models (the inhibition of forskolin-stimulated adenylyl cyclase in guinea-pig substantia nigra and the inhibition of K+-evoked [3H]-5-HT release from guinea-pig frontal cortex) do indeed reflect 5-HTID-mediated events.5. L-694,247 is a novel, highly potent 5-HTID/5-HTIB receptor ligand which should prove useful for the exploration of the physiological role of these receptors in animals.

Intrathecally administered endotoxin or cytokines produce allodynia, hyperalgesia and changes in spinal cord neuronal responses to nociceptive stimuli in the rat.

Inflammatory processes occurring within the central nervous system (CNS) can produce 'illness induced behaviours' which include fever, sleep and the development of allodynia and hyperalgesia. Here we demonstrate the effects of the pro-inflammatory mediators, bacterial endotoxin, and rat recombinant interleukin 1 beta (rrIL-1 beta) or tumour necrosis factor-alpha (rrTNF alpha) on the integration of somatosensory information at the single neuronal level, via recordings from wide-dynamic range neurones in the dorsal horn of the spinal cord in anaesthetized rats. Intrathecal administration of E. coli lipopolysaccharide (LPS, 10 and 100 microg, i.t.) enhanced the activity of dorsal horn neurones, including facilitation of neuronal post-discharge. Intrathecal administration of IL-1 beta (5-5000 pg) or TNF-alpha (5-5000 pg) enhanced dorsal horn neuronal responses, including the acute responses to C-fibre stimulation, wind-up and post-discharge, however, the effects of IL-1 beta were more robust than those of TNF alpha. Intrathecal administration of IL-1 beta (1-1000 pg) also leads to the development of mechanical allodynia and hyperalgesia. On the other hand intrathecal application of TNF alpha did not produce changes in sensitivity to mechanical stimuli. Changes in the activity of spinal wide-dynamic range neurones induced by local inflammation may provide a pathomechanism for the clinical pathology of central pain syndrome, which can accompany CNS disease or acute CNS injury.

Electrophysiological study on spinal antinociceptive interactions between adenosine and morphine in the dorsal horn of the rat.

The effects of intrathecal N6-cyclopentyladenosine (N6-CPA), an A1-receptor agonist, and morphine, a mu opioid-receptor agonist were studied on the electrically evoked responses of deep dorsal horn neurones in the rat. Both caused inhibitions, when applied alone, of the C-fibre evoked response and post-discharges. Enhanced inhibitory effects were observed when N6-CPA (5 micrograms) was given with morphine (1 microgram). Increasing the dose of morphine (5 micrograms) with the same dose of N6-CPA occluded the expected inhibitions. By contrast, the A delta-fibre evoked response, inhibited by morphine alone, was significantly facilitated by the combination. Parallel activation of purinergic and opioid inhibitory systems within the dorsal horn as well as complex interactions between them are likely.

Excitatory effects of galanin in the spinal cord of intact, anaesthetized rats.

The effects of intrathecal galanin on the activity of single wide dynamic range (WDR) spinal dorsal horn neurones were analyzed in anaesthetized adult rats. Abeta-, Adelta- and C-fibres were activated by transcutaneous electrical stimulation which also induced wind-up and post-discharge. Galanin dose-dependently (0. 15-15 nmol/50 microl) enhanced Adelta-and C-fibre evoked responses, post-discharge and wind-up. Application of (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15), a putative antagonist, did not attenuate the enhanced effects evoked by galanin. (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15) applied alone (0.3-30 nmol/50 microl) to a separate population of neurones inhibited Abeta responses but enhanced post-discharge and wind-up in a similar pattern to galanin. Thus, (D-Thr(6), D-Trp(8, 9), 15-ol)-galanin (1-15) behaved as an agonist in the present experiments. In conclusion, in the spinal cord of the anaesthetized rat, galanin has a purely pro-nociceptive role, since it enhanced responses evoked by high-threshold fibre stimulation, along with post-discharge and wind-up of WDR neurones.

Chronic secondary hypersensitivity of dorsal horn neurones following inflammation of the knee joint.

Intra-articular injection of Freund's complete adjuvant (FCA) into the rat knee joint produces a swelling of the joint and long lasting hypersensitivity. In this study we have used this model and in vivo electrophysiology to investigate the time course of spinal changes underlying chronic secondary hypersensitivity, by stimulating the ankle joint (an area outside the site of primary hypersensitivity), and have compared the results with behavioural data from the same population of animals at 4-8, 13-17 and 55-59 days following FCA injection. The magnitude of responses and the proportion of dorsal horn neurones receiving inputs from A beta- A delta- and C-fibre afferents were monitored. At all time points, there was a significant increase in the ongoing activity of deep dorsal horn neurones when compared to nai ve rats, correlating well with the behavioural hypersensitivity. Both the magnitude of neuronal responses, and the proportion of neurones responding to electrical or mechanical stimulation in an area of secondary hypersensitivity, were significantly increased 4-8 and 13-17 days following FCA injection. However, while there was still behavioural hypersensitivity at 55-59 days there was a substantial decline in the responses to mechanical stimulation and A-fibre responses to electrical stimulation, although the proportion of neurones responding in the C-fibre latency remained elevated. These results suggest that the behavioural hypersensitivity is due to hyperexcitability at the level of the dorsal horn reflected as an increase of both C-fibre responses and spontaneous activity.

Spinal effects of bicuculline: modulation of an allodynia-like state by an A1-receptor agonist, morphine, and an NMDA-receptor antagonist.

Single-unit recordings were made in the intact anesthetized rat of the responses of dorsal horn neurons to C-, Adelta-, and Abeta-fiber stimulation. The postdischarge and windup responses of the same cells along with responses to innocuous stimuli, prod and brush, also were measured. The effects of (-)-bicuculline-methobromide (0.5, 5, 50, and 250 microg) were observed on these neuronal responses. The C- and Adelta-fiber-evoked responses were facilitated significantly in a dose-dependent manner. The input was facilitated, but as the final overall response was not increased by the same factor, windup appeared to be reduced. However, postdischarge, resulting from the increase in the excitability produced by windup, tended to be facilitated. After doses of >/=5 microg bicuculline, stimulation at suprathreshold Abeta-fiber-evoked activity caused enhanced firing, mainly at later latencies corresponding to Adelta-fiber-evoked activity in normal animals. Few cells responded consistently to brush and so no significant change was observed. Responses evoked by innocuous pressure (prod) always were observed in cells that concurrently responded to electrical stimulation with a C-fiber response. This tactile response was facilitated significantly by bicuculline. The effects of N6-cyclopentyladenosine (N6-CPA), an adenosine A1-receptor agonist, was observed after pretreatment with 50 microg bicuculline, as were the effects of morphine and 7-chlorokynurenate (7-CK). N6-CPA inhibited prod, C- and Adelta-fiber-evoked responses as well as the initial and overall final response to the train of C-fiber strength stimuli. Inhibitions were reversed with 8(p-sulphophenyl) theophylline. Morphine, the mu-receptor agonist, also inhibited the postbicuculline responses to prod, C-, and Adelta-fiber responses and initial and final responses to a train of stimuli. Inhibitory effects of morphine were reversed partly by naloxone. 7-CK, an antagonist at the glycine site on the N-methyl-D-aspartate-receptor complex, inhibited the responses to C- and Adelta-fiber-evoked activity as well as prod. The postdischarges were inhibited by this drug. Again both the initial and overall responses of the cell were inhibited. To conclude, bicuculline caused an increase in the responses of deep dorsal horn cells to prod, Adelta-fiber-evoked activity, increased C-fiber input onto these cells along with the appearance of responses at latencies normally associated with Adelta fibers, but evoked by suprathreshold Abeta-fiber stimulation. These alterations may be responsible for some aspects of the clinical phenomenon of allodynia and hyperalgesia. These altered and enhanced responses were modulated by the three separate classes of drugs, the order of effectiveness being 7-CK, N6-CPA, and then morphine.