Monogenoideans (Platyhelminthes) from the gill lamellae of the spotted sea trout, Cynoscion nebulosus (Perciformes, Sciaenidae), from the western coast of the Yucatan Peninsula, Mexico, with redescription of Diplectanum bilobatus Hargis 1955 (Diplectanidae).

The gill lamellae ectoparasites of the spotted sea trout, Cynoscion nebulosus (Sciaenidae), in the western coast of the Yucatán Peninsula, Mexico, revealed three species of Monogenoidea: Cynoscionicola heteracantha (Manter 1938) Price, 1962 (Microcotylidae); Choricotyle cynoscioni (MacCallum, 1917) Llewellyn, 1941 (Diclidophoridae); and Diplectanum bilobatus Hargis, 1955 (Diplectanidae). Brief comments about the current taxonomic status as well as supplemental observations of all these monogenoids originally described and/or reported from the same host fish species found in the USA are provided. New illustrations, prevalence and mean intensity of infection, as well morphological and biometric data based on new specimens are shown. C. heteracantha and C. cynoscioni collected in this study represent the second and first records of the species of these genera for the Atlantic coast of Mexico. The specimens of D. bilobatus are provisionally retained within Diplectanum until an emendation of the genus and a formal revision of all named species of this monogenoidean genus are undertaken.

Drug-induced change in the distribution of sulfonamides in the mother rat and its fetus.

The distribution of a highly bound antibacterial sulfonamide was markedly altered in both the mother rat and its fetus by interfering with the binding of this drug to plasma protein in the mother. This effect was due to binding displacement, since the displacing agent had little or no effect on the distribution of another sulfonamide with very low binding to plasma protein.

Developmental expression and distribution of opioid receptors in zebrafish.

Zebrafish is a novel experimental model that has been used in developmental studies as well as in the study of pathological processes involved in human diseases. It has been demonstrated that the endogenous opioid system is involved in developmental mechanisms. We have studied the relationship between the different embryonic stages and opioid receptor expression for the four known opioid receptors in zebrafish (mu, delta 1, delta 2 and kappa). The mu opioid receptor is detected at higher levels than the other opioid receptors before the midblastula transition and during the segmentation period. The delta duplicate 2 exhibits only one peak of expression at 21 h postfertilization (hpf), when the motor nervous system is forming. The kappa receptor is expressed at very low levels. In situ hybridization studies at 24 hpf show that the opioid receptors are widely distributed in zebrafish CNS and at 48 hpf their localization is detected in more defined structures. Our results support specific implications of the opioid receptors in developmental processes such as morphogenesis of the CNS, neurogenesis, neuroprotection and development of neuromuscular and digestive system. Pain-related alterations can be a consequence of changes in the endogenous opioid system during development, hence we provide important information that might help to solve pain-related pathological situations.

Characterization of two duplicate zebrafish Cb2-like cannabinoid receptors.

Several cannabinoid receptors have been detected in many organisms. The best known are CB1, mainly expressed in the central nervous system and CB2 which is almost exclusively expressed in the periphery. Here we report the molecular characterization of two duplicate CB2-like cannabinoid receptors from zebrafish (Danio rerio) (zebrafish Cb2a and zebrafish Cb2b). The amino acid sequences of these receptors present 56% identity with Takifugu rubripes CB2 sequence and 39% with human CB2 sequence and conserve some specific key residues for cannabinoid receptor function. Both duplicate receptors are expressed in peripheral tissues (gills, heart, intestine and muscle), immune tissue (spleen) and also in the central nervous system. Using in situ hybridization techniques zebrafish Cb2 mRNA expression was observed for the first time in the adenohypophysial cells of the rostral pars distalis and proximal pars distalis of the pituitary gland. Given the importance of the existence of duplication of genes in teleosts, the combined analysis of these two new cannabinoid receptors opens a new exciting door to investigate and understand cannabinoid function throughout evolution.

Identification of dynorphin a from zebrafish: a comparative study with mammalian dynorphin A.

We report the cloning and molecular characterization of the zfPDYN. The complete open reading frame for this propeptide is comprised in two exons that are localized on chromosome 23. zfPDYN cDNA codes for a polypeptide of 252 amino acids that contains the consensus sequences for four opioid peptides: an Ile-enkephalin, the neo-endorphins, dynorphin A and dynorphin B. Upon comparison between zebrafish (zfDYN A) and mammalian dynorphin A (mDYN A) it has been stated that these two peptides only differ in two amino acids: the Leu(5) is replaced by Met(5) and the Lys(13) by Arg(13). Taking into consideration that mDYN A is able to bind to the three mammalian opioid receptors, we have compared the pharmacological profile of zfDYN A and mDYN A on the zebrafish opioid receptors. By means of radioligand binding techniques, we have established that these two dynorphins bind and activate all of the cloned opioid receptors from zebrafish (delta-, mu- and kappa-like), although with different affinities. zfDYN A and mDYN A displace [(3)H]-diprenorphine binding with K(i) values on the nanomolar range, showing greater affinity for zebrafish opioid receptor (ZFOR) 3 (kappa) receptor. ZFOR1 (delta) and ZFOR4 (delta) present higher affinity for zfDYN A than for mDYN A, while the opposing behavior is observed in ZFOR2 (mu). Functional [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) stimulation experiments indicate that these two peptides fully activate the zebrafish opioid receptors, although the mean effective dose (EC(50)) values obtained for ZFOR2 and ZFOR3 receptors are lower than those seen for ZFOR1 and ZFOR4. A comparative study indicates that mammalian and zebrafish opioid receptors might bind their corresponding dynorphin A in a similar fashion, hence suggesting an important role of the opioid system through the vertebrate evolution.

Genomic characterization of the human trkC gene.

The trkC gene encodes the high-affinity receptor for neurotrophin 3 and plays an important role in the regulation of the survival and differentiation of the mammalian nervous system and in heart development. Chromosomal rearrangements of trkC have been recently reported in congenital fibrosarcoma and it has been proposed that abnormal activation of this gene might be involved in tumor development. To facilitate the search for new mutations and rearrangements in the human trkC locus we have partially characterized its genomic organization by restriction mapping and have obtained the complete intron-exon structure. Our results show that human trkC consists of 20 exons, including two that encode the inserts present in the extracellular and tyrosine kinase domains, and another two that encode the carboxyl-terminal tail of the truncated TRKC isoform. Analysis of the 5' flanking region revealed the absence of TATA box, a very high content in C/G compatible with a CpG island and the presence of putative binding sites for the AP1, AP2, GC, ATF, BRN2, AML1 and Nkx2.5 transcription factors.

Cloning and characterization of a full-length pronociceptin in zebrafish: evidence of the existence of two different nociceptin sequences in the same precursor.

We report the cloning and molecular characterization of a zebrafish pronociceptin gene, which is expressed in brain and intestine. This fish precursor codes for two different nociceptin peptides, one of which presents an opioid sequence in its N-terminus, being more similar to dynorphin A than its mammalian counterparts. Our results represent a new tool to investigate the function of nociceptin and its receptor in relation to pain and drug addiction.

Ontogeny of pulsatile secretion of gonadotropin-releasing hormone in the bull calf during infantile and pubertal development.

During the infantile period of development in the bull calf (birth to 6 weeks of age), there is a virtual absence of episodic secretion of LH. Transition from infancy to the prepubertal period (6-10 weeks of age) is characterized by the onset of episodic LH release. This study was conducted to characterize the ontogeny of episodic GnRH release during these developmental periods. During the primary experiment, calves at 2, 5, 8, and 12 weeks of age (n = 4/age) were surgically fitted with cannulae for the collection of mixed hypophyseal portal and cavernous sinus blood. Hypophyseal portal and cavernous sinus and jugular blood samples were collected over a 9- to 12-h period at 10 min intervals. No pulses of LH were observed in calves at 2 or 5 weeks of age. At 8 and 12 weeks of age, pulsatile LH release became evident with a mean of 1.0 +/- 0.3 and 2.20 +/- 0.7 pulses/10 h, respectively. Unlike LH secretion, calves at both 2 and 5 weeks of age released GnRH in a pulsatile manner (3.5 +/- 0.2 and 5.0 +/- 0.6 pulses/10 h, respectively). The frequency of pulsatile GnRH release increased from 7.9 +/- 0.4 pulses/10 h at 8 weeks of age to 8.9 +/- 0.7 pulses/10 h at 12 weeks of age. These findings demonstrate the presence of pulsatile secretion of GnRH during the infantile period of development. Furthermore, the postnatal ontogeny of pulsatile LH release in this species is associated with an increase in the frequency of pulsatile GnRH secretion.

In vivo release of CCK-8 from the dorsal horn of the rat: inhibition by DAGOL.

There is evidence that CCK-8 may interact with opioids and that both systems are probably implicated in pain modulation. In order to elucidate this relationship we sought to examine factors governing the movement of CCK-8 from the spinal cord into the extracellular space. We report that CCK-8 like immunoreactivity, as measured by RIA, is released from the spinal cord of the rat in vivo, following potassium stimulation and by direct activation of high threshold peripheral afferents by stimulation of the sciatic nerve. Also, we show that CCK-8 release is inhibited by the mu-selective opioid receptor agonist DAGOL. Naloxone totally reversed the effect produced by DAGOL, implying an opiate mediated mechanism.

Expression of ZFOR1, a delta-opioid receptor, in the central nervous system of the zebrafish (Danio rerio).

Opioid receptors, besides mediating the effects of analgesic compounds, are involved in drug addiction. Although a large amount of work has been done studying these receptors in mammals, little information has been obtained from nonmammalian vertebrates. We have studied the regional distribution in the central nervous system (CNS) of the zebrafish of the recently cloned delta-opioid receptor homologue ZFOR1 using nonradioactive in situ hybridization. Our findings show that different nuclei within the main subdivisions of the brain displayed specific mRNA signal. The expression is widespread throughout the brain, but only specific cells within each nucleus displayed ZFOR1. Stained cells were abundant in the telencephalon, both in the olfactory bulb and telencephalic hemispheres, and in the diencephalon, where expression was observed in all the different subdivisions. In the mesencephalon, expression of ZFOR1 was abundant in the periventricular layer of the optic tectum. In the cerebellum, expression of ZFOR1 was detected in valvula cerebelli, corpus cerebelli, and lobus vestibulolateralis in both granule and Purkinje cells. In the myelencephalon, cells expressing ZFOR1 were also distributed in the octavolateralis area, the reticular formation, and the raphe nuclei, among others. Also, ZFOR1 was detected in cells of the dorsal and ventral horn of the spinal cord. This work presents the first detailed distribution of a delta-opioid receptor in the CNS of zebrafish. Distribution of ZFOR1 expression is compared with that of the delta-opioid receptor described in mammals.

Transient expression pattern of prolactin in Sparus aurata.

Using reverse transcription-polymerase chain reaction and in situ hybridization, the expression of the prolactin (PRL) gene was determined during development in gilthead sea bream (Sparus aurata) for the first time. The mRNA for PRL was detected from the second day of the larval stage onwards. This transcript was also located in the adenohypophysial cells, starting at four days post-hatching and was found to be pituitary-specific. Moreover, the possible involvement of PRL in asynchronous growth in the cultivation of gilthead sea bream was also examined. No differences in the distribution of PRL cells were observed in the three sizes of juvenile gilthead sea bream studied. These results suggest that the transcription of PRL is involved in the early development stages of sea bream and that the asynchronous growth-related changes are not due to distinct distribution of PRL cells.

The behavioural effects of intrathecally administered [D-PRO2, D-TRP7,9]-substance P, an analogue with presumed antagonist actions, in the rat.

[D-Pro2, D-Trp7,9]-substance P, an analogue of the putative nociceptive primary afferent neurotransmitter substance P, was administered to rats via chronically implanted intrathecal catheters (0.5-2.5 nmol per rat). Several animals showed large elevations of thermal and mechanical nociceptive thresholds, but these effects were always accompanied by profound, often long-lasting, impairments of motor function and thus cannot be regarded as a specific antinociceptive effect of the drug. These behavioural effects are considered to be probably due to direct spinal actions of the drug rather than antagonism of spinal substance P systems.

kappa-Opioid agonists produce antinociception after i.v. and i.c.v. but not intrathecal administration in the rat.

1. Nociceptive thresholds to noxious mechanical (paw pressure) and thermal (tail flick) stimuli were recorded in conscious rats. The effects of three selective kappa-opioid receptor agonists on the responses to these stimuli were determined following intravenous, intracerebroventricular or intrathecal administration. Results were compared with those obtained with morphine. 2. Following intravenous administration PD117302, U50488, U69593 and morphine produced steep parallel dose-response curves indicating antinociceptive activity when evaluated in the paw pressure test. When U50488 and U69593 were tested at a single dose of 3.3 mg kg-1 no effect was seen in the tail flick test. 3. When given by the intrathecal route only morphine was effective at increasing the nociceptive threshold. PD117302, U50488 and U69593 were without effect in either the paw pressure or tail flick tests when tested at doses up to 100 micrograms per rat. PD117302 caused flaccid paralysis of the hindlimbs following intrathecal administration at the top dose tested. This effect was not reversible by naloxone. 4. All three kappa-opioid receptor agonists produced naloxone-reversible antinociception in the paw pressure test, and to a lesser extent in the tail flick test, when injected directly into the third cerebral ventricle with the maximum effect occurring between 5 and 10 min after administration and declining back to control levels by 60 min. Morphine had a much slower onset of action with the peak effect being observed 30 min after dosing. 5. It is concluded that, under our experimental conditions in the rat, the antinociceptive effects of kappa-agonists are likely to be operated via an action at a supraspinal rather than a spinal site.

Delta-opioid receptor binding sites in rodent spinal cord.

1. The delta-opioid receptor agonist [D-Pen2,D-Pen5]enkephalin showed an antinociceptive effect in the mouse tail-flick test, following intrathecal administration. This action was reversed by naloxone and by the selective delta-opioid receptor antagonist ICI 174864. 2. High affinity, saturable binding of [3H]-[D-Pen2,D-Pen5]enkephalin has been demonstrated in spinal cord homogenates from guinea-pig, hamster, rat and both adult and young (18-20 g) mice. The binding was shown by autoradiography to be concentrated in the superficial laminae of the dorsal horn. 3. Competition studies confirmed that the binding of [3H]-[D-Pen2,D-Pen5]enkephalin was to the delta-opioid site. However, anomalies were seen with displacement assays using mu-ligands, which may suggest some common high affinity site for delta- and mu-opioid receptor agonists in the spinal cord. 4. The results add further evidence for a role of the delta-opioid receptor in spinally-mediated antinociception.

ZFOR2, a new opioid receptor-like gene from the teleost zebrafish (Danio rerio).

A new opioid receptor-like (ZFOR2) has been cloned and characterized in an anamniote vertebrate, the teleost zebrafish (Danio rerio). ZFOR2 encodes a 384-amino-acid protein with seven potential transmembrane domains, and its predicted amino acid sequence presents an overall 74% degree of identity to mammalian mu opioid receptors. Its inclusion in a dendrogram generated from the alignment of the opioid receptor's protein sequences, confirms its classification as a mu opioid receptor. Divergences in sequence are greater in the regions corresponding to extracellular loops, suggesting possible differences in ligand selectivity with respect to the classical mu opioid receptors. The genomic structure of ZFOR2 is also highly conserved throughout the phylogenetic scale, supporting the origin of opioid receptors early in evolution. Nevertheless, ZFOR2 lacks the fourth exon found in human and rodent mu opioid receptors, that is known to be involved in desensibilization and internalization processes.

Characterization of zebrafish proenkephalin reveals novel opioid sequences.

Cloning and molecular characterization of an homologous gene to proenkephalin in a teleost, the zebrafish Danio rerio is presented in this paper. The new zebrafish proenkephalin (zfPENK) encodes a 249 amino acid polypeptide that displays an identity of 40% to mammalian PENKs and which contains the consensus sequences for four Met-enkephalins, one Leu-enkephalin and one Met-enkephalin-Gly-Tyr, described for the first time in teleosts. Expression studies indicate that zfPENK is selectively expressed in zebrafish brain. Our findings support the concept that PENK genes might have been highly conserved throughout evolution and that its origins might be placed more than 400 million years ago. Moreover, we present evidence that the heptapeptide Met-enkephalin-Gly-Tyr present in fish might be anterior in evolution to the heptapeptide Met-enkephalin-Arg-Phe present in tetrapods. Also another homologous sequence to proenkephalin in zebrafish genome is presented. This sequence might stand for the third exon of a possible duplicate gene of zfPENK. Our findings not only present new data in relation to the evolution of opioid peptides in vertebrates, but also we present a new heptapeptide with putative differential activity than the other peptides derived from the mammalian proenkephalins. Future research will define the functional role of this new heptapeptide in the mechanism that describes opioid activity.

Antinociceptive activity of glycosidic enkephalin analogues.

The antinociceptive activity of two new enkephalin analogues: N1.5-(beta-D-glucopyranosyl)[D-Met2, Pro5]enkephalinamide and N1.5-(beta-D-galactopyranosyl)[D-Met2, Pro5]enkephalinamide was assessed using the tail immersion and paw pressure behavioural tests. Both enkephalin analogues appear to be more active than morphine when injected either into the fourth ventricle or intrathecally; the galactose analogue is more than 5000 times more active than morphine when injected into the fourth ventricle. The analgesic effects produced by the analogues are partially reversed by SC naloxone (0.1 mg/kg) and totally reversed when the dose of naloxone used was 1 mg/kg, suggesting that the analogues act upon more than one type of opiate receptor (mu/delta).

Interactions of exendin-(9-39) with the effects of glucagon-like peptide-1-(7-36) amide and of exendin-4 on arterial blood pressure and heart rate in rats.

This study was designed to determine the interactions of peptide exendin-(9-39) with the effect of glucagon-like peptide-1-(7-36) (GLP-1 (7-36)) amide and of exendin-4 on arterial blood pressure and heart rate in the rat. Both GLP-1 (7-36) amide and exendin-4 produced a dose-dependent increase in systolic, diastolic and mean arterial blood pressure, as well as in heart rate, although the effect of exendin-4 was more prolonged. These data indicate a longer functional half-life in vivo for exendin-4 as compared to GLP-1 (7-36) amide, which may have therapeutical applications. The antagonist effect of exendin-(9-39) on these cardiovascular parameters was also tested with 3000 ng of exendin-(9-39) intravenously administered 5 min before i.v. injection of 10 ng of either GLP-1 (7-36) amide or exendin-4. Under these experimental conditions the effect of the latter two peptides on arterial blood pressure and heart rate was blocked. By contrast, single administration of exendin-(9-39) did not modify cardiovascular parameters. These findings indicate that exendin-4 is an agonist and that exendin-(9-39) is an antagonist of the action of GLP-1 (7-36) amide on arterial blood pressure and heart rate. Therefore, the action of GLP-1 (7-36) amide on these parameters seems to be mediated through its own receptors.

Intrathecal administration of 5,6-DHT or 5,7-DHT reduces morphine and substance P-antinociceptive activity in the rat.

The administration of SP (15 and 50 ug) and morphine sulphate (10 ug) either into the IV ventricle or intrathecally produces an analgesic effect. This effect was attenuated when the rats received previously an intrathecal dose of 5,6-DHT or 5,7-DHT. The consequences of neurotoxin administration upon monoamine descending systems were evaluated by measuring 14C-5HT and 3H-NA synaptosomal uptake in different structures of the CNS. SP levels were also determined in the animals injected with the neurotoxins. Our results confirm the existence of a relationship injected with the neurotoxins. Our results confirm the existence of a relationship between the 5HT and SP descending systems, which control the nociceptive information at the level of the dorsal horn of the spinal cord.

CCK 8 analgesia and hyperalgesia after intrathecal administration in the rat: comparison with CCK-related peptides.

The C-terminal octapeptide of cholecystokinin (CCK 8) was administered intrathecally to rats. Doses in the nanogram range produced weak but significant antinociception in the paw pressure test five minutes after injection whereas microgram doses of CCK 8 produced hyperalgesia. The CCK 8-induced analgesia or hyperalgesia was not seen in the tail flick test and was not associated with motor incapacitation or any other noticeable side effects. The C-terminal tetrapeptide of CCK (CCK 4) and pentagastrin were found to be ineffective in all tests but caerulein and molluscan cardioexcitatory neuropeptide (FMRF-amide), like CCK 8, produced antinociception in the paw pressure test.