Raquel E. Rodríguez

Neural contribution to the effect of glucagon-like peptide-1-(7—36) amide on arterial blood pressure in rats

This study was designed to determine the contribution of the central nervous system (CNS) to the effects of glucagon-like peptide-1-(7-36) amide (tGLP-1) on arterial blood pressure and heart rate in rats. Accordingly, intracerebroventricular administration of the peptide produced an increase in cardiovascular parameters, which was blocked by previous administration of exendin-(9-39) through the same route, but not when it was intravenously injected. Intravenous administration of tGLP-1 produced a significant increase in arterial blood pressure and heart rate, which was blocked by the previous intracerebroventricular or intravenous administration of exendin-(9-39). Bilateral vagotomy blocked the stimulating effect of intracerebroventricular tGLP-1 administration on arterial blood pressure and heart rate. Also, bilateral vagotomy prevented the blocking effect of intracerebroventricular but not of intravenous exendin-(9-39) on cardiovascular parameters after intravenous administration of tGLP-1. These findings suggest that the action of tGLP-1 on cardiovascular parameters is under a dual control generated in the CNS and in peripheral structures and that the neural information emerging in the brain is transmitted to the periphery through the vagus nerve.This study was designed to determine the contribution of the central nervous system (CNS) to the effects of glucagon-like peptide-1-(7-36) amide (tGLP-1) on arterial blood pressure and heart rate in rats. Accordingly, intracerebroventricular administration of the peptide produced an increase in cardiovascular parameters, which was blocked by previous administration of exendin-(9-39) through the same route, but not when it was intravenously injected. Intravenous administration of tGLP-1 produced a significant increase in arterial blood pressure and heart rate, which was blocked by the previous intracerebroventricular or intravenous administration of exendin-(9-39). Bilateral vagotomy blocked the stimulating effect of intracerebroventricular tGLP-1 administration on arterial blood pressure and heart rate. Also, bilateral vagotomy prevented the blocking effect of intracerebroventricular but not of intravenous exendin-(9-39) on cardiovascular parameters after intravenous administration of tGLP-1. These findings suggest that the action of tGLP-1 on cardiovascular parameters is under a dual control generated in the CNS and in peripheral structures and that the neural information emerging in the brain is transmitted to the periphery through the vagus nerve.

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.

Morphine Regulates Dopaminergic Neuron Differentiation via miR-133b

Morphine is one of the analgesics used most to treat chronic pain, although its long-term administration produces tolerance and dependence through neuronal plasticity. The ability of morphine to regulate neuron differentiation in vivo has been reported. However, the detailed mechanisms have not yet been elucidated because of the inability to separate maternal influences from embryonic events. Using zebrafish embryos as the model, we demonstrate that morphine decreases miR-133b expression, hence increasing the expression of its target, Pitx3, a transcription factor that activates tyrosine hydroxylase and dopamine transporter. Using a specific morpholino to knock down the zebrafish μ-opioid receptor (zfMOR) in the embryos and selective mitogen-activated protein kinase inhibitors, we demonstrate that the morphine-induced miR-133b decrease in zebrafish embryos is mediated by zfMOR activation of extracellular signal-regulated kinase 1/2. A parallel morphine-induced down-regulation of miR-133b was observed in the immature but not in mature rat hippocampal neurons. Our results indicate for the first time that zebrafish embryos express a functional μ-opioid receptor and that zebrafish serves as an excellent model to investigate the roles of microRNA in neuronal development affected by long-term morphine exposure.

COMT (Val158Met) polymorphism is not associated to neuropathic pain in a Spanish population.

It is well known that the response to painful stimuli varies between individuals and this could be consequence of individual differences to pain sensitivity that may be related to genetic factors. Catechol‐O‐methyltransferase (COMT) is one of the enzymes that metabolize catecholamine neurotransmitters. Differences in the activity of COMT influence the functions of these neurotransmitters. A single nucleotide polymorphism (Val158Met) of COMT leads to a three to four fold reduction in the activity of the enzyme and has been associated to modifications in the response to a pain stressor. Neuropathic pain is a progressive nervous system disease due to an alteration of the peripheral or central nervous system. To elucidate the possible role of COMT polymorphism in the susceptibility to neuropathic pain, we have performed a case‐control study in a Spanish population. Analysis of the (Val158Met) COMT polymorphism was performed by PCR amplification and DNA digestion with restriction enzymes. Our study concludes that functional Val158Met polymorphism of COMT gene is not associated to increased susceptibility to neuropathic pain.

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 receptors 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 ZFOR1, a putative delta-opioid receptor from the teleost zebrafish (Danio rerio)

ZFOR1 is a putative opioid receptor from zebrafish brain which has 66% homology with the mammalian delta-opioid receptor. When expressed in HEK293 cells ZFOR1 bound the non-selective opioid antagonist [(3)H]diprenorphine with high affinity. However, the binding of this ligand was not readily displaced by opioids selective for mu, delta or kappa opioid receptors (affinities>1000 nM). Rather non-selective ligands showed good affinity, as did the non-peptide delta-ligand BW373U86 (Ki 69 nM), the delta-antagonist naltrindole (Ki 28 nM) and the peptide beta-endorphin (Ki 37 nM). Agonist binding to the receptor encoded by ZFOR1 receptor stimulated the binding of [(35)S]GTPgammaS confirming coupling to G proteins. Study of the receptor should contribute to understanding of the evolution of the opioid system.

New kappa opioid receptor from zebrafish Danio rerio.

A cDNA that encodes a kappa opioid receptor like from zebrafish (ZFOR3) has been cloned and characterized. The encoded protein is 377 residues long and presents 70% identity with the mammalian kappa receptors, although less homology is found in the amino- and carboxyl-terminus as well as in the extracellular loops. In situ hybridization studies have revealed that ZFOR3 mRNA is highly expressed in particular brain areas that coincide with the expression of the kappa opioid receptor in other species. When ZFOR3 is stably expressed in HEK293 cells, [(3)H]-diprenorphine binds with high affinity (K(D)=1.05+/-0.26 nM), being this value on the same range as those reported for mammalian kappa opioid receptors. On the other hand, the selective agonist for mammalian kappa receptors U69,593 does not bind to ZFOR3. [(3)H]-diprenorphine binding is readily displaced by the peptidic ligand dynorphin A and by the non-endogenous compounds bremazocine, naloxone and morphine, although with different affinities. Our results demonstrate that ZFOR3 is a unique model to study the kappa opioid receptor functionality.

New glycosylpeptides with high antinociceptive activity.

The antinociceptive activity of two new synthetic glucoside and galactoside enkephalinamide analogues was studied. The effects produced by the new analogues were compared with those obtained with [D-Met2,Hyp5]enkephalinamide and with morphine. The analogues were injected into the fourth ventricle and intrathecally. Tail immersion and paw pressure behavioural tests were used to assess antinociception. One of the analogues studied, O1,5-[beta-D-galactopyranosyl] [D-Met2,Hyp5]enkephalinamide appears to be 57,000 times more potent than morphine.

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.

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.