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I Kitchen

Publications and source records attributed to I Kitchen.

At least 37 records · Page 2Linked to original sources
PubMed1998

Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal.

***micro***-, delta- and kappa-opioid receptors are widely expressed in the central nervous system where they mediate the strong analgesic and mood-altering actions of opioids, and modulate numerous endogenous functions. To investigate the contribution of the kappa-opioid receptor (KOR) to opioid function in vivo, we have generated KOR-deficient mice by gene targeting. We show that absence of KOR does not modify expression of the other components of the opioid system, and behavioural tests indicate that spontaneous activity is not altered in mutant mice. The analysis of responses to various nociceptive stimuli suggests that the KOR gene product is implicated in the perception of visceral chemical pain. We further demonstrate that KOR is critical to mediate the hypolocomotor, analgesic and aversive actions of the prototypic kappa-agonist U-50, 488H. Finally, our results indicate that this receptor does not contribute to morphine analgesia and reward, but participates in the expression of morphine abstinence. Together, our data demonstrate that the KOR-encoded receptor plays a modulatory role in specific aspects of opioid function.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1998

The effect of acute kainic acid treatment on dopamine D2 receptors in rat brain.

Acute exposure to kainic acid (KA) induces neurochemical changes in dopaminergic systems in the brain and the aim of the present study was to investigate the acute toxicity of KA upon dopamine D2 receptors. Adult rats were injected intraperitoneally with either saline or 16 mg/kg KA. Brains were removed after 4 h. Membrane homogenates were prepared from seven brain regions and in addition, frozen coronal sections were sectioned for comparative quantitative autoradiographic analysis. Dopamine D2 receptors were characterised by saturation studies using [125I]iodosulpiride, [3H]raclopride and [3H]spiperone. KA produced a 2-fold decrease in receptor affinity for [125I]iodosulpiride and a 2-fold increase in receptor density in all regions studied except striatum. Quantitative autoradiography with [125I]iodosulpiride showed similar increases in D2 labelling following KA except in caudate putamen, nucleus accumbens and olfactory tubercle. In contrast, there was no change in [3H]spiperone binding in whole brain minus striatum nor in striatum alone after KA treatment. KA produced a significant increase in Bmax for [3H]raclopride in whole brain minus striatum and in striatum alone with minimal changes in affinity. These findings demonstrate acute changes in rat brain dopamine D2 receptors labelled with [125I]iodosulpiride and [3H]raclopride but not [3H]spiperone after KA treatment predominantly in extra striatal regions.

Animals↗
PubMed1998

Weaning-induced development of delta-opioid receptors in rat brain: differential effects of guanine nucleotides and sodium upon ligand-receptor recognition.

1. We have previously shown that weaning at day 21 increases delta-opioid receptor binding in the brain at day 25, which might be due to stimulation of the development of a delta-opioid receptor subtype or activation of G-protein coupling processes. 2. We have addressed the possibility that weaning stimulates coupling of the delta-receptor by homogenate binding studies with four agonist and one antagonist radioligand in the presence of a GTP analogue and Na+ in brain tissue from weaned and non-weaned animals. 3. Saturation studies with three agonist ligands ([3H]-deltorphin I, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II) showed higher levels of maximal binding in brains from 25-day weaned than in brains from non-weaned rats. The magnitude of the effects of GMPPNP and Na+ in decreasing this binding was ligand dependent and in each case was significantly more marked in brains from weaned animals. GMPPNP and Na+ were completely without effect on Bmax for, [3H]-S-Atc-Ile(5,6)deltorphin I and [3H]-R-Atc-Ile(5,6)deltorphin II in brains from non-weaned rats. 4. [3H]-Ile(5,6)deltorphin II and [3H]-naltrindole showed no differences in labelling between weaned and non-weaned groups and both groups responded similarly to the effects of GMPPNP and Na+ treatment. 5. GMPPNP and Na+ had small effects on binding affinity (K(D)) for some of the agonist radioligands which were similar in both weaned and non-weaned groups. 6. Weaning induced increases in binding of delta-receptors in 25-day rats can be explained in terms of the way delta-agonist radioligands recognize the receptor environment.

Animals↗
PubMed1998

Regional changes in 5-HT1A but not in 5-HT2A receptors in mouse brain after Semliki Forest virus infection: radioligand binding and autoradiographic studies.

Dysfunction of brain 5-hydroxytryptaminergic systems has been associated with several neurological and psychiatric diseases which may have a viral aetiology. The effect of Semliki Forest virus (SFV) on 5-hydroxytryptamine (5-HT1A and 5-HT2A) receptors in mouse brain has been assessed by membrane homogenate binding and autoradiography. Adult mice were injected with saline or virus and brains removed 2, 6, 14, 22 and 35 days after infection. 5-HT1A and 5-HT2A receptors were characterised by saturation studies using [3H] 8-OH-DPAT and [3H] Ketanserin respectively. SFV infection increased 5-HT1A receptor numbers by up to 80% in the cortex on days 6, 14, and 22 but had no effect on Bmax in the midbrain, pons/medulla and the hypothalamus. SFV infection did not affect 5-HT2A receptor number in any of the brain regions studied and the affinity (Kd) of either ligand for 5-HT1A or 5-HT2A receptors was unaffected. Autoradiographic mapping of 5-HT1A receptors in SFV-infected brain showed substantially higher binding in nucleus accumbens, tenia tecta, septohippocampal nucleus, septum, medial and basolateral amygdaloid nucleus, anterioventral preoptic nucleus, hippocampus, interpeduncular nucleus, frontal, lateral orbital, and entorhinal cortex and claustrum on days 6 and 14. Elevated binding persisted in tenia tecta, frontal, lateral orbital, entorhinal cortex, and hippocampal formation to day 22. Autoradiography of 5-HT2A receptors using [3H] Ketanserin showed no difference in the binding in SFV-infected brains. A decrease in plasma corticosterone levels in SFV-infected mice was observed on post infection days 6 and 22. These results show SFV infection induces a regionally selective upregulation of 5-HT1A but not 5-HT2A receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

Quantitative autoradiographic mapping of mu-, delta- and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene.

Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any mu-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. Mu-, delta-, kappa1- and total kappa-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin), [3H]DELTI (D-Ala2 deltorphinI), [3H]CI-977 and [3H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, mu-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from mu-knockout mice there were no detectable mu-receptors in any brain regions and no evidence for mu-receptors derived from another gene. Delta-, kappa1- and total kappa-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in kappa- or delta-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in delta- and kappa-receptors throughout the brains of animals deficient in mu-receptors.

Animals↗
PubMed1997

Ontogeny of P2-purinoceptors in the longitudinal muscle and muscularis mucosae of the rat isolated duodenum.

1. The ontogeny of P2-purinoceptors in the longitudinal muscle and the muscularis mucosae of the rat isolated duodenum was investigated by use of functional assays in tissues from neonatal animals. The degradation of purinoceptor agonists by the rat duodenum muscularis mucosae was also investigated. 2. In the rat duodenum muscularis mucosae adenosine 5'-(alpha, beta-methylene)triphosphonate (AMPCPP), adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP) and 2-methylthioadenosine 5'-triphosphate (2-Me-S-ATP) all caused a contraction from day 10 to day 40, day 10 being the earliest age it could be tested. The potency order of agonists above day 25 was AMPCPP > ATP = UTP > 2-Me-S-ATP and this is similar to the potency order previously obtained for the adult tissue. However, in the neonatal tissues below day 20, 2-Me-S-ATP was the most potent agonist and at days 10 and 15 the order was 2-Me-S-ATP > AMPCPP > ATP = UTP. 3. In the rat duodenum muscularis mucosae desensitization was observed with AMPCPP at day 30 but not at day 15. At day 30, cross-desensitization was also observed between AMPCPP and 2-Me-S-ATP but not between AMPCPP and ATP or UTP, whereas no cross-desensitization was observed at day 15 with AMPCPP and any of the agonists. At day 15 and below AMPCPP and 2-Me-S-ATP may therefore both activate P2Y-receptors (2-Me-S-ATP > AMPCPP, no desensitization with AMPCPP) whereas above day 20 the agonists activate P2X-receptors (AMPCPP > 2-Me-S-ATP, desensitization with AMPCPP) which is similar to the adult tissue. Since ATP and UTP were equipotent in the muscularis mucosae and as no cross-desensitization was observed with AMPCPP and UTP or ATP at days 15 or 30, it is likely that ATP and UTP both activate P2U-receptors throughout the ages, as in the adult. 4. The potency of all the agonists in causing contraction in the rat duodenum muscularis mucosae decreased with age. The potency of AMPCPP and 2-Me-S-ATP in causing contractions was highest in the neonates before day 25, and reached values not significantly different from adult by day 30, and the potency of ATP and UTP causing contractions in this tissue was also highest in the neonates at days 10 and 15, and reached values not significantly different from adult by day 20. This suggests either that the receptor populations mediating contraction are highest in the neonates below day 20 or that the agonists are degraded by the muscularis mucosae to a greater extent after day 20. 5. In the rat duodenum muscularis mucosae the degradation of ATP, UTP, 2-Me-S-ATP and AMPCPP was followed by high pressure liquid chromatography at days 15 and 30. ATP was degraded to adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) and inosine with no adenosine being detected, 2-Me-S-ATP was degraded to 2-methylthioadenosine 5'-diphosphate (2-Me-S-ADP), 2-methylthioadenosine 5'-monophosphate (2-Me-S-AMP) and 2-methylthioadenosine (2-Me-S-adenosine), and UTP was degraded to uridine 5'-diphosphate (UDP), uridine 5'-monophosphate (UMP) and uridine. The rate of degradation of these agonists was much faster at day 30 than at day 15, probably due to the increase in the size of the tissue. AMPCPP was also degraded with adenosine 5'-(alpha,beta-methylene)diphosphonate (AMPCP) being detected at both ages. However, at day 30 the rate of degradation of AMPCPP was much slower than for ATP, UTP or 2-Me-S-ATP. 6. In the rat duodenum longitudinal muscle 2-Me-S-ATP and AMPCPP both caused a relaxation with a potency order of 2-Me-S-ATP > AMPCPP, suggesting the activation of P2Y-receptors, as previously found for the adult tissue. Weak relaxations were observed to both the agonists at day 15 (the earliest age it could be studied), and the potency of the agonists reached values not significantly different from adult tissues by day 25. 7. Overall, these results suggest that in the neonatal rat duodenum longitudinal muscle there are P2Y-receptors mediating relaxation and that the receptor population i

Adenosine Triphosphate↗
PubMed1996

Differential ontogeny of adenosine receptors in the longitudinal muscle and muscularis mucosae of the rat isolated duodenum.

The ontogeny of P1 purinoceptors in the separated layers of the rat duodenum was investigated using functional assays. In the longitudinal muscle N6-cyclopentyladenosine (CPA) caused relaxations from day 20 that were inhibited by 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) (10 nM) indicating an action via adenosine A1 receptors. 5'-N-ethylcarboxamidoadenosine (NECA) caused relaxations at day 15 that were inhibited by DPCPX (1 microM) while 2-p-(2-carboxyethl)phenylethylamino-5'-N-ethylcarboxamidoade nosine (CGS 21680) was almost inactive, indicating an action at adenosine A2B receptors. From day 20 NECA was inhibited by DPCPX (10 nM) but was not antagonised by DPCPX (1 microM) to the extent expected for an adenosine A1 receptor, suggesting activation of adenosine A1 and adenosine A2B receptors. In the muscularis mucosae, CPA and NECA caused contractions from day 10 inhibited by DPCPX (1 microM) while CGS 21680 was less potent, indicating activation of adenosine A2B receptors. These results show that adenosine A2B receptors are present early in the postnatal period, whereas adenosine A1 receptors develop after day 20.

Adenosine↗
PubMed1996

Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.

Despite tremendous efforts in the search for safe, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Opioids exert their pharmacological actions through three opioid-receptor classes, mu, delta and kappa, whose genes have been cloned. Genetic approaches are now available to delineate the contribution of each receptor in opioid function in vivo. Here we disrupt the mu-opioid-receptor gene in mice by homologous recombination and find that there are no overt behavioural abnormalities or major compensatory changes within the opioid system in these animals. Investigation of the behavioural effects of morphine reveals that a lack of mu receptors abolishes the analgesic effect of morphine, as well as place-preference activity and physical dependence. We observed no behavioural responses related to delta- or kappa-receptor activation with morphine, although these receptors are present and bind opioid ligands. We conclude that the mu-opioid-receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component of the opioid system for morphine action.

Analgesics↗
PubMed1996

Differential distribution of adenosine A2 receptors in the epididymal and prostatic portions of the rat vas deferens.

In the rat vas deferens there are prejunctional A1 receptors mediating inhibition of transmitter release and post-junctional A1 and A2 receptors mediating enhancement and inhibition of contractions respectively. In this study the distribution of adenosine receptors in the prostatic and epididymal portions of the bisected rat vas deferens was investigated. The pre- and post-junctional A1 receptors were present on both portions of the bisected tissue. However, post-junctional A2 receptors appear to be present only in the prostatic region, showing that adenosine receptors are differentially distributed along the length of the rat vas deferens.

Adenosine↗
PubMed1996

Selective enhancement by an adenosine A1 receptor agonist of agents inducing contraction of the rat vas deferens.

The adenosine analogue N6-cyclopentyladenosine (CPA), acting via postjunctional A1 receptors, has been shown to enhance contractions of the rat vas deferens induced by adenosine 5'-triphosphate (ATP), the sympathetic cotransmitter in this tissue. The aim of the present study was to examine the ability of CPA to enhance contractions induced by other contractile agents. CPA (0.01-0.3 microM) enhanced contractions induced by exogenous ATP (10 microM), 5-hydroxytryptamine (5-HT) (3 microM), tyramine (10 microM), 2-methyl-5-hydroxytryptamine (2-Me-5-HT) (10 microM) and KCl (35 mM) and this enhancement was blocked by an A1-selective concentration (3 nM) of 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX). CPA failed to enhance contractions induced by exogenous noradrenaline (NA) (1 microM or 10 microM), bradykinin (0.1 microM), phenylephrine (3 microM) or carbachol (10 microM). The contractions induced by ATP (10 microM), 5-HT (3 microM), 2-Me-5-HT (10 microM) and KCl (35 mM) were unaffected by tetrodotoxin (1 microM) as well as by desensitisation of the P2x-purinoceptors with the ATP analogue adenosine 5'-(alpha, beta-methylene) triphosphonate. The contractions induced by tyramine (10 microM) and 2-Me-5-HT (10 microM) were blocked by prazosin (100 nM) or by imipramine (1 microM). Ketanserin (10 nM) antagonised the response to 5-HT giving a dose-ratio of 12.9 corresponding to an apparent pA2 of 9.1. In conclusion, the A1-mediated effect was clearly selective for certain contractile agents and not due to a non-specific increase in contractility of the tissue. CPA enhanced contractions induced by both ATP and indirect sympathomimetics which release endogenous NA, and this enhancement of the two sympathetic cotransmitters may have a functional significance, and demonstrates the complexity of the neuromodulatory effects of adenosine in the rat vas deferens.

Adenosine↗
PubMed1996

Differential development of adenosine A1 and A2b receptors in the rat duodenum.

1. The development of the adenosine A1 and A2b receptors inducing relaxation of the rat duodenum was studied by use of a combination of functional and radioligand binding assays on rats aged between 5 and 30 days and compared with results previously found in adult rat duodenum. 2. 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) bound with high affinity to a single site in duodenum preparations from rats aged 20, 25 and 30 days. At 10 and 15 days there was no detectable specific binding of [3H]-DPCPX. 3. The affinity (KD) of the binding site for [3H]-DPCPX was similar in membrane preparations from 20, 25 and 30 day old animals (1.58-2.27 nM), but the density (Bmax) of binding sites was found to increase up to 25 days where peak levels (72.0 +/- 9.5 fmol mg-1 protein) were observed and then decline at 30 days (45.5 +/- 2.9 fmol mg-1 protein) to levels commensurate with those previously determined in the adult rat duodenum. 4. In duodenum from 10 day old rats no responses to N6-cyclopentyladenosine (CPA, 1 nM-10 microM) were observed, at 15 days the duodenum responded to the highest concentration of CPA (3 microM) only, and at 20-30 days concentration-related responses were observed, with the potency of CPA increasing with an increase in age. DPCPX (10 nM) abolished the responses to CPA except at the highest concentration of CPA (3 microM) where the response was markedly attenuated, suggesting the presence of an A1, receptor. 5. In rat duodenum from animals of all ages (5-30 days), concentration-related responses to 5'-N-ethylcarboxamidoadenosine (NECA) were observed. The potency of NECA remained constant with an increase in age, whereas the maximum relaxation response increased from 20% at 5 days to 110% at 25 and 30 days. In the presence of 1 microM DPCPX a right-ward shift in the concentration-response curve to NECA was observed at all ages. In the presence of 10 nM DPCPX, the response to NECA was unaffected in the duodenum from animals aged 10 and 15 days. However, in duodenum from animals aged 20-30 days the concentration-response curve to NECA was shifted to the right suggesting that there is an A1 component to the action of NECA at these ages. Schild analysis of the effects of increasing concentrations of DPCPX versus NECA on the duodenum from 25 day old animals generated a slope of 0.62 suggesting that NECA acts at A1 and A2b receptors as in the adult. 6. The A2b-selective analogue, 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) (10 nM-10 microM) was without effect on the carbachol-contracted duodenum from 15 day old rats and the duodenum from 25 day old rats responded to the highest concentration of CGS 21680 only, suggesting that the A2 receptors here, as in the adult, are not of the A2a subtype. The adenosine antagonist, 8-phenyltheophylline (8-PT) (10 microM), abolished the inhibitory effects of NECA (100 nM-100 microM) on 10, 15 and 25 day old rat duodenum indicating that the responses to NECA were not mediated via an adenosine A3 receptor. 7. These results show that adenosine A1 receptors in rat duodenum are present and functionally viable from day 20 onwards and that the density of A1 receptors varies with age, increasing up to day 25 and then declining at day 30 to a density commensurate with that found in the adult. The responses to CPA, mediated via the A1 receptor, increase with age in a similar fashion. In contrast however, the response to NECA was evident from day 5, the earliest age studied, and from days 5-15 NECA acted via the A2b receptor subtype. However, from day 20 onwards NECA acted at a mixed population of A1 and A2b receptors. These results demonstrate the differential development of the A1 and the A2b receptors in the rat duodenum.

Adenosine↗
PubMed1996

The ontogenetic profiles of the pre- and postjunctional adenosine receptors in the rat vas deferens.

1. The ontogenetic profiles of the prejunctional A1 and postjunctional A1 and A2 receptors on the rat vas deferens were investigated, using a combination of functional and radioligand binding assays to follow the A1 receptors and functional assays alone to follow the development of the A2 receptors. 2. The prejunctional A1 receptor, assessed by the inhibitory action of N6-cyclopentyladenosine (CPA) (3 nM-3 microM) on nerve-mediated contractions, was present from day 15 onwards, day 15 being the earliest age at which nerve-mediated contractions could be detected. The potency of CPA was constant across the ages studied, with pD2 values ranging from 6.4-7.1, not significantly different from that previously observed in adult rat vas deferens. 3. The postjunctional A2 receptors, assessed by the inhibitory action of 5'-N-ethylcarboxamidoadenosine (NECA) (10 nM-30 microM) on KCl-induced contractions were present from day 10 onwards, day 10 being the earliest age at which responses to KCl could be observed. The potency of NECA remained constant with an increase in age, with potency values, expressed as pEC25 values, ranging from 6.5-7.0. 4. The postjunctional A1 receptor displayed a different development profile from that of the prejunctional A1 and postjunctional A2 receptors. Postjunctional A1 receptors were identified by the enhancement of KCl-induced contractions by CPA (10 nM-0.3 microM). At 10 and 15 days, CPA failed to enhance KCl-induced contractions. From day 20 to day 40, this enhancement increased with an increase in age and the level of enhancement achieved statistical significance from day 30. 5. Radioligand binding studies using 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX) revealed binding sites characteristic of A1 receptors on the vas deferens from rats aged 20 days onwards. The density (Bmax) of A1 receptors expressed relative to protein content was greatest at day 20 (153 +/- 33 fmol mg-1 protein) and declined at day 30 (43.9 +/- 3.7 fmol mg-1 protein) to a level commensurate with that previously determined in adult rat vas deferens (43.3 +/- 12 fmol mg-1 protein). However, when expressed relative to tissue wet weight little variation in receptor density was observed between these ages (Bmax 0.13 +/- 0.02 fmol mg-1 wet weight at 20 days; 0.17 +/- 0.01 fmol mg-1 wet weight at 30 days). The binding affinity (KD) remained constant with an increase in age and was similar to the KD value previously generated for adult rat vas deferens (approximately 1 nM). At ages 10 and 15 days no reproducible binding could be detected. 6. These results show the differential development of the adenosine receptors on the rat vas deferens with postjunctional A1 receptors demonstrating delayed development, while prejunctional A1 and postjunctional A2 receptors were present from the earliest ages studied. In addition, comparison of binding studies and functional studies suggests that the binding studies detect only the A1 receptors present on the smooth muscle and not those present on the nerve terminals.

Adenosine↗
PubMed1995

Passage of Japanese encephalitis virus in HeLa cells results in attenuation of virulence in mice.

Of four wild-type strains (Nakayama-original, SA14, 826309 and Beijing-1) of Japanese encephalitis (JE) virus that were passaged six times in HeLa cells (HeLa p6), two (Nakayama-original and 826309) became attenuated for mice. In the case of strain Nakayama-original, the virulence for mice was markedly reduced and attenuation was retained on passage in primary chicken embryo fibroblast, LLC-MK2 and C6/36 cells. The binding of non-HeLa-passaged Nakayama virus to mouse brain membrane receptor preparations could be differentiated from binding by Nakayama HeLa p6 virus, suggesting that the envelope (E) protein is involved in the attenuated phenotype. Both of the attenuated viruses can be distinguished from the virulent non-HeLa-passaged parental viruses by examination with E protein reactive vaccine and wild-type-specific monoclonal antibodies (MAbs). The vaccine-specific MAb V23, which is only reactive with the SA14 series of live vaccine viruses, recognized the HeLa cell-attenuated Nakayama-original and 826309 viruses, whereas two wild-type-specific MAbs (MAbs K13 and K39) lost reactivity. Comparison of the nucleotide sequences of the structural protein genes of the 826309 and Nakayama-original virulent parent and attenuated HeLa p6 viruses revealed that the viruses differed by 37 and 46 nucleotides coding for eight and nine amino acid mutations, respectively. However, other than one amino acid in the E protein, the membrane and E protein amino acid sequences of the two attenuated HeLa p6 viruses were identical.

Animals↗
PubMed1995

Development of delta-opioid receptor subtypes and the regulatory role of weaning: radioligand binding, autoradiography and in situ hybridization studies.

Evidence from behavioral studies suggests that the process of weaning activates the development of a delta-opioid receptor subtype. We now report the influence of weaning on the development of delta receptors in the central nervous system assessed by membrane homogenate binding and autoradiography with selective delta radioligands and by in situ hybridization using a cRNA probe for the delta receptor. Binding was carried out by using [3H][D-Ala2]deltorphin I (DELT I), [3H]IIe5,6-deltorphin II (IIe5,6-DELT II) and [3H]naltrindole (NTI). [3H]IIe5,6-DELT II and [3H]NTI labeled an equivalent number of sites in brain and spinal cord from both weaned and nonweaned 25-day-old rats. The number of sites labeled by [3H]DELT I was similar in nonweaned rats but significantly higher in the brain and cord from weaned animals. Furthermore, the ontogenetic profile of these three ligands was distinct. Quantitative autoradiography showed identical levels of [3H]IIe5,6-DELT II binding in all brain regions in weaned and nonweaned rats. In contrast, levels of [3H]DELT I binding were significantly higher in weaned rats and this difference was localized to the deep layers of the frontal-parietal cortex and to the pontine nucleus. In situ hybridization experiments showed no differences in delta-opioid receptor mRNA density between weaned and nonweaned groups in the regions in which binding differences were observed. Weaning stimulates the development of a subpopulation of delta receptors recognized by [3H]DELT I but not by [3H]IIe5,6-DELT II or NTI. This effect is localized to specific brain regions and does not appear to reflect increased synthesis of mRNA coding for the delta receptor.

Animals↗
PubMed1994

Evidence that weaning stimulates the developmental expression of a delta-opioid receptor subtype in the rat.

To study whether weaning stimulates the development of a subtype of the delta-opioid receptor we have measured antinociceptive activity of putative delta 1 (DELT I) and delta 2 (DSLET) agonists in weaned and non-weaned 25-day-old rats. In weaned rats, DSLET produced antinociception reversed by the delta 2-antagonist naltriben, but in non-weaned rats DSLET had no effect. Responses to DELT I were evident in both weaned and non-weaned rats and were not antagonised by naltriben. This is the first evidence that delta-receptor subtypes develop independently and suggests that weaning is the stimulus for the expression of the delta 2-subtype.

Analgesics↗
PubMed1994

Effect of chronic maternal diazepam treatment on the development of stress-induced antinociception in young rats.

The use and abuse of benzodiazepines is widespread and we have begun to address whether maternal exposure to these drugs influences the development of opioid systems. We have studied the effect of maternal diazepam treatment on stress-induced antinociception in the neonatal offspring. Diazepam (1 or 10 mg/kg) was administered twice daily to mothers from conception. Pre- and postweanling rat pups were assessed for opioid-mediated stress-induced antinociception by 3-min swimming and measuring nociception using the tail immersion test. In preweanling rats there was stress-induced antinociception in both vehicle- and diazepam-treated animals but in diazepam-treated groups (1 and 10 mg/kg) this was insensitive to reversal by the opioid antagonist naloxone, suggesting that nonopioid systems are operating this response. In postweanling rats a similar insensitivity to naloxone was observed in 1 mg/kg diazepam-treated groups; with 10 mg/kg diazepam there was no significant antinociception. The results suggest that maternal diazepam treatment interferes with the development of stress-mediated responses and that part of this toxicity is due to actions on opioid systems in the CNS.

Animals↗
PubMed1994

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor.4. The existence of Al binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies.However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine Al receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.

Animals↗