The myelin basic protein-specific T cell repertoire in Lewis rats: T cell receptor diversity is influenced both by intrathymic milieu and by extrathymic peptide presentation.

In the Lewis rat, the T lymphocyte response to guinea pig myelin basic protein (MBP) is focused almost exclusively on epitopes nested in the MBP peptide sequence p68-88, and is dominated by T cell receptors (TCR) using Vbeta8.2 gene elements, together with short N(D)N regions. Here we analyzed MBP-specific TCR from Lewis T cells differentiating in chimeric thymuses of Lewis rat/SCID mouse chimeras, in the absence of an intact rat thymic microenvironment (SCID(FL) mice). In these T cells, the TCR Vbeta repertoire is broad, N(D)N regions are significantly longer, and contain regular rates of template-independent N nucleotides. In striking contrast, a Vbeta8.2 biased TCR repertoire and few N-region inserts are seen in p68-88-specific, Lewis rat-derived T cells differentiating in the complete rat thymic microenvironment provided by chimeric SCID mice bearing embryonic Lewis thymus grafts (SCID(FL/FT) mice). A T cell repertoire resembling the one in SCID(FL) mice is used by T cells of intact Lewis rats following immunization with a truncated epitope of MBP, p69-86. Also this selection generates a broad TCR Vbeta pattern with long N(D)N regions, and higher numbers of N nucleotides. These results show that both intrathymic repertoire selection, and extrathymic peptide priming exert profound effects on the TCR usage in the anti-MBP response of Lewis rats.

Prodynorphin gene expression in spinal cord is enhanced after traumatic injury in the rat.

Levels of mRNAs coding for prodynorphin (Pro-Dyn) and proenkephalin (Pro-Enk) as well as the levels of immunoreactive (ir)-dynorphin (Dyn) and (ir)-Met-enkephalin (Met-Enk) were measured in the spinal cord of rats, 65 h following transection or injury of the spinal cord at the T6 spinal segment. Levels of both Pro-Dyn mRNA and of ir-Dyn were significantly increased between 60 and 150%, above control levels in the whole spinal cord, whereas those of Pro-Enk mRNA and of ir-Met-Enk remained unchanged. The increase in spinal levels of Pro-Dyn mRNA were highest in the areas close to the side of transection and indicate an involvement of the Pro-Dyn opioid system in the response to spinal injury and transection.

Prodynorphin gene expression is enhanced in the spinal cord of chronic arthritic rats.

The influence of chronic arthritic pain upon the levels of mRNA encoding prodynorphin (mRNADYN) in the spinal cord of rats was evaluated by use of the RNA blot technique. Rats were rendered arthritic by inoculation of the tail-base with a suspension of Mycobacterium butyricum. Three weeks post inoculation, levels of mRNADYN revealed a pronounced alteration in arthritic rats by a factor of greater than or equal to 2.5 as compared to control animals. This rise was specific in that there was no change in total RNA content. These data indicate that the biosynthetic activity of the dynorphin system is facilitated under chronic pain. Together with our previous biochemical and behavioural data, a functional role of this system in the response to chronic pain is suggested.

Localization of prodynorphin messenger rna in rat brain by in situ hybridization using a synthetic oligonucleotide probe.

An in situ hybridization technique has been developed to study the distribution of messenger RNA (mRNA) encoding prodynorphin (proenkephalin B) in sections of rat brain. A 100-mer oligonucleotide was used as a template to synthesize a 32P-labelled DNA probe complementary to the coding region of rat prodynorphin mRNA. Using this probe, dense labelling was observed in the supraoptic and paraventricular nuclei of the hypothalamus, in the striatum and in the dentate gyrus. The results show for the first time the localization of prodynorphin mRNA in rat brain, and additionally demonstrate the usefulness of oligonucleotides to detect rare mRNAs by in situ hybridization.

Stress-induced alterations in the levels of messenger RNA coding for proopiomelanocortin and prolactin in rat pituitary.

Acute stress promotes the secretion of prolactin (PRL) and of proopiomelanocortin (POMC)-derived peptides, adrenocorticotropic hormone and beta-endorphin, from the pituitary into the systemic circulation. The present study evaluates the influence of recurrent stress upon the biosynthetic activity of cells secreting these hormones in the rat. Chronic, intermittent, electrical foot-shock (3 mA,1 s duration, every 5 s for 30 min, twice daily) over a period of 1, 3 or 7 days caused an increase in messenger ribonucleic acid (mRNA) levels coding for POMC in the anterior pituitary. Maximally elevated mRNA levels were achieved after 3 days treatment (about 80% in excess of control values) which showed no further change at 7 days. These elevated levels of POMC mRNA were associated with increased levels of immunoreactive (ir)-beta-endorphin in the adenohypophysis following 7 days of stress treatment. In contrast, this treatment did not significantly alter mRNA levels coding for PRL in the anterior pituitary. Similarly, POMC mRNA levels in the intermediate/posterior pituitary were also not significantly altered during exposure to repeated stress. Similar changes in the biosynthesis of the pituitary hormones were seen in rats suffering from chronic arthritic pain for 3 weeks: there was an approximately 80% increase in POMC mRNA levels in the anterior pituitary which was associated with an increase in the levels of ir-beta-endorphin in this lobe and an increase in the plasma levels of ir-beta-endorphin. In contrast, there were no changes in the levels of mRNA coding for PRL in the adenohypophysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential alterations by chronic treatment with morphine of pro-opiomelanocortin mRNA levels in anterior as compared to intermediate pituitary lobes of rats.

Chronic treatment of rats with morphine by implantation of pellets over a period of 10 days resulted in a differential alteration of pro-opiomelanocortin (POMC) mRNA levels in individual pituitary lobes: Hybridisation studies using a 32P-labelled mouse POMC cDNA fragment of 150 bases as a probe revealed that chronic morphine treatment causes about 50% enhancement of POMC mRNA levels in the anterior lobe and about 40% decrease in the intermediate lobe of the pituitary. Significant changes were observed 3 days following commencement of administration and persisted over the rest of the morphine treatment.

Corticotropin-releasing factor differentially regulates proopiomelanocortin messenger ribonucleic acid levels in anterior as compared to intermediate pituitary lobes of rats.

Chronic subcutaneous infusion of small doses (0.1 microgram/h) of ovine corticotropin-releasing factor (oCRF) into rats for 8 days resulted in differential alteration of proopiomelanocortin (POMC) mRNA levels in the individual pituitary lobes: In the anterior lobe POMC mRNA levels, quantitated by hybridisation using a 32P-labelled POMC cDNA probe, increased by about 80%, whereas in the intermediate/posterior lobe a marked decrease to about 30% of the initial levels was observed. Significant changes were found not earlier than 3 days following commencement of administration.

Pro-enkephalin intermediates in bovine brain and adrenal medulla: characterization of immunoreactive peptides related to BAM-22P and peptide F.

Using antibodies against the synthetic opioid peptides BAM-22P and peptide F, immunoreactive (ir-) peptides were measured in bovine brain and adrenal medulla. In addition to the high levels in the adrenal medulla, both ir- peptides were measurable in various areas of the brain with highest concentrations in the anterior hypothalamus. Analysis of the ir- components by gel filtration revealed molecular heterogeneity. Besides peptides with the size of BAM-22P or peptide F, various higher molecular weight species were found. These forms were found in the adrenal medulla in much higher concentrations than in the brain indicating a different processing mechanism for proenkephalin. Synthetic BAM-22P injected intracerebroventricularly into mice produces a substantial analgesia (ED50 6.4 nmole) which is almost as high as that of morphine (ED50 2.8 nmole). This finding and the presence of BAM-22P-like compounds in the brain suggests a role of the enkephalinergic system in pain transmission.

Chronic haloperidol treatment increases the level of in vitro translatable messenger ribonucleic acid coding for the beta-endorphin/adrenocorticotropin precursor proopiomelanocortin in the pars intermedia of the rat pituitary.

Chronic treatment of rats with haloperidol (1.5 mg/kg, once daily) over a period of 7--21 days resulted in a 80--100% increase in the tissue levels of immunoreactive beta-endorphin and in the in vitro release of immunoreactive beta-endorphin from the neurointermediate pituitary. Incorporation of [3H]phenylalanine into isolated neurointermediate pituitaries of haloperidol-treated rats revealed an increase in the amount of label incorporated into the beta-endorphin/ACTH precursor proopiomelanocortin (POMC) to a similar extent (about 80%) but had essentially no effect on the conversion of the precursor into beta-lipotropin and beta-endorphin. Extraction of messenger (m) RNA from neurointermediate pituitaries followed by cell-free translation in a reticulocyte system showed an increase in the total level of translatable mRNA (about 25%). The content of translatable mRNA coding for POMC, however, was increased by 100-150%. Time-course studies revealed a parallelism between the effect of haloperidol on the level of in vitro translatable mRNA coding for POMC and the ability of the drug to increase the concentrations of beta-endorphin in the neurointermediate pituitary. A complete reversal of the effects of haloperidol was seen 2 weeks after discontinuation of the drug. These findings suggest that the chronic blockade of dopaminergic receptors by haloperidol causes a reversible increase in the beta-endorphin biosynthesis in the rat intermediate pituitary at the pretranslational level by markedly increasing the level of translatable mRNA coding for POMC.

Levels of dynorphin-(1-13) immunoreactivity in rat neurointermediate pituitaries are concomitantly altered with those of leucine enkephalin and vasopressin in response to various endocrine manipulations.

The levels of dynorphin-(1-13), leucine enkephalin, beta-endorphin and vasopressin immunoreactivity (ir-DYN, ir-1-ENK, ir-beta-END, ir-VP) have been determined in the anterior and in the neurointermediate lobes of the pituitary of rats subjected to a variety of manipulations. Dehydration of rats by 5 days enforced inhibition of a 2% solution of NaCl resulted in a significant decrease in the levels of ir-DYN, ir-1-ENK and ir-VP, but not in those of ir-beta-END in the neurointermediate lobe of the pituitary. In contrast, substitution of drinking water by a solution containing 20 microgram/ml dexamethasone for 5 days produced a significant increase in the neurointermediate pituitary content of ir-DYN, ir-1-ENK and ir-VP, whereas levels of ir-beta-END remained unaffected. This treatment, however, resulted in a significant fall in the ir-beta-END content of the adenopituitary without changing levels of ir-DYN in this structure. Adrenalectomy was associated with a significant decrease in the ir-DYN, ir-VP and ir-1-ENK content of the neurointermediate lobe of the pituitary and a pronounced elevation in the ir-beta-END but not ir-DYN content of the adenohypophysis. These observations are indicative that the regulation mechanisms of the functional state of particular endorphins differ between the anterior and neurointermediate lobes of the pituitary. The concomitant alterations in levels of ir-DYN, ir-1-ENK and ir-VP detected suggest that a common or similar mechanism of regulation may exist for these peptides. A common biosynthetic origin, however, appears to be unlikely, since Brattleboro rats which are unable to synthesize vasopressin possess unchanged ir-DYN- and ir-1-ENK- levels in the pituitary.

Long-term treatment of rats with morphine reduces the activity of messenger ribonucleic acid coding for the beta-endorphin/ACTH precursor in the intermediate pituitary.

Chronic administration of morphine to rats for a period of 4 weeks resulted in a 50-60% decrease in the tissue concentrations of beta-endorphin and in the in vitro release from the neurointermediate pituitary. Incorporation of [3H]phenylalanine into isolated intermediate/posterior pituitaries in vitro revealed a reduction in the amount of label incorporated into the beta-endorphin/ACTH precursor to a similar extent (about 45%), but essentially no effect on the conversion of the precursor into beta-lipotropin and beta-endorphin. Extraction of mRNA from intermediate/posterior pituitaries followed by cell-free translation in a reticulocyte system showed no significant decrease in the total level of translatable mRNA. In contrast, the content of translatable mRNA coding for the beta-endorphin/ACTH precursor was significantly reduced by 50-60%. Thus, long-term treatment with morphine appears to depress beta-endorphin formation in the rat intermediate pituitary at the pretranslational level by markedly decreasing the activity of mRNA coding for the beta-endorphin/ACTH precursor without any alteration in the processing of this precursor.

Dynorphin-related immunoreactive peptides in rat brain and pituitary.

Antisera recognizing dynorphin 1-13, but not other opioid peptides with a high avidity, have been generated in rabbits. Using a radioimmunoassay technique levels of dynorphin-immunoreactivity (dyn-ir) have been measured in the brain and pituitary of rats. The concentrations of dyn-ir ranged from a maximum in the intermediate/posterior lobe of the pituitary (about 1200 pmol/g) to a minimum in the cerebellum (about 1 pmol/g) as follows: pituitary intermediate/posterior lobe greater than adenohypophysis greater than hypothalamus greater than hippocampus = striatum = midbrain = thalamus = medulla/pons greater than cortex greater than cerebellum. Gel-filtration of hypothalamic extracts revealed 4 immunoreactive components with apparent molecular weights (MW) of 3500, 2400, 1300 and less than 1000 daltons, respectively. No dyn-ir was found to elute as dynorphin1-13 (MW = 1700). The 2400 and 1300 dalton materials showed opiate-like activity on the guinea-pig ileum longitudinal muscle preparation, indicating that a substantial part of the dyn-ir measured represented biologically active material.

Identification of opiate/receptor binding in vivo.

The displacement of small amounts of tritiumlabbelled antagonists or agonists by increasing amounts of unlabelled antagonists in mouse brain in vivo offered the possibility of characterizing properties of opiate receptors in the intact animal. The displacement effect was stereospecific, saturable and dependent upon the affinity of the substance investigated. At brain concentrations of 0.3 nM, 75% of 3H-diprenorphine, 60% of 3H-naltrexone and 50% of 3H-naloxone were displaced by high amounts of the respective unlabelled drug. Comparison of the in vivo data with receptor binding in vitro revealed similar results in respect to binding sites and receptor affinity. The displacement was different in various brain areas. The time course of the displacement was also different for the various substances used and seems to reflect differences in the speed of association and dissociation to and from the receptors. The displacement of 3H-etorphine by naltrexone could be correlated with the reversal of analgesia.