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.
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.
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.
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.
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.
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 3Hphenylalanine 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.
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.
