Levels Of ir-Dyn Research Articles

Substance P N-terminal fragment SP(1–7) attenuates chronic morphine tolerance and affects dynorphin B and nociceptin in rats

The N-terminal substance P fragment SP1–7 is known to modulate hyperalgesia and opioid withdrawal in animal models. This study examined the effects of intraperitoneal (i.p.) injections of SP1–7 on chronic morphine tolerance and on the levels of dynorphin B (DYN B) and nociceptin/orphanin FQ (N/OFQ) in various brain areas of male Sprague–Dawley rats. Morphine tolerance was induced by subcutaneous injections of the opioid (10mg/kg) twice daily for 7 days. SP1–7 injected i.p. (185nmol/kg) 30min prior to morphine reduced the development of morphine tolerance. Immunoreactive (ir) DYN B and N/OFQ peptide levels were measured in several areas of the central nervous system. Levels of ir DYN B in rats treated with SP1–7 and morphine were decreased in the nucleus accumbens, substantia nigra and ventral tegmental area and increased in the frontal cortex. The ir N/OFQ levels were increased in the periaqueductal gray and decreased in the nucleus accumbens. Since the concentration profiles of the two peptides were altered by SP1–7 in the areas that are implicated in the modulation of opioid tolerance and analgesia, it is suggested that DYN B and N/OFQ systems may be involved in the effects of SP1–7 on opioid tolerance.

Regional distribution of immunoreactive dynorphin A in the human gastrointestinal tract

Immunoreactive dynorphin A (ir-Dyn A) was detected throughout the human gastrointestinal tract by a validated radioimmunoassay. Moreover, the stability of 125I-Dyn A during extraction procedures was confirmed by high performance liquid chromatography. Levels of ir-Dyn A were higher in the stomach and in the small bowel. In tissue samples separated into the main layers composing the gut wall (muscularis externa, submucosa and mucosa) ir-Dyn A was uniformly distributed. An exception was the colon, where concentrations were higher in the muscular portion. Gel permeation chromatography on samples of mucosa and muscalaris externa extracts of ileum and gastric fundus, showed immunoreactive material eluting in several forms of apparently higher molecular weight than Dyn A, while only a minor peak was found to coelute with authentic Dyn A.

Response of rat pituitary anterior lobe prodynorphin products to changes in gonadal steroid environment.

The total content of rat pituitary anterior lobe (AL) immunoreactive (ir) dynorphin A (ir-Dyn A) and ir-dynorphin B (Dyn B) increased in male rats between 15 and 58 days of age, but there was little alteration in the concentration of ir-Dyn A or B expressed relative to protein content. Adult rats (90 days of age) had lower concentrations of these peptide immunoreactivities in the AL. Castration of 58-day-old male rats produced a testosterone-reversible loss of ir-Dyn A and B by 50-60% 3 days after surgery. Thereafter, the levels of these peptides gradually increased to 2.5 times the levels found in control animals at 1 month after castration. These effects of castration on AL dynorphin were not seen in 15-day-old rats and were much less marked in adults. Similar changes were seen in the levels of other prodynorphin products, alpha- and beta-neo-endorphin (ir-alpha-nEnd and ir-beta-nEnd), and ir-[Leu5]enkephalin (ir-LE). Administration of testosterone (100 micrograms/100 g BW) to castrated rats for 2 days largely prevented the drop in the levels of AL ir-Dyn A and B. Ovariectomy produced an increase in the levels of ir-Dyn A, Dyn B, alpha-nEnd, beta-nEnd, and LE 2 weeks after surgery, but, in contrast to castration, no significant decrease was seen 3 days after ovariectomy. These changes in AL content of dynorphin-related peptides after castration or ovariectomy directly reflect those previously reported for AL content of LH. The mechanisms regulating storage (and perhaps secretion) of AL peptides derived from prodynorphin may be similar to those regulating storage and secretion of LH and FSH in rat AL. AL ir-LE could potentially arise from proenkephalin A or prodynorphin (proenkephalin B). Ir-LE levels in AL were approximately 10 times higher than the levels of ir-[Met5]-enkephalinyl-Arg-Gly-Leu (ME-RGL) in male rat AL, and changes in ir-LE content after castration were very similar to those observed in other prodynorphin-derived peptides, but different from the effects of castration on ir-ME-RGL. It is possible that prodynorphin is a major source of AL ir-LE.

Lesions of the hypothalamic arcuate nucleus modify discrete brain and pituitary pools of dynorphin in addition to β-endorphin in the rat

Radiofrequency destruction of the hypothalamic arcuate nucleus, the exclusive central location of β-endorphin (β-EP)-synthesizing neurones, greatly depressed levels of immunoreactive (ir)-β-EP in the hypothalamus. However, the content of ir-dynorphin (DYN) therein was also decreased, in parallel with that of ir-β-EP. Levels of ir-Met-enkephalin (ME) were, in contrast, unaffected. In the midbrain and septum, the content of ir-β-EP was greatly depleted; in the former but not latter tissue, levels of ir-DYN were also diminished whilst levels of ir-ME were not affected in either of these structures. Further, in the neurointermediate pituitary, the content of ir-β-EP was depressed while that of ir-DYN was not changed, whereas, in the anterior pituitary, levels of ir-β-EP were not altered in contrast to those of ir-DYN which were decreased. Destruction of the arcuate nucleus is not, therefore, restricted to selective effects upon central β-EP neurones: this finding is of importance for studies of the role of central β-EP via lesioning techniques. The data suggest an interaction of the arcuate nucleus and, possibly, β-EP with particular brain and pituitary pools of DYN.

Dynorphin and enkephalins in adrenal paraneurones. Opiates in the adrenal medulla

Immunoreactive dynorphin (ir-Dyn), immunoreactive leucine-enkephalin (ir-Leu-Enk) and various other neuropeptides were measured in acid extracts of bovine adrenal medulla and isolated adrenal chromaffin cells. Their respective levels ranged as follows: Leu-Enk greater than Dyn greater than bombesin greater than vasoactive intestinal peptide (VIP) greater than neurotensin greater than substance P. Comparisons of the total catecholamine levels with the levels of Leu-Enk in both extracts gave ratios in the same order of magnitude (2600, tissue extract and 5000, cell extract). However, the catecholamine/Dyn ratio in the tissue extract (138 000) was much higher than that found in the cell extract (20 180), suggesting a possible selective degradation of Dyn in tissue extract as compared with cell extract or an induction of Dyn biosynthesis in cells which have been isolated from their natural microenvironment. Immunofluorescence staining of isolated chromaffin cell sections revealed the presence of ir-Dyn in 5 to 10% of the total cell population. To localize ir-Dyn in regard to Leu-Enk and catecholamines, adrenal chromaffin cells were separated into three populations (I, II, and III) on a stepwise bovine serum albumin (BSA) gradient. Relative high levels of ir-Dyn were measured in cell layer I (4 pmol/10(6) cells), a cell population enriched in noradrenaline. However, ir-Leu-Enk was more concentrated in cell layers II and III (5.3 and 8.3 pmol/10(6) cells), two populations enriched in adrenaline. Isolation and high pressure liquid chromatography (HPLC) analysis of adrenomedullary Dyn indicated the presence of at least five molecular forms corresponding to Dyn-(1-11), Dyn-(1-12), Dyn-(1-13), Ala-containing-Dyn-(1-13) and a nonidentified molecule eluting closely to Dyn-(1-13). These data indicate that adrenal ir-Dyn and ir-Leu-Enk have distinct cellular distributions. In addition, the identification of Dyn fragments in bovine adrenal medulla indicates that these short peptides may be considered as natural active forms of Dyn.

Contrasting interactions of the locus coeruleus as compared to the ventral noradrenergic bundle with CNS and pituitary pools of vasopressin, dynorphin and related opioid peptides in the rat

The present study examines the influences of selective destruction of the locus coeruleus (LC) or of the ventral noradrenergic bundle (VB) upon discrete CNS and pituitary pools of vasopressin, dynorphin and related opioid peptides in the rat. The selectivity of the lesions was indicated by the fact that destruction of the LC strongly depressed levels of noradrenaline in the cortex in contrast to the hypothalamus, whereas destruction of the VB decreased noradrenaline in hypothalamus but not cortex. Rats sustaining VB lesions displayed a parallel depletion neurointermediate, but not anterior, lobe levels of immunoreactive- (irdynorphin 1–17 (DYN), ir-DYN 1–8, ir-α-neo-endorphin (ir-α-NE) and ir-vasopressin (ir-VP) whereas those of ir-Met-enkephalin (ir-ME) were unaffected. In the hypothalamus, the content of ir-DYN and ir-VP tended to rise and that of ir-DYN 1–8 and ir-α-NE was significally elevated, whereas that of ir-ME was not altered. LC destruction failed, in contrast, to modify levels of ir-VP, ir-DYN, ir-DYN 1–8, ir-α-NE or ir-ME in any of the above structure. It was found to, however, result in depression in levels of ir-DYN and ir-α-NE, but not of ir-ME or ir-VP, in both the hippocampus and striatum whereas VB lesions were, in this respect, ineffective. Further, in the spinal cord, LC lesions resulted in a significant elevation in levels of ir-DYN and ir-α-NE in comparison to those of ir-DYN 1–8, ir-VP and ir-ME. Neither type of lesion significantly altered the content of any opioid peptide examined in thalamus, cortex, septum or midbrain. These data indicate that: (1) the LC as compared to the VB interact differently with discrete pools of ir-DYN, ir-DYN 1–8, ir-α-NE and ir-VP in brain, pituitary and spinal cord; (2) it is the VB rather than the LC which modulates the activity of magnocellular neurones projecting to the neural lobe of the pituitary; (3) ir-DYN, ir-DYN 1–8 and ir-α-NE are, in all tissues, regulated independently of ir-ME; (4) levels of ir-DYN, ir-DYN 1–8 and ir-α-NE are co-regulated with those of ir-VP in the hypothalamus-neural obbe axis but not in extrahypothalamic brain tissues for the spinal cord; and (5) DYN, DYN 1–8 and α-NE might, in certain cases, be modulated differentially of one another, possibly reflecting alterations in precursor processing.

The influence of foot-shock stress upon brain, pituitary and spinal cord pools of immunoreactive dynorphin in rats

The distribution of immunoreactive-dynorphin (ir-dyn) in the pituitary, discrete regions of brain and the spinal cord, and the influence of 5 min foot-shock stress (FS) upon levels of ir-dyn in these structures, were examined in rats. FS produced a significant fall in the anterior pituitary lobe (AL) content of ir-dyn but no significant change in its neurointermediate (NIL) counterpart. In the hypothalamus, in contrast, a significant elevation in levels of ir-dyn was observed. With the exception of the frontal cortex, in which a decrease in levels of ir-dyn was found, in all other brain regions examined no significant changes emerged. A significant diminution in concentrations of ir-dyn in both the lumbosacral and thoracic sections of the spinal cord was, however, detected.

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.