Ryszard Przewłocki

Opiate receptor binding sites in human spinal cord

Opiate receptor binding sites were analyzed in various regions of human spinal cord and compared to results obtained in spinal cord and brain of certain animals. mu-, delta- and kappa binding sites were individually monitored by the overall labeling of opiate binding sites with [3H]diprenorphine followed by the sequential elimination of binding to particular sites by the use of selective ligands. kappa-Receptors were the predominant type (approximately 50%), followed by mu-receptors (approximately 40%), and, in rather small amounts, delta-receptors. A similar proportion of receptor types was found in the spinal cord of guinea pigs.

Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord

Abstract By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), α-neo-endorphin (ir-α-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral > lumbar > cervical > thoracic. Concentrations of ir-DYN, ir-α-NEO and ir-SP were 2–10-fold, but of ir-MET 1–2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord. The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter. Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin 1–17 . ir-α-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-α-NEO were also detected in these tissue. These results suggest that dynorphin and α-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.

Distribution of methionine- and leucine-enkephalin within the rat pituitary gland measured by highly specific radioimmunoassays

Abstract The distribution of methionine- and leucine-enkephalin within the rat pituitary gland was measured using highly specific antisera in conjunction with purification by high performance liquid chromatography. The highest concentrations were found in the pars intermedia (7 pmole/mg for methionine-enkephalin, 4 pmole/mg for leucine-enkephalin), whilst the pars nervosa contained 2.2 pmole/mg of each and the pars anterior the least (methionine-enkephalin: 0.51 pmole/mg, leucine-enkephalin: 0.36 pmole/mg).

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.

Modulation of in vitro release of β-endorphin from the separate lobes of the rat pituitary

Abstract The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10−7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe. Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue. These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.

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)

Lesions of the hypothalamic arcuate nucleus produce a temporary hyperalgesia and attenuate stress-evoked analgesia

Abstract In an attempt to elucidate the role of β-endorphin in the modulation of ‘basal’ nociceptive threshold and in the mediation of the antinociception (analgesia) evoked by stress, a series of lesions of the arcuate nucleus, the origin of the central system of β-endorphinergic neurones, were performed. These lesions produced an ∼80% depression in the level of β-endorphin immunoreactivity in both the hypothalamus and periventricular β-endorphinergic fibre-containing tissue. A 50% decrease in the neurointermediate lobe content of immunoreactivity, but no change in the levels of this in the anterior lobe was also observed. Arcuate lesioned rats were significantly hyperalgesic in comparison to sham animals on day 4 post-operation, but on days 10 and 12, the basal nociceptive threshold of lesioned and sham groups did not differ significantly. On day 12 post-surgery upon exposure to 5 min foot-shock stress, lesioned rats developed a significantly smaller increase in tail-flick latency than did sham animals. These data are evidential of the importance of the arcuate nucleus in the determination of basal nociceptive threshold and in the generation of the analgesia which accompanies stress and are, further, suggestive of a role of central β-endorphin in the mediation of these processes.

Pertussis toxin abolishes the antinociception mediated by opioid receptors in rat spinal cord

Intrathecal injection of pertussis toxin (1 microgram) in rats produced a marked decrease in the antinociceptive effect of the intrathecally administered opioid agonists [D-Ala2,D-Leu5]enkephalin, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and bremazocine. The effect of the toxin was time-dependent, since it was more pronounced at 6 than at 2 days after its injection. The pertussis toxin-catalyzed ADP ribosylation of a 40 KDa substrate in membranes prepared from the spinal cord of toxin-injected rats was strongly reduced as compared to controls. The data indicate that the antinociceptive effect produced by opioid agonists with different receptor preference is initiated at receptor sites which interact with G-protein substrates of pertussis toxin.

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.

Endogenous opioids, circadian rhythms, nutrient deprivation, eating and drinking.

Immunoreactive (ir) beta-endorphin (b-END) and dynorphin (DYN) in rat brain and pituitary were measured after food and water deprivation and from brains taken during either day or night. In other rats, eating and drinking were measured following lesions in the arcuate n. Ir-DYN levels are higher in hypothalamus and lower in pituitary at night. Deprivation, particularly water deprivation, increases hypothalamic, day-time ir-DYN. Water deprivation decreases pituitary levels of ir-DYN. Arcuate-lesions, depleting both ir-b-END and ir-DYN, do not modify total daily intake of water or food but does modify circadian rhythmicity of eating and drinking. These data support the conclusion that b-END and DYN are involved in maintaining day-night patterns of eating and drinking.