dynorphins and dynorphin-(1-32)

Animal models are useful to evaluate pharmacological therapies to alleviate joint pain. The present study characterized central neuropeptides modulation in the monoiodoacetate (MIA) rat model. Animals receiving a single 3mg MIA injection were euthanized at 3, 7, 14, 21 and 28 days post injection. Spinal cords were analyzed by liquid chromatography ion trap mass spectrometry. Up-regulations of the calcitonin gene-related peptide and substance P were observed starting on days 7 and 28 respectively, whereas big dynorphin(₁₋₃₂) content decreased significantly on day 14 in comparison to control animals (P<0.05). Preclinical drug evaluations using this model should be conducted between 7 and 21 days post injection when the lesions resemble most to human osteoarthritis.

Topics: Animals; Calcitonin Gene-Related Peptide; Chromatography, Liquid; Disease Models, Animal; Dynorphins; Enzyme Inhibitors; Humans; Iodoacetic Acid; Knee Joint; Male; Neuropeptides; Osteoarthritis; Pain; Rats; Rats, Sprague-Dawley; Spinal Cord; Substance P; Tandem Mass Spectrometry; Time Factors

We measured the release of immunoreactive (ir) dynorphin (dyn) A-(1-17) and dyn B from the rat hypothalamus by an in vitro superfusion technique. The system was validated on the basis of the recovery and stability of radiolabeled peptides added to the superfused hypothalami. These were detected as authentic peptides by reverse-phase high-performance liquid chromatography (rp-HPLC) only in the presence of a cocktail of peptidase inhibitors added to the superfusion medium. We observed spontaneous release of ir-dyn B, evaluated by a validated radioimmunoassay in the superfusates, that was increased by potassium and veratridine depolarization. It was calcium-dependent and tetrodotoxin-sensitive. We could not evaluate ir-dyn A-(1-17) directly in the superfusates, because the peptidase inhibitors added to the medium significantly altered the tracer-antibody reaction. To obviate this problem, pooled superfusate samples were purified on C18 cartridges and assayed by rp-HPLC. Rp-HPLC analysis of superfusates revealed two molecular forms with the same retention time as authentic dyn A-(1-17) and dyn B which were four times higher in K(+)-stimulated fractions. We could not detect dyn A-(1-32), comprising dyn A-(1-17) and dyn B, even though this peptide is recognized by the antibodies used in this study and is detected in acetic acid extracts of the rat hypothalamus. The spontaneous and K(+)-evoked release of ir-dyn A-(1-17) and ir-dyn B were significantly higher in 2-week ovariectomized rats, in parallel with the increase of their content in the anterior hypothalamus preoptic area.

Topics: Animals; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Female; Hypothalamus; In Vitro Techniques; Male; Neuromuscular Depolarizing Agents; Ovariectomy; Ovary; Perfusion; Radioimmunoassay; Rats; Rats, Inbred Strains

Prodynorphin-derived peptides were tested for their effects on body temperature after intracerebroventricular administration to unrestrained male rats. Dynorphin A (Dyn A) (5 and 10 nmol) and Dynorphin A-(1-32) (Dyn A-(1-32) (2.5 and 5 nmol) lowered body temperature with a maximum approximately 30 min after administration. Dyn B (up to 50 nmol) did not induce hypothermia. Lower doses of all peptides did not alter body temperature. The hypothermic effect was significantly, but not completely prevented by MR1452 (30 nmol), a preferential antagonist of the kappa receptor, administered intracerebroventricularly. Naloxone, a mu receptor antagonist, naltrexone, its long acting analog up to doses of 100 nmol, as well as MR1453, the (+)-enantiomer of kappa antagonist MR1452 with no opioid binding properties, did not prevent the hypothermic effect. Moreover, episodic barrel rolling and bizarre postures elicited by Dyn A and Dyn A-(1-32) were reduced in rats pretreated i.c.v. with MR1452 (30 nmol), but not with naloxone (up to 100 nmol). Interestingly, des-Tyr-Dynorphin A (Dyn A-(2-17)), a fragment with virtually no opioid binding potential, was 4 times less potent that Dyn A in inducing hypothermia. These findings are consistent with the hypothesis that prodynorphin-derived peptides effects are not exclusively opioids in nature.

Topics: Animals; Benzomorphans; Body Temperature; Dynorphins; Endorphins; Hypothermia; Kinetics; Male; Motor Activity; Naloxone; Naltrexone; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Opioid

The pituitary and hypothalamic content of dynorphin was determined by radioimmunoassay and characterized by high-performance liquid chromatography (HPLC) in adult female Sprague-Dawley rats, intact and ovariectomized with and without estrogen treatment. Animals were given estradiol benzoate, or vehicle (oil) by six daily intramuscular injections. Anterior pituitary content of immunoreactive (ir)-dynorphin in ovariectomized rats was approximately twice that of intact animals, and consisted of a single HPLC peak co-eluting with dynorphin 32. Administration of estradiol benzoate (0.06-6 micrograms/day) caused a marked decrease of ir-dynorphin in the anterior lobe of castrate female rats, with a half-maximal effect at 0.2 microgram/day; levels were restored to those seen in intact animals with 6 micrograms estradiol benzoate per day, an effect which was not influenced by concomitant administration of progesterone (1 mg/day), or bromocriptine (100 micrograms/day). In the hypothalamus and neuro-intermediate lobe multiple peaks of immunoreactive dynorphin were seen, coeluting with dynorphin A 1-8, dynorphin A 1-17 and dynorphin 32. Neither castration nor estrogen treatment altered ir-dynorphin content in these tissues. These findings suggest that the ovary exerts a specific modulating influence on AP ir-dynorphin in the rat, and that in addition this inhibition appears to be mediated by ovarian estrogen.

Topics: Animals; Chromatography, High Pressure Liquid; Dynorphins; Estradiol; Female; Hypothalamus; Injections, Intramuscular; Molecular Weight; Ovariectomy; Peptide Fragments; Pituitary Gland, Anterior; Radioimmunoassay; Rats; Rats, Inbred Strains

Topics: Animals; Central Nervous System; Chromatography, High Pressure Liquid; Dynorphins; Endorphins; Hypothalamus; Male; Mice; Peptide Fragments; Pituitary Gland

The opioid peptide dynorphin1-32 (DYN1-32, 25 nmol) intrathecally administered causes, in the rat, an elevation of nociceptive threshold of longer duration than that of DYN A, as ascertained by vocalization test. Comparative findings obtained with tail flick test allow to differentiate antinociception from motor dysfunction. The breakdown of DYN A at spinal level is very rapid. The electrical stimulation of the tail associated to a restraint condition of the rat produces a significant increase of immunoreactive DYN in cervical, thoracic and lumbar segments of spinal cord, therefore indicating a correlative, if not causal, relationship between the spinal dynorphinergic system and aversive stimuli.

Topics: Analgesics; Animals; Dynorphins; Male; Pain; Rats; Rats, Inbred Strains; Sensory Thresholds; Spinal Cord; Synaptic Transmission