Effects of aging on proteasomal-ubiquitin system, oxidative stress balance and calcium homeostasis in middle-aged female rat colon.

Aging is a multifactorial process, which is considered as a decline over time. It is increasingly clear that there is a gender difference in aging and in the prevalence of age-related diseases as well. We aimed to examine the effects of the aging process in the colonic tissue of female Wistar rats aged 10 weeks (young) and 13 months (middle-aged) at an early stage, according to three main symptoms associated with aging: a decrease in the efficacy of the proteasome and muscle function and an increase in oxidative stress. The aging process was found to cause a significant decrease in ubiquitin C-terminal hydrolase ligase (UCHL-1) and a significant increase in 3-nitrotyrosine (3-NT), total glutathione (GSH), calcium (Ca2+), calcitonin gene-related peptide (CGRP) and superoxide dismutase (SOD) activity in middle-aged animals. In summary, it is suggested that the reduced activity of the proteasomal degradation system may be the result of the diminished expression of the UCHL-1 enzyme and the decreased levels of ubiquitin; furthermore, we found some key targets which may help to better understand the fundamental aging process.

A novel non-opioid binding site for endomorphin-1.

Endomorphins are natural amidated opioid tetrapeptides with the following structure: Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2). Endomorphins interact selectively with the µ-opioid or MOP receptors and exhibit nanomolar or sub-nanomolar receptor binding affinities, therefore they suggested to be endogenous agonists for the µ-opioid receptors. Endomorphins mediate a number of characteristic opioid effects, such as antinociception, however there are several physiological functions in which endomorphins appear to act in a fashion that does not involve binding to and activation of the µ-opioid receptor. Our recent data indicate that a radiolabelled [3H]endomorphin-1 with a specific radioactivity of 2.35 TBq/mmol - prepared by catalytic dehalogenation of the diiodinated peptide precursor in the presence of tritium gas - is able to bind to a second, naloxone insensitive recognition site in rat brain membranes. Binding heterogeneity, i.e., the presence of higher (Kd = 0.4 nM / Bmax = 120 fmol/mg protein) and lower (Kd = 8.2 nM / Bmax = 432 fmol/mg protein) affinity binding components is observed both in saturation binding experiments followed by Schatchard analysis, and in equilibrium competition binding studies. The signs of receptor multiplicity, e.g., curvilinear Schatchard plots or biphasic displacement curves are seen only if the non-specific binding is measured in the presence of excess unlabeled endomorphin-1 and not in the presence of excess unlabeled naloxone. The second, lower affinity non-opioid binding site is not recognized by heterocyclic opioid alkaloid ligands, neither agonists such as morphine, nor antagonists such as naloxone. On the contrary, endomorphin-1 is displaced from its lower affinity, higher capacity binding site by several natural neuropeptides, including methionine-enkephalin-Arg-Phe, nociceptin-orphanin FQ, angiotensin and FMRF-amide. This naloxone-insensitive, consequently non-opioid binding site seems to be present in nervous tissues carrying low density or no µ-opioid receptors, such as rodent cerebellum, or brain of µ-opioid receptor deficient (MOPr-/-) transgenic or 'knock-out' (K.O.) mice. The newly described non-opioid binding component is not coupled to regulatory G-proteins, nor does it affect adenylyl cyclase enzyme activity. Taken together endomorphin-1 carries opioid and, in addition to non-opioid functions that needs to be taken into account when various effects of endomorphin-1 are evaluated in physiological or pathologic conditions.

Behavioral and neuroendocrine actions of endomorphin-2.

The effects of intracerebroventricularly administered endomorphin-2 (EM2) on open-field activity and the hypothalamo-pituitary-adrenal (HPA) system were investigated. EM2 (0.25-1 microg) significantly increased both the locomotor and the rearing activity, resulting in a bell-shaped dose-response curve. EM2 also enhanced corticosterone release, with an even more profound downturn phase at higher concentrations. The corticotropin-releasing hormone (CRH) antagonist alpha-helical CRH9-41 completely abolished the EM2-evoked endocrine and behavioral responses. These findings reinforce the hypothesis that the endomorphins may play a significant role in the regulation of locomotion, rearing activity and the HPA system through the release of CRH.

Influence of degradation on binding properties and biological activity of endomorphin 1.

The recently-isolated endogenous peptide endomorphin 1 has high affinity for the mu opioid receptor and plays an important role in analgesia. Several of its degradation products have been isolated from the central nervous system. Degradation products present structural similarities and may influence the receptor binding properties and biological activity of the parent compound. Therefore, we investigated how degradation of endomorphin 1 might influence ligand binding to the mu opioid receptor, the consequent activation of G proteins and its antinociceptive effect. Both N- and C-terminal truncation of endomorphin 1 resulted in peptides presenting considerably lower opioid receptor binding potency. None of these peptides had an effect on GTP binding, nor was able to produce analgesia, suggesting that degradation destroys the biological activity of endomorphin 1.

Effects of endomorphin-1 on open-field behavior and on the hypothalamic-pituitary-adrenal system.

The effects of endomorphin-1 (EM1) on behavioral responses and on the hypothalamic-pituitary-adrenal system were investigated in mice. Locomotor activity was measured in an "open-field" apparatus, with parallel recording of the numbers of rearings and groomings. Different doses of the peptide (250 ng to 5 microg) were administered to the animals intracerebroventricularly 30 min before the tests. EM1 caused significant increases in the locomotor activity and the number of rearings. The effect of EM1 on the basal corticosterone secretion was also investigated. At a dose of 5 microg, the peptide significantly increased plasma corticosterone level. The corticotropin-releasing hormone (CRH) antagonist alpha-helical CRH9-41, applied 30 min prior to EM1 administration, completely abolished the increases in both locomotion and the number of rearings and attenuated the corticosterone release evoked by EM1. These results suggest that the EM1 -induced increases in locomotion and rearing activity as well as the pituitary-adrenal activation are mediated by CRH.

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

Liquid chromatographic study of the enzymatic degradation of endomorphins, with identification by electrospray ionization mass spectrometry.

The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.

Antinociceptive effects of intrathecal endomorphin-1 and -2 in rats.

Endomorphin-1 and endomorphin-2 were recently postulated to be endogenous mu-opioid receptor agonists. We have investigated the antinociceptive and antihyperalgesic effects of intrathecally administered endomorphins in cumulative doses (0.1-100 microg) on acute and inflammatory pain sensations in awake rats. In the tail-flick test, both peptides caused a dose-dependent short-lasting antinociception, except at the highest dose, which caused motor impairment also. The dose-response curves revealed the development of acute tolerance (tachyphylaxis) to endomorphin. Similarly in the carrageenan-injected paw, the endomorphins (10 microg) exerted transient antinociceptive effects. These are the first data to demonstrate decreased responsivity in models of both acute and inflammatory pain after intrathecal administration of endomorphin-1 and -2 in awake rats.

Pain inhibition by endomorphins.

Spinal analgesic effects of endomorphin-1 and endomorphin-2 were studied during acute, inflammatory, and neuropathic pain in rats chronically implanted with intrathecal cannulas. Endomorphin-1 and endomorphin-2 (2.5-10 micrograms i.t.), as well as their analogues, increased the tail-flick and the paw pressure latencies. In a model of inflammatory pain, the formalin-induced behavior was attenuated by endomorphins; however, the effect studied was not dose-dependent and was less pronounced in comparison with that evoked by morphine. On the other hand, in rats with a sciatic nerve injury (crush), endomorphins antagonized allodynia in a dose-dependent manner, whereas morphine was found to be ineffective in a similar dose range. Endomorphins also exhibited an antinociceptive potency in rats tolerant to morphine. In conclusion, our results show a powerful analgesic action of endomorphins at the spinal level. The most interesting finding is a strong effect of endomorphins in neuropathic pain, which opens up a possibility of using these compounds in pain therapy.

In vitro binding and signaling profile of the novel mu opioid receptor agonist endomorphin 2 in rat brain membranes.

The recently discovered endogenous mu receptor selective endomorphin 2 was prepared in tritiated form by a catalytic dehalogenation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), and used for in vitro labelling of rat brain membranes. The binding was saturable, stereospecific and of high affinity (Kd: 0.97 and 1.12 nM based on kinetic and equilibrium binding studies, respectively). The maximal number of binding sites (Bmax) was found to be 114.8 fmol/mg protein. [3H]Endomorphin 2 was displaced by mu-receptor selective specific peptides and heterocyclic compounds with high affinity, whereas kappa and delta receptor specific ligands were much less potent. The Ki values of endomorphin 1 and 2 in inhibiting [3H]naloxone binding increased by 15-fold in the presence of 100 mM NaCl which indicates the agonist property of these peptides. Endomorphins stimulated [35S]GTPgammaS binding and inhibited adenylyl cyclase activity which also provides evidence for the agonist character of endomorphins.