Detection of a novel immunoreactive endomorphin 2-like peptide in rat brain extracts.

To pursue further the possible de novo biosynthetic pathway of endomorphins in rat brain we raised antibodies to endomorphin-2 conjugate in rabbits. Antiserum R1 recognized endomorphin-2 with good selectivity as compared to endomorphin-1 with a median detection value of 65.5+/-7.5 pg/tube (n=7), whereas R4 antiserum recognized both endomorphins with similar sensitivity. Neither antisera recognized YP-related di- or tripeptides or YGGF-related opioid sequences (enkephalins, beta-endorphin, dynorphin). Using the same rat brain extraction-RP-HPLC-gradient separation paradigm as previously, antisera detected 144.6+/-40.0 (n=3) pg/g wet brain weight endomorphin-2-like immunoreactivity in the fraction corresponding to standard endomorphin-2 retention time and also in the fraction matching endomorphin-2-OH standard retention time (179.1+/-30.1 pg/g). Since R1 failed to recognize authentic endomorphin-2-OH, the second immunoreactive species must be different from both endomorphin-2 and endomorphin-2-OH. Possible biosynthetic intermediates to endomorphins, synthetic YPFFG and YPWFG had retention times close to the parent endomorphin standards in RP-HPLC gradient separation profile. The former was a mu-opioid receptor agonist of medium potency in the in vitro assays (rat brain RBA>P gamma S binding and mouse vas deferens), whereas the latter was a weak mu-opioid receptor agonist with a significant delta-opioid receptorial action as well and a definite indication of partial agonism.

alpha2B-adrenoceptor agonist ST-91 antagonizes beta2-adrenoceptor-mediated relaxation in rat mesenteric artery rings.

We sought an isolated vascular preparation and experimental setting where the function of alpha2B-adrenoceptors could be demonstrated by non-recombinant technique. ST-91 (2-[2,6-diethylphenylamino]-2-imidazoline), an alpha 2B-adrenoceptor agonist with a mixed alpha adrenergic receptor type/subtype selection profile antagonized the relaxant effect of isoproterenol in endothelium-denuded rat mesenteric artery rings precontracted with phenylephrine. At 10(-7) M of ST-91, the antagonism was characterized by a rightward shift of isoproterenol dose-response curve (A50=6.81+/-1.40 e-7 (n=4) vs the control 1.29+/-0.25 e-7 M (n=4)) with no E(max) depression. At 10(-6) M the Emax depression was prevalent (36.1+/-7.0% (n=4) vs the control 79.9+/-5.1% (n=4)); both actions could be antagonized by the alpha2-adrenoceptor antagonist yohimbine. The not subtype-selective alpha(2)-adrenoceptor agonist xylazine (10(-7) M) did not affect the relaxant action of isoproterenol. Present findings are discussed in the light of previously reported hemodynamic effects attributed to alpha2B-adrenoceptors in receptor subtype-knockout animals.

Partial and full agonism in endomorphin derivatives: comparison by null and operational model.

The partial mu-opioid receptor pool inactivation strategy in isolated mouse vas deferens was used to determine partial agonism of endomorphins and their analogs (endomorphin-1-ol, 2',6'-dimethyltyrosine (Dmt)-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2) using morphine, normorphine, morphiceptin, (D-Ala2,MePhe4,Gly5-ol)-enkephalin (DAMGO) and its amide (DAMGA) as reference opioid agonists. Agonist affinities (KA) and efficacies were assessed both by the "null" and the "operational" method. The KA values determined by the two methods correlated significantly with each other and also with the displacing potencies against 3H-naloxone in the receptor binding assay in the presence of Na+. DAMGO and DAMGA were full agonist prototypes, morphine, endomorphin-1, endomorphin-1-ol, Dmt-endomorphin-1, endomorphin-2-ol and (D-Met2)-endomorphin-2 were found by both methods to be partial agonists whereas the parameters for normorphine, morphiceptin and endomorphin-2 were intermediate.

Endomorphin synthesis in rat brain from intracerebroventricularly injected [3H]-Tyr-Pro: a possible biosynthetic route for endomorphins.

In spite of concentrated efforts, the biosynthetic route of mu-opioid receptor agonist brain tetrapeptide endomorphins (Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2), discovered in 1997, is still obscure. We report presently that 30 min after intracerebroventricular injection of 20 or 200 microCi [3H]Tyr-Pro (49.9 Ci mmol(-1)) the incorporated radioactivity was found in endomorphin-related tetra- and tripeptides in rat brain extracts. As detected by the combination of HPLC with radiodetection, a peak corresponding to endomorphin-2-OH could be identified in two of four extracts of "20 microCi" series. Radioactive peaks in position of Tyr, Tyr-Pro, Tyr-Pro-Phe or Tyr-Pro-Trp appeared regularly in both series and also in the "tetrapeptide cluster" constituted by endomorphins and their free carboxylic forms. In one of the four extracts in the "200 microCi" series a robust active peak in the position of endomorphin 2 could be detected. Intracerebroventricularly injected 100 nmol, but not 10 or 1000 nmol cold Tyr-Pro (devoid of opioid activity in vitro), caused a naloxone-reversible prolongation of tail-flick latency in rats, peaking between 15 and 30 min. We suggest that Tyr-Pro may serve as a biosynthetic precursor to endomorphin synthesis.

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides.

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.

Characterisation of alpha2-adrenoceptor subtypes involved in gastric emptying, gastric motility and gastric mucosal defence.

The effect of clonidine on ethanol-induced gastric mucosal damage, gastric emptying and gastric motility was compared. The clonidine-induced gastroprotective effect (0.03-0.09 micromol/kg, s.c.) was antagonised by yohimbine (5 micromol/kg, s.c.), prazosin (0.23 micromol/kg; alpha2B-adrenoceptor antagonist) and naloxone (1.3 micromol/kg, s.c.). Clonidine also inhibited the gastric emptying of liquid meal (0.75-3.75 micromol/kg, s.c.) and gastric motor activity (0.75 micromol/kg, i.v.) stimulated by 2-deoxy-D-glucose (300 mg/kg, i.v.). Inhibition of gastric emptying and motility was reversed by yohimbine (5 and 10 micromol/kg, s.c., respectively), but not by prazosin (0.23 micromol/kg, s.c.) or naloxone (1.3 micromol/kg, s.c.). Oxymetazoline-an alpha2A-adrenoceptor agonist-inhibited both gastric emptying (0.67-6.8 micromol/kg, s.c.) and motility (0.185-3.4 micromol/kg, i.v.), whereas it failed to affect gastric mucosal lesions. The results indicate that in contrast to the gastroprotective effect, which is mediated by alpha2B-adrenoceptor subtype, alpha2A-adrenoceptor subtype may be responsible for inhibition of gastric emptying and motility. However, the site of action (central, peripheral, both) remains to be established.

Structure-activity study on the Phe side chain arrangement of endomorphins using conformationally constrained analogues.

Endomorphins-1 and -2 were substituted with all the beta-MePhe stereoisomers in their Phe residues to generate a conformationally constrained peptide set. This series of molecules was subjected to biological assays, and for beta-MePhe(4)-endomorphins-2, a conformational analysis was performed. Incorporation of (2S,3S)-beta-MePhe(4) resulted in the most potent analogues of both endomorphins with enhanced enzymatic stability. Their micro opioid affinities were 4-times higher than the parent peptides, they stimulated [(35)S]GTPgammaS binding, and they were found to be full agonists. NMR experiments revealed that C-terminal (2S,3S)-beta-MePhe in endomorphin-2 strongly favored the gauche (-) spatial orientation which implies the presence of the chi(1) = -60 degrees rotamer of Phe(4) in the binding conformer of endomorphins. Our results emphasize that the appropriate orientation of the C-terminal aromatic side chain of endomorphins is substantial for binding to the micro opioid receptor.

The effect of endomorphins on the release of 3H-norepinephrine from rat nucleus tractus solitarii slices.

We used two, 3-min field stimulation cycles 30 min apart (S1, S2) in 3H-norepinephrine-loaded, superfused rat nucleus tractus solitarii-dorsal motor vagal nucleus (NTS-DVN) slices. The stimulation-induced release was expressed as the area above the baseline. Drugs were introduced 12 min before S2 and drug actions were characterized in terms of alterations of S2/S1 ratios. The S2/S1 ratio was 1.047 (0.946-1.159, n = 4, geometric mean and 95% confidence interval) in controls and 0.336 (0.230-0.490, n = 3), 0.726 (0.590-0.892, n = 4), 0.613 (0.594-0.683, n = 4) and 0.665 (0.500-0.886, n = 4) in the presence of 10(-6) M clonidine, D-Ala(2),MePhe(4),Gly(5)-ol-enkephalin (DAMGO), endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2), EM-1) and -2 (Tyr-Pro-Phe-Phe-NH(2), EM-2) [the latter two in the presence of 10(-4) M diprotin A, an inhibitor of dipeptidyl-aminopeptidase IV (DAP-IV) enzyme]. The effect of DAMGO at 10(-5) M was significantly higher than at 10(-6) M, whereas the effect of endomorphins did not differ at the two concentration levels. Diprotin A potentiated only very modestly the action of endomorphins. These data (a) confirm the presence of functional mu-opioid receptors in the vagal complex, (b) render it likely that the enzymic degradation of endomorphins is not a highly effective process in brain slices and (c) may suggest that the apparent ceiling in the effect of endomorphins might be related to their partial agonist property.

Gyrus cinguli transection abolishes delta-opioid receptor-induced gastroprotection and alters alpha 2 adrenoceptor activity in the lower brainstem in rats.

Previously, using the acidified ethanol-induced ulcer model in rats, we demonstrated that the mainly vagus-dependent gastroprotective effect of intracerebroventricularly injected clonidine was mediated by beta-endorphin release in the lower brainstem. Presently, retroarcuate transections were used to evaluate the contribution of forebrain beta-endorphinergic projection in this mechanism. Since the transection trajectory affected the cingulate cortex and other forebrain structures, matching lesions were also performed. In control and sham-operated rats intracisternal injection of clonidine and the direct opioid receptor (delta type) stimulant peptide (D-Ala(2), D-Leu(5))-enkephalin caused a potent and fully naloxone-reversible (i.e. opioid receptor-mediated) protection against acidified ethanol-induced mucosal damage. In gyrus cinguli-transected rats (as well as in groups with midline hippocampal, thalamic and hypothalamic lesions) gastric mucosal protection induced centrally by direct delta-opioid receptor stimulation in the lower brainstem was completely abolished. The protective effect of clonidine was significantly reduced but it was still present in these animals. The residual protection by clonidine was naloxone-resistant, i.e. independent of an opioid mediation. Transections of the cingulate gyrus as well as thalamic but not the retroarcuate transections elevated plasma corticosterone levels. The changes seen in the clonidine/opioid-induced gastroprotection did not show any correlation with the changes in plasma corticosterone levels. It was concluded that (i) the transection of the cingulate cortex strongly influences the neural input to the nucleus tractus solitarii-dorsal motor vagal nucleus complex that is required for the activation of gastroprotective vagus outflow by delta-opioid receptor stimulation; (ii) the transection uncovers a direct, clonidine-induced gastroprotective pathway which is probably suppressed in intact animals.

Age and monosodium glutamate treatment cause changes in the stimulation-induced [3H]-norepinephrine release from rat nucleus tractus solitarii-dorsal vagal nucleus slices.

In nucleus tractus solitarii-dorsal vagal nucleus slices prepared from young adult rats (180-260 g) 10(-3) M L-glutamate and 10(-5) M baclofen caused a 2-3-fold increase of field stimulation-induced [3H]-norepinephrine release without affecting the resting release. In slices prepared from rats treated neonatally with monosodium glutamate neither L-glutamate nor baclofen had any effect on stimulation-induced norepinephrine release, tested between postnatal days 74-99 (350-530 g). In untreated littermates used in the same period (460-580 g) L-glutamate was fully effective whereas baclofen was ineffective. The tritium content in tissue extracts did not differ significantly in the three experimental groups. It is concluded that i) the loss of GABA(B) receptor-mediated disinhibitory stimulation of norepinephrine release is an age-related phenomenon and ii) neonatal monosodium glutamate treatment causes a damage in the local neural circuitry characterized by the loss of glutamate receptor-mediated mechanism that stimulates the release of norepinephrine.

Angiotensin II-induced activation of central AT1 receptors exerts endocannabinoid-mediated gastroprotective effect in rats.

The aim of the present study was to analyze whether angiotensin II via the endocannabinoid system can induce gastric mucosal protection, since transactivation of cannabinoid CB1 receptors by angiotensin AT1 receptor in CHO cells was described. Experimental ulcer was induced by acidified ethanol given orally in male Wistar rats, CB1(+/+) wild type and CB1(-/-) knockout mice. The compounds were administered intracerebroventricularly. It was found, that 1. Angiotensin II inhibited the ethanol-induced gastric lesions (11.9-191pmol); the effect of angiotensin II (191pmol) was inhibited by the CB1 receptor inverse agonist AM 251 (1.8nmol) and the inhibitor of diacylglycerol lipase (DAGL), tetrahydrolipstatin (0.2nmol). 2. Angiotensin II exerted gastroprotection in wild type, but not in CB1(-/-) mice. 3. The gastroprotective effect of angiotensin II (191pmol) was reduced by atropine (1mg/kg i.v.) and bilateral cervical vagotomy. In conclusion, stimulation of central angiotensin AT1 receptors via activation of cannabinoid CB1 receptors induces gastroprotection in a DAGL-dependent and vagus-mediated mechanism.

The dipeptidyl peptidase IV (CD26, EC 3.4.14.5) inhibitor vildagliptin is a potent antihyperalgesic in rats by promoting endomorphin-2 generation in the spinal cord.

We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall-Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an "euglycemic" agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1mg/kg) is a potent antihyperalgesic also when given subcutaneously.

Intrathecally injected Ile-Pro-Ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase IV, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 2.

We have found recently that membrane-bound dipeptidyl peptidase IV (DPP-IV) generated extracellularly immunoreactive endomorphin-2 from Tyr-Pro precursor in a depolarisation-sensitive manner in rat isolated L4,5 dorsal root ganglia when the enzyme was switched to synthase mode by the hydrolase inhibitor Ile-Pro-Ile. Presently, we induced hyperalgesia in rats by injecting carrageenan into the right hindpaw and measured the reduction in nociceptive threshold (hyperalgesia) to pressure (Randall-Selitto test). The hyperalgesia, peaking at 180 min after injection, was fully reversed by intrathecal administration of 30 nmol/rat Ile-Pro-Ile. The antihyperalgesic action was antagonized by s.c. naloxone (1 mg/kg) and intrathecally injected specific antiserum to endomorphin-2 indicating that the opioid receptor-mediated effect was produced by an endogenously generated endomorphin-2-like immunoreactive substance. Intrathecal Ile-Pro-Ile was ineffective as an analgesic in the acute nociceptive test such as the rat tail-flick, whereas endomorphin-2 (EC(50)=13.3 nmol/rat), endomorphin-1 (6.8 nmol/rat), morphine (0.11 nmol/rat) and DAMGO (0.0059 nmol/rat) exerted opioid receptor-mediated analgesia given by the same route. We concluded that carrageenan-induced C-fiber barrage (wind-up) may create ideal conditions for the de novo synthesis of endomorphin-2 in rat spinal cord dorsal horns if the DPP-IV enzyme is switched to the synthase functional mode by Ile-Pro-Ile.

Immunoreactive endomorphin 2 is generated extracellularly in rat isolated L4,5 dorsal root ganglia by DPP-IV.

BACKGROUND AND AIMS: The gene(s) encoding for endomorphin precursor(s) is/are still unknown. We have raised the possibility of and did find some evidence for a potential de novo biosynthetic route starting from Tyr-Pro precursor. To pursue further this possibility we measured the generation of immunoreactive endomorphin-2 (E2-IR) in adult rat isolated L4,5 dorsal root ganglia. RESULTS AND CONCLUSIONS: In rat isolated dorsal root ganglia the combination of presumed biosynthetic precursor of endomorphin 2 (E2), Tyr-Pro with the dipeptidyl peptidase IV (DPP-IV) inhibitor Ile-Pro-Ile generated 1.60+/-0.37 pg/mg Wet Tissue Weight_30 min E2-IR in the bathing fluid (n=4) with an 8-fold increase upon depolarization whereas the tissue content was low (0.50+/-0.08 pg/mg_WTW). Substance P, as determined by ELISA in the pilot experiments, was found almost exclusively within the tissues. It is concluded that E2-IR was generated extracellularly by a membrane-bound DPP-IV, which was switched to "synthase" mode by the hydrolase inhibitor Ile-Pro-Ile. DPP-IV was depolarization-sensitive in "synthase" functional mode.

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.