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2',6'-Dimethyl substitution of the Tyr(1) residue of opioid agonist peptides and deletion of the positively charged N-terminal amino group or its replacement with a methyl group has recently been shown to represent a general structural modification to convert opioid peptide agonists into antagonists. This conversion requires the syntheses of opioid peptide analogues containing either 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) or (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Tyr(1). Using this approach, delta-, kappa- and mu-selective opioid peptide agonist peptides were successfully converted into corresponding delta-, kappa- and mu-selective antagonists, whereby receptor selectivity was often maintained or even improved. Thus, two (2S)-Mdp(1)-analogues of the delta-selective cyclic enkephalin analogue H-Tyr-c[D-Pen-Gly-Phe(pF)-Pen]-Phe-OH turned out to be potent and selective delta antagonists. Most successful was the development of kappa antagonists derived from dynorphin A (Dyn A), including the highly potent and selective kappa-antagonist [(2S)-Mdp(1)]Dyn A(1-11)-NH(2) (dynantin) and the enzymatically stable octapeptide analogue [(2S)-Mdp(1),MeArg(7),D-Leu(8)]Dyn A(1-8)-NH(2). The (2S)-Mdp(1)-analogues of dynorphin B and alpha-neoendorphin also were kappa antagonists and may be useful as pharmacological tools in studies of kappa receptor subtypes. Finally, the Dhp(1)-analogues of the mu-selective cyclic enkephalin analogue H-Tyr-c[N(epsilon ),N(beta)-carbonyl-D-Lys(2),Dap(5)]enkephalinamide and of endomorphin-2 were moderately potent mu opioid antagonists.

Mice made cold water swimming (CWS: 4 degrees C, 3 min) produced an opioid-mediated antinociception. Experiments were designed to determine what types of opioid receptors and endogenous opioid peptides in the spinal cord are involved in the CWS-induced antinociception in male ICR mice. Antinociception was measured by the tail-flick test. CWS-induced antinociception was blocked by intrathecal (i.t.) pretreatment with antiserum to [Met5]enkephalin (100 microg, 1 hr), but not by antiserum (100 microg, 1 hr) to [Leu5]enkephalin, beta-endorphin or dynorphin A (1-17). Moreover, i.t. pretreatment with delta2-opioid receptor antagonist naltriben (NTB: 10 microg, 10 min) blocked the antinociception induced by CWS or i.t.-administered [Met5]enkephalin (10 microg). However, the antinociception induced by CWS or i.t.-administered [Met5]enkephalin was not blocked by i.t. pretreatment with delta1-opioid receptor antagonist 7-benzylidene naltrexone (BNTX: 1 microg, 10 min), mu-opioid receptor antagonist D-Phe-Cys-Try-D-Try-Om-Thr-Phe-Thr-NH2 (CTOP: 50 ng, 10 min), or kappa-opioid receptor antagonist norbinaltorphimine (norBNI: 5 microg, 24 hr). These data indicate that [Met5]enkephalin and delta2-opioid receptor in the spinal cord are involved in antinociception induced by CWS.

Nociceptin (orphanin FQ) is a novel peptide isolated from brain tissue that has an amino acid sequence most similar to that of the endogenous opioid peptide dynorphin A. Aside from this similarity, the association of nociceptin to the endogenous opioid peptide systems and the functional importance of this new peptide in vivo are not completely known. Here we report that nociceptin is physiologically active in vivo and produces marked changes in the renal excretion of water and sodium. In conscious Sprague-Dawley rats, intravenous infusion of nociceptin produced a profound increase in urine flow rate and decrease in urinary sodium excretion. In further studies, intracerebroventricular (i.c.v.) injection of nociceptin into conscious rats produced a concurrent diuresis (dose-dependent) and antinatriuresis. The magnitude and pattern of the central nociceptin-induced water diuresis was similar to that produced by i.c.v. dynorphin A. Whereas i.c.v. pretreatment with the selective kappa-opioid receptor antagonist, nor-binaltorphimine, completely prevented the renal responses produced by dynorphin A, this antagonist did not alter the diuresis or antinatriuresis produced by central nociceptin. Thus, these results indicate that in conscious rats, nociceptin produces a selective water diuresis via a central nervous system mechanism independent of kappa-opioid receptors. Together, these observations suggest that endogenous nociceptin may be a novel peptide involved in the central control of water balance and ultimately in the regulation of arterial blood pressure. In the future, analogues of nociceptin may prove to be the first clinically useful water diuretics for patients with water-retaining diseases.

Receptor binding studies were performed to characterize the properties of subtypes of k opioid receptors in membrane preparations of human cerebral cortex. [ 3 H]U69,593 ([ 3 H]U69), a selective κ 1 -agonist, and [ 3 H]diprenorphine ([ 3 H]DIP), a non-selective opioid antagonist, in the presence of 1 μM each of DAMGO, DPDPE and U-69 to block μ -, δ -, and κ 1 -sites, labeled single population of binding sites, respectively. [ 3 H]U-69 binding sites (Kd = 3.8 ± 0.2 nM, B max = 6.3 ± 0.2 fmol/mg protein) had a binding profile that correspond to κ 1 -receptor. That is, dynorphin A (1–13) (Dyn A), bremazocine (BZC), U50,488H (U50), (−)ethylketocyclazocine (EKC) and nor-binaltorphimine (nor-BNI) bound to this site with high affinities. [ 3 H]DIP labeled binding sites (Kd = 7.3 ± 0.2 nM, B max = 102 ± 9 fmol/mg protein) that were not sensitive to U-50, but to BZC, EKC and nor-BNI. These results indicate that κ 1 , and κ 2 opioid receptors exist in human cerebral cortex with different ligand binding profiles.

Proteolytic processing of prodynorphin-derived peptides in rat brain was studied with the help of high performance size exclusion chromatography (SEC) connected to electrospray ionization mass spectrometry. Extracts from rat striatum were incubated with individual synthetic dynorphin peptides. Dynorphin A was the most resistant to proteolytic cleavage, converting slowly to Leu-enkephalin (0.3 pmol/min), whereas dynorphin B was processed to this pentapeptide at a 10 4 -fold higher rate. Minor cleavage was also observed between Arg 6 - Arg 7 . Alphaneoendorphin was also rapidly metabolized to Leu-enkephalin (6 nmol/min) and, to a lesser extent, to Leu-enkephalinArg 6 . This new strategy for studying peptidases can easily be adapted to identification of components present in body fluids.

Immunoreactive dynorphin A-like material (ir-dyn A) in human plasma was measured by a validated radioimmunoassay. In peripheral plasma extracts mean concentrations between 20 and 40 fmol/ml were determined in volunteers and in patients with pituitary adenomas. In this latter group superimposable levels were detected three days before and during transsphenoidal microsurgery. Interestingly, ir-dyn A levels evaluated in extracts of hypothalamic-hypophysial plasma obtained during surgery, just after tumor removal, were 4–5 times higher than in peripheral plasma. Reverse-phase high performance liquid chromatography (rp-HPLC) of extracts of peripheral plasma samples revealed two immunoreactive peaks. The major form had the same retention time of dyn A-(1–32); whereas a second, more lipophilic, peak eluted later and was not further characterized. In contrast, rp-HPLC analysis of extracts of plasma collected from the suprapituitary region displayed only one peak eluting in the position of synthetic dyn A-(1–17). The presence of dyn-related peptides in hypothalamic-hypophysial plasma supports the hypothesis that they may play a part in the regulation of hypothalamic and/or pituitary functions in humans.

This paper describes a study on a dynorphin converting enzyme in spinal cord homogenates from rats with experimental arthritis after adjuvant injection into one hindpaw. The enzyme resembles a neutral cysteine endopeptidase which cleaves the opioid peptide dynorphin B and generates its N-terminal fragment, Leu-enkephalin-Arg 6 with opioid activity. It exhibits considerably lower activity against dynorphin A and α-neoendorphin, the two other prodynorphin derived peptides. The enzyme showed significantly higher activity in the dorsal part than in the ventral part of the spinal cord. A significant decrease in enzyme activity was observed in the dorsal spinal cord during inflammation as compared to vehicle-injected controls. This decrease paralleled a decrease in the tissue level of Leu-enkephalin-Arg 6 . These data thus indicate that adjuvant-induced arthritis may generate an important change in a converting enzyme acting on peptide structures, which may be involved in pain modulation. Therefore, a functional role of the present enzyme in the regulation of pain-related peptides is suggested.

Opioid drugs and peptides were investigated for their effect on uterine DNA synthesis induced by a single injection of 17 beta-oestradiol given to ovariectomized rats 24 h prior to decapitation. [D-Met2,Pro5]-enkephalinamide administered 12, 2 or 1 h before killing resulted in a significant (approximately 50%) inhibition of in vitro [3H]-thymidine incorporation into DNA, while injections given 24 or 6 h before decapitation were ineffective. Non-linear regression of the dose-effect curves resulted in an ED50 of approximately 0.26 and approximately 0.45 microgram/100 g b.wt. for the opioid treatments given 12 or 2 h before killing, respectively. These effects could be completely reversed by the opioid antagonist naloxone injected 30 min prior to the agonist treatment, while naloxone itself had no effect. Morphine and [D-Ala2,D-Leu5]-enkephalin administered 12 h, as well as dynorphin A fragment 1-13 given 2 h before decapitation also inhibited oestradiol-induced uterine DNA synthesis. In ovariectomized animals without 17 beta-oestradiol priming no significant effect of [D-Met2,Pro5]-enkephalinamide or naloxone on [3H]TdR incorporation was found.

The modulatory effects of intrathecally (i.t.) administered dynorphin A(1-17) and dynorphin A(1-13) on morphine antinociception have been studied previously in rats by other investigators. However, both potentiating and attenuating effects have been reported. In this study, the modulatory effects of i.t. administered dynorphin A(1-17) as well as the smaller fragment, dynorphin A(1-8), were studied in mice. In addition, nor-binaltorphimine (nor-BNI), a highly selective kappa opioid receptor antagonist, and naltrindole (NTI), a highly selective delta opioid receptor antagonist, were used to characterize the possible involvement of spinal kappa and delta opioid receptors in the modulatory effects of the dynorphins. Dynorphin A(1-17) and dynorphin A(1-8) administered i.t. at doses that did not alter tail-flick latencies, were both able to antagonize in a dose-dependent manner, the antinociceptive action of s.c. administered morphine sulfate. The antinociceptive ED50 of morphine sulfate was increased 3.9- and 5.3-fold by 0.4 nmol/mouse of dynorphin A(1-17) and dynorphin A(1-8), respectively. Injections of 0.4 and 0.8 nmol/mouse of nor-BNI i.t., but not its inactive enantiomer (+)-1-nor-BNI, inhibited dose-dependently the antagonistic effects of the dynorphins. These doses of nor-BNI alone did not affect the antinociceptive action of morphine sulfate. Intrathecal administration of 5 nmol/mouse of NTI also did not affect the modulatory effects of dynorphins. These observations that dynorphins exert their antagonistic effects on morphine-induced antinociception stereoselectively through spinal kappa opioid receptors may suggest a coupling between spinal kappa and mu opioid receptors.

The effects of the kappa-selective ligands dynorphin A (1-13) (DYN) and U50, 488H (U50) on free intracellular calcium were evaluated using synaptosomes prepared from the cerebellum of the guinea pig, an area with a high density of kappa receptors. DYN (10 microM) produced small nonsignificant decreases in basal free intracellular calcium (5-7%). U50 (10 microM) produced significant 15-20% decreases in basal free intracellular calcium which were reversed by nor-BNI (1 microM). When intracellular calcium levels were increased 8-10% by the administration of c-AMP or forskolin, DYN (10 microM) produced significant decreases in intracellular calcium of 10%. The effects of U50, 488H were not enhanced by increasing the synaptosomal levels of c-AMP. Neither DYN nor U50 (1 microM) significantly blocked the rise in free intracellular calcium induced by 50 mM KCI. When intracellular calcium concentrations were increased by the administration of 50 mM KCI prior to the administration of DYN or U50 (10 microM), the kappa ligands decreased intracellular calcium concentrations. These data indicate that DYN and U50 interact with kappa receptors resulting in a decrease in free intracellular calcium possibly via an enhancement of the efflux of calcium. The modulation of intracellular free calcium by the kappa opioids may be a mechanism by which these opioids produce their biological effects.

Beside the well known actions of opioid peptides on mu-, delta- and kappa-opioid receptors, increasing amount of pharmacological and biochemical evidence has recently been published about non-opioid actions of various opioid peptides. These effects are not abolished by naloxone treatments. Such non-opioid effects are observed both in nervous tissues and in the cellular elements of the immune system. Peptides exhibiting non-opioid effects include beta-endorphin, dynorphin A, nociceptin/OFQ, endomorphins, hemorphins and a number of Proenkephalin A derived peptides, such as Met-enkephalin, Met-enkephalin-Arg-Phe (MERF) and bovine adrenal medullary peptide (BAM22). Non-opioid actions are exerted through different neuronal receptors, e.g., dynorphin hyperalgesia through NMDA receptor, Met-enkephalin induced regulation of cell growth through zeta receptors, pain modulation by nociceptin through ORL-1 or NOP receptors, while BAM22 acts through sensory neuron specific G protein-coupled receptors (SNSR). We have investigated Met-enkephalin-Arg-Phe (MERF) and its analogues by the means of direct and indirect radioligand binding assays. It has been found that in addition to kappa(2) and delta-opioid receptors, MERF can act also through sigma(2)- or probably via FMRF-NH(2) receptors in rat cerebellum. A role of functionally assembling heterodimer receptors in mediating the non-conventional actions of these peptide ligands can not be excluded as well.

The administration of dynorphin A(1-17), Dyn, intrathecally (i.t.) or of nociceptin, intracerebroventricularly (i.c.v.) produces antianalgesic actions against i.t. morphine in the tail flick test in mice. The antianalgesic action of nociceptin is mediated by spinal PGE 2 and attenuated by i.t. PGD 2 or indomethacin. The Dyn response is mediated by release of IL 1β in the spinal cord to activate an ascending pathway to the brain and in turn releases IL 1β in the brain which activates a descending pathway to the spinal cord. The present work investigated the possibility that the action of IL 1β in the Dyn system might release prostaglandins so that the Dyn and nociceptin antianalgesic systems would overlap at these points. The results indicated that in the Dyn system neither the IL 1β in the spinal cord or brain implicated prostaglandin release because i.t. and i.c.v. PGD 2 and indomethacin did not affect Dyn-induced antianalgesia. In addition, nociceptin-induced antianalgesia did not involve components in the Dyn system. Thus, the Dyn and nociceptin antianalgesic systems did not overlap and each were independent systems.

Opioids inhibit the release of catecholamines in the nervous system. Normal adrenal chromaffin cells produce delta opioids and they respond to them by suppressing the release of their catecholamines. Chromaffin cell tumors, the pheochromocytomas, produce mainly kappa opioids. The aim of this work was: (a) to test if pheochromocytomas retain the response of normal chromaffin cell catecholamines to delta opioids and to naloxone (a general opioid antagonist), and (b) to test if kappa opioids exert any specific effect on catecholamine release from these tumors. Since we have previously shown that, in common with human pheochromocytomas, the PC12 rat pheochromocytoma cells express the prodynorphin gene and secret its kappa opioid products, we used these cells to examine the effect of several opioid agonists and of naloxone on basal, nicotine-, and KCI-induced dopamine release. Dopamine is the main PC12 catecholamine. We have found that the specific kappa opioid agonist U-69593 inhibited the release of dopamine in a dose-dependent manner (DICD 50 = 0.5 × 10 −8 DMD). Under basal conditions the mean concentration of dopamine in the culture media was 11.25±0.57 ng/mg of total cellular protein (n = 13). A 30 min exposure to U-69593 at 10 −6 M suppressed basal dopamine release to 58±2%. (n = 7) of controls. A 12 hr pre-incubation with U-69593 caused the same degree of suppression. The effect of the synthetic kappa opioid agonist dynorphin A was indistinguishable from that of U-69593. DADLE (a mu and delta synthetic opioid agonist) was significantly less effective in suppressing dopamine release (IC50 = 10 −7 M). The concentration of dopamine following exposure to 10 −6 M of DADLE for 30 min was 74±5% of the controls (n = 4). The mu opioid agonist DAGO was ineffective. The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved.

When rat ventricular myocytes were stimulated with dynorphin A(1-13), a transient and rapid increase followed by a sustained and prolonged elevation in the intracellular levels of inositol 1,4,5-trisphosphate ¿Ins(1,4,5)P3¿ was observed. The responses were dose-related and abolished by nor-binaltorphimine. In the presence of lithium and absence of extracellular free inositol, the initial rapid elevation in Ins(1,4,5)P3 remained the same, but the second phase of sustained and prolonged elevation was abolished. Under this condition, the elevation in cytosolic free Ca2+ ([Ca2+]i) was reduced significantly although there was still a detectable elevation over a time period when the Ins(1,4,5)P3 was at the basal level. The responses in Ins(1,4,5)P3 and [Ca2+]i were not affected by lithium when stimulation of ventricular myocytes with dynorphin A(1-13) was performed in the presence of extracellular inositol. The data suggest that in rat ventricular myocytes, the kappa-opioid receptor agonist stimulated mobilization of [Ca2+]i was mediated mainly by Ins(1,4,5)P3.

Previously, we have demonstrated that intrathecally (i.t.) administered corticotropin-releasing factor (CRF) in mice produces stimulus-specific antinociception and modulation of morphine-induced antinociception by mechanisms involving spinal kappa opioid receptors. Recently, we also have found that CRF releases immunoreactive dynorphin A, a putative endogenous kappa opioid receptor agonist, from superfused mice spinal cords in vitro. Dynorphin A administered intracerebroventricularlly (i.c.v.) to mice has been shown to modulate the expression of morphine tolerance. In the present study, the possible modulatory effects of i.t. administered CRF as well as dynorphin A on morphine tolerance were studied in an acute tolerance model. Subcutaneous administration of 100 mg/kg of morphine sulfate (MS) to mice caused an acute tolerance to morphine-induced antinociception. The antinociceptive ED50 of MS was increased from 4.4 mg/kg (naive mice) to 17.9 mg/kg (4 hours after the injection of 100 mg/kg MS). To study the modulatory effects of spinally administered CRF and dynorphin A on the expression of morphine tolerance, CRF and dynorphin A were injected i.t. at 15 min and 5 min, respectively, before testing the tolerant mice by the tail-flick assay. The antinociceptive ED50 of MS in tolerant mice was decreased to 8.8 mg/kg and 7.1 mg/kg, respectively, after i.t. administration of CRF (0.1 nmol) and dynorphin A (0.2 nmol). In contrast, 0.5 nmol of alpha-helical CRF (9-41), a CRF antagonist and 0.4 nmol of norbinaltorphimine, a highly selective kappa opioid receptor antagonist, when administered i.t. at 15 min before the tail-flick test in tolerant mice, increased the antinociceptive ED50 of MS to 56.6 mg/kg and 88.8 mg/kg, respectively. These data confirmed the modulatory effect of dynorphin A on morphine tolerance and suggested that CRF, which releases dynorphin A in several central nervous system regions, also plays a modulatory role in the expression of morphine tolerance.

Methionine-enkephalin (Met-enkephalin), leucine-enkephalin (Leu-enkephalin) and dynorphin A (1-17) (dynorphin A) concentrations in discrete brain areas were determined in the mice showing behavioral changes induced by stress using radioimmunoassay (RIA). In the present experiment, we used environment-induced conditioned suppression of motility and forced swimming-induced immobility. In the environment-induced conditioned suppression of motility, Met-enkephalin concentration in the striatum and hypothalamus significantly decreased. Leu-enkephalin concentration in the hypothalamus also decreased. Dynorphin A concentration in the striatum decreased, but significantly increased in the hypothalamus and pituitary. In the forced swimming-induced immobility, Met-enkephalin concentration in the striatum significantly decreased. Leu-enkephalin concentration in the hypothalamus and pituitary significantly decreased. Dynorphin A concentration in the pituitary decreased, but significantly increased in the hypothalamus. Our results indicated that the concentrations of Met-enkephalin, Leu-enkephalin and dynorphin A in the discrete brain areas changed in two different stressful situations. These findings suggested that these peptides might modulate the behavioral changes induced by stressors.

Three peptide neuromodulators that are found in high concentration in the subtantia nigra: dynorphin A 1–8, met5-enkephalin-arg6-gly7-leu8 and substance P, were measured by specific radioimmunoassays in nigral tissue from normals and schizophrenics postmortem. Substance P and dynorphin were unchanged between the two groups. However, the proenkephalin-derived peptide was significantly elevated in the schizophrenic group. The immunoreactivity was identified as authentic met5-enkephalin-arg6-gly7-leu8 by high pressure liquid chromatography. The data suggest that a different set of regulatory controls exists for nigral enkephalin peptides as compared to dynorphin and substance P, and that the former system may be disordered in schizophrenia.

The development of proopiomelanocortin (POMC) derived peptides was compared to that of leucine-enkephalin ([Leu]ENK) and dynorphin A (DYN-A) immunoreactivity (i.r.) in the rat CNS and pituitary gland. POMC i.r. appeared first in hypothalamic neurons on embryonic day E12, in pituitary anterior lobe (AL) cells on E15, in pituitary intermediate lobe (IL) cells on E16, and in perikarya of the nucleus tractus solitarius on E17. In the fetal stages (E19-22), all POMC systems appeared adult-like; however, peak i.r. occurred between postnatal days P21-28. The development of alpha-MSH (a-MSH) i.r. was dissimilar to that of other POMC peptides including beta-endorphin (B-End). In contrast, both [Leu]ENK and DYN-A i.r. appeared in later embryonic stages (E16-17), and their maturation lagged behind that of POMC peptides. Peak i.r. for these latter peptides also occurred between P21-28.

Intrathecally injected dynorphin A (1–13) in rats results in a reversible hindlimb paralysis and an irreversible loss of the tail-flick reflex. Histologic examination of the spinal cords of dynorphin treated rats demonstrated dead and/or dying neurons predominately localized in the central area which approximates Rexed lamina VII and X. In this area a maximum effect of the dynorphin-induced neurotoxicity is evident. Thus, the dynorphin-induced neuron death is suggestive of an anatomical selectivity.

Using combined autoradiography-immunocytochemistry, the anatomical distribution of [ 3 H]naloxone-labelled opiate receptors was compared to the loci of neuronal systems immunoreactive for β -endorphin, [Leu]enkephalin and dynorphin A in rhesus monkey brain. High densities of binding were observed in relation to each of the systems, consistent with findings that each opioid precursor can synthesize one or more peptides with substantial (though not selective) activity at μ receptors.

To examine the possible involvement of multiple opioid receptors in animal hibernation, we infused opioids selective for mu , kappa , and delta opioid receptors into summer-active ground squirrels ( Citellus tridecemlineatus ). The effects of those opioid treatments on the hibernation induced by HIT (Hibernation Induction Trigger) were also examined. Mu opioids morphine (1.50 mg/kg/day) and morphiceptin (0.82 mg/kg/day) and kappa opioid peptide dynorphin A (0.82 mg/kg/day) did not induce hibernation. On the contrary, morphine, morphiceptin and dynorphin A antagonized HIT-induced hibernation in summer-active ground squirrels. Infusion of delta opioid DADLE (D-Ala2-D-Leu5 enkephalin; 1.50 mg/kg/day), however, induced summer hibernation in a manner comparable to that induced by HIT. It is concluded therefore that delta opioid receptor and its ligand may be intimately involved in animal hibernation. In view of the fact that HIT was obtained from winter hibernating animals and might therefore be responsible for natural hibernation, our results also suggest that naturally occuring mu and kappa opioids may play an important role in the arousal state of hibernation.

Intracerebroventricular (i.c.v.) administration of dynorphin A [1-17] induced significant place preference conditioning in male, Sprague-Dawley rats. Place preferences were induced by 2.3 and 3.5 nmole, but not 1.2 nmole of dynorphin A. Co-administration of naloxone, 27.5 nmole but not 5.5 nmole, antagonized the reward response induced by 2.3 nmole of dynorphin A. Leu-enkephalin, 5 or 25 nmole, and dynorphin A [2-17], 2.3 or 3.5 nmole, had no effect in the place conditioning paradigm.

Intraventricular injection of morphine sulfate, 40 micrograms, released an enzyme from the spinal cord into the perfusate which degraded dynorphin A (1-8) and, to a lesser extent, dynorphin A (1-13) in urethane anesthetized rats. The enzyme did not degrade dynorphin A (1-17), Met-enkephalin, Leu-enkephalin, substance P and neurotensin. This dynorphin A (1-8) degrading enzyme was inhibited by aprotinin, thiorphan, and, to a lesser extent, by bacitracin but was not inhibited by bestatin. A kinetic study of the interaction between dynorphin A (1-8) and aprotinin with the enzyme indicated that it is competitive in nature. The pharmacological significance of the findings is still unknown.

The effects of dynorphin A (1–13) (DYN), injected into the preoptic area, was investigated on water intake in rats. DYN at both doses of 2 and 10 nmoles significantly increased water intake for two and four hours after the injection in a dose related fashion. However, no significant change was observed in food intake. Naloxone pretreatment (0.3 mg/kg, s.c.) completely attenuated the DYN-induced stimulation of water intake. The present studies suggest that DYN in the preoptic area may play an important role in the regulation of drinking behavior, but have no effect on food intake.

Two dynorphins of 24 and 32 residues, respectively, were isolated from porcine pituitary and sequenced. They both contain the dynorphin heptadecapeptide, now designated dynorphin A, at the amino terminus, followed immediately by the putative processing signal Lys-Arg. In the 24-residue peptide the subsequent five amino acids comprise the [Leu] enkephalin sequence. In the 32-residue peptide this [Leu] enkephalin sequence is part of a novel opioid tridecapeptide designated dynorphin B.

To investigate the role of multiple opiate receptors in spinal mechanisms of antinociception the activity of various opiate receptor agonists was determined against different nociceptive stimuli in the mouse and rat. Intrathecal administration of the putative kappa-receptor agonists dynorphin A (DYN), bremazocine, and ethylketocyclazocine, as well as the delta-agonist D-Ala2,D-Leu5-enkephalin and the mu-agonists, Tyr-D-Ala-Gly-MePhe-NH-(CH2)2OH and morphine resulted in a dose-dependent antinociceptive effect. The order of potencies was similar for visceral and thermal pain. Inhibition of writhing in mice was much more strongly antagonized by MR 2266 than by naloxone, and des-Tyr1-DYN1-13 was 50 times less potent than DYN in this test suggesting that DYN acts through the kappa-receptor. It is concluded that mu-, delta- and kappa-opiate receptors are involved in spinally mediated antinociception related to kinds of noxious stimuli.

The effects of dynorphin-A (Dyn) and [Leu5]-enkephalin (Enk) were compared in the in vitro hippocampal slice preparation, using extracellular field potential and intracellular voltage recordings. In the CAl region, Dyn, like Enk, consistently increased the size of the extracellularly recorded population spike (PS) evoked by stratum radiatum (StR) stimulation of the Schaffer collaterals. These responses were naloxone reversible. In contrast, in the CA3 region, Dyn both increased and decreased the PSs evoked by mossy fiber stimulation, whereas Enk slightly enhanced the PS. Intracellular rrecordings from CA3 pyramidal neurons (HPNs) revealed both excitatory and inhibitory actions of Dyn of spontaneous activity. Associated membrane potential changes were variable. In contrast, Enk had only weak effects on spontaneous activity and no effect on membrane potential. These data suggest regional differences in the effect of Dyn and Enk on hippocampal activity.

Gamma-hydroxybutyrate administered in anaesthetic doses produced a profound increase in the levels of dynorphin A (DYN) in the hippocampus and pituitary. Simultaneously, a significant, short-lasting decrease in beta-endorphin (B-E) levels occurred in the brain and pituitary. Antiepileptic drugs strongly, though in various ways, influenced the brain DYN and B-E concentrations; though most of them caused a decrease in the hypothalamic opioid peptide content.

The effect of various opioids on Na + , K + -ATPase partially purified from rat heart was examined. Dynorphin-A(1–13), dynorphin-A(1–17) and ethylketocyclazocine (EKC), which are κ-type opiate agonists, markedly inhibited the enzyme activity in a dose-dependent manner; IC 50 values were 12 μM, 21 μM and 0.38 mM, respectively. Morphine (μ-type agonist), methionine- and leucine-enkephalin (δ-type agonist) at the concentration of 1 mM did not affect the enzyme activity. The effect of dynorphin-A(1–13) and EKC was not antagonized by naloxone. Dynorphin-A(1–13) mainly decreased Vmax value without the change of Km value in the activation of Na + , K + -ATPase by ATP, Na + and K + . Dynorphin-A(1–13) inhibited the partial reactions of Na + , K + -ATPase at the different degree of the potency; the inhibition of K + -stimulated phosphatase was greater than that of Na + -dependent phosphorylation. The present study suggests that dynorphin-A and EKC have an effect on cardiovascular system which is mediated by the inhibition of Na + , K + -ATPase in the heart.

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.

The molecular forms of Dynorphin A immunoreactivity (Dyn A-IR) detected in hippocampus were resolved by reverse-phase high pressure liquid chromatography (C18-HPLC). Peptide co-eluting with synthetic Dyn A(1–17) represents 20% of the total Dyn A-IR. Dyn A-IR was released from hippocampal slices in vitro by a Ca++-dependent mechanism stimulated by superfusion with K+, kainate or veratrine. Released peptides were resolved by C18-HPLC and shown to contain Dyn A-IR co-eluting with synthetic Dyn A(1–17). These observations are consistent with the hypothesis that Dyn A(1–17) acts as a neurotransmitter at synapses in hippocampus.

[3H]ethylketocyclazocine (EKC) and [3H]etorphine bind to an apparent single class of sites in the rat kidney (kd = 7.3 and 5.2 nM; Bmax = 33.2 and 59.6 fmol/mg protein, respectively). No detectable binding was observed for [3H]dihydromorphine and [3H]D-Ala2,D-Leu5 enkephalin. Ligand selectivity pattern strongly suggests that both [3H]EKC and [3H]etorphine label a kappa opiate binding site since (-)bremazocine greater than EKC greater than etorphine greater than naloxone greater than dynorphin A much greater than beta-endorphin. In the adrenal gland, [3H]EKC also labels a single class of sites (Kd = 20.1 nM; Bmax = 74.7 fmol/mg protein) while no specific binding was seen with mu and delta ligands. Autoradiographically, [3H]EKC and [3H]etorphine binding sites are highly localized in kidney cortex compared to outer and inner medulla. Rather surprisingly, [3H]EKC binding sites in the adrenal are highly localized in the cortex with only low densities in the medulla. We are currently investigating the possible physiological roles of kappa agonists in kidney and adrenal gland functions.

[3H]-Dynorphin A (1-9) is rapidly degraded in brain homogenates, even at 0 degree C. Although protection from degradation at O degree C, and to a lesser extent at 25 degrees C, was obtained with a combination of bestatin, L-leucyl-L-leucine, L-arginyl-L-arginine and captopril, these peptidase inhibitors had little effect on binding at 0 degree C. At the kappa-site, the maximum binding capacity of [3H]-dynorphin A (1-9) at 0 degree C was similar to that at 25 degrees C but it is suggested that, even after suppression of mu- and delta-binding, [3H]-dynorphin A (1-9) may bind to more than one site for which it has differing affinities at 0 degree C.

A recent neuropathological study has reported decreased levels of dynorphin A immunoreactivity in striato-pallidal fibers in the brain of a patient with severe Gilles de la Tourette's syndrome (TS). This observation, taken with the neuroanatomic distribution of dynorphin and its broad range of motor and behavioral effects, has led to speculation concerning its role in the pathobiology of TS. We report on the presence of elevated concentrations of dynorphin A [1–8] in the CSF of 7 TS patients, aged 20 to 45 years. The increase in CSF dynorphin was found to be associated with the severity of the obsessive compulsive symptoms but not with tic severity in these patients. Although CSF studies lack the precision necessary to address questions of selective involvement of neuronal systems in specific CNS locations, these findings suggest that endogenous opioids are involved in the pathobiology of TS and related disorders. Tourette's syndrome (TS) is a chronic neuropsychiatric disorder of childhood onset that is characterized by multiple motor and phonic tics that wax and wane in severity and an array of behavioral problems including some forms of obsessive compulsive disorder (OCD) (1). Once thought to be a rare condition, the prevalence of TS is now estimated to be on case per 1, 000 boys and one case per 10, 000 girls, and milder variants of the syndrome are likely to occur in a sizeable percentage of the population (2). Although the etiology of TS remains unknown, the vertical transmission of TS within families follows a pattern consistent with an autosomal dominant form of inheritance (3, 4). Neurobiologic and pharmacological data have implicated central monoaminergic and neuropeptidergic systems in the pathophysiology of TS, and basal ganglia structures remain the prime candidates as the neuroanatomical origin for TS and related conditions (1). Endogenous opioids, including dynorphin and met-enkephalin are concentrated in structures of the basal ganglia (5), are known to interact with central dopaminergic neurons (6, 7), and may play an important role in the control of motor functions (8). Post-mortem brain studies have directly implicated opioids in the pathophysiology of Parkinson's disease (9), Huntington's disease (10), and most recently in TS (11). The neuropathological study of Haber et al. (11) reported decreased levels of dynorphin A [1–17] immunoreactivity in striatal fibers projecting to the globus pallidus in the brain of a patient with severe TS. This observation, taken with the neuroanatomic distribution of dynorphin and its broad range of motor and behavioral effects, has led to speculation concerning its role in the pathobiology of TS. Additional evidence supporting the role of endogenous opioids comes from reports of dramatic, but poorly documented, effects of opiate antagonists in the treatment of TS patients (12, 13).

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin)-sensitive GTP-binding proteins was investigated by studying effects of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) binding by increasing the dissociation constant of [3H]DADLE and membranes, and enhanced slightly [3H]diprenorphine binding. IAP treatment of membranes reduced [3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [3H]DADLE binding. Treatment of membranes with IAP and [32P]NAD resulted in radio-labeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, beta-endorphin, levorphanol and dynorphin A(1-13) did not show any significant inhibitory action on bovine adrenal medullary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.