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PubMed1996

Role of vasopressin in altered pial artery responses to dynorphin and beta-endorphin following brain injury.

Pial artery constriction following fluid percussion brain injury (FPI) is associated with elevated CSF dynorphin and beta-endorphin concentration in newborn pigs. Additionally, dynorphin is a dilator under control conditions and a vasoconstrictor under decreased cerebrovascular tone conditions. Vasopressin contributes to beta-endorphin-induced pial constriction and the constrictor potential for dynorphin. Recently, it has been observed that FPI reverses vasopressin from a dilator to a constrictor. The present study was designed to characterize the effect of FPI on beta-endorphin-induced constriction and the role of vasopressin in that constriction as well as in the reversal of dynorphin's vascular response following FPI. Brain injury of moderate severity (1.9 - 2.3 atm) was produced in anesthetized newborn pigs equipped with a closed cranial window. Dynorphin in physiologic and pharmacologic concentrations (10(-10), 10(-8), 10(-6) M) was reversed from a dilator to a constrictor following FPI (7 +/- 1, 11 +/- 1, and 16 +/- 1 vs -4 +/- 1, -7 +/- 1, and -11 +/- 1% before and after FPI, respectively). Dynorphin-induced vascular changes were accompanied by increased cortical periarachnoid CSF vasopressin and these biochemical changes were potentiated following FPI (24 +/- 4 vs 134 +/- 7 and 53 +/- 7 vs 222 +/- 14 pg/mliter for control and dynorphin (10(-6) M) before and after FPI, respectively). In contrast, in animals pretreated with the vasopressin receptor antagonist [1-(beta-mercapto-beta beta-cyclopentamethylene propionic acid) 2-(O-methyl)-Tyr-AVP] (MEAVP, 5 micrograms/kg iv), dynorphin-induced constriction following FPI was attenuated (6 +/- 1, 12 +/- 1, and 16 +/- 1, vs -2 +/- 1, -4 +/- 1, and -7 +/- 1% before and after FPI, respectively). Additionally, beta-endorphin-induced pial constriction was potentiated following FPI (-7 +/- 1, -10 +/- 1, -15 +/- 1 vs -10 +/- 1 -15 +/- 2, and -21 +/- 2% for beta-endorphin (10(-10), 10(-8), 10(-6) M) before and after FPI, respectively). beta-endorphin-induced CSF vasopressin release was similarly potentiated following FPI. Further, MEAVP blunted the augmented constrictor responses to beta-endorphin observed following FPI (-5 +/- 1, -9 +/- 1, -14 +/- 1 vs -2 +/- 1, -5 +/- 1, and -8 +/- 1% before and after FPI, respectively). These data indicate that FPI potentiates beta-endorphin-induced pial construction and reverses dynorphin from a dilator to a constrictor. Additionally, these data show that vasopressin contributes to augmented beta-endorphin pial constriction and the reversal of dynorphin's vascular effects following FPI. Further, since CSF dynorphin and beta-endorphin concentrations are increased following FPI, these data suggest that these two opioids contribute to pial artery constriction observed following FPI, at least, in part, via the release of vasopressin.

Animals↗
PubMed2000

Dynorphin A elicits an increase in intracellular calcium in cultured neurons via a non-opioid, non-NMDA mechanism.

The opioid peptide dynorphin A is known to elicit a number of pathological effects that may result from neuronal excitotoxicity. An up-regulation of this peptide has also been causally related to the dysesthesia associated with inflammation and nerve injury. These effects of dynorphin A are not mediated through opioid receptor activation but can be effectively blocked by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists, thus implicating the excitatory amino acid system as a mediator of the actions of dynorphin A and/or its fragments. A direct interaction between dynorphin A and the NMDA receptors has been well established; however the physiological relevance of this interaction remains equivocal. This study examined whether dynorphin A elicits a neuronal excitatory effect that may underlie its activation of the NMDA receptors. Calcium imaging of individual cultured cortical neurons showed that the nonopioid peptide dynorphin A(2-17) induced a time- and dose-dependent increase in intracellular calcium. This excitatory effect of dynorphin A(2-17) was insensitive to (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) pretreatment in NMDA-responsive cells. Thus dynorphin A stimulates neuronal cells via a nonopioid, non-NMDA mechanism. This excitatory action of dynorphin A could modulate NMDA receptor activity in vivo by enhancing excitatory neurotransmitter release or by potentiating NMDA receptor function in a calcium-dependent manner. Further characterization of this novel site of action of dynorphin A may provide new insight into the underlying mechanisms of dynorphin excitotoxicity and its pathological role in neuropathy.

Animals↗
PubMed1990

Effect of hypertonic saline infusion on the level of immunoreactive dynorphin in extracted human plasma.

Dynorphin-A and its related peptides are derived from prodynorphin, one of the three known endogenous opioid precursors. The prodynorphin gene is expressed in the vasopressinergic magnocellular neurons of the hypothalamus, while its peptide products are present in the vasopressin (AVP) neurosecretory vesicles of the neurohypophysis. The concentration of immunoreactive (IR) dynorphin is orders of magnitude higher in the neurohypophysis than in any other tissue, suggesting that perhaps the prodynorphin-derived peptides are secreted from the hypothalamic-neurohypophyseal unit into the general circulation. Experiments in rats have shown that osmotic stimuli increase both AVP and prodynorphin in the hypothalamus. To determine whether human hypothalamic prodynorphin is also under osmotic regulation, we measured plasma IR-dynorphin, plasma IR-AVP, and serum sodium immediately before and during the infusion of normal or hypertonic saline in normal human volunteers. Because of the unusual susceptibility of the prodynorphin-derived peptides to cleavage by endopeptidases, we also developed an appropriate plasma dynorphin extraction technique. We found that the IR-dynorphin present in human plasma was composed of 6K- and 4K-sized peptides and that no larger than 6K or smaller than 4K dynorphins were present. The infusion of normal saline did not have any significant effect on plasma IR-dynorphin, while 3% hypertonic saline increased its plasma levels. Thus, the mean IR-dynorphin level in the plasma of the volunteers infused with normal saline was 40.3 +/- 6.4 fmol/mL (mean +/- SE; n = 6) at zero time; after 30 min of infusion, plasma IR-dynorphin was 36.0 +/- 6.3, after 60 min it was 29.9 +/- 5, after 90 min it was 36.0 +/- 4.7, after 120 min it was 36.8 +/- 3.2, and after 150 min it was 36.0 +/- 6.1. The plasma IR-dynorphin level in the volunteers infused with hypertonic saline was 31.7 +/- 3.5 fmol/mL (mean +/- SE; n = 10) at zero time. After 30 min of infusion it increased to 37.4 +/- 3.8, after 60 min to 46.4 +/- 7.7, after 90 min to 56.2 +/- 9.1, after 120 min to 53.6 +/- 8.7, and after 150 min to 99.0 +/- 14.2. The increase in plasma IR-dynorphin with time was significant (P less than 0.0001) and correlated positively with serum sodium and plasma AVP. The physiological role of the prodynorphin-derived peptides of the hypothalamic-neurohypophyseal unit is not yet known.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromatography, Affinity↗
PubMed1995

Comparative study of the analgesic and paralytic effects induced by intrathecal dynorphin a in rats.

Intrathecal injection of dynorphin A produced dual effects on sensory and motor functions in the spinal cord of the rat. At a dose of 5 nmol, dynorphin A produced an increase in tail flick latency (TFL) as well as a reversible motor paralysis as assessed by change in the angle of inclined plane. At a dose of 10 or 20 nmol, dynorphin produced a motor paralysis lasting for up to 24 hours. The effect of dynorphin A on the sensory function of the spinal cord was shown by an increase in the vocalization threshold induced by electrical stimulation of the tail, at dose range of 1.25-10 nmol, with a quick onset (5 min) and relatively short duration (within 60 min). Unlike tail flick reaction which involves spinal motor function, tail stimulation-induced vocalization threshold is a relatively pure index for spinal nociceptive activities. The differential effect of dynorphin on sensory and motor function was supported by the evidence that (1) dynorphin-induced analgesic effect (increase in vocalization threshold) was naloxone reversible, whereas dynorphin-induced motor paralysis was naloxone resistant. (2) Nor-BNI, a specific antagonist for kappa opioid receptor, blocked the sensory effect of dynorphin, but had no influence on motor effect of dynorphin. It is thus concluded that dynorphin has both analgesic and paralytic effects in spinal cord. The analgesia shown by an increase of vocalization threshold is an opioid effect, most probably mediated by kappa opioid receptor; the paralytic effect, however, is a non-opioid effect. The increase of TFL induced by dynorphin involves both sensory (analgesia) and motor (paralysis) effects.

Analgesics↗
PubMed1989

Dynorphin-selective inhibition of adenylyl cyclase in guinea pig cerebellum membranes.

Guinea pig cerebellum, which contains kappa opioid receptors uncontaminated by other opioid receptor types, was chosen to examine whether kappa receptors are coupled to adenylyl cyclase. Membranes were prepared from guinea pig cerebellum and pretreated at pH 4.5 to increase inhibitory activity, and adenylyl cyclase was assayed in the presence of dynorphin analogs as prototypical kappa agonists. Results showed that several dynorphin analogs inhibited adenylyl cyclase by 30-50%, whereas mu- and delta-preferring agonists had no effect. Dynorphin A and the kappa-selective compounds D-Pro10-dynorphin(1-11) and U-50,488H were the most potent agonists, with IC50 values of 0.03-0.05 microM, whereas other dynorphin gene products like dynorphin B and alpha-neo-endorphin were approximately 10-fold less potent. Like other Gi-coupled responses, dynorphin-inhibited adenylyl cyclase required GTP and sodium. Naloxone was a competitive antagonist for dynorphin-inhibited adenylyl cyclase, with 1 microM naloxone shifting the IC50 value of dynorphin A by 20-fold. The kappa-selective antagonist nor-binaltorphimine was even more potent, with 0.1 microM nor-binaltorphimine shifting the dynorphin IC50 value by 50-fold. These results suggest that dynorphin A and its analogs inhibit adenylyl cyclase by binding to a guanine nucleotide-binding protein-coupled opioid receptor whose pharmacological specificity matches those of kappa receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1994

Effect of dynorphin microinjection in the paraventricular nucleus on the hemodynamic response to hemorrhage in the rat.

We studied the effect of dynorphin A-(1-13), dynorphin A-(1-17), des-tyr dynorphin A-(2-17) (inactive at opioid receptor) or normal saline (NS) microinjected into the paraventricular nucleus (PVN) (n = 9/treatment) on mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), and left ventricular stroke work (LVSW) during fixed-volume hemorrhage in conscious rats. Microinjection of dynorphin A-(1-13) (6 nmol) into PVN at 15 min following the termination of fixed volume hemorrhage (8 ml/300 g) significantly decreased MAP from 50 min to 2 hr postinjection (P < 0.05 compared to animals receiving NS), while dynorphin A-(1-17) (6 nmol) significantly decreased MAP from 30 min up to 2 hr postinjection (P < 0.05). Microinjection of des-tyr dynorphin A-(2-17) (6 nmol) into the PVN did not significantly affect MAP following hemorrhage. Recovery of MAP in the dynorphin A-(1-13) and dynorphin A-(1-17) groups following hemorrhage was found to be significantly attenuated compared to the NS group (P < 0.05 and P < 0.01, respectively). Dynorphin A-(1-13) increased heart rate at 20 min and decreased stroke volume at 60 min after microinjection directly into the PVN following hemorrhage when compared with the NS group (P < 0.05). Both dynorphin A-(1-13) and dynorphin A-(1-17) significantly decreased LVSW after PVN injection following hemorrhage compared to NS group (both P < 0.05). No significant effects were observed on CO following microinjection of active or inactive opioid peptides into the PVN following hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in Sprague-Dawley rats.

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg6 levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg6 levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg6 and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg6 levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg6 levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointermediate lobe were 0ven lower in the withdrawal state.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1986

Chronic haloperidol and clozapine differentially affect dynorphin peptides and substance P in basal ganglia of the rat.

The effect of chronic neuroleptic treatment, using haloperidol or clozapine, on immunoreactive dynorphin peptide and substance P levels in basal ganglia of rats was examined. The drugs were administered i.p. in daily doses for 10 days (haloperidol 1 mg/kg and clozapine 10 mg/kg). Dynorphin A, dynorphin B and substance P were measured in substantia nigra, striatum, globus pallidus and hypothalamus using specific radioimmunoassays. The most prominent effects were observed with with clozapine which increased levels of all measured peptides in substantia nigra. Haloperidol only affected nigral substance P levels which declined, while nigral dynorphin peptide levels remained unchanged. In striatum, haloperidol slightly reduced dynorphin peptides while substance P was unaffected. Clozapine increased striatal substance P but the dynorphin peptides were not affected. Minor changes in dynorphin peptides found in globus pallidus and hypothalamus were not statistically reliable. Substance P was not changed in these structures after either of the two drugs. High molecular weight fragments (greater than or equal to 5,000) from the dynorphin precursor, proenkephalin B, were measured in substantia nigra and striatum using trypsin digestion and subsequent analysis of generated Leu-enkephalin-Arg6. These high molecular weight fragments were found to be affected in the same manner as the dynorphin peptides. This study indicates that the two types of neuroleptic drugs have different modes of interaction on peptide systems in basal ganglia of rats. Dynorphin peptides and substance P were also differentially affected.

Animals↗
PubMed1982

A specific radioimmunoassay for the opioid peptide dynorphin B in neural tissues.

Dynorphin-32 was recently isolated from porcine pituitary and shown to consist of dynorphin A (the originally isolated dynorphin heptadecapeptide) at the amino terminus, followed by Lys-Arg (a putative signal for proteolytic cleavage) and then a tridecapeptide, dynorphin B, at the carboxyl terminus. Dynorphin B, like dynorphin A, contains leucine enkephalin. The present report describes and validates a radioimmunoassay for dynorphin B using an antiserum, "13S", that does not crossreact with other known opioid peptides. Immunoreactive dynorphin B was estimated in rat and porcine neural tissues and found to be 2-3 fold higher than reported values for dynorphin A. Distribution based on tissue concentration was similar to that of dynorphin A, with very high concentrations in neurointermediate pituitary. Small quantities of antiserum "13S" are available to investigators upon request.

Animals↗
PubMed1992

Dynorphin A (1-13) in the brain suppresses epinephrine-induced ventricular premature complexes and ventricular tachyarrhythmias.

The objectives of this study were to test the hypothesis that dynorphin in the central nervous system modulates epinephrine-induced cardiac arrhythmias and that central cholinergic mechanisms are operative in this action of dynorphin. Cardiac arrhythmias were produced by continuous intravenous infusion of epinephrine, in Wistar rats, previously instrumented with catheters in the lateral cerebral ventricle, femoral vein and femoral artery. Epinephrine produced ventricular premature complexes and later the development of fatal ventricular fibrillation. Dynorphin A (1-13), 5 or 20 micrograms (3 or 12 nM) administered into the lateral cerebral ventricle (ICV), significantly (P less than 0.05) increased the threshold for development of cardiac arrhythmias. Dynorphin A (1-13), 20 micrograms, increased the epinephrine dose at the occurrence of ventricular premature beats to 171 +/- 8 (mean +/- 1 S.E.M.) compared to 120 +/- 5 micrograms epinephrine/kg in the control group and increased the dose at the onset of fatal arrhythmias to 186 +/- 8 compared to 141 +/- 10 micrograms epinephrine/kg in the control group. The action of dynorphin was significantly (P less than 0.05) antagonized by the kappa opioid antagonist MR2266. Atropine sulfate, administered ICV or intravenously, produced a dose dependent antagonism of this action of dynorphin A (1-13). This was not due to the peripheral effects of atropine, as atropine methylnitrate, which does not cross the blood brain barrier, did not oppose the effects of dynorphin A (1-13). These data indicate (i) dynorphin A (1-13) increases the threshold for or suppresses the manifestations of epinephrine-induced ventricular arrhythmias, (ii) dynorphin's action on cardiac arrhythmias is mediated through central cholinergic rather than peripheral parasympathetic mechanisms (iii) dynorphin may play a role as an endogenous opioid within the brain that modulates cardiac arrhythmias in circumstances of elevated circulating epinephrine concentration.

Animals↗
PubMed1997

Opiate modulation of dynorphin conversion in primary cultures of rat cerebral cortex.

Rat brain cortical cells in primary culture were used to investigate long-term effects of opiates on endopeptidases acting on dynorphin peptides. Enzyme activity in the soluble fraction of the cells converted dynorphin B to Leu-enkephalin-Arg6 and to a lesser extent to Leu-enkephalin. Five day treatment with 10 microM morphine increased the conversion to Leu-enkephalin-Arg6 by 370%. This effect was prevented by the presence of naloxone in the culture medium. The opiate-inducible activity was directed to the Arg-Arg bond in dynorphins with preference for dynorphin B > alpha-neoendorphin > > dynorphin A. The Km for the generation of Leu-enkephalin-Arg6 from dynorphin B was 40 microM. Enzyme activity was inhibited by dynorphin fragments, in the following order of potency: dynorphin A(1-13) > A(2-13) > A(1-17) > A(2-17) and by SH-reagents, suggesting the presence of a cysteine-protease. The opiate-stimulated dynorphin-converting enzyme (DCE)-activity affects the balance between dynorphin peptides (selective for kappa-opioid receptors) and enkephalin peptides (selective for delta-opioid receptors). Since both types of opioid peptides can influence the development of opiate tolerance, the change in the extent of this transformation may be functionally important.

Animals↗
PubMed1996

Dynorphin and epilepsy.

Studies on dynorphin involvement in epilepsy are summarised in this review. Electrophysiological, biochemical and pharmacological data support the hypothesis that dynorphin is implicated in specific types of seizures. There is clear evidence that this is true for complex partial (limbic) seizures, i.e. those characteristic of temporal lobe epilepsy, because; (1) dynorphin is highly expressed in various parts of the limbic system, and particularly in the granule cells of the hippocampus; (2) dynorphin appears to be released in the hippocampus (and in other brain areas) during complex partial seizures; (3) released dynorphin inhibits excitatory neurotransmission at multiple synapses in the hippocampus via activation of kappa opioid receptors; (4) kappa opioid receptor agonists are highly effective against limbic seizures. Data on generalised tonic-clonic seizures are less straightforward. Dynorphin release appears to occur after ECS seizures and kappa agonists exert a clear anticonvulsant effect in this model. However, more uncertain biochemical data and lack of efficacy of kappa agonists in other generalised tonic-clonic seizure models argue that the involvement of dynorphin in this seizure type may not be paramount. Finally, an involvement of dynorphin in generalised absence seizures appears unlikely on the basis of available data. This may not be surprising, given the presumed origin of absence seizures in alterations of the thalamo-cortical circuit and the low representation of dynorphin in the thalamus. In conclusion, it may be suggested that dynorphin plays a role as an endogenous anticonvulsant in complex partial seizures and in some cases of tonic-clonic seizures, but most likely not in generalised absence. This pattern of effects may coincide with the antiseizure spectrum of selective kappa agonists.

Animals↗
PubMed1995

Dynorphins modulate DNA synthesis in fetal brain cell aggregates.

Previously, opioid peptide analogues, beta-endorphin, and synthetic opiates were found to inhibit DNA synthesis in 7-day fetal rat brain cell aggregates via kappa- and mu-opioid receptors. Here dynorphins and other endogenous opioid peptides were investigated for their effect on DNA synthesis in rat and guinea pig brain cell aggregates. At 1 microM, all dynorphins tested and beta-endorphin inhibited [3H]thymidine incorporation into DNA by 20-38% in 7-day rat brain cell aggregates. The putative epsilon-antagonist beta-endorphin (1-27) did not prevent the effect of beta-endorphin, suggesting that the epsilon-receptor is not involved in opioid inhibition of DNA synthesis. The kappa-selective antagonist norbinaltorphimine blocked dynorphin A or B inhibition of DNA synthesis, implicating a kappa-opioid receptor. In dose-dependency studies, dynorphin B was three orders of magnitude more potent than dynorphin A in the attenuation of thymidine incorporation, indicative of the mediation of its action by a discrete kappa-receptor subtype. The IC50 value of 0.1 nM estimated for dynorphin B is in the physiological range for dynorphins in developing brain. In guinea pig brain cell aggregates, the kappa-receptor agonists U50488, U69593, and dynorphin B reduced thymidine incorporation by 40%. When 21-day aggregates were treated with dynorphins, a 33-86% enhancement of thymidine incorporation was observed. Because both 7- and 21-day aggregates correspond to stages in development when glial cell proliferation is prevalent and glia preferentially express kappa-receptors in rat brain, these findings support the hypothesis that dynorphins modulate glial DNA synthesis during brain ontogeny.

Animals↗
PubMed1999

Antianalgesic action of dynorphin A mediated by spinal cholecystokinin.

Previous work indicates that the antianalgesic action of pentobarbital and neurotensin administered intracerebroventricularly in mice arises from activation of a descending system to release cholecystokinin (CCK) in the spinal cord where CCK is known to antagonize morphine analgesia. Spinal dynorphin, like CCK, has an antianalgesic action against intrathecally administered morphine. This dynorphin action is indirect; even though it is initiated in the spinal cord, it requires the involvement of an ascending pathway to the brain and a descending pathway to the spinal cord where an antianalgesic mediator works. The aim of the present investigation was to determine if the antianalgesic action of intrathecal dynorphin A involved spinal CCK. All drugs were administered intrathecally to mice in the tail flick test. Morphine analgesia was inhibited by dynorphin as shown by a rightward shift of the morphine dose-response curve. The effect of dynorphin was eliminated by administration of the CCK receptor antagonists lorglumide and PD135 158. One hour pretreatment with CCK antiserum also eliminated the action of dynorphin. On the other hand, the antianalgesic action of CCK was not affected by dynorphin antiserum. Thus, CCK did not release dynorphin. Both CCK and dynorphin were antianalgesic against DSLET but not DPDPE, delta 2 and delta 1 opioid receptor peptide agonists, respectively. The results suggest that the antianalgesic action of dynorphin occurred through an indirect mechanism ultimately dependent on the action of spinal CCK.

Analgesia↗
PubMed2002

Relationship between NOC/oFQ, dynorphin, and COX-2 activation in impaired NMDA cerebrovasodilation after brain injury.

Previous studies have observed that the recently described endogenous opioid, nociceptin/orphanin FQ (NOC/oFQ), contributes to impairment of N-methyl-D-aspartate (NMDA)-induced cerebrovasodilation following fluid percussion brain injury (FPI) via a cyclooxygenase (COX)-dependent generation of superoxide anion (O(2)(-)). This study was designed to investigate the relationship between NOC/oFQ, another opioid, dynorphin, and activation of the COX-2 isoform of the enzyme in such impaired dilation to NMDA after FPI in piglets equipped with a closed cranial window. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O(-)(2) generation. Under non-brain injury conditions, NOC/oFQ (10(-10) M), the CSF concentration observed after FPI, increased CSF dynorphin, while the NOC/oFQ antagonist [F/G] NOC/oFQ (1-13) NH(2) attenuated the stimulated release of dynorphin following FPI (34 +/- 3 and 97 +/- 6 vs. 36 +/- 3 and 68 +/- 8 pg/mol for CSF dynorphin before and after FPI in untreated and NOC/oFQ antagonist-pretreated animals). FPI increased SOD-inhibitable NBT reduction, but pretreatment with norbinaltorphimine, a dynorphin antagonist, or NS398, a COX-2 inhibitor, blunted such reduction (1 +/- 1 vs. 19 +/- 3 vs. 4 +/- 1 vs. 4 +/- 1 pmol/mm(2) for control, FPI, FPI-norbinaltorphimine and FPI-NS398, respectively). Under non-brain injury conditions, dynorphin, in a concentration observed in CSF after FPI, also increased SOD-inhibitable NBT reduction, which was blunted by NS398. NMDA-induced pial artery dilation was reversed to vasoconstriction following FPI, but pretreatment with norbinaltorphimine or NS398 partially protected such responses (9 +/- 1 and 16 +/- 1, control; - 8 +/- 1 and - 13 +/- 2, FPI; 6 +/- 1 and 12 +/- 1% FPI-norbinaltorphimine for NMDA 10(-8), 10(-6) M, respectively). These data show that NOC/oFQ modulates the CSF release of dynorphin after FPI. These data also show that dynorphin contributes to O(2)(-) generation after FPI via COX-2 activation. These data additionally indicate that dynorphin and COX-2 activation contribute to impairment of NMDA pial artery dilation after FPI. Finally, these data suggest that NOC/oFQ impairs NMDA dilation postinsult via the sequential release of dynorphin, activation of COX-2, and generation of O(2)(-).

Animals↗
PubMed1991

Subcellular localization, partial purification, and characterization of a dynorphin processing endopeptidase from bovine pituitary.

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.

4-Chloromercuribenzenesulfonate↗
PubMed1990

Specific binding and clearance of [3H]dynorphin (1-13) in the perfused rat lung: an application of the multiple-indicator dilution method.

The clearance and binding of a kappa-selective opioid peptide, dynorphin (1-13), in the perfused rat lung has been examined, using the multiple indicator dilution method. More than 50% of [3H]dynorphin (1-13) entering the pulmonary circulation was eliminated by the lung during a single passage of a tracer dose. By contrast, when a high dose (100 microM) of dynorphin (1-13) was concomitantly injected, [3H]dynorphin (1-13) behaved like [14C]sucrose, an extracellular marker. The kinetic analyses of the pulmonary venous outflow curves of [3H]dynorphin (1-13) and [14C]sucrose at low and high doses of dynorphin (1-13) indicated that the initial uptake rate constant, extraction ratio and distribution volume of [3H]dynorphin (1-13) decreased significantly in the presence of a high concentration of unlabelled dynorphin (1-13). These results suggest that [3H]dynorphin (1-13) is eliminated by a saturable process and binds to a specific binding site in the perfused lung, which may be the kappa-type binding site. The multiple indicator dilution technique, in combination with a moment analysis, was successfully applied to demonstrate the specific binding and clearance of dynorphin (1-13) in the perfused lung.

Analgesics↗
PubMed1986

Dynorphin A decreases voltage-dependent calcium conductance of mouse dorsal root ganglion neurones.

The actions of the opioid peptides dynorphin A and (Leu)enkephalin were assessed on calcium-dependent action potentials and inward calcium currents recorded from somata of mouse dorsal root ganglion (d.r.g.) neurones grown in primary dissociated cell culture. Dynorphin A and (Leu)enkephalin decreased the duration of somatic calcium-dependent action potentials in a portion of d.r.g. neurones impaled with potassium acetate-filled micropipettes. When substantial potassium conductance was blocked by intracellular injection of caesium acetate, d.r.g. neurones continued to respond to dynorphin A but responses to (Leu)enkephalin were abolished. In voltage-clamp experiments, dynorphin A but not (Leu)enkephalin reduced the magnitude of inward calcium currents. Dynorphin A responses were blocked by the opiate antagonist naloxone. The dynorphin A effect was due to reduction of voltage-dependent calcium conductance since dynorphin A reduced depolarization-evoked inward currents but did not alter membrane conductance following blockade of calcium channels by cadmium, and because dynorphin A reduced the instantaneous current-voltage slope (chord conductance) during step commands that produced maximal activation of voltage-dependent calcium conductance. Dynorphin A binds with high affinity to kappa-opioid receptors. (Leu)enkephalin, which has affinity for both mu- and delta-receptors but not for kappa-opioid receptors, was without effect on calcium conductance. Therefore, we suggest that kappa-receptors are coupled to voltage-dependent calcium-channels and that binding of dynorphin A produces a decrease of calcium current.

Action Potentials↗