beta-Casomorphins induce apnea and irregular breathing in adult rats and newborn rabbits

Bovine beta-casomorphin, beta-casomorphin, morphiceptin or morphine were administered systemically (i.v. or i.p.) and intracerebroventricularly (lateral or IVth ventricles) to anesthetized adult rats and preterm newborn rabbits. All agents caused dose-related depressions of respiratory frequency and tidal volume. Morphiceptin and beta-casomorphin were approximately equipotent to morphine while beta-casomorphin was 10 times as potent after intracerebroventricular injection. The beta-casomorphins decreased inspiratory drive and prolonged the expiratory phase by delaying the setpoint for inspiration. A respiratory depression could be elicited by systemic administration of morphiceptin but not by beta-casomorphin or beta-casomorphin. All ventilatory effects induced by the beta-casomorphins could be readily reversed or prevented by naloxone. Intracerebroventricular but not intraperitoneal injection of beta-casomorphin depressed ventilation in preterm newborn rabbits in a similar pattern with apnoic periods to that seen in the adult rats. In addition, an irregular breathing pattern was elicited. Thus, the bovine beta-casomorphins possess potent central respiratory depressive effects. However, after systemic administration, only morphiceptin which is more metabolically stable induced a shortlasting effect on ventilation in adult rats.     

Interactions between the antiopiate Tyr-MIF-1 and the mu opiate morphiceptin at their respective binding sites in brain

The interactions between Tyr-MIF-1, a brain peptide with antiopiate activity, and the beta-casomorphins, a family of peptides derived from milk protein with opiate activity, were investigated by in vitro binding assays. Specific binding of 125I-Tyr-MIF-1 to rat brain membranes was displaced with high potency by beta-casomorphin, morphiceptin, and the morphiceptin analog PL017 but not by the analgesically inactive analog D-Pro2-morphiceptin or by several other ligands for classical delta, kappa, or sigma opiate receptors. In addition, Tyr-MIF-1 displaced 125I-morphiceptin from its binding sites in brain with affinities similar to those of unlabeled morphiceptin and PL017. These results, which include the first demonstration of a binding site in brain for labeled morphiceptin, indicate that brain antiopiate Tyr-MIF-1 and the beta-casomorphin derived peptides with opiate activity may share a common binding site or cross-react at each other's site. This suggests a possible mechanism of action for endogenous antiopiate-opiate interactions.     

Conformational analysis of morphiceptin by NMR spectroscopy

Three exorphins, beta-casomorphin-5, morphiceptin and its D-Pro4 analog, were studied in DMSO by means of 1H and 13C NMR spectroscopy, with the aim of detecting conformational features of potential biological significance for the mu opioid activity since the presence of two Pro residues restricts the accessible conformational space more than in all other peptides. It is found that the conformational mixtures present in solution contain relevant fractions of folded conformers, a feature that assures the observation of four different Tyr OH signals in the 500 MHz spectrum of morphiceptin. The conformer distribution of (very active) (D-Pro4)-morphiceptin is different from those of its (less active) congeners.     

Absorption of intact morphiceptin by diisopropylfluorophosphate-treated rabbit ileum

The amidated beta-casomorphin morphiceptin Tyr-Pro-Phe-Pro-NH2 is an opioid peptide isolated from bovine milk beta-casein digests whose physiological significance remains unclear. Opiates are known to modify intestinal electrolyte transport by acting on receptors located on the serosal side of the intestine. The aim of the present study was to determine under what conditions morphiceptin can act from the luminal side. When added to the serosal side of untreated rabbit ileum in an Ussing chamber in vitro, 10(-3) M morphiceptin acted through an opiate mechanism to reduce simultaneously short-circuit current (delta Isc = 0.33 +/- 0.07 muEq.hr-1.cm-2) and stimulate net Na and Cl absorption (delta JnetNa = 1.62 +/- 0.11 and delta JnetCl = 2.07 +/- 0.08 muEg.hr-1.cm-2). After mucosal addition under the same conditions, morphiceptin was degraded without any opiate action on electrolyte transport. Pretreatment of the ileum by 10(-3) M diisopropylfluorophosphate, which inhibited brush-border dipeptidylpeptidase IV, prevented mucosal degradation of morphiceptin. Under these conditions, morphiceptin was able, when added mucosally, to cross the epithelium intact (Jm----s = 1.8 +/- 0.16 nmole.hr-1.cm-2) and to stimulate electrolyte absorption by means of an opioid mechanism (delta Isc = 0.22 +/- 0.02 muEq.hr-1.cm-2). These results showed that the action of morphiceptin from the lumen depends on its transfer intact to the serosal side of the intestine where the opiate receptors are located. The limiting step in this transfer is at the brush-border membrane, where dipeptidylpeptidase IV in particular seems to play a major role.     

Opioid peptides derived from wheat gluten: their isolation and characterization.

Four opioid peptides were isolated from the enzymatic digest of wheat gluten. Their structures were Gly-Tyr-Tyr-Pro-Thr, Gly-Tyr-Tyr-Pro,Tyr-Gly-Gly-Trp-Leu and Tyr-Gly-Gly-Trp, which were named gluten exorphins A5, A4, B5 and B4, respectively. The gluten exorphin A5 sequence was found at 15 sites in the primary structure of the high molecular weight glutenin and was highly specific for delta-receptors. The structure-activity relationships of gluten exorphins A were unique in that the presence of Gly at their N-termini increased their activities. Gluten exorphin B5, which corresponds to [Trp4,Leu5]enkephalin, showed the most potent activity among these peptides. Its IC50 values were 0.05 microM and 0.017 microM, respectively, on the GPI and the MVD assays.     

Effect of beta-casomorphin on neonatal sleep in rats

The effects of bovine beta-casomorphin(1-7) (Tyr-Pro-Phe-Pro-Gly-Pro-Ile) on neonatal sleep in rats were studied. The pups received intraperitoneal injections of beta-casomorphin(1-7) (1 mg, 5 mg, 10 mg, 50 mg, or 100 mg/kg) or a corresponding volume of sodium chloride. In any of the doses used, beta-casomorphin(1-7) had no effect on waking. Only 100 mg/kg caused significant changes in sleep: the percentage of quiet state of the total recording time (TRT) increased and the percentage of active sleep decreased. Beta-casomorphin(1-7) did not cause significant respiratory depression. Naloxone pretreatment (1 mg/kg IP) reversed the effects of beta-casomorphin(1-7) on sleep, a finding which suggests that opiate mu-receptors are involved in mediating the sleep effects of beta-casomorphin.     

Soluble dipeptidyl peptidase IV from terminal differentiated rat epidermal cells: purification and its activity on synthetic and natural peptides

In terminally differentiated epidermal cells dipeptidyl peptidase IV (EC 3.4.14.5) (DPP IV) is present mainly in a soluble form. We purified the enzyme from 2-day-old rat cornified cells to homogeneity by Sephadex G-200 and Mono-Q column chromatography and finally HPLC gel filtration on G3000SW. The enzyme was estimated to be Mr 190,000 by HPLC gel filtration and Mr 90,000 by sodium dodecyl sulfate-electrophoresis. The enzyme showed general properties reported for detergent-solubilized DPP IV from other tissues. It was Con A binding and almost completely inhibited by 1 mM diisopropyl fluorophosphate and Diprotin A. The pI was 5.6 and the pH optimum was 7.5. The specific activity for Gly-Pro-p-nitroanilide was 31.9 units/mg. HPLC analysis demonstrated the release of dipeptides of the N-terminal of substance P, beta-casomorphin, and their related peptides. A stoichiometric reaction of the enzyme on substance P was observed. The epidermal DPP IV had a Km of 0.3 mM and a kcat of 50.3 s-1 for substance P and the Km value decreased by shortening the peptide from the carboxyl-terminal amino acids. The enzyme hydrolyzed human and bovine beta-casomorphin with Km values of 0.025 and 0.05 mM, respectively. Shortening the bovine beta-casomorphin peptide chain did not affect enzyme affinity.     

Selective activation of opioid receptors differentially affects lordosis behavior in female rats

The effects of opioid peptides that are highly selective ligands for mu receptors (morphiceptin). delta receptors (delta-receptor peptide), kappa receptors (dynorphin 1-9), and the mu/delta complex (beta-endorphin), were tested on lordosis behavior in ovariectomized rats primed with estrogen and progesterone. Intracerebroventricular infusions of beta-endorphin or morphiceptin both inhibited and facilitated lordosis in a dose-dependent fashion whereas all doses of delta-receptor peptide facilitated lordosis. Dynorphin 1-9 had no significant effect at any dose, although a trend toward increased lordosis quotients was observed 30 min after infusion. The effects of beta-endorphin, morphiceptin, and delta-receptor peptide were reversed with naloxone, although naloxone alone had no effect on lordosis behavior. These results indicate that the specific activation of opioid receptor subtypes differentially affects lordosis behavior. It appears that binding to high-affinity mu 1 receptors exerts an inhibitory influence on lordosis, whereas binding to low-affinity mu 2 receptors or delta receptors exerts a facilitatory influence. Binding to kappa receptors does not appear to affect lordosis behavior.     

Antisense oligodeoxynucleotide targeting distinct exons of the cloned mu-opioid receptor distinguish between endomorphin-1 and morphine supraspinal antinociception in mice.

Antisense oligodeoxynucleotides (ODN) were used to investigate the supraspinal antinociceptive effects of endomorphin-1, an endogenous peptide whose analgesic profile suggests that it is a ligand at the mu-opioid receptor. To selectively restrict the expression of this receptor, five ODN targeting distinct exons of the gene sequence were injected subchronically by the intracerebroventricular route (i.c.v.) into mice. The antinociception induced by endomorphin-1 was greatly reduced in animals receiving the ODN directed to nucleotides 677-697, which code for a sequence located on the second extracellular loop of the mu receptor. ODN-mu(un), one of the two antisense ODN directed to exon 1, also impaired endomorphin-1 antinociception. ODN targeting exons 2 and 4 were totally inactive. In contrast, all five ODN blocked the antinociception induced by morphine and beta-casomorphin. The analgesic potency of endomorphin-1, morphine, and beta-casomorphin remained unaltered by administration of an ODN to nucleotides 29-46 of the murine delta-opioid receptor gene sequence of a random-sequence ODN. This suggest the existence of diverse molecular forms for the mu-opioid receptor that mediate the antinociceptive effects of endomorphin-1 and morphine/beta-casomorphin.     

Dmt1, d-1-Nal3]morphiceptin, a novel opioid peptide analog with high analgesic activity

The morphiceptin-derived peptide [Dmt1, d-1-Nal3]morphiceptin, labeled mu-opioid receptor (MOP) with very high affinity and selectivity in the receptor binding assays. In the mouse hot plate test, [Dmt1, d-1-Nal3]morphiceptin given intracerebroventricularly (i.c.v.) produced profound supraspinal analgesia, being approximately 100-fold more potent than the endogenous MOP receptor ligand, endomorphin-2. The antinociceptive effect of this new analog lasted up to 120min. Thus, [Dmt1, d-1-Nal3]morphiceptin is an interesting and extraordinarily potent analgesic, raising the possibility of novel approaches in the design of clinically useful drugs for pain treatment.     

Prolactin and growth hormone response to intracerebroventricular administration of the food opioid peptide gluten exorphin B5 in rats

Although it has long been known that opioid peptides cause marked changes of pituitary hormone secretion in both animals and humans, little is known about the possible effect(s) of food-derived opioids (exorphins) on pituitary function. In order to investigate the possible role of exorphins derived from wheat gluten on pituitary function, we gave the following treatments to four groups of male rats: intracerebroventricular (ICV) vehicle, Gluten Exorphin B5 (GE-B5) 200 microg ICV, naloxone intraperitoneally (IP) followed by vehicle ICV, naloxone IP followed by GE-B5 ICV. Blood samples for Prolactin (PRL) and Growth Hormone (GH) were taken at intervals for 90 minutes after vehicle or GE-B5 administration. GE-B5 strongly stimulated PRL secretion; its effect was completely abolished by naloxone administration. GH secretion was unaffected by GE-B5 under these experimental conditions. The present study shows for the first time that an opioid peptide derived from wheat gluten, GE-B5, has an effect on pituitary function when administered ICV; its mechanism of action appears to be mediated via classical opioid receptors.     

Intragastric beta-casomorphin(1-7) attenuates the suppression of fat intake by enterostatin

The current experiments were designed to compare the feeding response to enterostatin and beta-casomorphin(1-7) injected intragastrically. Sprague-Dawley rats with a gastric cannula were allowed to chose from high-fat diet (HF) or low-fat diet (LF) in separate jars. Enterostatin injected intragastrically into overnight fasted rats caused a U-shaped dose-dependent reduction in the intake of the HF diet for the first two hours after infusion but had no effect on the LF intake. beta-Casomorphin(1-7) stimulated the intake of the HF diet but had no effect on the LF diet. Finally, beta-casomorphin(1-7) blocked the inhibitory effect of enterostatin on HF intake in fasted rats.     

Opioid pharmacology in the rat hippocampal slice

The ability of several opioids in potentiating the synaptic activation of CA1 pyramidal cells in the rat hippocampal slice were compared. Morphine and the opioid peptides, (D-ala2, D-leu5)-enkephalin (DADL), morphiceptin, beta-endorphin, and Tyr-D-Ser-Gly-Phe-Leu-Thr (DSThr) caused a concentration-dependent, naloxone-reversible shift to the left in the input-output (IO) curve constructed by plotting the population spike as a function of the field EPSP. These opioids then produced an increase in the size of the population spike while leaving the EPSP unaffected. In contrast, the kappa agonist prototype, ethylketazocine, had no effect on the IO curve when perfused in concentrations up to 10 microM. The rank order of potency for the opioids in the CA1 region of the hippocampus was DADL greater than DSThr greater than beta-endorphin greater than morphiceptin greater than morphine much greater than ethylketazocine. Thus, opioids that are more specific for delta opiate receptors were the most potent and mu receptor agonists, the least potent in this action. Taken together with previous studies suggesting that morphine and DADL may interact with a common opiate receptor in the CA1 region, the results are consistent with the notion that these epileptiform effects may be primarily mediated by delta opiate receptors in this area although the potency of morphiceptin indicates that mu receptors play some role in this effect.     

Comparison of mu, delta, and kappa opiate binding sites in rat brain and spinal cord

The binding characteristics of mu, delta, and kappa opiate sites were studied in rat brain and spinal cord membrane homogenates. Scatchard analysis of 3H-Dihydromorphine, 3H-D-Ala2-D-Leu5-Enkephalin (in the presence of morphiceptin), and 3H-Ethylketocyclazocine (in the presence of morphiceptin and D-Ala2-D-Leu5-Enkephalin) binding sites revealed similar high affinities of these ligands for their respective sites in brain and spinal cord. The majority of binding in brain and spinal cord was attributed to mu and delta sites, with only about 10% of the combined total binding capacity being kappa.     

Cyclic AMP Protects Against Staurosporine and Wortmannin-Induced Apoptosis and Opioid-Enhanced Apoptosis in Both Embryonic and Immortalized (F-11κ7) Neurons

Abstract: The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11κ7 cell line (an immortalized mouse neuroblastoma × dorsal root ganglion hybrid stably transfected to overexpress κ-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the μ-specific opioid agonist morphiceptin (E7CH) or the κ-specific opioid agonist U69,593 (F-11κ7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with nonpretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16–24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp -adenosine 3',5'-cyclic monophosphothioate and increased killing with the antagonist Rp -adenosine 3',5'-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.     

Characterization of Opioid Receptors Modulating Noradrenaline Release in the Hippocampus of the Rabbit

Noradrenaline (NA) release and its modulation via presynaptic opioid receptors were studied in rabbit hippocampal slices, which were preincubated with [3H]NA, continuously superfused in the presence of 30 microM cocaine and stimulated electrically. The evoked release of [3H]NA was strongly reduced by the preferential kappa-agonists ethylketocyclazocine, dynorphin A1-13, dynorphin A, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] -benzeneacetamide (U-50,488), and (-)-5,9-dimethyl-2'-OH-2-tetrahydrofurfuryl-6,7-benzomorphan [(-)-MR 2034], whereas (+)-MR 2035 [the (+)-enantiomer of (-)-MR 2034] was ineffective. In contrast, the preferential delta-agonists Leu-enkephalin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalin (DADLE) as well as the mu-agonists morphine, normorphine, D-Ala2-Gly-ol5-enkephalin (DAGO), and beta-casomorphin 1-4 amide (morphiceptin) were much less potent. However, in similar experiments on rat hippocampal slices DAGO (1 microM) was much more potent than ethylketocyclazocine (1 microM) or DADLE (1 microM). (-)-N-(3-furylmethyl)-alpha-noretazocine [(-)-MR 2266], 1 microM, a preferential kappa-antagonist, antagonized the effect of ethylketocyclazocine more potently than (-)-naloxone or (+)-MR 2267 [the (+)-enantiomer of (-)-MR 2266]. Given alone, (-)-MR 2266 slightly and (+)-MR 2267 (1 microM each) greatly enhanced NA release, apparently due to alpha 2-adrenoceptor blockade since their effects were completely abolished in the presence of yohimbine (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid assay to detect possible natural substrates of proteases in living cells

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.     

Saturable transport of peptides across the blood-brain barrier

Peptides can be transported across the blood-brain barrier by saturable transport systems. One system, characterized with radioactively labeled Tyr-MIF-1 (Tyr-Pro-Leu-Gly-amide), is specific for some of the small peptides with an N-terminal tyrosine, including Tyr-MIF-1, the enkephalins, beta-casomorphin, and dynorphin (1-8). Another separate system transports vasopressin-like peptides. The choroid plexus has at least one system distinguishable from those above that is capable of uptake and possibly transport of opiate-like peptides. The possibility of saturable transport of other peptides has been investigated to a varying degree. Specificity, stereo-specificity, saturability, allosteric regulation, modulation by physiologic and pharmacologic manipulations, and noncompetitive inhibition have been demonstrated to occur in peptide transport systems and suggest a role for them in physiology and disease.     

Delayed effect of exorphins on learning of albino rat pups

The delayed effect of food-derived opioid peptides (exorphins) after chronic administration on postnatal days 1–14 on the learning of albino rat pups has been studied. Heptapeptide YPFPGPI (β-casomorphin-7), pentapeptide YPLDL (rubiscolin-5) and pentapeptide YPISL (exorphin C) improved the development of the conditioned foraging reflex in a complex maze. Hexapeptide PFPGPI lacking the N-terminal tyrosine proved inefficient. Only β-casomorphin-7 had an effect (negative) on passive avoidance conditioning. The obtained data confirm that exorphins (particularly, milk-derived β-casomorphins) can have significant and long-term effects on the environmental adaptation of young mammals.     

Effects of oral casokefamide on plasma levels, tolerance, and intestinal transit in man

Food-derived opioid peptides such as beta-casomorphins are of interest for treatment of chronic diarrhea. The beta-casomorphin analog casokefamide was administered orally at doses of 5.5, 8.0, and 16.0 mg to 10 healthy male volunteers, respectively. Dose-dependent increases of plasma levels with a maximum of 350 fmol/l were determined. No side-effects due to casokefamide has been observed. In comparison to placebo, casokefamide showed a trend toward prolongation of oro-caecal transit time. Orally applied casokefamide is well tolerated and may represent a useful tool for treatment of diarrhea in the future.