Tyrosyl-tRNA synthetase: A potential kyotorphin synthetase in mammals.

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 µM and 200 µM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.

Synthesis and activity of opioid peptidomimetics with ß2- and ß3-amino acids.

Morphiceptin (Tyr-Pro-Phe-Pro-NH2) is a selective ligand of the mu opioid receptor, an important target in pain regulation. In this study, morphiceptin was modified at positions 2 or 3 by introduction of ß2- or ß3-amino acids and additionally in position 1 by replacing Tyr by Dmt (2',6'-dimethyltyrosine), which resulted in obtaining enzymatically stable analogs with mixed opioid receptor affinity profiles. An analog of the sequence Dmt-d-Ala-(R)-ß2-1-Nal-Pro-NH2 [Nal=3-(1-naphthyl)-alanine] showed very high activity at the mu and delta receptors in the calcium mobilization functional test but did not cross the artificial membrane imitating the blood-brain barrier. In the in vivo test this analog induced strong antinociceptive effect in the writhing test in mice after intraperitioneal but also oral administration and inhibited diarrhea similarly to loperamide. Therefore, it may become an interesting lead compound in the development of peripherally restricted drugs for the treatment of gastrointestinal disorders.

Comparative evaluation of cow ß-casein variants (A1/A2) consumption on Th2-mediated inflammatory response in mouse gut.

PURPOSE: Recently, apprehension has been raised regarding "A1/A2 hypothesis" suggesting relationship between consumption of A1 "like" variants of cow ß-casein and various physiological disorders. The information available is based on either the human epidemiological data of milk consumption or in vitro trials on cell lines with ß-casomorphin peptides. The direct scientific evidence establishing the link between consumption of A1/A2 "like" milk and health is scanty. Thus, under present investigation, in vivo trials in mice were undertaken to study the effect of feeding three genetic variants (A1A1, A1A2 and A2A2) of cow ß-casein milk on gastrointestinal immune system as it is the first and foremost site of immunological interactions. METHODS: Animals were divided into four groups for feeding with basal diet (control) and ß-casein isolated from milk of genotyped (A1A1, A1A2 and A2A2) dairy animals, respectively. Gut immune response was analyzed by spectrophotometric assessment of MPO activity, quantitative sandwich ELISA of inflammatory cytokines (MCP-1 and IL-4), antibodies (total IgE, IgG, sIgA, IgG1 and IgG2a) and qRT-PCR of mRNA expression for toll-like receptors (TLR-2 and TLR-4). Histological enumeration of goblet cells, total leukocytes and IgA(+) cells was also carried out. RESULTS: It was observed that consumption of A1 "like" variants (A1A1 and A1A2) significantly increased (p < 0.01) the levels of MPO, MCP-1, IL-4, total IgE, IgG, IgG1, IgG2a and leukocyte infiltration in intestine. TLR-2 and TLR-4 mRNA expression was also up-regulated (p < 0.01) on administration of A1 "like" variants. However, no changes in sIgA, IgA(+) and goblet cell numbers were recorded on consumption of any of the ß-casein variants. CONCLUSION: It is reasonable to conclude that consumption of A1 "like" variants of ß-casein induced inflammatory response in gut by activating Th2 pathway as compared to A2A2 variants. The present study thus supports the purported deleterious impacts of consumption of A1 "like" variants of ß-casein and suggests possible aggravation of inflammatory response for etiology of various health disorders.

Milk from cows of different ß-casein genotypes as a source of ß-casomorphin-7.

The aim of this study was to quantify ß-casomorphin-7 in raw, hydrolyzed and processed milk in different stages of the cow lactation. The obtained results lead to the following conclusion: the highest amount of ß-casomorphin-7 released from the hydrolyzed and processed milk is related to the ß-casein A1 allele, irrespective of a lactation period. Some traces of ß-casomorphin-7 in milk from cows with the ß-casein A2 variant are probably a result of the acid hydrolysis of ß-casein during its digestion with pepsin. It has been shown that processing of raw milk at high temperatures affects, in a slight degree, the differences between ß-casomorphins-7 originating from different ß-casein genotypes. The obtained results suggest a possibility to provide a new nutritional factor for milk quality based on the content of ß-casomorphin-7 liberated in vivo from milk digested by a mixture of the gastrointestinal enzymes.

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such ‘gene doping’, exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

The use of genes for performance enhancement: doping or therapy?

Recent biotechnological advances have permitted the manipulation of genetic sequences to treat several diseases in a process called gene therapy. However, the advance of gene therapy has opened the door to the possibility of using genetic manipulation (GM) to enhance athletic performance. In such 'gene doping', exogenous genetic sequences are inserted into a specific tissue, altering cellular gene activity or leading to the expression of a protein product. The exogenous genes most likely to be utilized for gene doping include erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor type 1 (IGF-1), myostatin antagonists, and endorphin. However, many other genes could also be used, such as those involved in glucose metabolic pathways. Because gene doping would be very difficult to detect, it is inherently very attractive for those involved in sports who are prepared to cheat. Moreover, the field of gene therapy is constantly and rapidly progressing, and this is likely to generate many new possibilities for gene doping. Thus, as part of the general fight against all forms of doping, it will be necessary to develop and continually improve means of detecting exogenous gene sequences (or their products) in athletes. Nevertheless, some bioethicists have argued for a liberal approach to gene doping.

Sweet preference, sugar addiction and the familial history of alcohol dependence: shared neural pathways and genes.

Contemporary research has shown that a high number of alcohol-dependent and other drug-dependent individuals have a sweet preference, specifically for foods with a high sucrose concentration. Moreover, both human and animal studies have demonstrated that in some brains the consumption of sugar-rich foods or drinks primes the release of euphoric endorphins and dopamine within the nucleus accumbens, in a manner similar to some drugs of abuse. The neurobiological pathways of drug and "sugar addiction" involve similar neural receptors, neurotransmitters, and hedonic regions in the brain. Craving, tolerance, withdrawal and sensitization have been documented in both human and animal studies. In addition, there appears to be cross sensitization between sugar addiction and narcotic dependence in some individuals. It has also been observed that the biological children of alcoholic parents, particularly alcoholic fathers, are at greater risk to have a strong sweet preference, and this may manifest in some with an eating disorder. In the last two decades research has noted that specific genes may underlie the sweet preference in alcohol- and drug-dependent individuals, as well as in biological children of paternal alcoholics. There also appears to be some common genetic markers between alcohol dependence, bulimia, and obesity, such as the A1 allele gene and the dopamine 2 receptor gene.

Reward processing by the opioid system in the brain.

The opioid system consists of three receptors, mu, delta, and kappa, which are activated by endogenous opioid peptides processed from three protein precursors, proopiomelanocortin, proenkephalin, and prodynorphin. Opioid receptors are recruited in response to natural rewarding stimuli and drugs of abuse, and both endogenous opioids and their receptors are modified as addiction develops. Mechanisms whereby aberrant activation and modifications of the opioid system contribute to drug craving and relapse remain to be clarified. This review summarizes our present knowledge on brain sites where the endogenous opioid system controls hedonic responses and is modified in response to drugs of abuse in the rodent brain. We review 1) the latest data on the anatomy of the opioid system, 2) the consequences of local intracerebral pharmacological manipulation of the opioid system on reinforced behaviors, 3) the consequences of gene knockout on reinforced behaviors and drug dependence, and 4) the consequences of chronic exposure to drugs of abuse on expression levels of opioid system genes. Future studies will establish key molecular actors of the system and neural sites where opioid peptides and receptors contribute to the onset of addictive disorders. Combined with data from human and nonhuman primate (not reviewed here), research in this extremely active field has implications both for our understanding of the biology of addiction and for therapeutic interventions to treat the disorder.

Now or Later? An fMRI study of the effects of endogenous opioid blockade on a decision-making network.

Previously, we found that distinct brain areas predict individual selection bias in decisions between small immediate ("Now") and larger delayed rewards ("Later"). Furthermore, such selection bias can be manipulated by endogenous opioid blockade. To test whether blocking endogenous opioids with naltrexone (NTX) alters brain activity during decision-making in areas predicting individual bias, we compared fMRI BOLD signal correlated with Now versus Later decision-making after acute administration of NTX (50 mg) or placebo. We tested abstinent alcoholics and control subjects in a double-blind two-session design. We defined regions of interest (ROIs) centered on activation peaks predicting Now versus Later selection bias. NTX administration significantly increased BOLD signal during decision-making in the right lateral orbital gyrus ROI, an area where enhanced activity during decision-making predicts Later bias. Exploratory analyses identified additional loci where BOLD signal during decision-making was enhanced (left orbitofrontal cortex, left inferior temporal gyrus, and cerebellum) or reduced (right superior temporal pole) by NTX. Additional analyses identified sites, including the right lateral orbital gyrus, in which NTX effects on BOLD signal predicted NTX effects on selection bias. These data agree with opioid receptor expression in human frontal and temporal cortices, and suggest possible mechanisms of NTX's therapeutic effects.

Increased adiposity on normal diet, but decreased susceptibility to diet-induced obesity in mu-opioid receptor-deficient mice.

The mu-opioid receptor encoded by the Oprm1 gene plays a crucial role in the mediation of food reward and drug-induced positive reinforcement, but its genetic deletion has been shown to provide food intake-independent, partial protection from diet-induced obesity. We hypothesized that mu-opioid receptor-deficient mice would show an even greater, intake-dependent, resistance to high-fat diet-induced obesity if the diet comprises a sweet component. We generated an F2 population by crossing the heterozygous offspring of homozygous female Oprm1(-/-) mice (on a mixed C57BL/6 and BALB/c genetic background) with male inbred C57BL/6 mice. Groups of genotyped wild-type (WT) and homozygous mutant (KO) males and females were fed either control chow or a high caloric palatable diet consisting of sweet, liquid chocolate-flavored Ensure together with a solid high-fat diet. Food intake, body weight, and body composition was measured over a period of 16 weeks. Unexpectedly, male, and to a lesser extent female, KO mice fed chow for the entire period showed progressively increased body weight and adiposity while eating significantly more chow. In contrast, when exposed to the sweet plus high-fat diet, male, and to a lesser extent female, KO mice gained significantly less body weight and fat mass compared to WT mice when using chow fed counterparts for reference values. Male KO mice consumed 33% less of the sweet liquid diet but increased intake of high-fat pellets, so that total calorie intake was not different from WT animals. These results demonstrate a dissociation of the role of mu-opioid receptors in the control of adiposity for different diets and sex. On a bland diet, normal receptor function appears to confer a slightly catabolic predisposition, but on a highly palatable diet, it confers an anabolic metabolic profile, favoring fat accretion. Because of the complexity of mu-opioid gene regulation and tissue distribution, more selective and targeted approaches will be necessary to fully understand the underlying mechanisms.

Dysregulation of dynorphins in Alzheimer disease.

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.

Gene doping.

Together with the rapidly increasing knowledge on genetic therapies as a promising new branch of regular medicine, the issue has arisen whether these techniques might be abused in the field of sports. Previous experiences have shown that drugs that are still in the experimental phases of research may find their way into the athletic world. Both the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) have expressed concerns about this possibility. As a result, the method of gene doping has been included in the list of prohibited classes of substances and prohibited methods. This review addresses the possible ways in which knowledge gained in the field of genetic therapies may be misused in elite sports. Many genes are readily available which may potentially have an effect on athletic performance. The sporting world will eventually be faced with the phenomena of gene doping to improve athletic performance. A combination of developing detection methods based on gene arrays or proteomics and a clear education program on the associated risks seems to be the most promising preventive method to counteract the possible application of gene doping.

Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor.

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.

Time course of opioid and cannabinoid gene transcription alterations induced by repeated administration with fluoxetine in the rat brain.

This study examined the time course effects (8, 16 and 31 days) of fluoxetine administration (1 mg/kg, p.o./day) on serotonin transporter (5-HTT), opioid, tyrosine hydroxylase (TH) and cannabinoid CB1 receptor gene expressions in selected regions of the rat brain. Treatment with fluoxetine progressively decreased (35-55%) 5-HTT gene expression in dorsal raphe nucleus at 8, 16 and 31 days. The results revealed that fluoxetine administration decreased (30%) proenkephalin gene expression in nucleus accumbens shell (AcbS) and caudate-putamen (CPu) (31 days) but was without effect in nucleus accumbens core AcbC. A pronounced and time related decrease (25-65%) in prodynorphin gene expression was detected in AcbC, AcbS, CPu, hypothalamic supraoptic and paraventricular nuclei at all time points as well as in proopiomelanocortin gene expression (20-30%) in the arcuate nucleus (ARC) of the hypothalamus. On days 16 and 31, tyrosine hydroxylase gene expression in ventral tegmental area and substantia nigra and cannabinoid CB1 receptor gene expression in the CPu decreased (approximately 45-50% from vehicle). In conclusion, fluoxetine by inhibiting the reuptake of serotonin produced pronounced and time related alterations in genes involved in the regulation of emotional behaviour, suggesting that these neuroplastic changes may be involved, at least in part, in the clinical efficacy of this drug in neuropsychiatric disorders.

Characterization of antinociceptive activity of novel endomorphin-2 and morphiceptin analogs modified in the third position.

In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in the D configuration: 3-(1-naphthyl)-D-alanine (D-1-Nal), 3-(2-naphthyl)-D-alanine (D-2-Nal), 3-(4-chlorophenyl)-D-alanine (D-ClPhe), 3-(3,4-dichlorophenyl)-D-alanine (D-Cl2Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in the mouse hot-plate test, performed after i.c.v. administration of the peptides at a dose of 10 microg/animal. The best results were obtained for two morphiceptin analogs, [D-Phe3]morphiceptin and [D-1-Nal3]morphiceptin, which showed greatly improved analgesic activity, as compared to morphiceptin. In the endomorphin-2 series none of the modifications produced analogs more potent than the parent compound, but [D-1-Nal3]endomorphin-2 was the best analog. Antinociception induced by endomorphin-2 was reversed by concomitant i.c.v. administration of [D-Phe3]endomorphin-2, [D-2-Nal3]endomorphin-2, and [D-2-Nal3]morphiceptin, indicating that these analogs were weak mu-opioid antagonists.

Opioids and alcoholism.

Although far from conclusive, evidence implicating the endogenous opioid system in the development and maintenance of alcoholism is growing. Currently available data suggest that ethanol increases opioid neurotransmission and that this activation is part of the mechanism responsible for its reinforcing effects. Findings from preclinical research indicate that ethanol consumption and ethanol-induced dopamine (DA) release are both reduced by opioid antagonists. Individual differences in endogenous opioid activity have been linked to inherited risks for alcoholism in studies comparing ethanol-preferring and nonpreferring rats, as well as in studies using targeted gene mutation (knockout) strategies. To a large extent, findings from human studies have paralleled those from the preclinical work. Persons who differ in family history of alcoholism have been shown to also differ in basal beta-endorphin activity, beta-endorphin response to alcohol, and subjective and HPA axis hormonal response to opioid antagonists. Findings from clinical trials indicate that opioid antagonists may reduce ethanol consumption in alcoholics, particularly in persons who have resumed drinking. Nevertheless, many questions remain unanswered about the use of opioid antagonists in alcoholism treatment and about the exact role of the opioid system in ethanol preference and reward. The progression of knowledge in this field suggests that many of these questions are imminently answerable, as our ability to characterize relationships between opioid activity and human behavior continues to develop. This paper summarizes both the progress that has been made and the gaps that remain in our understanding of the interactions between the endogenous opioid system and risk for alcoholism.

Protein kinase C signaling transduces endorphin-primed cardiogenesis in GTR1 embryonic stem cells.

The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor-encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface kappa opioid receptors, as well as protein kinase C (PKC)-alpha, -beta1, -beta2, -delta, -epsilon, and -zeta. Cardiac differentiation was associated with a marked increase in the Bmax value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-alpha, -beta1, -beta2, -delta, and -epsilon all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-delta and -epsilon were mainly expressed at the nuclear level. The nuclear increase of PKC-alpha, -beta1, and -beta2 was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-delta and PKC-epsilon in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-zeta expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-alpha, PKC-beta1, and PKC-beta2 and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-delta and -epsilon. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes.

Identification of a fourth opioid core sequence in a prodynorphin cDNA cloned from the brain of the amphibian, Bufo marinus: deciphering the evolution of prodynorphin and proenkephalin.

In mammals, prodynorphin codes for three C-terminally extended forms of leu-enkephalin. This is not the case for the anuran amphibian, Bufo marinus. A combination of 3'RACE, RT-PCR and 5'RACE protocols was used to clone and characterize a prodynorphin cDNA from the brain of this amphibian that contained two met-enkephalin sequences. One met-enkephalin sequence was located at the N-terminal of Met(5)-dynorphin A(1-17), and the other met-enkephalin sequence was located in the N-terminal region of B. marinus prodynorphin in a position that aligned with a pentapeptide met-enkephalin site in mammalian proenkephalin. The latter B. marinus met-enkephalin sequence is flanked by sets of paired basic proteolytic cleavage sites. In addition to the extra met-enkephalin sequence and the Met(5)-dynorphin A(1-17) sequence, the B. marinus prodynorphin contained two C-terminally extended forms of leu-enkephalin [alpha-neo-endorphin and dynorphin B(1-13)]. In the toad precursor the alpha-neo-endorphin sequence is identical to human alpha-neo-endorphin. The B. marinus dynorphin B(1-13) sequence differs from human dynorphin B(1-13) by one amino acid (Thr(12) vs. Val(12)). Steady-state analysis suggests that dynorphin B(1-13) and possibly alpha-neo-endorphin may be cleaved to yield leu-enkephalin as an end-product in the amphibian brain. Finally, the alignment of the extra met-enkephalin sequence in the N-terminal of B. marinus prodynorphin with the corresponding met-enkephalin site in mammalian proenkephalin adds support to the hypothesis that the prodynorphin gene arose as a duplication of the proenkephalin gene.

Opioid peptide gene expression primes cardiogenesis in embryonal pluripotent stem cells.

Zinc finger-containing transcription factor GATA-4 and homeodomain Nkx-2.5 govern crucial developmental fates and have been found to promote cardiogenesis in embryonic cells exposed to the differentiating agent DMSO. Nevertheless, intracellular activators of these transcription factors are largely unknown. In this study, pluripotent P19 cells expressed the prodynorphin gene, an opioid gene encoding for the dynorphin family of opioid peptides. P19 cells were also able to synthesize and secrete dynorphin B, a biologically active end product of the prodynorphin gene. DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion. The DMSO effect occurred at the transcriptional level. In the absence of DMSO, dynorphin B triggered GATA-4 and Nkx-2.5 gene expression and led to the appearance of both alpha-myosin heavy chain and myosin light chain-2V transcripts, two markers of cardiac differentiation. Moreover, dynorphin B-exposed cells were positively stained in the presence of MF 20, a mouse monoclonal antibody raised against the alpha-myosin heavy chain. Opioid receptor antagonism and inhibition of opioid gene expression by a prodynorphin antisense phosphorothioate oligonucleotide blocked DMSO-induced cardiogenesis, suggesting an autocrine role of an opioid gene in developmental decisions.

Opioid peptides encrypted in intact milk protein sequences.

Opioid agonistic and antagonistic peptides which are inactive within the sequence of the precursor milk proteins can be released and thus activated by enzymatic proteolysis, for example during gastrointestinal digestion or during food processing. Activated opioid peptides are potential modulators of various regulatory processes in the body. Opioid peptides can interact with subepithelial opioid receptors or specific luminal binding sites in the intestinal tract. Furthermore, they may be absorbed and then reach endogenous opioid receptors.