Opioid Precursor Research Articles

Progesterone receptor distribution in the human hypothalamus and its association with suicide

The human hypothalamus modulates mental health by balancing interactions between hormonal fluctuations and stress responses. Stress-induced progesterone release activates progesterone receptors (PR) in the human brain and triggers alterations in neuropeptides/neurotransmitters. As recent epidemiological studies have associated peripheral progesterone levels with suicide risks in humans, we mapped PR distribution in the human hypothalamus in relation to age and sex and characterized its (co-) expression in specific cell types. The infundibular nucleus (INF) appeared to be the primary hypothalamic structure via which progesterone modulates stress-related neural circuitry. An elevation of the number of pro-opiomelanocortin+ (POMC, an endogenous opioid precursor) neurons in the INF, which was due to a high proportion of POMC+ neurons that co-expressed PR, was related to suicide in patients with mood disorders (MD). MD donors who died of legal euthanasia were for the first time enrolled in a postmortem study to investigate the molecular signatures related to fatal suicidal ideations. They had a higher proportion of PR co-expressing POMC+ neurons than MD patients who died naturally. This indicates that the onset of endogenous opioid activation in MD with suicide tendency may be progesterone-associated. Our findings may have implications for users of progesterone-enriched contraceptives who also have MD and suicidal tendencies.

Localization and characterization of proenkephalin-A as a potential biomarker for kidney disease in murine and human kidneys

Introduction Exact measurement of renal function is essential for the treatment of patients. Elevated serum-creatinine levels, while established, are influenced by other parameters and show a significant time-lag. This drives the search for novel biomarkers of renal function and injury. Beside Lipocalin-2 and kidney-injury-molecule-1 (KIM-1), the endogenous opioid precursor proenkephalin-A (Penk) has recently emerged as a promising marker for renal function. But the cellular origin and regulation of Penk outside the brain has not yet been investigated in depth. Materials and methods This study characterizes the cellular origin of Penk expression with high-resolution in situ hybridization in two models of renal fibrosis in mice and human tissue. Results Interstitial cells are the main expression site for renal Penk. This classifies Penk as biomarker for interstitial damage as opposed to tubular damage markers like Lipocalin-2 and KIM-1. Furthermore, our data indicate that renal Penk expression is not regulated by classical profibrotic pathways. Discussion This study characterizes changing Penk expression in the kidneys. The similarity of Penk expression across species gives rise to further investigations into the function of Penk in healthy and injured kidneys. Conclusion Penk is a promising biomarker for interstitial renal damage that warrants further studies to utilize its predictive potential. Clinical significance Knowledge of real-time renal function is essential for proper treatment of critically ill patients and in early diagnosis of acute kidney injury (AKI). Proenkephalin-A has been measured in a number of patient cohorts as a highly accurate and predictive biomarker of renal damage. The present study identifies Penk as a biomarker for interstitial damage in contrast to the tubular biomarkers such as Lipocalin-2 or KIM-1. Our data show that Penk is regulated independently of classical profibrotic or proinflammatory pathways, indicating it might be more robust against extra-renal influences. Data presented in this study provide fundamental information about cell type-specific localization and regulation of the potential new biomarker Penk across species as foundation for further research.

Cellular Profiles of Prodynorphin and Preproenkephalin mRNA-Expressing Neurons in the Anterior Olfactory Tubercle of Mice.

The olfactory tubercle (OT) is a striatal region that receives olfactory inputs. mRNAs of prodynorphin (Pdyn) and preproenkephalin (Penk), precursors of dynorphins and enkephalins, respectively, are strongly expressed in the striatum. Both produce opioid peptides with various physiological effects such as pain relief and euphoria. Recent studies have revealed that OT has anatomical and cytoarchitectonic domains that play different roles in odor-induced motivated behavior. Neuronal subtypes of the OT can be distinguished by their expression of the dopamine receptors D1 (Drd1) and D2 (Drd2). Here, we addressed whether and which type of opioid peptide precursors the D1- and D2-expressing neurons in the OT express. We used multiple fluorescence in situ hybridization for mRNAs of the opioid precursors and dopamine receptors to characterize mouse OT neurons. Pdyn was mainly expressed by Drd1-expressing cells in the dense cell layer (DCL) of the OT, whereas Penk was expressed primarily by Drd2-expressing cells in the DCL. We also confirmed the presence of a larger population of Pdyn-Penk-Drd1 co-expressing cells in the DCL of the anteromedial OT compared with the anterolateral OT. These observations will help understand whether and how dynorphins and enkephalins in the OT are involved in diverse odor-induced motivated behaviors.

The mechanism of action of pulsed radiofrequency in reducing pain: a narrative review

Pain from nervous or musculoskeletal disorders is one of the most common complaints in clinical practice. Corticosteroids have a high pain-reducing effect, and their injection is generally used to control various types of pain. However, they have various adverse effects including flushing, hyperglycemia, allergic reactions, menstrual changes, immunosuppression, and adrenal suppression. Pulsed radiofrequency (PRF) is known to have a pain-reducing effect similar to that of corticosteroid injection, with nearly no major side effects. Therefore, it has been widely used to treat various types of pain, such as neuropathic, joint, discogenic, and muscle pain. In the current review, we outlined the pain-reducing mechanisms of PRF by reviewing previous studies. When PRF was first introduced, it was supposed to reduce pain by long-term depression of pain signaling from the peripheral nerve to the central nervous system. In addition, deactivation of microglia at the level of the spinal dorsal horn, reduction of proinflammatory cytokines, increased endogenous opioid precursor messenger ribonucleic acid, enhancement of noradrenergic and serotonergic descending pain inhibitory pathways, suppression of excitation of C-afferent fibers, and microscopic damage of nociceptive C- and A-delta fibers have been found to contribute to pain reduction after PRF application. However, the pain-reducing mechanism of PRF has not been clearly and definitely elucidated. Further studies are warranted to clarify the pain-reducing mechanism of PRF.

The Opioid System in Rainbow Trout Telencephalon Is Probably Involved in the Hedonic Regulation of Food Intake.

We hypothesize that opioids are involved in the regulation of food intake in fish through homeostatic and hedonic mechanisms. Therefore, we evaluated in rainbow trout (Oncorhynchus mykiss) hypothalamus and telencephalon changes in precursors, endogenous ligands and receptors of the opioid system under different situations aimed to induce changes in the homeostatic (through fasted/fed/refed fish) and hedonic (through feeding fish a control or a palatable high-fat diet) regulation of food intake. No major changes occurred in parameters assessed related with the nutritional condition of fish (fasted/fed/refed), allowing us to suggest that the opioid system seems not to have an important role in the homeostatic regulation of food intake in rainbow trout. The responses observed in telencephalon of rainbow trout fed the palatable high-fat diet included a decrease in mRNA abundance of the opioid precursor penka, in a way similar to that known in mammals, and increased mRNA abundance of the opioid receptors oprd1 and oprk1 supporting a role for telencephalic opioid system in the hedonic regulation of food intake in fish.

Abstract P085: Plasma Pro-enkephalin A And Incident Cognitive Impairment In The Regards Cohort

Background: Cardiovascular disease is a major risk for cognitive impairment. The opioid precursor peptide pro-enkephalin A (PENK-A) is a circulating hormone associated with several cardiovascular diseases and possibly vascular dementia. The objective of this study was to test the association of plasma PENK-A with incident cognitive impairment and describe potential differences by age, race, and sex. Methods: REGARDS is a prospective population-based cohort of 30,239 Black and White adults. Baseline plasma PENK-A was measured in 462 participants who developed cognitive impairment over a median 4.7 years and 556 who did not develop this. PENK-A’s association with incident cognitive impairment was assessed by logistic regression models adjusted for confounders. We tested for differences by age, sex, and race with interaction terms. Results: PENK-A concentration was comparable between cases and controls, but higher with White race, female sex, greater age, coronary artery disease, and chronic kidney disease. Categorical and linear models did not reveal an association with cognitive impairment. Spline plots showed a non-linear association with decreased odds at very high PENK-A values (adjusted OR for 95 th vs 50 th percentiles 0.66, 95% CI 0.46-0.95). This association was only apparent after final adjustment for kidney function. Interaction testing showed substantial interactions by each age and sex (all P < 0.06). In subgroup analyses, shapes of association differed but no independent trends were discernable (Figure). Conclusions: Circulating PENK-A was not particularly associated with cognitive impairment odds. High levels may be protective; this finding could be spurious. Differences by sex and age may exist, expanding upon prior findings in REGARDS of differing non-linear associations with stroke by sex and race. Further work is needed to characterize the importance of endogenous opioid pathways to cardiovascular health and differences by demographic factors.

Ethanol consumption activates a subset of arcuate nucleus pro-opiomelanocortin (POMC)-producing neurons: a c-fos immunohistochemistry study.

Ethanol activates various opioid peptide‐containing circuits within the brain that may underlie its motivational and rewarding effects. One component of this circuitry consists of neurons located in the arcuate nucleus (ArcN) of the hypothalamus which express pro‐opiomelanocortin (POMC), an opioid precursor peptide that is cleaved to form bioactive fragments including β‐endorphin and α‐melanocyte stimulating hormone. In this study, we sought to determine if ethanol intake activates ArcN POMC neurons as measured by expression of the immediate early gene c‐fos. Male and female POMC‐EGFP mice underwent drinking‐in‐the‐dark (DID) procedures for 3 consecutive days (2 h/day) and were allowed to consume either ethanol (20% v/v), saccharin (0.2% w/v), or water. On the fourth day of DID procedures, animals were allowed to consume their respective solutions for 20 min, and 1 h following the session brains were harvested and processed for c‐fos immunohistochemistry and co‐localization with EGFP. Our results indicate that ethanol intake activates a subset (~15–20%) of ArcN POMC neurons, whereas saccharin or water intake activates significantly fewer (~5–12%) of these neurons. The percent of activated POMC neurons did not correlate with blood ethanol levels at the time of tissue collection, and activation appeared to be distributed throughout the rostrocaudal axis of the ArcN. No sex differences were observed in the degree of neuronal activation across drinking solutions. These findings indicate a preferential activation of ArcN POMC neurons by ethanol consumption, strengthening the notion that ethanol activates endogenous opioid systems in the brain which may underlie its motivational properties.

Heme oxygenase-1 in the spinal cord plays crucial roles in the analgesic effects of pregabalin and gabapentin in a spared nerve-injury mouse model

IntroductionNeuropathic pain remains one of the most intractable types of pain; although calcium channel α2δ ligands, such as pregabalin and gabapentin, are classified as first-line drugs, they have only modest efficacy. Heme oxygenase-1 (HO-1) signaling attenuates glial activation during neuropathic pain. Thus, this study aimed to investigate the effects of the blood–brain barrier (BBB)-permeable HO-1 inhibitor, tin protoporphyrin IX (SnPP), or the BBB-impermeable HO-1 inhibitor, zinc (II) protoporphyrin IX (ZnPP), on the analgesic efficacy of pregabalin and gabapentin. Additionally, we examined the effects of co-administration of SnPP with pregabalin or gabapentin on the expression of glial markers or other genes. MethodsNeuropathic pain was induced by spared nerve injury (SNI) of the sciatic nerve. The mechanical threshold was tested using the von Frey filaments. The expression of spinal glial markers or other genes was examined using reverse transcription polymerase chain reaction. ResultsSystemic HO-1 inhibition reversed the mechanical antiallodynic effects of pregabalin and gabapentin, although peripheral HO-1 inhibition did not alter the mechanical antiallodynic effects of either pregabalin or gabapentin. Intrathecal injection of SnPP or ZnPP abolished the mechanical antiallodynic effects of pregabalin and gabapentin. Pregabalin and gabapentin increased HO-1, arginase-1, and endogenous opioid precursor preproenkephalin gene expression and decreased the expression of glial markers, interleukin-1β, and inducible nitric oxide synthase. ConclusionsThis study suggests that spinal HO-1 plays a crucial role in the analgesic effects of calcium channel α2δ ligands through the attenuation of glial activation and endogenous opioid release.

Abstract P611: Plasma Pro-Enkephalin a and Ischemic Stroke Risk in the Regards Cohort

Background: The opioid precursor peptide pro-enkephalin A (PENK-A) is believed to be a plasma marker of blood brain barrier dysfunction or cardiovascular risk. PENK-A is previously described in acute ischemic stroke, but PENK-A’s impact on stroke development is unknown. The objective of this study was to test the independent association of plasma PENK-A concentration with incident stroke and describe potential differences by race and sex. Methods: The REGARDS cohort is a population-based cohort of 30,239 black and white adults. Baseline plasma PENK-A was measured in 547 participants who developed stroke and 857 who did not. Participants were followed for a median of 5.6 years (interquartile range 4.4-7.2). PENK-A’s association with incident stroke was examined with multivariable adjusted Cox models. Results: Cross-sectionally, PENK-A was higher with female sex, white race, older age, lower body mass index, and antihypertensive medication use. Continuously, higher PENK-A was independently associated with stroke risk (fully adjusted hazard ratio 1.20 per standard deviation higher, 95% confidence interval 1.01-1.42). Restricted cubic spline plots revealed a U-shaped relationship (Figure). Among subgroups, this U-shaped association was most apparent in white men and black women. Adjustment for demographics and established stroke risk factors had minimal impact. PENK-A concentration was not associated with stroke severity or subtype. Conclusions: Higher plasma PENK-A concentration was independently associated with incident stroke in REGARDS. This association was U-shaped, and PENK-A may be most relevant to white men and black women. PENK-A’s association with stroke was not impacted by established stroke risk factor suggesting a possible causal role in stroke etiology. Further work is needed to understand the role of PENK-A in stroke pathogenesis.

Loss of POMC-mediated antinociception contributes to painful diabetic neuropathy

Painful neuropathy is a frequent complication in diabetes. Proopiomelanocortin (POMC) is an endogenous opioid precursor peptide, which plays a protective role against pain. Here, we report dysfunctional POMC-mediated antinociception in sensory neurons in diabetes. In streptozotocin-induced diabetic mice the Pomc promoter is repressed due to increased binding of NF-kB p50 subunit, leading to a loss in basal POMC level in peripheral nerves. Decreased POMC levels are also observed in peripheral nervous system tissue from diabetic patients. The antinociceptive pathway mediated by POMC is further impaired due to lysosomal degradation of μ-opioid receptor (MOR). Importantly, the neuropathic phenotype of the diabetic mice is rescued upon viral overexpression of POMC and MOR in the sensory ganglia. This study identifies an antinociceptive mechanism in the sensory ganglia that paves a way for a potential therapy for diabetic neuropathic pain.

Genetic and functional analysis of a Pacific hagfish opioid system.

The actions of endogenous opioids and nociceptin/orphanin FQ are mediated by four homologous G protein-coupled receptors that constitute the opioid receptor family. However, little is known about opioid systems in cyclostomes (living jawless fish) and how opioid systems might have evolved from invertebrates. Here, we leveraged de novo transcriptome and low-coverage whole-genome assembly in the Pacific hagfish (Eptatretus stoutii) to identify and characterize the first full-length coding sequence for a functional opioid receptor in a cyclostome. Additionally, we define two novel endogenous opioid precursors in this species that predict several novel opioid peptides. Bioinformatic analysis shows no closely related opioid receptor genes in invertebrates with regard either to the genomic organization or to conserved opioid receptor-specific sequences that are common in all vertebrates. Furthermore, no proteins analogous to vertebrate opioid precursors could be identified by genomic searches despite previous claims of protein or RNA-derived sequences in several invertebrate species. The presence of an expressed orthologous receptor and opioid precursors in the Pacific hagfish confirms that a functional opioid system was likely present in the common ancestor of all extant vertebrates some 550 million years ago, earlier than all previous authenticated accounts. We discuss the premise that the cyclostome and vertebrate opioid systems evolved from invertebrate systems concerned with antimicrobial defense and speculate that the high concentrations of opioid precursors in tissues such as the testes, gut, and activated immune cells are key remnants of this evolutionary role.

Proenkephalin+ regulatory T cells expanded by ultraviolet B exposure maintain skin homeostasis with a healing function

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK+ UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis.

Dynorphin and Enkephalin Opioid Peptides and Transcripts in Spinal Cord and Dorsal Root Ganglion During Peripheral Inflammatory Hyperalgesia and Allodynia

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PerspectiveThis is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

BMP-7 protects male and female rodents against neuropathic pain induced by nerve injury through a mechanism mediated by endogenous opioids

Neuropathic pain is highly prevalent in pathological conditions such as diabetes, herpes zoster, trauma, etc. The severity and refractoriness to treatments make neuropathic pain a significant health concern. The transforming growth factor (TGF-β) family of cytokines is involved in pain modulation. Bone morphogenetic proteins (BMPs) constitute the largest subgroup within the TGF-β family. BMP-7 induces the transcription of genes coding endogenous opioid precursors in vitro. However, a nociception modulatory function for this cytokine remains unexplored in vivo. Herein, we show that BMP-7 and its type I receptors were detected in regions of the nervous system involved in pain transmission, processing, and modulation. BMP-7 haploinsufficiency confers to male and female mice a tactile hyperalgesia phenotype to mechanical stimuli, both at baseline and after sciatic nerve injury (SNI). The administration of recombinant BMP-7 (rBMP-7) reduced the severity of the allodynia after SNI in rodents without sexual dimorphism. Central administration of rBMP-7 delayed allodynia development after SNI and reduced the severity of allodynia. The opioid antagonist naloxone antagonized the antinociceptive effect of rBMP-7 in rats. The analgesic effect of morphine was significantly attenuated in BMP-7+/− mice. The antiallodynic effect of voluntary exercise after SNI, whose mechanism involves the endogenous opioid system, was hampered by BMP-7 deficiency while potentiated by rBMP-7. Our results suggest that BMP-7 may constitute a novel therapeutic target for the treatment of neuropathic pain, which improves the function of the endogenous pain-resolution mechanisms to alleviate chronic pain.

Maternal manipulation during late gestation (GDs 17-20) enhances ethanol consumption and promotes changes and opioid mRNA expression in infant rats

Fetal ethanol experience generates learning and memories capable to increase ethanol consummatory behaviors during infancy. Opioid system seems to be involved in mediating those alcohol-related behaviors. In this work, we proposed to study the impact of prenatal exposure to a moderate ethanol dose, upon ingestion of the drug and possible ethanol-induced molecular changes on opioid precursor peptides (POMC, Pro-enk and Pro-DYN) and receptors (MOR, DOR and KOR) mRNA expression, in hypothalamus. Pregnant rats received during gestational days (GDs) 17-20, a daily intragastric (i.g.) administration with 2g/kg ethanol or water. A third group of dams was left undisturbed during pregnancy (Unmanipulated group). Intake test was conducted at postnatal days (PDs) 14-15. Three groups of pups were performed: control (no intake test), water (vehicle) and 5% ethanol. At the end of intake test blood samples were taken to quantify blood ethanol concentrations (BECs) and hypothalamus sections were obtained to perform qRT-PRC assessment of opioid precursor peptides and receptors. The analysis of the consummatory responses (% of consumption) and pharmacokinetic profiles (BECs) suggested that maternal manipulation induced by i.g. intubations, during the last four days of gestation (whenever ethanol or water), are sufficient to induce infantile ethanol intake during infancy. Gene expression from the hypothalamus of unmanipulated group revealed that infantile ingestive experiences with ethanol can down-regulate expression of mRNA Pro-Dyn and up-regulate mRNA expression of MOR and KOR. Finally, MOR mRNA expression was attenuated by prenatal i.g. manipulation in pups exposed to 5% ethanol.

Proving the synergistic effect of Alcalase, PepX and PepN during casein hydrolysis by complete degradation of the released opioid precursor peptide VYPFPGPIPN

A casein solution was hydrolyzed with Alcalase 2.4 L (EC 3.4.21.62) and the recombinantly produced aminopeptidases PepX (EC 3.4.14.11) and PepN (EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 in various combinations to analyze the synergistic effect of these peptidases during casein hydrolysis. The sequential application of PepX or PepN after prehydrolysis with Alcalase resulted in an relative degree of hydrolysis (rDH) increase of 1.12- or 2.00-fold, respectively, compared to only using Alcalase. By a combined application of PepX and PepN the rDH increased ~ 2.32-fold. Using Alcalase, PepX and PepN simultaneously from the beginning the rDH increased ~ 2.42-fold. Compared to the single application of PepX or PepN after an Alcalase treatment, the combined usage led to an increased amount of small peptides (< 1.1 kDa) and free amino acids. After the sequential application of first Alcalase and then PepX and PepN, only 14 peptides, which originated mainly from the C-terminal end of the β-casein chain remained. Even the opioid precursor peptide VYPFPGPIPN [β-casein, ƒ(59–68); V-β-casomorphine-9], generated by the Alcalase treatment was fully hydrolyzed after adding PepX and PepN. Therefore, the synergistic effect of PepX and PepN during casein hydrolysis was confirmed. The simultaneous application of Alcalase, PepX and PepN from the beginning showed similar results as the sequential application, but only three remaining peptides were observed by the mass spectrometric analysis. Additionally, the hydrolysis time was reduced from 16 h (sequential approach) to 6.5 h (simultaneous approach). This indicated a further synergism between Alcalase and the two aminopeptidases.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain

BackgroundNeuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. MethodsNovel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. ResultsTwo novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. Conclusions and general significanceHigh levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

Spatiotemporal expression of endogenous opioid processing enzymes in mouse uterus at peri-implantation.

Successful implantation requires intimate interactions between a competent blastocyst and a receptive uterus. We recently demonstrated that the aberrant activation of opioid signaling by exogenous ligands adversely affects preimplantation embryonic development and subsequent implantation in mice. However, the underlying machinery governing the dynamic homeostasis of the endogenous opioid system in the uterus during early pregnancy remains elusive. We now show that all three major endogenous opioid precursors are spatiotemporally expressed in the uterus during early pregnancy. Moreover, we observe the well-coordinated expression of the synthetic enzyme prohormone convertases 1/3 (PC1/3) at lower levels and of its inhibitor proprotein convertase subtilisin/kexin type 1 inhibitor (Pcsk1n) and the degrading enzyme membrane metallo-endopeptidase (MME) at higher levels in the receptive uterus. Both estrogen and progestin tend to reduce the uterine levels of opioid ligand precursors in the ovariectomized mouse model. This tight regulation of the endogenous opioid system by PC1/3, Pcsk1n and MME has been further confirmed in physiologically related pseudopregnancy and delayed implantation mouse models. The coordinated regulation of opioid precursor biosynthesis and metabolism helps to create appropriate opioid signaling ensuring uterine receptivity for implantation. Thus, endogenous uterine opioid levels are primarily determined by the coordinated expressions of PC1/3, Pcsk1n and MME under the influence of ovarian progestin and estrogen. Our findings raise an additional cautionary note regarding the effects of opioid abuse on early pregnancy events.

Stable Peptide of the Endogenous Opioid Enkephalin Precursor and Breast Cancer Risk.

In experimental studies, enkephalins (ENKs) and related opioids have been implicated as negative regulators of breast cancer development by enhancing immune-mediated tumoral defense as well as directly inhibiting cancer cells. We hypothesized that plasma levels of ENKs are predictive of the long-term breast cancer risk. Therefore, our objective was to measure pro-ENK A, a surrogate for mature ENK, and evaluate its predictive value for the development of breast cancer in a large population of middle-aged women and an independent study population. We related pro-ENK in fasting plasma samples from 1,929 healthy women (mean age, 57.6 ± 5.9 years) of the Malmö Diet and Cancer study to breast cancer incidence (n = 123) during a median follow-up of 14.7 years. For replication, pro-ENK was related to risk of breast cancer (n = 130) in an older independent sample from the Malmö Preventive Project consisting of 1,569 women (mean age, 70.0 ± 4.4 years). In the Malmö Diet and Cancer study, pro-ENK was inversely related to the risk of incident breast cancer, with a hazard ratio per each standard deviation increment of logarithm-transformed pro-ENK of 0.72 (95% CI, 0.62 to 0.85; P < .001). The linear elevation of risk over pro-ENK quartiles 3, 2, and 1, with the fourth quartile as a reference, was 1.38 (95% CI, 0.73 to 2.64), 2.29 (95% CI, 1.26 to 4.15), and 3.16 (95% CI, 1.78 to 5.60; for the trend, P < .001), respectively. These results were replicated in the Malmö Preventive Project, where the continuous odds ratio for incident breast cancer was 0.63 (95% CI, 0.52 to 0.76; P < .001) and the risk over pro-ENK quartiles 3, 2, and 1, where the fourth quartile was the reference, was 2.48 (95% CI, 1.25 to 4.94), 2.94 (95% CI, 1.50 to 5.77), and 4.81 (95% CI, 2.52 to 9.18; for the trend, P < .001), respecitvely. Low fasting plasma concentration of the opioid precursor peptide pro-ENK is associated with an increased risk of future breast cancer in middle-aged and postmenopausal women.

The expression of mRNAs for opioid precursors in endometrium of cyclic and early pregnant pigs; effects of IL-1β, IL-6 and TNFα

The expression of mRNAs for opioid precursors in endometrium of cyclic and early pregnant pigs; effects of IL-1β, IL-6 and TNFα