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A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1–13) potently inhibits the Nln activity while dynorphin A (1–8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln. [ABSTRACT FROM AUTHOR]

Abstract A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1–13) potently inhibits the Nln activity while dynorphin A (1–8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.

[Display omitted] • Dexamethasone and membrane-impermeable Dex-BSA alleviated neuropathic pain in rats. • The precise mechanisms of glucocorticoids on neuropathic pain remain elusive. • Dexamethasone alleviated neuropathic pain via upregulated microglial dynorphin A. • Dexamethasone alleviated neuropathic pain through membrane glucocorticoid receptor. • The cAMP/PKA/p38 MAPK/CREB pathway mediated the upregulation of dynorphin A. This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway. [ABSTRACT FROM AUTHOR]

The neuropeptides kisspeptin, neurokinin B, and dynorphin A are imperative for the pulsatile secretion of gonadotropin-releasing hormone and luteinizing hormone to ultimately regulate reproductive cyclicity. A population of neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy status from afferent neuronal and glial cells. We hypothesized that KNDy expressing neurons in the ARC of mature ewes are influenced by energy balance. To test this hypothesis, ovary-intact, mature ewes were fed to lose, maintain, or gain body weight and hypothalamic tissue harvested during the luteal phase of the estrous cycle. Fluorescent, multiplex immunohistochemistry with direct antibody conjugation was employed to identify and quantify neurons expressing a single neuropeptide, as well as for the first time report co-expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC. Previous reports using this population of ewes demonstrated that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone. Moreover, within this population of ewes tanycyte density and cellular penetration into the ARC was increased in ewes fed to gain body weight. Within the current report we have revealed that the number of neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin A protein was increased in ewes fed to gain body weight. Moreover, the number of KNDy neurons in the ARC expressing all three neuropeptides within a single neuron was decreased in ewes fed to lose body weight and increased in ewes fed to gain body weight when compared to ewes fed to maintain body weight. The cumulative findings of this experimental model suggest that expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC during the luteal phase of the estrous cycle are influenced by energy balance-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes to ultimately regulate central perception of global energy status. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu via proopiomelanocortin afferents. [ABSTRACT FROM AUTHOR]

The neuropeptides kisspeptin, neurokinin B, and dynorphin A are imperative for the pulsatile secretion of gonadotropin-releasing hormone and luteinizing hormone to ultimately regulate reproductive cyclicity. A population of neurons co-expressing these neuropeptides, KNDy neurons, within the arcuate nucleus of the hypothalamus (ARC) are positioned to integrate energy status from afferent neuronal and glial cells. We hypothesized that KNDy-expressing neurons in the ARC of mature ewes are influenced by energy balance. To test this hypothesis, ovary-intact, mature ewes were fed to lose, maintain, or gain body weight and hypothalamic tissue harvested during the luteal phase of the estrous cycle. Fluorescent, multiplex immunohistochemistry with direct antibody conjugation was employed to identify and quantify neurons expressing a single neuropeptide, as well as for the first time report co-expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC. Previous reports using this population of ewes demonstrated that concentrations of insulin and leptin differed between ewes fed to achieve different body weights and that ewes fed to gain body weight had increased concentrations of progesterone. Moreover, within this population of ewes tanycyte density and cellular penetration into the ARC was increased in ewes fed to gain body weight. Within the current report we have revealed that the number of neurons in the ARC expressing kisspeptin, neurokinin B, and dynorphin A protein was increased in ewes fed to gain body weight. Moreover, the number of KNDy neurons in the ARC expressing all three neuropeptides within a single neuron was decreased in ewes fed to lose body weight and increased in ewes fed to gain body weight when compared to ewes fed to maintain body weight. The cumulative findings of this experimental model suggest that expression of kisspeptin, neurokinin B, and dynorphin A protein in the ARC during the luteal phase of the estrous cycle are influenced by energy balance-induced alterations in circulating concentrations of progesterone that drive changes in morphology and density of tanycytes to ultimately regulate central perception of global energy status. Moreover, these results demonstrate that changes in KNDy neurons within the ARC occur as an adaptation to energy balance, potentially regulated divergently by metabolic milieu via proopiomelanocortin afferents.

Uncovering the central mechanism underlying mammalian reproduction is warranted to develop new therapeutic approaches for reproductive disorders in humans and domestic animals. The present study focused on the role of arcuate kisspeptin neurones (also known as KNDy neurones) as an intrinsic gonadotropin‐releasing hormone (GnRH) pulse generator, which plays a fundamental role in mammalian reproduction via the stimulation of pituitary gonadotropin synthesis and release and thereby in gametogenesis and steroidogenesis in the gonads of mammals. We also discuss the mechanism that inhibits pulsatile GnRH/gonadotropin release under a negative energy balance, considering that reproductive disorders often occur during malnutrition in humans and livestock. [ABSTRACT FROM AUTHOR]

Panax notoginseng (Chinese ginseng, Sanqi), one of the major ginseng species, has been traditionally used to alleviate different types of chronic pain. The raw P. notoginseng powder is commonly available in China as a non-prescription drug to treat various aliments including arthritic pain. However, strong scientific evidence is needed to illustrate its pain antihypersensitive effects, effective ingredients and mechanism of action. The oral P. notoginseng powder dose-dependently alleviated formalin-induced tonic hyperalgesia, and its total ginsenosides remarkably inhibited neuropathic pain hypersensitivity. Ginsenoside Rb1, the most abundant ginsenoside of P. notoginseng , dose-dependently produced neuropathic pain antihypersensitivity. Conversely, ginsenosides Rg1, Re and notoginseng R1, the other major saponins from P. notoginseng , failed to inhibit formalin-induced tonic pain or mechanical allodynia in neuropathic pain. Ginsenoside Rb1 metabolites ginsenosides Rg3, Compound-K and protopanaxadiol also had similar antineuropathic pain efficacy to ginsenoside Rb1. Additionally, intrathecal ginsenoside Rb1 specifically stimulated dynorphin A expression which was colocalized with microglia but not neurons or astrocytes in the spinal dorsal horn and primary cultured cells. Pretreatment with microglial metabolic inhibitor minocycline, dynorphin A antiserum and specific κ-opioid receptor antagonist GNTI completely blocked Rb1-induced mechanical antiallodynia in neuropathic pain. Furthermore, the specific glucocorticoid receptor (GR) antagonist Dex-21-mesylate (but not GPR30 estrogen receptor antagonist G15) also entirely attenuated ginsenoside Rb1-related antineuropathic pain effects. All these results, for the first time, show that P. notoginseng alleviates neuropathic pain and ginsenoside Rb1 is its principal effective ingredient. Furthermore, ginsenoside Rb1 inhibits neuropathic pain by stimulation of spinal microglial dynorphin A expression following GR activation. [Display omitted] • Novel therapies are required to treat neuropathic pain due to limited efficacy of available treatments. • Panax notoginseng powder and its total ginsenosides remarkably inhibit pain hypersensitive states. • Ginsenoside Rb1 is the principal saponin in Panax notoginseng to ameliorate neuropathic pain. • Ginsenoside Rb1 stimulates spinal microglial dynorphin A expression to alleviate neuropathic pain probably by membrane glucocorticoid receptor activation. [ABSTRACT FROM AUTHOR]

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0–2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0–2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals. [ABSTRACT FROM AUTHOR]

Abstract Background Isotalatizidine is a representative C19-diterpenoid alkaloid extracted from the lateral roots of Aconitum carmichaelii, which has been widely used to treat various diseases on account of its analgesic, anti-inflammatory, anti-rheumatic, and immunosuppressive properties. The aim of this study was to evaluate the analgesic effect of isotalatizidine and its underlying mechanisms against neuropathic pain. Methods A chronic constrictive injury (CCI)-induced model of neuropathic pain was established in mice, and the limb withdrawal was evaluated by the Von Frey filament test following isotalatizidine or placebo administration. The signaling pathways in primary or immortalized microglia cells treated with isotalatizidine were analyzed by Western blotting and immunofluorescence. Results Intrathecal injection of isotalatizidine attenuated the CCI-induced mechanical allodynia in a dose-dependent manner. At the molecular level, isotalatizidine selectively increased the phosphorylation of p38 and ERK1/2, in addition to activating the transcription factor CREB and increasing dynorphin A production in cultured primary microglia. However, the downstream effects of isotalatizidine were abrogated by the selective ERK1/2 inhibitor U0126-EtOH or CREB inhibitor of KG-501, but not by the p38 inhibitor SB203580. The results also were confirmed in in vivo experiments. Conclusion Taken together, isotalatizidine specifically activates the ERK1/2 pathway and subsequently CREB, which triggers dynorphin A release in the microglia, eventually leading to its anti-nociceptive action.

Background and Purpose: New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment. Experimental Approach The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies. Key Results: Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long‐term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage‐related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ‐opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone‐21‐mesylate, but not GPR‐30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol‐induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane‐impermeable glucocorticoid receptor agonist dexamethasone‐BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone‐21‐mesylate. Conclusion and Implications: All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain. [ABSTRACT FROM AUTHOR]

Increasing evidence shows that puberty onset is largely dependent on body weight rather than chronological age. To investigate the mechanism involved in the energetic control of puberty onset, the present study examined effects of chronic food restriction during the prepubertal period and the resumption of ad libitum feeding for 24 and 48 h on estrous cyclicity, Kiss1 (kisspeptin gene), Tac3 (neurokinin B gene) and Pdyn (dynorphin A gene) expression in the hypothalamus, luteinizing hormone (LH) secretion and follicular development in female rats. When animals weighed 75 g, they were subjected to a restricted feeding to retard growth to 70–80 g by 49 days of age. Then, animals were subjected to ad libitum feeding or remained food-restricted. The growth-retarded rats did not show puberty onset associated with suppression of both Kiss1 and Pdyn expression in the arcuate nucleus (ARC). 24-h ad libitum feeding increased tonic LH secretion and the number of Graafian and non-Graafian tertiary follicles with an increase in the numbers of ARC Kiss1- and Pdyn-expressing cells. 48-h ad libitum feeding induced the vaginal proestrus and a surge-like LH increase with an increase in Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV). These results suggest that the negative energy balance causes pubertal failure with suppression of ARC Kiss1 and Pdyn expression and then subsequent gonadotropin secretion and ovarian function, while the positive energetic cues trigger puberty onset via an increase in ARC Kiss1 and Pdyn expression and thus gonadotropin secretion and follicular development in female rats.

Bulleyaconitine A (BAA), a C19-diterpenoid alkaloid, has been prescribed as a nonnarcotic analgesic to treat chronic pain over four decades in China. The present study investigated its inhibition in morphine-induced withdrawal symptoms, conditioned place preference (CPP) and locomotor sensitization, and then explored the underlying mechanisms of actions. Multiple daily injections of morphine but not BAA up to 300 μg/kg/day into mice evoked naloxone-induced withdrawal symptoms (i.e., shakes, jumps, genital licks, fecal excretion and body weight loss), CPP expression, and locomotor sensitization. Single subcutaneous BAA injection (30–300 μg/kg) dose-dependently and completely attenuated morphine-induced withdrawal symptoms, with ED50 values of 74.4 and 105.8 μg/kg in shakes and body weight loss, respectively. Subcutaneous BAA (300 μg/kg) also totally alleviated morphine-induced CPP acquisition and expression and locomotor sensitization. Furthermore, subcutaneous BAA injection also specifically stimulated dynorphin A expression in microglia but not astrocytes or neurons in nucleus accumbens (NAc) and hippocampal, measured for gene and protein expression and double immunofluorescence staining. In addition, subcutaneous BAA-inhibited morphine-induced withdrawal symptoms and CPP expression were totally blocked by the microglial metabolic inhibitor minocycline, dynorphin A antiserum, or specific KOR antagonist GNTI, given intracerebroventricularly. These results, for the first time, illustrate that BAA attenuates morphine-induced withdrawal symptoms, CPP expression, and locomotor sensitization by stimulation of microglial dynorphin A expression in the brain, suggesting that BAA may be a potential candidate for treatment of opioids-induced physical dependence and addiction.

Visceral pain is one of the leading causes for abdominal pain in gastroenterological diseases and is still hard to treat effectively. Bulleyaconitine A (BAA) is an aconitine analog and has been used for the treatment of pain. Our previous work suggested that BAA exerted analgesic effects on neuropathic pain through stimulating the expression of dynorphin A in spinal microglia. Here, we investigated the inhibitory effect of BAA on visceral pain and examined whether the expression of dynorphin A in spinal microglia was responsible for its effects. We found that BAA produced significant antivisceral pain effect induced by acetic acid through stimulating dynorphin A expression in spinal microglia. In addition, anxiety and chronic visceral pain are highly prevalent comorbid conditions in clinical research, which is still a problem to be solved. We also aimed to evaluate the effects of BAA on anxiety. A comorbidity model with characteristics of both chronic visceral pain and anxiety was developed by colorectal injection of 2,4,6-trinitrobenzene sulfonic acid and the induction of heterotypic intermittent chronic stress protocol. In comorbid animals, BAA exerted great antianxiety effects. Meanwhile, the antianxiety mechanism of BAA was different with the antivisceral pain mechanism of BAA. In conclusion, our study demonstrated, for the first time, that BAA exerted marked antivisceral pain and antianxiety effects, which expands the analgesic spectrum and clinical application of BAA. Furthermore, it also it provides a better guidance for the clinical use of BAA.

Kisspeptin plays a critical role in governing gonadotrophin‐releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co‐express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary‐cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T‐antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone‐derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17β‐oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants. [ABSTRACT FROM AUTHOR]

Visceral pain is one of the leading causes for abdominal pain in gastroenterological diseases and is still hard to treat effectively. Bulleyaconitine A (BAA) is an aconitine analog and has been used for the treatment of pain. Our previous work suggested that BAA exerted analgesic effects on neuropathic pain through stimulating the expression of dynorphin A in spinal microglia. Here, we investigated the inhibitory effect of BAA on visceral pain and examined whether the expression of dynorphin A in spinal microglia was responsible for its effects. We found that BAA produced significant antivisceral pain effect induced by acetic acid through stimulating dynorphin A expression in spinal microglia. In addition, anxiety and chronic visceral pain are highly prevalent comorbid conditions in clinical research, which is still a problem to be solved. We also aimed to evaluate the effects of BAA on anxiety. A comorbidity model with characteristics of both chronic visceral pain and anxiety was developed by colorectal injection of 2,4,6-trinitrobenzene sulfonic acid and the induction of heterotypic intermittent chronic stress protocol. In comorbid animals, BAA exerted great antianxiety effects. Meanwhile, the antianxiety mechanism of BAA was different with the antivisceral pain mechanism of BAA. In conclusion, our study demonstrated, for the first time, that BAA exerted marked antivisceral pain and antianxiety effects, which expands the analgesic spectrum and clinical application of BAA. Furthermore, it also it provides a better guidance for the clinical use of BAA. [ABSTRACT FROM AUTHOR]

Background: Isotalatizidine is a representative C19-diterpenoid alkaloid extracted from the lateral roots of Aconitum carmichaelii, which has been widely used to treat various diseases on account of its analgesic, anti-inflammatory, anti-rheumatic, and immunosuppressive properties. The aim of this study was to evaluate the analgesic effect of isotalatizidine and its underlying mechanisms against neuropathic pain.Methods: A chronic constrictive injury (CCI)-induced model of neuropathic pain was established in mice, and the limb withdrawal was evaluated by the Von Frey filament test following isotalatizidine or placebo administration. The signaling pathways in primary or immortalized microglia cells treated with isotalatizidine were analyzed by Western blotting and immunofluorescence.Results: Intrathecal injection of isotalatizidine attenuated the CCI-induced mechanical allodynia in a dose-dependent manner. At the molecular level, isotalatizidine selectively increased the phosphorylation of p38 and ERK1/2, in addition to activating the transcription factor CREB and increasing dynorphin A production in cultured primary microglia. However, the downstream effects of isotalatizidine were abrogated by the selective ERK1/2 inhibitor U0126-EtOH or CREB inhibitor of KG-501, but not by the p38 inhibitor SB203580. The results also were confirmed in in vivo experiments.Conclusion: Taken together, isotalatizidine specifically activates the ERK1/2 pathway and subsequently CREB, which triggers dynorphin A release in the microglia, eventually leading to its anti-nociceptive action. [ABSTRACT FROM AUTHOR]

Estrogen produced by ovarian follicles plays a key role in the central mechanisms controlling reproduction via regulation of gonadotropin-releasing hormone (GnRH) release by its negative and positive feedback actions in female mammals. It has been well accepted that estrogen receptor α (ERα) mediates both estrogen feedback actions, but precise targets had remained as a mystery for decades. Ever since the discovery of kisspeptin neurons as afferent ERα-expressing neurons to govern GnRH neurons, the mechanisms mediating estrogen feedback are gradually being unraveled. The present article overviews the role of kisspeptin neurons in the arcuate nucleus (ARC), which are considered to drive pulsatile GnRH/gonadotropin release and folliculogenesis, in mediating the estrogen negative feedback action, and the role of kisspeptin neurons located in the anteroventral periventricular nucleus-periventricular nucleus (AVPV-PeN), which are thought to drive GnRH/luteinizing hormone (LH) surge and consequent ovulation, in mediating the estrogen positive feedback action. This implication has been confirmed by the studies showing that estrogen-bound ERα down- and up-regulates kisspeptin gene (Kiss1) expression in the ARC and AVPV-PeN kisspeptin neurons, respectively. The article also provides the molecular and epigenetic mechanisms regulating Kiss1 expression in kisspeptin neurons by estrogen. Further, afferent ERα-expressing neurons that may regulate kisspeptin release are discussed.

The pulsatile secretion of hormones is an efficient way of coding a large variety of chemical messages. The GnRH pulse pattern determines which gonadotropin is released when and at what concentration, prescribing a detailed set of instructions to the gonads that produce changes in the steroid hormone milieu. Although GnRH neurons possess some inherent rhythmicity, they are diffusely situated within the hypothalamus and in isolation are only capable of generating physiologically irrelevant messages, hence a synchronization module exists upstream. The identity of the neural unit comprising the GnRH pulse generator is now generally thought to include KNDy neurons in the arcuate nucleus. These neurons coexpress the neuropeptides kisspeptin, neurokinin B and dynorphin A, as well as other transmitters, and are in intimate contact with the GnRH network. The GnRH pulse generator’s function is the precise control of GnRH neuron excitability, coordinated activation, stimulation of neurosecretory events, modulation of gene transcription and the mediation of the negative feedback effect of gonadal steroids. Additionally, the GnRH pulse generator is an ideal venue for the integration of various sensory and homeostatic cues that regulate reproductive functions. In this chapter we provide a historical perspective of the elegant science that sparked interest in the central mechanisms underlying the functions of the reproductive system, explain how hypotheses surrounding GnRH pulse generation have evolved and describe the current state of knowledge within the dynamic field of GnRH pulse generator research.

Pubertal onset is thought to be timed by an increase in pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in mammals. The underlying mechanism of pubertal onset in mammals is still an open question. Evidence accumulated in the last 15 years suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus play a key role in pubertal onset by triggering pulsatile GnRH/gonadotropin secretin in mammals. Specifically, KNDy neurons are now considered a part of GnRH pulse generator, in which neurokinin B facilitates and dynorphin A inhibits, the synchronized discharge of KNDy neurons in autocrine and/or paracrine manners. Kisspeptin serves as a potent secretagogue of GnRH secretion and thus its release is fundamental to pubertal increase in GnRH/gonadotropin secretion in mammals. Proposed mechanisms inhibiting Kiss1 (kisspeptin gene) expression during childhood to juvenile varies from species to species: we envisage that negative feedback action of estrogen plays a key role in the inhibition of Kiss1 expression in KNDy neurons in rodents and sheep, whereas estrogen-independent inhibition of kisspeptin secretion by γ-amino butyric acid or neuropeptide Y are suggested to be responsible for the pre-pubertal suppression of GnRH/gonadotropin secretion in primates. Taken together, the timing of pubertal onset is postulated to be controlled by upstream regulators for kisspeptin biosynthesis and secretion in mammals.

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and β-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of β-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants.

• Dynorphin A increment contributed to the initiation and maintenance of neuropathic pain after SNL surgery. • Dynorphin A stimulated IL-1β and TNF-α release in the acute phase of neuropathic pain. • Activation of alpha7 n acetylcholine receptor improved neuropathic pain behaviors by decreasing dynorphin A release. Dynorphin A is increased in neuropathic pain models. Activation of α7 n acetylcholine receptor (nAchR) reduces inflammation and pain. Whether activation of α7 nAchR affects dynorphin A release is unknown. The experiments evaluated the proinflammatory effect of dynorphin A in the spinal nerve ligation-induced neuropathic pain models and the effect of α7 nAchR activation on the dynorphin A content. α7 nAchR agonist, PHA-543613 and its antagonist, methyllycaconitine citrate were used and dynorphin A content was measured after spinal nerve ligation and in microglia cultures to test the analgesic mechanisms of α7 nAchR activation. The results showed that dynorphin A content peaked 3 to 7 days after nerve injury, and dynorphin A anti-serum intrathecal injection decreased IL-β and TNF-α content a week after nerve injury. Activation of α7 nAchR by PHA-543613 alleviated neuropathic pain behaviors and decreased dynorphin A concentration in the ipsilateral spinal cords. Also, PHA-543613 decreased dynorphin A release from the microglia cultures to LPS stimulation by activation of α7 nAchR. Our results suggest that dynorphin A contribute to the development and maintenance of neuropathic pain and that decreasing dynorphin A content by activation of α7 AchR of microglia is a potential therapeutic target for treating neuropathic pain. [ABSTRACT FROM AUTHOR]

Pubertal onset is thought to be timed by an increase in pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in mammals. The underlying mechanism of pubertal onset in mammals is still an open question. Evidence accumulated in the last 15 years suggests that kisspeptin/neurokinin B/dynorphin A (KNDy) neurons in the hypothalamic arcuate nucleus play a key role in pubertal onset by triggering pulsatile GnRH/gonadotropin secretin in mammals. Specifically, KNDy neurons are now considered a part of GnRH pulse generator, in which neurokinin B facilitates and dynorphin A inhibits, the synchronized discharge of KNDy neurons in autocrine and/or paracrine manners. Kisspeptin serves as a potent secretagogue of GnRH secretion and thus its release is fundamental to pubertal increase in GnRH/gonadotropin secretion in mammals. Proposed mechanisms inhibiting Kiss1 (kisspeptin gene) expression during childhood to juvenile varies from species to species: we envisage that negative feedback action of estrogen plays a key role in the inhibition of Kiss1 expression in KNDy neurons in rodents and sheep, whereas estrogen-independent inhibition of kisspeptin secretion by γ-amino butyric acid or neuropeptide Y are suggested to be responsible for the pre-pubertal suppression of GnRH/gonadotropin secretion in primates. Taken together, the timing of pubertal onset is postulated to be controlled by upstream regulators for kisspeptin biosynthesis and secretion in mammals. [ABSTRACT FROM AUTHOR]

Lactational anestrus, characterized by the suppression of pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release, would be a strategic adaptation to ensure survival by avoiding pregnancy during lactation in mammals. In the present article, we first provide a current understanding of the central regulation of reproduction in mammals, i.e., a fundamental role of arcuate kisspeptin neurons in mammalian reproduction by driving GnRH/LH pulses. Second, we discuss the central mechanism inhibiting arcuate Kiss1 (encoding kisspeptin) expression and GnRH/LH pulses during lactation with a focus on suckling stimulus, negative energy balance due to milk production, and the role of circulating estrogen in rats. We also discuss upper regulators that control arcuate kisspeptin neurons in rats during the early and late lactation periods based on the findings obtained by a lactating rat model. Finally, we discuss potential reproductive technology for the improvement of reproductive performance in milking cows. • Suckling stimulus suppresses ARC Kiss1 expression and GnRH pulse generation. • SST neurons mediate lactational suppression of ARC Kiss1 expression and GnRH pulses. • Negative energy balance due to milk production also suppresses GnRH pulses. • Dyn and Enk neurons mediate lactational and malnutritional suppression of GnRH pulses. [ABSTRACT FROM AUTHOR]

Lappaconitine is a representative C18-diterpenoid alkaloid extracted from Aconitum sinomontanum Nakai and has been prescribed as a pain relief medicine in China for more than 30 years. This study evaluated its antihypersensitivity activity in the rat models of neuropathic and cancer pains and explored its underlying mechanisms. Subcutaneous injection of cumulative doses of lappaconitine produced dose-dependent mechanical antiallodynia and thermal antihyperalgesia in spinal nerve ligation-induced neuropathic rats. The cumulative dose-response analysis exhibited their Emax values of 53.3 and 58.3% MPE, and ED50 values of 1.1 and 1.6 mg/kg. Single intrathecal lappaconitine dose in neuropathy also dose- and time-dependently blocked mechanical allodynia, with an Emax of 66.1% MPE and an ED50 of 0.8 μg. Its multiple twice-daily intrathecal administration over 7 days did not induce mechanical antiallodynic tolerance. Subcutaneous cumulative doses of lappaconitine also produced dose-dependent blockade of mechanical allodynia in the rat bone cancer pain model induced by tibia implantation of cancer cells, with the Emax of 57.9% MPE and ED50 of 2.0 mg/kg. Furthermore, lappaconitine treatment stimulated spinal dynorphin A expression in neuropathic rats, and in primary cultures of microglia but not neurons or astrocytes. Intrathecal pretreatment with the specific microglia depletor liposome-encapsulated clodronate, dynorphin A antibody, and κ-opioid receptor antagonist GNTI totally suppressed intrathecal and subcutaneous lappaconitine-induced mechanical antiallodynia. This study suggests that lappaconitine exhibits antinociception through directly stimulating spinal microglial dynorphin A expression.ᅟ [ABSTRACT FROM AUTHOR]

Guinea pigs are important zoo animals and have been recommended for animal-assisted activities or therapy, however there are problems concerning testosterone inducing aggressive or sexual behaviors in male guinea pigs. Testicular testosterone secretion is regulated by pulsatile gonadotropin releasing hormone (GnRH)/luteinizing hormone (LH) release in mammals. The mechanism generating GnRH/LH pulses is thought to be governed by kisspeptin neurons, which coexpress neurokinin B (NKB) and dynorphin A (Dyn), in the arcuate nucleus (ARC). Kisspeptin neurons in the ARC are frequently referred to as KNDy neurons. The purpose of this study was to examine whether the antagonization of NKB-neurokinin-3 receptor (NK3R) signaling can manipulate testosterone secretion in male guinea pigs. A single subcutaneous administration or 7 days of oral administration of an NK3R-selective antagonist, SB223412 (50 mg/body), significantly decreased plasma testosterone levels in male guinea pigs. In vitro binding assays confirmed that SB223412 has a high affinity to guinea pig NK3R. These results suggest that SB223412 could be used as an orally-available compound to suppress testosterone levels in male guinea pigs. Double labeling in situ hybridization of kisspeptin and either NKB or Dyn showed that kisspeptin-expressing neurons contained NKB (77.9%) or Dyn (62.3%) in the ARC, suggesting the presence of KNDy neurons in the ARC of guinea pigs. In conclusion, the present study shows that SB223412 could be a candidate compound to suppress testosterone secretion in male guinea pigs for controlling sexual and aggressive behaviors in the species. [ABSTRACT FROM AUTHOR]

We show here that the intraplantar administration of CCL5 in mice produces hyperalgesia at low doses but activates compensatory antinociceptive mechanisms at doses slightly higher. Thus, the injection of 3-10 ng of CCL5 evoked thermal hyperalgesia through the activation of CCR1 and CCR5 receptors, as demonstrated by the inhibitory effect exerted by the selective antagonists J113863 (0.01-0.1 μg) and DAPTA (0.3-3 μg), respectively. The prevention of this hyperalgesia by diclofenac (1-10 μg), the inhibitors of COX-1 SC-560 (0.1-1 μg) or COX-2 celecoxib (1-5 μg), the TRPV1 antagonist capsazepine (0.03-0.3 μg) or the TRPA1 antagonist HC030031 (10-50 μg) demonstrates the involvement of prostaglandin synthesis and TRP sensitization in CCL5-evoked hyperalgesia. Doses of CCL5 higher than 17 μg did not evoke hyperalgesia. However, this effect was restored by the administration of naloxone-methiodide (5 μg), nor-binaltorphimine (10 mg/kg) or an anti-dynorphin A antibody (0.62-2.5 ng). The administration of 30 ng of CCL5 also induced hyperalgesia in mice with reduced number of circulating white blood cells in response to cyclophosphamide or with selective neutrophil depletion induced by an anti-Ly6G antibody. In fact, the number of neutrophils present in paws treated with 30 ng of CCL5 was greater than in paws receiving the administration of the hyperalgesic dose of 10 ng. Finally, the expression of the endogenous opioid peptide dynorphin A was demonstrated by double immunofluorescence assays in these neutrophils attracted by CCL5. These results support previous data describing the hyperalgesic properties of CCL5 and constitute the first indication that a chemokine of the CC group can activate endogenous analgesic mechanisms. [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevance Bullatine A, a C 20 -diterpenoid alkaloid and one of the major effective ingredients in Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao), can block pain hypersensitivity in a variety of rodent models through expression of spinal microglial dynorphin A. Aim of the study To assess the interaction between bullatine A and morphine on antinociception in acute nociception and pain hypersensitivity states, with the exogenous synthetic dynorphin A as a comparison Materials and methods Spinal nerve ligation-induced neuropathic rats and naïve mice were used for assessing the acute and chronic interactions of bullatine A/dynorphin A with morphine. Results Single subcutaneous injection of bullatine A or dynorphin A(1−17) did not either alter formalin- and thermally (hot-plate and water immersion tests)-induced acute nociception or potentiate morphine antinociception in naïve mice. In contrast, bullatine A dose-dependently inhibited formalin-induced tonic pain with the efficacy of 54% inhibition and the half-effective dose of 0.9 mg/kg. Concurrent bullatine A additively enhanced morphine antinociception. In neuropathic rats, the antinociceptive effects of multiple bidaily intrathecal injections of bullatine A and dynorphin A remained consistent over 13 days, whereas morphine produced progressive and complete tolerance to antinociception, which was completely inhibited by concurrent bullatine A and dynorphin A. A single intrathecal injection of bullatine A and dynorphin A immediately reversed established morphine tolerance in neuropathic rats, although the blockade was a less degree in the thermally induced mouse acute nociceptive tests. The inhibitory effects of bullatine A and dynorphin A on morphine tolerance were immediately and completely attenuated by intrathecal dynorphin A antibody and/or selective κ-opioid receptor antagonist GNTI. Conclusion These results suggest that bullatine A produces antinociception without induction of tolerance and inhibits morphine antinociceptive tolerance, and provide pharmacological basis for concurrent bullatine A and morphine treatment for chronic pain and morphine analgesic tolerance. [ABSTRACT FROM AUTHOR]

Previously study showed κ-opioid receptor stimulation with exogenous κ-opioid receptor agonist elicited a protective effect against hypoxic pulmonary hypertension (HPH). However, the effect of endogenous κ-opioid receptor agonist dynorphin A on HPH remains unclear. This study was to determine the role of dynorphin in HPH. Hypoxia for 2 weeks induced HPH. Compared with the HPH group, the HPH + nor-BNI (a selective κ-opioid receptor antagonist) group showed a significant increase in mean pulmonary arterial pressure (mPAP). Exogenous treatment with dynorphin A 1–13 significantly decreased mPAP in HPH rat. In addition, we evaluated the effect of exogenous κ-opioid receptor agonist U50,488H on mPAP. The anti-HPH effect of dynorphin A was less than that of U50,488H. Meanwhile, level of dynorphin A in serum and lung was increased during hypoxia for 2 weeks, while it decreased after hypoxia for 4 weeks. In addition, both the level of ET-1 and AngII were increased during hypoxia. Dynorphin A 1–13 and U50,488H time-dependently relaxed pulmonary artery from both normal and HPH rats. The relaxation of dynorphin A was less than that of U50,488H. Dynorphin A 1–13 inhibited the proliferation of pulmonary artery smooth muscle cells (PASMCs) during hypoxia, which was blocked by nor-BNI. κ-opioid receptor expression increased in PASMCs in both normoxia exposed to dynorphin A 1–13 and during hypoxia. Hypoxia-induced increase was enhanced by dynorphin A 1–13 and abolished by nor-BNI. In conclusion, endogenous dynorphin A released in the early stage of hypoxia plays a protective effect against HPH via stimulation of κ-opioid receptor. [ABSTRACT FROM AUTHOR]

Aconitines, including bulleyaconitine A, probably the most bioactive and abundant alkaloids in Aconitum plant, are a group of diester C19-diterpenoid alkaloids with one acetylester group attached to C8 of the diterpenoid skeleton and one benzoylester group to C14. Hydrolysis of both groups is involved in the processing of Aconitum, a traditional Chinese medicinal approach. We recently demonstrated that bulleyaconitine A produced anti-hypersensitivity, which was mediated by stimulation of spinal microglial dynorphin A expression. This study aimed to elucidate whether the acetylester and benzoylester groups are involved in aconitine-induced dynorphin A expression, anti-hypersensitivity, neurotoxicity in neuropathic rats. Intrathecal administration of aconitine and benzoylaconine (but not aconine) attenuated mechanical allodynia and heat hyperalgesia, with normalized ED50 values of 35 pmol and 3.6 nmol, respectively. Aconitine and benzoylaconine anti-allodynia was completely blocked by the microglial inhibitor, dynorphin A antiserum, and κ-opioid receptor antagonist. Aconitine and benzoylaconine, but not aconine, stimulated dynorphin A expression in cultured primary spinal microglia, with EC50 values of 32 nM and 3 μM, respectively. Intrathecal aconitine, benzoylaconine and aconine induced flaccid paralysis and death, with normalized TD50 values of 0.5 nmol, 0.2 μmol, and 1.6 μmol, respectively. The TD50/ED50 ratios of aconitine and benzolyaconine were 14:1 and 56:1. Our results suggest that both the C8-acetyl and C14-benzoyl groups are essential for aconitine to stimulate spinal microglial dynorphin A expression and subsequent anti-hypersensitivity, which can be separated from neurotoxicity, because both benzoylaconine and aconine differentially produced anti-hypersensitivity and neurotoxicity due to their different stimulatory ability on dynorphin A expression. Our results support the scientific rationale for Aconitum processing, but caution should be taken to avoid overprocessing and excess hydrolysis of benzolyaconine to aconine.

We used Neuromolecular Imaging (NMI) and trademarked BRODERICK PROBE® mini-implantable biosensors, to selectively and separately detect neurotransmitters in vivo, on line, within seconds in the dorsal striatal brain of the Parkinson’s Disease (PD) animal model. We directly compared our results derived from PD to the normal striatal brain of the non-Parkinson’s Disease (non-PD) animal. This advanced biotechnology enabled the imaging of dopamine (DA), serotonin (5-HT), homovanillic acid (HVA) a metabolite of DA, L-tryptophan (L-TP) a precursor to 5-HT and peptides, dynorphin A 1-17 (Dyn A) and somatostatin (somatostatin releasing inhibitory factor) (SRIF). Each neurotransmitter and neurochemical was imaged at a signature electroactive oxidation/half-wave potential in dorsal striatum of the PD as compared with the non-PD animal. Both endogenous and bromocriptine-treated neurochemical profiles in PD and non-PD were imaged using the same experimental paradigm and detection sensitivities. Results showed that we have found significant neurotransmitter peptide biomarkers in the dorsal striatal brain of endogenous and bromocriptine-treated PD animals. The peptide biomarkers were not imaged in dorsal striatal brain of non-PD animals, either endogenously or bromocriptine-treated. These findings provide new pharmacotherapeutic strategies for PD patients. Thus, our findings are highly applicable to the clinical treatment of PD.

The pulsatile secretion of hormones is an efficient way of coding a large variety of chemical messages. The GnRH pulse pattern determines which gonadotropin is released when and at what concentration, prescribing a detailed set of instructions to the gonads that produce changes in the steroid hormone milieu. Although GnRH neurons possess some inherent rhythmicity, they are diffusely situated within the hypothalamus and in isolation are only capable of generating physiologically irrelevant messages, hence a synchronization module exists upstream. The identity of the neural unit comprising the GnRH pulse generator is now generally thought to include KNDy neurons in the arcuate nucleus. These neurons coexpress the neuropeptides kisspeptin, neurokinin B and dynorphin A, as well as other transmitters, and are in intimate contact with the GnRH network. The GnRH pulse generator's function is the precise control of GnRH neuron excitability, coordinated activation, stimulation of neurosecretory events, modulation of gene transcription and the mediation of the negative feedback effect of gonadal steroids. Additionally, the GnRH pulse generator is an ideal venue for the integration of various sensory and homeostatic cues that regulate reproductive functions. In this chapter we provide a historical perspective of the elegant science that sparked interest in the central mechanisms underlying the functions of the reproductive system, explain how hypotheses surrounding GnRH pulse generation have evolved and describe the current state of knowledge within the dynamic field of GnRH pulse generator research. [ABSTRACT FROM AUTHOR]

Background: Aconiti brachypodi Radix (Xue-shang-yi-zhi-hao) has been prescribed to manage chronic pain, arthritis, and traumatic injuries. Bullatine A, a C20-diterpenoid alkaloid, is one of its principle effective compounds. This study aimed to investigate the anti-hypersensitivity of bullatine A in a variety of rat pain models and explore its mechanisms of action.Methods: Rat neuropathic pain, inflammatory pain, diabetic neuropathic pain, and bone cancer pain models were used. Dynorphin A and pro-inflammatory cytokines were measured in the spinal cord and cultured primary microglia. Double immunofluorescence staining of dynorphin A and glial and neuronal cellular markers was also measured in the spinal cord.Results: Subcutaneous and intrathecal injection of bullatine A dose-dependently attenuated spinal nerve ligation-, complete Freud's adjuvant-, diabetes-, and bone cancer-induced mechanical allodynia and thermal hyperalgesia, with the efficacies of 45-70 % inhibition, and half-effective doses of 0.9-1.9 mg/kg for subcutaneous injection. However, bullatine A was not effective in blocking acute nociceptive response in the normal condition. Bullatine A specifically stimulated dynorphin A expression in microglia in the spinal cord in vivo and cultured primary microglia in vitro; the stimulatory effects were completely inhibited by the microglial inhibitor minocycline. In contrast, bullatine A did not have an inhibitory effect on peripheral nerve injury- or lipopolysaccharide-induced pro-inflammatory cytokine expression. The spinal anti-allodynic effects of bullatine A were entirely blocked by intrathecal injection of minocycline, the specific dynorphin A antiserum, and the selective k-opioid receptor antagonist.Conclusions: We, for the first time, demonstrate that bullatine A specifically attenuates pain hypersensitivity, regardless of the pain models employed. The results also suggest that stimulation of spinal microglial dynorphin A expression mediates bullatine A anti-nociception in pain hypersensitivity conditions. [ABSTRACT FROM AUTHOR]

Dynorphins are important neuropeptides with a central role in nociception and pain alleviation. Many mechanisms regulate endogenous dynorphin concentrations, including proteolysis. Proprotein convertases (PCs) are widely expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous big dynorphin (BDyn) and dynorphin A (Dyn A) levels has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis control of BDyn and Dyn A levels using cellular fractions of spinal cords from wild-type (WT), PC1 −/+ and PC2 −/+ animals and mass spectrometry. Our results clearly demonstrate that both PC1 and PC2 are involved in the proteolysis regulation of BDyn and Dyn A with a more important role for PC1. C-terminal processing of BDyn generates specific peptide fragments dynorphin 1–19, dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, and C-terminal processing of Dyn A generates dynorphin 1–13, dynorphin 1–11 and dynorphin 1–7, all these peptide fragments are associated with PC1 or PC2 processing. Moreover, the proteolysis of BDyn leads to the formation of Dyn A and Leu-Enk, two important opioid peptides. The rate of formation of both is significantly reduced in cellular fractions of spinal cord mutant mice. As a consequence, even the partial inhibition of PC1 or PC2 may impair the endogenous opioid system. [ABSTRACT FROM AUTHOR]

Unlabelled: Aconitine and its structurally-related diterpenoid alkaloids have been shown to interact differentially with neuronal voltage-dependent sodium channels, which was suggested to be responsible for their analgesia and toxicity. Bulleyaconitine A (BAA) is an aconitine analogue and has been prescribed for the management of pain. The present study aimed to evaluate the inhibitory effects of BAA on pain hypersensitivity and morphine antinociceptive tolerance, and explore whether the expression of dynorphin A in spinal microglia was responsible for its actions. Single intrathecal or subcutaneous (but not intraventricular or local) injection of BAA blocked spinal nerve ligation-induced painful neuropathy, bone cancer-induced pain, and formalin-induced tonic pain by 60 to 100% with the median effective dose values of 94 to 126 ng per rat (intrathecal) and 42 to 59 μg/kg (subcutaneous), respectively. After chronic treatment, BAA did not induce either self-tolerance to antinociception or cross-tolerance to morphine antinociception, and completely inhibited morphine tolerance. The microglial inhibitor minocycline entirely blocked spinal BAA (but not exogenous dynorphin A) antinociception, but failed to attenuate spinal BAA neurotoxicity. In a minocycline-sensitive and lidocaine- or ropivacaine-insensitive manner, BAA stimulated the expression of dynorphin A in the spinal cord, and primary cultures of microglia but not of neurons or astrocytes. The blockade effects of BAA on nociception and morphine tolerance were totally eliminated by the specific dynorphin A antiserum and/or κ-opioid receptor antagonist. Our results suggest that BAA eliminates pain hypersensitivity and morphine tolerance through directly stimulating dynorphin A expression in spinal microglia, which is not dependent on the interactions with sodium channels.Perspective: The newly illustrated mechanisms underlying BAA antinociception help us to better understand and develop novel dynorphin A expression-based painkillers to treat chronic pain. [ABSTRACT FROM AUTHOR]