u-50488 and rimorphin

The present studies were undertaken to examine the influence of mu (beta-endorphin, DAMGO, FK 33-824), delta (met-enkephalin, leu-enkephalin, DPLPE) and kappa opioid receptor agonists (dynorphin A, dynorphin B, U 50488) used at different doses (1-1000 nM) alone and in combination with LH (100 ng/ml) on steroidogenesis in porcine granulosa cells derived from large follicles. The effects of mu, delta and kappa receptor agonists on both basal and LH-induced progesterone (P4) secretion were negligible. Agonists of mu opioid receptors reduced basal androstenedione (A4), testosterone (T) and oestradiol (E2) release. Co-treatment with LH entirely abolished the inhibitory effect of these agonists on A4 and E2 secretion and resulted in an increase in T release. The addition of delta receptor agonists was followed by a decrease in basal A4, T and E2 secretion. The cells incubated in the presence of LH increased the androgen production and abrogated the inhibitory effect of delta agonists on E2 output. Basal A4, T and E2 release was also suppressed by kappa receptor agonists. The presence of LH in culture media extended the inhibitory effect of these opioids on E2 output and caused either abolition of the inhibitory influence of kappa agonists or even augmentation of both androgen release in response to the opioids. In conclusion, these data support the involvement of three major types of opioid receptors in the regulation of porcine granulosa cell steroidogenesis.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; beta-Endorphin; D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; Dose-Response Relationship, Drug; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Female; Granulosa Cells; Luteinizing Hormone; Opioid Peptides; Ovarian Follicle; Swine

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; beta-Endorphin; Cells, Cultured; D-Ala(2),MePhe(4),Met(0)-ol-enkephalin; Dynorphins; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Methionine; Estradiol; Female; Opioid Peptides; Progesterone; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Steroids; Swine; Testosterone; Theca Cells

In binding assays, both dynorphin B and alpha-neoendorphin are relatively selective for the kappa1b site, unlike U50,488H which has high affinity for both kappa1a and kappa1b sites. In vivo, U50,488H, dynorphin B and alpha-neoendorphin analgesia are reversed by the kappa1-selective antagonist, nor-binaltorphimine (norBNI). Antisense mapping the three exons of KOR-1 revealed that probes targeting all three exons blocked U50,488H analgesia, as expected. However, the selectivity profile of dynorphin B and alpha-neoendorphin analgesia towards the various antisense oligodeoxynucleotides differed markedly from U50,488H, implying a different receptor mechanism of action.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Antisense Elements (Genetics); Binding Sites; Chromosome Mapping; Dynorphins; Endorphins; Ion Channel Gating; Male; Mice; Mice, Inbred ICR; Protein Precursors; Receptors, Opioid, kappa

Prodynorphin mRNA and dynorphin B expression have been previously shown to be greatly increased in cardiac myocytes of BIO 14.6 cardiomyopathic hamsters. Here we report that exogenous dynorphin B induced a dose-dependent increase in prodynorphin mRNA levels and stimulated prodynorphin gene transcription in normal hamster myocytes. Similar responses were elicited by the synthetic selective kappa opioid receptor agonist U-50,488H. These effects were counteracted by the kappa opioid receptor antagonist Mr-1452 and were not observed in the presence of chelerythrine or calphostin C, two specific protein kinase C (PKC) inhibitors. Treatment of cardiomyopathic cells with Mr-1452 significantly decreased both prodynorphin mRNA levels and prodynorphin gene transcription. In control myocytes, dynorphin B induced the translocation of PKC-alpha to the nucleus and increased nuclear PKC activity without affecting the expression of PKC-delta, -epsilon, or -zeta. Acute release of either U-50,488H or dyn B over single normal or cardiomyopathic cells transiently increased the cytosolic Ca2+ concentration. A sustained treatment with each opioid agonist increased the cytosolic Ca2+ level for a more prolonged period in cardiomyopathic than in control myocytes and led to a depletion of Ca2+ from the sarcoplasmic reticulum in both groups of cells. The possibility that prodynorphin gene expression may affect the function of the cardiomyopathic cell through an autocrine mechanism is discussed.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Calcium; Cardiomyopathy, Hypertrophic; Cell Compartmentation; Cell Nucleus; Cricetinae; Cytosol; Dynorphins; Endorphins; Enkephalins; Enzyme Inhibitors; Gene Expression; Mesocricetus; Myocardium; Protein Kinase C; Protein Precursors; Pyrrolidines; Receptors, Opioid, kappa; Ryanodine; Sarcoplasmic Reticulum

Opioid peptides, and especially the dynorphins, have been localized to several circuits in the CA3 hippocampal region, yet electrophysiological studies often find mixed effects of opiates on the excitability of CA3 neurons. Reasoning that these mixed effects might involve voltage-dependent actions, we tested the effect of several opiates on CA3 pyramidal neurons using single-electrode voltage-clamp recording in a slice preparation of rat hippocampus. In most CA3 neurons, the voltage-dependent K+ current known as the M-current (IM) was uniquely sensitive to the opioid peptides, with the direction of response dependent upon the opiate type and concentration. Thus, an opiate selective for kappa receptors, U-50,488H, significantly augmented IM. The kappa-selective agonists dynorphin A and dynorphin B, which exist in mossy fiber afferents to CA3 pyramidal neurons, also markedly augmented IM at low concentrations (20-100 nM). By contrast, dynorphin A at higher concentrations (1-1.5 microM) often reduced IM. Similarly, several opiates [e.g., D-Ala2,D-Leu5-enkephalin: (DADL), [D-Pen2,5]-enkephalin (DPDPE)] known to act on the delta receptor subtypes reduced the M-current, with partial reversal of this effect by naloxone. Neither the selective mu-receptor agonist [D-Ala2, NMe-Phe4, Gly-ol]-enkephalin (DAMGO) nor the nonopioid fragment of dynorphin, des-Tyr-dynorphin, consistently altered IM. These opiate effects on IM were accompanied by changes in conductance and holding current consistent with their respective effects on IM. Dynorphin A did not measurably affect the Q-current, a conductance known to contribute to inward rectification in hippocampal pyramidal neurons. The opiate effects on IM were not altered by pretreatment with Cs+ (which blocks IQ) or Ca2+ channel blockers. The opposing effects of the dynorphins (both A and B) and DADL on IM were antagonized by naloxone (1-3 microM), and the dynorphin-induced augmentations of IM were usually reversed by the kappa receptor antagonist norbinaltorphimine. These results suggest that the opiates can have opposing effects on the same voltage-dependent K+ channel type (the M channel) in the rat CA3 pyramidal neuron, with the direction of the response depending on which receptor subtype is activated. These data not only help explain the mixed effects of opiates seen in other studies, but also suggest a potential postsynaptic function for the endogenous opiates contained in the CA3 mossy fibers.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Barium Compounds; Cadmium; Carbachol; Chlorides; Computer Simulation; Dynorphins; Electrophysiology; Endorphins; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Models, Neurological; Naloxone; Narcotics; Neurons; Pyramidal Tracts; Pyrrolidines; Rats; Rats, Sprague-Dawley