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OBJECTIVES: Well-established clinical practice for assessing progress in labor involves routine abdominal palpation and vaginal examination (VE). However, VE is subjective, poorly reproducible and painful for most women. In this study, our aim was to evaluate the feasibility of systematically integrating transabdominal and transperineal ultrasound assessment of fetal position, parasagittal angle of progression (psAOP), head-perineum distance (HPD) and sonographic cervical dilatation (SCD) to monitor the progress of labor in women undergoing induction of labor (IOL). We also aimed to determine if ultrasound can reduce women's pain during such examinations. METHODS: Women were recruited as they presented for IOL in three maternity units. Ultrasound assessments were performed in 100 women between 37 + 0 and 41 + 6 weeks' gestation. A baseline combined transabdominal and transperineal scan was performed, including assessment of fetal biometry, umbilical artery and fetal middle cerebral artery Doppler, amniotic fluid index, fetal spine and occiput positions, psAOP, HPD, SCD and cervical length. Intrapartum scans were performed instead of VE, unless there was a clinical indication to perform a VE, according to protocol. Participants were asked to indicate their level of pain by verbally giving a pain score between 0 and 10 (with 0 representing no pain) during assessment. Repeated measures data were analyzed using mixed-effect models to identify significant factors that affected the relationship between psAOP, HPD, SCD and mode of delivery. RESULTS: A total of 100 women were included in the study. Of these, 20% delivered by Cesarean section, 65% vaginally and 15% by instrumental delivery. There were no adverse fetal or maternal outcomes. A total of 223 intrapartum ultrasound scans were performed in 87 participants (13 women delivered before intrapartum ultrasound was performed), with a median of two scans per participant (interquartile range (IQR), 1-3). Of these, 76 women underwent a total of 151 VEs with a median of one VE per participant (IQR, 0-2), with no significant difference between vaginal- or Cesarean-delivery groups. After excluding those with epidural anesthesia during examination, the median pain score for intrapartum scans was 0 (IQR, 0-1) and for VE it was 3 (IQR, 0-6). Cesarean delivery was significantly associated with a slower rate of change in psAOP, HPD and SCD. CONCLUSIONS: Comprehensive transabdominal and transperineal ultrasound assessment can be used to assess progress in labor and can reduce the level of pain experienced during examination. Ultrasound assessment may be able to replace some transabdominal and vaginal examinations during labor. © 2024 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

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OBJECTIVE: To determine whether maternal serum glycosylated fibronectin (GlyFn) level in the first trimester increases the sensitivity of the Fetal Medicine Foundation (FMF) triple test, which incorporates mean arterial pressure, uterine artery pulsatility index and placental growth factor, when screening for pre-eclampsia (PE) in an Asian population. METHODS: This was a nested case-control study of Chinese women with a singleton pregnancy who were screened for PE at 11-13 weeks' gestation as part of a non-intervention study between December 2016 and June 2018. GlyFn levels were measured retrospectively in archived serum from 1685 pregnancies, including 101 with PE, using an enzyme-linked immunosorbent assay (ELISA), and from 448 pregnancies, including 101 with PE, using a point-of-care (POC) device. Concordance between ELISA and POC tests was assessed using Lin's correlation coefficient and Passing-Bablok and Bland-Altman analyses. GlyFn was transformed into multiples of the median (MoM) to adjust for maternal and pregnancy characteristics. GlyFn MoM was compared between PE and non-PE pregnancies, and the association between GlyFn MoM and gestational age at delivery with PE was assessed. Risk for developing PE was estimated using the FMF competing-risks model. Screening performance for preterm and any-onset PE using different biomarker combinations was quantified by area under the receiver-operating-characteristics curve (AUC) and detection rate (DR) at a 10% fixed false-positive rate (FPR). Differences in AUC between biomarker combinations were compared using the DeLong test. RESULTS: The concordance correlation coefficient between ELISA and POC measurements was 0.86 (95% CI, 0.83-0.88). Passing-Bablok analysis indicated proportional bias (slope, 1.08 (95% CI, 1.04-1.14)), with POC GlyFn being significantly higher compared with ELISA GlyFn. ELISA GlyFn in non-PE pregnancies was independent of gestational age at screening (P = 0.11), but significantly dependent on maternal age (P < 0.003), weight (P < 0.0002), height (P = 0.001), parity (P < 0.02) and smoking status (P = 0.002). Compared with non-PE pregnancies, median GlyFn MoM using ELISA and POC testing was elevated significantly in those with preterm PE (1.23 vs 1.00; P < 0.0001 and 1.18 vs 1.00; P < 0.0001, respectively) and those with term PE (1.26 vs 1.00; P < 0.0001 and 1.22 vs 1.00; P < 0.0001, respectively). GlyFn MoM was not correlated with gestational age at delivery with PE (P = 0.989). Adding GlyFn to the FMF triple test for preterm PE increased significantly the AUC from 0.859 to 0.896 (P = 0.012) and increased the DR at 10% FPR from 64.9% (95% CI, 48.7-81.1%) to 82.9% (95% CI, 66.4-93.4%). The corresponding DRs at 10% FPR for any-onset PE were 52.5% (95% CI, 42.3-62.5%) and 65.4% (95% CI, 55.2-74.5%), respectively. CONCLUSIONS: Adding GlyFn to the FMF triple test increased the screening sensitivity for both preterm and any-onset PE in an Asian population. Prospective non-intervention studies are needed to confirm these initial findings. © 2023 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

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Exhaust air treatment has gained importance as an essential factor in intensive livestock areas due to the rising emissions in the environment. Wet filter walls of multi-stage exhaust air treatment systems precipitate gaseous ammonia and dust particles from exhaust air in washing water. Microbial communities in the biomass developed in the washing water of five large-scale exhaust air treatment units of pig housing facilities, were investigated by fluorescence in situ hybridization (FISH) and 16S rDNA sequence analyses. No "standard" nitrifying bacteria were found in the washing water. Instead mainly α-Proteobacteria, aggregating ß- and χ-Proteobacteria, a large number of Actinobacteria, as well as individual Planctomycetales and Crenarchaeota were detected after more than twelve months' operation. The main Proteobacteria species present were affiliated to the families Alcaligenaceae, Comamonadaceae and Xanthomonadaceae. Furthermore, we investigated the consumption of inorganic nitrogen compounds in the washing water of one exhaust air treatment unit during a fattening period with and without pH control. Maintaining the pH at 6.0 resulted in a ca. fivefold higher ammonium concentration and a ca. fourfold lower concentration of oxidized nitrogen compounds after the fattening period was finished.

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Pharmacogenomics links individual drug response variation to genetic differences, such as single nucleotide polymorphisms (SNPs). In particular, pharmacogenomics will allow clinicians to use genetic diagnostics to predict the response of a patient to a drug. We investigated whether SNPs in opioid receptors correlated with the development of morphine tolerance in mouse strains that showed either high or low tolerance to morphine. Sequencing identified five silent SNPs in the delta opioid receptor that varied from the published sequence in some strains, but which were found in both high and low tolerance strains. The mu and kappa opioid receptor sequences had no SNPs. Taken together, these data definitively demonstrate that morphine tolerance development in mice is independent of opioid receptor sequence.

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Apoptosis of eosinophils is of increasingly important value in modulating allergic inflammatory airway diseases, such as asthma, and is suppressed by interleukin-5 (IL-5) in in vitro culture. In this study, we examined the effects of theophylline on survival/apoptosis, intracellular cAMP concentration, and Bcl-2 protein expression. Treatment with theophylline protected eosinophils against IL-5-mediated inhibition of apoptosis with a simultaneous suppression of survival in a dose-dependent manner. Theophylline caused an increase in the intracellular cAMP levels of IL-5-stimulated eosinophils. Enhancement of eosinophil apoptosis was consistent with an increase in DNA fragmentation in eosinophils treated with theophylline. On the other hand, the Bcl-2 protein appeared to be expressed constitutively in freshly isolated eosinophils. Bcl-2 expression was augmented by IL-5 stimulation, yet it was considerably inhibited by theophylline treatment. These data suggest that intracellular cAMP levels and Bcl-2 expression are involved in the suppression of eosinophil survival by theophylline.

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The object of this study is to investigate the routes of the introduction of the western psychiatric knowledges and practices in Korea. The historical documents including newspapers and governmental bulletins as well as articles and books on the history of the Korean medicine were examined and the results are as follows: The western knowledge about the brain anatomy and physiology were introduced from China by the enlightened Confucian and Taoistic scholars of Korea in the mid seventeenth century through the Chinese translations of the western science and medicine. Due to the lack of support for the scholars and even persecution by the ruling power to those who had great interests in the western thoughts including sciences, the western medical knowledges could not be actualized in practice. Thus, the active practices of western medicine were started in the late 19th century in Korea through the two routes; one, via Japanese military physicians and the other one, via the western missionary physicians. The psychiatry was lectured by Japanese psychiatrist in 1910 at the medical school of Tai-Han Unwon, the Korean governmental clinic and 1913 at the Severance medical school of Tai-Han Uiwon, the Korean governmental clinic and in 1913 at the Severance medical school by the Australian psychiatrist, McLaren. As the independent department with the psychiatric ward, the first Dept. of Psychiatry was established in 1913 at the colonial governmental clinic, Chosun Chondokbu-Uiwon, the former Tai-Han Ui-won. Medicine as well as psychiatry was introduced into Korea under the political atmosphere of one sided admiration for the western science. The attempts to combine the western medicine with the traditional Korean medicine could not be tolerated by both missionary physicians and the colonial regime.

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The influence of pregnancy on the dilator effects of acetylcholine in the isolated human uterine artery was investigated. Acetylcholine (0.1 nM to 0.1 microM) produced concentration- and endothelium-dependent relaxation of norepinephrine (3 microM)-induced contraction. The relaxation was greater in arteries from pregnant patients (P arteries) than from non-pregnant patients (NP arteries). The maximal relaxation was 53.5+/-3.4% (n=21) in P arteries and 23.5+/-2.5% (n=35) in NP arteries. In both P and NP arteries the cholinergic relaxation was increased in the presence of superoxide dismutase and greatly reduced in the presence of the nitric oxide synthase inhibitors, NG-mono-methyl L-arginine (L-NMMA) and L-nitro-arginine-methylester (L-NAME). The effect of these nitric oxide synthase inhibitors was reversed by L-arginine. We conclude that pregnancy enhances acetylcholine-induced nitric oxide synthesis and release in the human uterine artery.

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Although the mu selective agonist [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAMGO) and the delta selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE) are both antinociceptive when administered directly into the spinal cord of mice, 50% of antinociceptive dose (AD50) of DAMGO is about 2 orders of magnitude lower than the AD50 of DPDPE. In contrast, the two ligands show similar affinities for their respective receptors in in vitro binding assays. One possible explanation for this discrepancy is that DPDPE antinociception in the spinal cord is mediated through not delta but mu receptors, for which it has an several hundred-fold lower affinity than DAMGO. In support of this, we found that DPDPE-mediated antinociception was blocked by the mu selective antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). The pA2 value of CTAP for DPDPE was virtually identical with that for DAMGO. However, because its action also was blocked by naltrindole, an antagonist selective for delta receptors, the latter must also play a role in antinociception. When DAMGO and DPDPE were administered i.t. together at ratios ranging from 1:200 to 1:500, the AD50 of DAMGO was lowered as much as 10-fold relative to its AD50 when given alone. Thus DPDPE had a potentiating effect on DAMGO, although the reverse was not observed. This potentiation was lost in animals made tolerant to systemic morphine. The loss of potentiation seemed to be caused by changes in the delta receptors, because a) the AD50 of DAMGO (i.t.) given alone to tolerant animals was virtually the same as for naive animals, whereas the AD50 of DPDPE given alone increased by 4-fold; and b) the AD50 of DPDPE given alone in the tolerant animal was increased only slightly by naltrindole, whereas CTAP was still a very potent antagonist. We conclude that DPDPE, a selective delta agonist, mediates antinociception in the spinal cord through mu receptors, consistent with results of recent studies of "knock-out" mice lacking mu receptors. At the same time, however, the delta agonist acting through delta receptors can potentiate the mu receptor-mediated antinociceptive action of either mu or delta agonists. This potentiating effect, like the synergistic effect observed between mu receptors at spinal and supraspinal sites, is lost during tolerance.

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Accumulating evidence indicates that the endogenous opioid peptides dynorphinA-(1-17) and dynorphinA-(1-13) interact not only with opioid but also with yet poorly characterized non-opioid receptors. The latter have been implicated in a number of the effects of dynorphins including induction of ACTH release in sheep and in AtT 20 cells, a pituitary-derived mouse cell line. AtT 20 cells do not express opioid receptors and therefore are particularly suitable for search of non-opioid dynorphin receptors. We report here that 3H-dynorphinA-(1-13)-NH2 associates specifically with AtT 20 cells, apparently through an uptake process and a binding site. Within the cell, it binds preferentially to fractions containing secretory vesicles, with a Kd of about 100 nM. DynorphinA-(1-17), and several non-opioid fragments of dynorphin, including A-(2-17), A-(2-16) and A-(2-13), compete with 3H-dynorphinA-(1-13)-NH2 for that site with IC50s ranging from 200 nM to 2 microM. ACTH(1-39) also competes with 3H-dynorphinA-(1-13)-NH2 for the site with an IC50 of about 300 nM. DynorphinA-(2-17) at microM concentrations stimulates release of ACTH from the isolated vesicles. The results indicate the presence of a non-opioid dynorphin binding site on the secretory vesicle fractions of AtT20 cells that might be involved in ACTH release. The ability of ACTH itself to compete for the binding sites associated with the vesicles suggest that those sites may be involved in an autocrine loop.

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Morphine administered simultaneously to intracerebroventricular (i.c.v.) and intrathecal (i.t.) sites exhibits synergism, with the antinociceptive potency much greater than would be predicted from a simple addition of the potencies of the same dose administered to either site alone. This synergism was quantified in mice using both a fixed dose method, in which the morphine dose at one site was fixed while the AD50 (antinociceptive dose at 50% effectiveness) of morphine at the other site was determined; and a variable dose method, in which different doses of morphine were administered simultaneously to both sites at a fixed ratio, and the AD50 determined and compared to the AD50 at a single site alone. When animals were made tolerant to morphine by implantation of a 75-mg morphine pellet for 3 days, this synergism was eliminated, so that morphine administered simultaneously to i.c.v. and i.t. sites had an additive effect. However, administration of the peptide DynorphinA-(2-17) i.v. simultaneously to the test doses of morphine in morphine-tolerant animals resulted in a partial restoration of synergism. These results suggest that morphine-induced antinociception is highly dependent on an intact integrated central nervous system system and that the initial tolerance development is the result of a disruption of synergism between the central nervous system sites. Morphine tolerance results not from a reduced sensitivity to morphine at discrete central nervous system sites, but rather from a reduced synergistic interaction of morphine at spinal and supraspinal sites. In support of this conclusion, there was no tolerance observed in morphine-pelleted animals to morphine administered to i.c.v. or i.t. sites alone. DynorphinA-(2-17), a nonopioid peptide has previously been shown to enhance the antinociceptive potency of morphine in morphine-tolerant animals, appears to act by restoring this synergism.

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Several lines of evidence link the opioid binding cell adhesion molecule (OBCAM) to opioid function. When delta-opioid receptor cDNA (DOR-1) was expressed in CHO cells, OBCAM immunoreactivity (OBCAM-ir) was detected. Transfected cell lines which displayed high opioid binding also expressed high cell surface OBCAM-ir, while untransfected CHO and vector control cells did not. The positive control, neural cell adhesion molecule (NCAM), a protein with structural homology to OBCAM, displayed the same levels of immunofluorescence in transfected and nontransfected cell lines. Membranes from CHO cells transfected with and expressing a variety of muscarinic and dopamine receptors were tested for immunoreactivity. No significant OBCAM-ir was detected in any of these cell membranes. When anti-OBCAM peptide antibodies were used for immunoblots of CHO cells, untransfected, non-binding transfected, and high binding transfected cells revealed the same banding patterns with approximately equal intensity. These observations suggest that in untransfected cells OBCAM is either not present on the extracellular side of the CHO cell membrane or that it exists in an altered conformation which changes upon transfection with opioid receptors to allow recognition of the non-denatured protein by anti-OBCAM antibodies.

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Despite the recent cloning of mu, delta and kappa opioid receptors, a role in opioid receptor function for an opioid binding cell adhesion molecule is supported by several lines of evidence, including inhibition of opioid binding by opioid binding cell adhesion molecule antibodies, down-regulation of opioid binding cell adhesion molecule by chronic opioid agonist treatment of cultured NG108-15 cells, and reduction of opioid binding in NG108-15 cells by transfection of opioid binding cell adhesion molecule antisense cDNA. In the present study, we report that chronic in vivo treatment of mice with morphine results in down-regulation of opioid binding cell adhesion molecule immunoreactivity in primary afferent neurons in dorsal root and trigeminal ganglia as well as their axons. This effect was blocked by the opioid antagonist naloxone. Down-regulation of opioid binding cell adhesion molecule immunoreactivity was not observed in other areas of the central nervous system. Taken together, the previous studies which demonstrated the role played by opioid receptors in regulating release of transmitters from primary afferent neurons and the present findings of a specific regulation of opioid binding cell adhesion molecule expression by chronic exposure to morphine, provides evidence from an in vivo perspective which advances the notion that opioid binding cell adhesion molecule plays a role in the action of opioids.

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Dynorphin A(1-13) blocks opiate withdrawal in rats without producing dependence, and enhances analgesia in morphine-tolerant animals. Its potential use in humans is therefore of interest. Dynorphin A(1-13) has little toxicity when administered at modest doses IV but has been reported to cause hindlimb paralysis and necrosis of the spinal cord in rats, at the catheter tip, when administered intrathecally. To further evaluate its potential neurotoxicity, we administered dynorphin A(1-13) to rats at very high doses IV. Rats (n = 6-10 per group) received dynorphin A(1-13) as bolus IV doses of 5 mg/kg, or as continuous IV infusions of 40 mg/kg/day for 1 day, with saline controls. The appearance and behavior of all animals was normal. Tail flick latencies remained unchanged (p > 0.5). There were no histologic abnormalities of the spinal cord or brain when examined by light microscopy. Two additional groups received bolus injections of dynorphin A(1-13) 50 or 100 mg/kg IV. Animals receiving 50 mg/kg showed cutaneous flushing, labored respirations, and decreased spontaneous movement, which resolved within 10 min. Histology at 1 week was normal. All six animals receiving 100 mg/kg convulsed and died within minutes. Three animals that received dynorphin A(1-13) 40 mg/kg/day for 7 days had normal behavior and histology. We conclude that the previously observed neurotoxicity of intrathecally administered dynorphin A(1-13) is a local effect that does not occur when dynorphin A(1-13) is administered IV, even at very high doses.

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The dynorphin family of peptides stands out among the opioids in that its members are not antinociceptive after central administration in the common antinociceptive assays. In addition, reports of spinal antinociception have been conflicting. We have tested the antinociceptive activity of i.v. dynorphin A-(1-13) in the writhing assay and have found it to be very potent, with an ED50 of 1.0 (0.99-1.02) mumol/kg. Remarkably, [des-tyr1]dyn A-(2-17) was equally active with an ED50 of 1.1 (0.99-1.20). This activity was also retained by several smaller, non-opioid dynorphin A fragments and was not affected by the presence of either 50 mumol/kg naloxone or 20 mumol/kg Nor-BNI. Further, ED50 values were not different in morphine-dependent mice. The peak effect of dyn A-(1-13) and A-(2-17) was observed 5 min after administration and the effect of dyn A-(1-13) or dyn A-(2-17) was still measurable 1 hr after i.v. administration with a 5- to 6-fold increase in ED50 at this time. The ED50 values after i.c.v. and i.t. administration of dyn A-(1-13) were similar to those reported previously. Dyn A(2-17) was also effective by these routes with ED50 values not significantly different from those of dyn A-(1-13). Both dyn A-(1-13) and A-(2-17) were also active when injected i.p., whereas ED50 values increased substantially after s.c. administration.(ABSTRACT TRUNCATED AT 250 WORDS)

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Oligodeoxyribonucleotide (oligo) primers derived from rat opioid-binding cell adhesion molecule (OBCAM)-encoding cDNA sequence were used to amplify a 403-bp fragment from a human brain cDNA library using the polymerase chain reaction (PCR). The fragment was cloned, sequenced and used as a hybridization probe to screen the library. lambda plaque clones were isolated which contained a 1.5-kb cDNA fragment, including a complete open reading frame (ORF) of 1038 bp. Sequence analysis of the ORF revealed 93% identity to the rat OBCAM cDNA at the nucleotide level, and the deduced amino-acid sequences shared 98% identity. Percentages of identity between human and bovine OBCAM ORFs were within 2% of these values. OBCAM was mapped to human chromosome 11 by hybridizing the probe with a somatic cell hybrid panel.

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A single dose of dynorphin A-(1-13) [dyn A(1-13)] is effective in suppressing the expression of opioid withdrawal and tolerance in morphine-dependent mice. In addition, this modulatory activity is retained by the corresponding non-opioid [des-Tyr1]-dynorphin A peptide [dynA(2-17)]. We have further investigated the non-opioid nature of this activity by comparing the efficacies of dyn A(1-13) and (2-17) under different experimental protocols with a variety of dosing regimens. The effect of dyn A(1-13) on withdrawal and tolerance expression was dose-dependent and could be enhanced by repeated dosing. Thus, the ED50 of naloxone to precipitate withdrawal jumping was increased 1.8-fold when morphine-dependent mice were treated with 4.2 mumol/kg dyn A(1-13) on the fourth day after pellet implantation and 2.4-fold on the sixth day with continued daily dyn A(1-13) treatment. The maximal effect was observed on day 6 when the ED50 of mice treated with 8.4 mumol/kg of dyn A(1-13) was increased nearly 6-fold over that of saline controls. Dyn A(2-17) proved to be nearly as effective as dyn A(1-13).

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A subtraction cDNA library was constructed from control hybrid NG108-15 (mouse neuroblastoma x rat glioma) cells and NG108-15 cells which had been treated for 48 h with the delta-opioid agonist D-Ala2-D-Leu5 enkephalin (DADLE) to down-regulate the delta-opioid receptor on these cells. Among the clones isolated from this library was NGD16-4, a 2768-bp clone encoding a putative 64-kDa protein containing 14 tandemly repeated zinc fingers (Zf) with high homology to the Krüppel family of Zf proteins. NGD16-4 also contains a region homologous to the A element of the Krüppel Associated Box (KRAB) domain, a domain recently linked to transcriptional repression. Southern and Northern analyses indicate that NGD16-4 is derived from the mouse genome. Northern analysis also demonstrates that expression of NGD16-4 mRNA is much higher in several mouse neuroblastoma cell lines than in mouse brain or other tissues. Although the function of NGD16-4 is unclear, the expression pattern of NGD16-4 indicates a possible association with the processes of differentiation or transformation in the mouse.

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Opioids elicit an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in neuroblastoma x glioma hybrid NG108-15 cells, which, depending upon growth conditions, results from either Ca2+ influx in differentiated cells or Ca2+ release from internal stores in undifferentiated cells (Jin et al., 1992). In this report we describe fura-2-based digital imaging studies that demonstrate that opioid-evoked Ca2+ release in these cells results from the activation of phospholipase C (PLC) and subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive store. D-Ala2-D-Leu5-enkephalin (DA-DLE) evoked concentration-dependent increases in [Ca2+]i (EC50 approximately equal to 4 nM). The response was blocked by naloxone (1 microM). In single cells, sequential application of selective opioid agonists (10 nM) evoked responses of the rank order DADLE = D-Pen2, D-Pen5-enkephalin (DPDPE) > trans-(+/-) 3,4-dichloro-N-methyl-N-(2-[1- pyrrolidinyl]cyclohexyl) benzeneacetamide (U50488) > D-ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO), consistent with activation of a delta-opioid receptor. Forty percent (n = 198) of the cells responded to 100 nM DADLE with a net [Ca2+]i increase of 483 +/- 40 nM. Bradykinin (100 nM) elicited a response in 91% of the cells with a mean net amplitude of 707 +/- 36 nM. The DADLE-evoked responses were not blocked by removal of extracellular Ca2+; instead, they were abolished by treatment with 10 nM thapsigargin, an agent that depletes and prevents refilling of IP3-sensitive Ca2+ stores. A 1 microM concentration of U73122, an aminosteroid inhibitor of PLC, completely blocked the DADLE-evoked [Ca2+]i increase, while an inactive analog, U73433, was without effect. To explore the possible role of G-proteins in mediating opioid-induced [Ca2+]i increases in NG108-15 cells, we pretreated cells with pertussis or cholera toxin; pertussis toxin blocked the opioid-induced response while cholera toxin was without effect, consistent with a Gi- or Go-mediated effect. Activation of the opioid inhibitory pathway previously described for these cells appears to stimulate the phosphoinositide (PI) cascade as well. Including the PI cascade among the multiple second messenger systems modulated by opioids may be key to understanding the biochemical events that underlie acute and chronic opioid action.

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