Early life stress induces sex-dependent increases in phosphorylated extracellular signal-regulated kinase in brains of mice with neuropathic pain.

BACKGROUND: Both early life stress and neuropathic pain induce morphological and functional abnormalities of the nervous system that are associated with emotional regulation. In our previous study, early life stress enhanced nerve injury-induced hyperalgesia in adult male and female mice. In the present study, using phosphorylated extracellular signal-regulated kinase (p-ERK) as a marker of neuronal activation, we examined the effect of early life stress on neuronal function following partial sciatic nerve ligation (PSL). METHODS: Early life stress was induced by maternal separation from 2 to 3 weeks of age and by social isolation after weaning (MSSI). Neuropathic pain was induced by PSL at 9 weeks of age, and p-ERK expression after light touch stimulation to the ipsilateral paw was measured using immunohistochemistry 1 week after nerve injury. RESULTS: Although MSSI increased p-ERK expression in the paraventricular nucleus (PVN) and amygdala of male mice, PSL did not affect p-ERK expression in control and MSSI mice. In female mice, increased p-ERK expression was observed in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Furthermore, p-ERK expression in the PVN and amygdala was increased in MSSI-PSL mice. CONCLUSIONS: The present data suggest that early life stress sex-dependently and site-specifically increases neuronal activity in the brain. In addition, increased neuronal activity in multiplebrain regions of mice subjected to early life stress may enhance hyperalgesia after nerve injury. WHAT DOES THIS STUDY ADD?: Maternal separation and social isolation (MSSI) increased p-ERK in the paraventricular nucleus (PVN) and amygdala of male mice. MSSI increased p-ERK in the medial prefrontal cortex and nucleus accumbens of female mice. Neuropathic pain increased p-ERK in the PVN and amygdala of female MSSI mice.

Activation of cerebral sodium-glucose transporter type 1 function mediated by post-ischemic hyperglycemia exacerbates the development of cerebral ischemia.

The regulation of post-ischemic hyperglycemia plays an important role in suppressing neuronal damage in therapeutic strategies for cerebral ischemia. We previously reported that the cerebral sodium-glucose transporter (SGLT) was involved in the post-ischemic hyperglycemia-induced exacerbation of cerebral ischemic neuronal damage. Cortical SGLT-1, one of the cerebral SGLT isoforms, is dramatically increased by focal cerebral ischemia. In this study, we focused on the involvement of cerebral SGLT-1 in the development of cerebral ischemic neuronal damage. It was previously reported that activation of 5'-adenosine monophosphate-activated protein kinase (AMPK) increases SGLT-1 expression. Moreover, ischemic stress-induced activation of AMPK exacerbates cerebral ischemic neuronal damage. Therefore, we directly confirmed the relationship between cerebral SGLT-1 and cerebral AMPK activation using in vitro primary culture of mouse cortical neurons. An in vivo mouse model of focal cerebral ischemia was generated using a middle cerebral artery occlusion (MCAO). The development of infarct volume and behavioral abnormalities on day 3 after MCAO were ameliorated in cerebral SGLT-1 knock down mice. Cortical and striatal SGLT-1 expression levels were significantly increased at 12h after MCAO. Immunofluorescence revealed that SGLT-1 and the neuronal nuclear antigen (NeuN) were co-localized in the cortex and striatum of MCAO mice. In the in vitro study, primary cortical neurons were cultured for five days before each treatment with reagents. Concomitant treatment with hydrogen peroxide and glucose induced the elevation of SGLT-1 and phosphorylated AMPK/AMPK ratio, and this elevation was suppressed by compound C, an AMPK inhibitor in primary cortical neurons. Moreover, compound C suppressed neuronal cell death induced by concomitant hydrogen peroxide/glucose treatment in primary cortical neurons. Therefore, we concluded that enhanced cerebral SGLT-1 function mediated by post-ischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage. One of the mechanisms of cerebral SGLT-1 up-regulation may be involved in the AMPK activation after cerebral ischemia.

The activation of supraspinal GPR40/FFA1 receptor signalling regulates the descending pain control system.

BACKGROUND AND PURPOSE: The ω-3 polyunsaturated fatty acids exert antinociceptive effects in inflammatory and neuropathic pain; however, the underlying mechanisms remain unclear. Docosahexaenoic acid-induced antinociception may be mediated by the orphan GPR40, now identified as the free fatty acid receptor 1 (FFA1 receptor). Here, we examined the involvement of supraspinal FFA1 receptor signalling in the regulation of inhibitory pain control systems consisting of serotonergic and noradrenergic neurons. EXPERIMENTAL APPROACH: Formalin-induced pain behaviours were measured in mice. Antinociception induced by FFA1 receptor agonists was examined by intrathecal injections of a catecholaminergic toxin, 5-HT lowering drug or these antagonists. The expression of FFA1 receptor protein and c-Fos was estimated by immunohistochemistry, and the levels of noradrenaline and 5-HT in the spinal cord were measured by LC-MS/MS. KEY RESULTS: FFA1 receptors colocalized with NeuN (a neuron marker) in the medulla oblongata and with tryptophan hydroxylase (TPH; a serotonergic neuron marker) and dopamine ß-hydroxylase (DBH; a noradrenergic neuron marker). A single i.c.v. injection of GW9508, a FFA1 receptor agonist, increased the number of c-Fos-positive cells and the number of neurons double-labelled for c-Fos and TPH and/or DBH. It decreased formalin-induced pain behaviour. This effect was inhibited by pretreatment with 6-hydroxydopamine, DL-p-chlorophenylalanine, yohimbine or WAY100635. Furthermore, GW9508 facilitated the release of noradrenaline and 5-HT in the spinal cord. In addition, GW1100, a FFA1 receptor antagonist, significantly increased formalin-induced pain-related behaviour. CONCLUSION AND IMPLICATIONS: Activation of the FFA1 receptor signalling pathway may play an important role in the regulation of the descending pain control system.

Sodium-glucose transporter type 3-mediated neuroprotective effect of acetylcholine suppresses the development of cerebral ischemic neuronal damage.

Cerebral ischemia can be exacerbated by post-ischemic hyperglycemia, which may involve the cerebral sodium-glucose transporter (SGLT). However, the contribution of each SGLT isoform in cerebral ischemia is still unclear. SGLT-1, -3, -4, and -6 have been reported to be expressed in various brain regions. Among these isoforms, only SGLT-3 does not transport glucose, but depolarizes the plasma membrane when glucose is bound, suggesting that SGLT-3 is a glucose sensor. Therefore, in this study, we investigated the involvement of cerebral SGLT-3 in the development of ischemia. The mouse model of focal ischemia was generated by middle cerebral artery occlusion (MCAO). Neuronal damage was assessed by histological and behavioral analyses. Fasting blood glucose levels on day 1 after MCAO were not affected in SGLT-3 siRNA-mediated knockdown of SGLT-3. The development of infarct volume and behavioral abnormalities on day 1 after MCAO were exacerbated in SGLT-3 knockdown mice (control group: n=7, 94.2 ± 21.8 mm(3), 2 (1.6-2.4), SGLT-3 knockdown group: n=6, 1414.8 ± 492.4 mm(3), 6 (5.8-6.3), P<0.05). Moreover, SGLT-3 expression levels were significantly decreased in the striatum (65.0 ± 8.1%, P<0.05) on day 1, and in the hippocampus (67.6 ± 7.2%, P<0.05) and hypothalamus (47.5 ± 5.1%, P<0.01) on day 3 after MCAO (n=12-13). These effects were significantly inhibited by donepezil (DPZ) treatment (SGLT-3 knockdown group: n=6, 1419.0 ± 181.5 mm(3), 3.6 (3.4-3.7), SGLT-3 knockdown and 3mg/kg DPZ-treated group: n=5, 611.3 ± 205.3 mm(3), 1.5 (1.4-1.8), P<0.05). Immunofluorescence revealed that SGLT-3 and choline acetyltransferase were co-localized in the cortex. Our results indicated that cerebral SGLT-3 suppressed neuronal damage by the activation of cholinergic neurons, which are neuroprotective. In contrast, other cerebral SGLT isoforms may be involved in the development of ischemia.

I.c.v administration of an endothelin ET(B) receptor agonist stimulates vascular endothelial growth factor-A production and activates vascular endothelial growth factor receptors in rat brain.

Vascular endothelial growth factors (VEGFs), a family of angiogenic factors, are upregulated by nerve injuries. To clarify the extracellular signals involved in VEGF production in the brain, the effects of endothelins (ETs), a family of vasoconstricting peptides, were examined. I.c.v. administration of 500 pmol/d Ala(1,3,11,15)-ET-1, an ET(B) receptor agonist, increased the level of VEGF-A mRNA in the rat cerebrum, whereas those of VEGF-B, placental growth factor (PLGF), angiopoietin (ANG)-1, and ANG-2 mRNAs were not largely affected by Ala(1,3,11,15)-ET. The ET-induced increases in cerebrum VEGF-A mRNA were reduced by coadministration of 1 nmol/d BQ788, an ET(B) antagonist. Ala(1,3,11,15)-ET-1 also stimulated the production of VEGF-A proteins in the cerebrum. Immunohistochemical observations in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats showed that glial fibrillary acidic protein (GFAP)-positive astrocytes had VEGF-A immunoreactivity. Neurons, microglia, and brain capillary endothelial cells in the Ala(1,3,11,15)-ET-1-infused rats did not show VEGF-A reactivity. The i.c.v. administration of Ala(1,3,11,15)-ET-1 stimulated tyrosine phosphorylations of VEGF-R1 and R2 receptors in the rat cerebrum, whereas expression levels of total VEGF-R1 and R2 proteins were not largely changed. Immunoreactivity of tyrosine-phosphorylated VEGF-R1 was selectively shown in GFAP-positive astrocytes in the cerebrum of Ala(1,3,11,15)-ET-1-infused rats. Tyrosine-phosphorylated VEGF-R2 proteins were present in astrocytes and brain capillary endothelial cells. These findings indicate that activation of brain ET(B) receptors increases production of VEGF-A and stimulates VEGF receptor signaling in the brain.

Induction and repression of beta-xylanase of Thermomyces lanuginosus TISTR 3465.

The effect of carbon sources on the production of beta-xylanase by Thermomyces lanuginosus TISTR 3465 was investigated. Xylan showed the highest inductive effect on the enzyme formation whereas xylobiose and xylooligosaccharides resulted in lesser effect. beta-Xylanase was also produced at low level with xylose as well as other sugars tested. Xylan concentration at 15 g L(-1) gave the maximum inductive effect on beta-xylanase formation, whereas xylooligosaccharides and xylose were effective at a lower concentration of 2.5 g L(-1). High concentrations of these sugars significantly repressed the enzyme formation. Crude enzyme from the supernatants, without and with other sugars produced a single xylanase band on non-denaturing PAGE gels. However, an intense xylanase activity band was observed from the supernatant of media with xylan, xylobiose and xylooligosaccharides as the carbon sources. An intense protein band of 24.9 kDa from the culture filtrate of xylan medium was observed. Xylan increased beta-xylanase production by the fungus 16-fold when it was added to the xylose medium after cultivation for 3 days. In contrast, addition of xylose to the xylan medium decreased beta-xylanase production 3-fold. A distinct appearance and disappearance of a 24.9 kDa protein and the activity band coincided with an increase and decrease of xylanase activity, respectively. This indicated the synthesis of xylanase by this strain was most induced by xylan. Moreover, the level of xylanase induction has no related to amino acid sequence of the enzyme.

Over-expression system for secretory phospholipase D by Streptomyces lividans.

The structural gene for phospholipase D (PLD) of an actinomycete, Streptoverticillium cinnamoneum, together with its promoter region was introduced into Streptomyces lividans using a shuttle vector-pUC702-for Escherichia coli and S. lividans. The transformant was found to secrete a large amount of PLD (about 2.0x10(4) U/l, 42 mg/l) when cultured in a jar fermentor. Both an initial glucose concentration of 17.5 g/l and the feeding of carbon and nitrogen sources are effective for efficient secretion of PLD; under these culture conditions, the amount of PLD secreted reached a maximum level (about 5.5x10(4) U/l, 118 mg/l) after about 60 h. In contrast to the original producer, Stv. cinnamoneum, which secretes only a small amount of PLD (about 1.1x10(3) U/l, 2 mg/l) along with other extracellular proteins, this heterologous expression system is markedly more efficient in production of secretory PLD.

[Retrospective evaluation of PSA density for selection of biopsy candidates with prostate specific antigen in the gray zone].

PURPOSE: We examined the usefulness of prostate specific antigen density (PSAD) for selection of biopsy candidate with prostate specific antigen levels between 4.1 and 10.0 ng./ml. in prostate cancer screening retrospectively. MATERIALS AND METHODS: The screening was conducted on male candidates in Natori city, aged 55 years or older, for 6 years from 1994 through 1999. We could analyze serum PSA levels and PSA density in 118 men with PSA levels between 4.1 and 10.0 ng./ml. All of 118 men underwent ultrasound guided systematic prostate biopsy regardless of findings of digital rectal examination and transrectal ultrasound. Prostate volume was estimated by transrectal ultrasound measurements using the prolate ellipse formula (pi/6 x length x width x height). PSAD was calculated by dividing serum PSA level by prostate volume. Serum PSA levels were determined by Tandem-R assay. RESULTS: In 118 men, twenty-five men had prostate cancer. There was no significant difference in mean PSA between those with prostate cancer and those without prostate cancer, but the difference was significant in the mean PSA density (mean 0.26 and 0.16, respectively, p < 0.0001). Receiver operating characteristic curves for PSA and PSAD demonstrated superior benefit for PSAD in 118 men. A sensitivity, a specificity, a positive predictive value and a negative predictive value of PSAD cut-off of 0.15 were 88%, 52.7%, 33.3% and 94.2%. PSAD cut-off of 0.18 showed the highest sum of sensitivity and specificity, which gave a sensitivity of 80%, a specificity of 72%, a positive predictive value of 43.5% and a negative predictive value of 93.1%. PSAD cut-off of 0.15 would seem to be preferable to cut-off of 0.18 because of less cancer missing. CONCLUSIONS: Although further studies are needed to determine optimal cut-off value to be used in clinical practice, PASD seems to be useful for the selection of biopsy candidates with PSA levels of 4.1 to 10.0 ng./ml. in the prostate cancer screening.

Neurosteroids stimulate G protein-coupled sigma receptors in mouse brain synaptic membrane.

Dehydroepiandrosterone, its sulfate (DHEAS) and pregnenolone sulfate, representative neurosteroids as well as (+)-pentazocine concentration-dependently stimulated the [35S]GTPgammaS binding in synaptic membranes of mouse prefrontal cortex. These stimulations were blocked by NE-100, a sigma-receptor antagonist, and by progesterone, another type of neurosteroid. The DHEAS-induced stimulation was blocked by the pertussis toxin (PTX)-treatment, and completely recovered by reconstitution of PTX-treated membranes with recombinant G(i1), but not with G(oA). DHEAS also stimulated the [35S]GTPgammaS binding in the coronal sections of mouse brain in NE-100- or progesterone-reversible manner. These findings suggest that some neurosteroids may act on metabotropic sigma receptors, and this study may be the first to show the coupling of neurosteroid binding site and G(i).

Changes of tissue factor-dependent coagulant activity mediated by adhesion between polymorphonuclear leukocytes and endothelial cells.

The purpose of this study was to examine whether polymorphonuclear leukocytes (PMNs) facilitate a tissue factor, a physiologic initiator of coagulation in endothelial cells, -dependent coagulant activity (TF activity). The TF activity in bovine endothelial cells (BAECs) was significantly increased in a concentration-dependent manner by PMNs (1 x 10(5) - 1 x 10(7) cells/ml) without affecting the treatment of N-formyl-methionyl-leucyl-phenylalanine, a selective activator of PMNs, and the addition of PMNs finally resulted in cell damage as evaluated by the lactate dehydrogenase leakage method. In the same conditions, an increase of adhesion between PMNs and BAECs was also observed in a time-dependent manner. However, since direct adhesion of PMNs to BAECs was impossible by using the transwell, PMNs failed to induce any changes in the TF activity. Hence, the change of TF activity found here might be closely related to the PMNs adhesion to BAECs. Indeed, anti-intercellular adhesion molecule-1 (anti-ICAM-1) antibody blocked the increase of TF activity in BAECs. These findings suggest that PMNs could increase TF activity in endothelial cells, which is triggered by adhesion to endothelial cells through ICAM-1.

Climbazole is a new potent inducer of rat hepatic cytochrome P450.

We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver.

Further evidence for a role of NMDA receptors in the locus coeruleus in the expression of withdrawal syndrome from opioids.

To examine a role of N-methyl-D-aspartate (NMDA) receptors in the locus coeruleus (LC) in the expression of the withdrawal signs from opioids, rats were continuously infused with morphine (a mu-opioid agonist, 26 nmol/microl per h) or butorphanol (a mu/delta/kappa-mixed opioid agonist, 26 nmol/microl per h) intracerebroventricularly (i.c.v.) through osmotic minipumps for 3 days. An LC injection of NMDA (0.1 and 1 nmol/5 microl) induced withdrawal signs in opioid-dependent animals. However, it did not precipitate any abnormal behaviors in saline-treated control rats. The expression of the withdrawal signs precipitated by NMDA (1 nmol/5 microl), glutamate (10 nmol/5 microl), or naloxone (an opioid antagonist, 24 nmol/5 microl) was completely blocked by pretreatment with a NMDA antagonist, MK-801 (5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine), 0.1 mg/kg, i.p. In animals that had been infused with opioids in the same manner, naloxone (48 nmol/5 microl, i.c.v.) precipitated withdrawal signs and increased extracellular glutamate levels in the LC of opioid-dependent rats measured by in vivo microdialysis method. Pretreatment with MK-801, however, did not affect the increases of glutamate levels in the LC. These results further demonstrate that the expression of opioid withdrawal induced by an expeditious release of glutamate in the LC region of opioid-dependent animals might be mainly mediated by the postsynaptic NMDA receptors.

Effect of 4-(4-chlorobenzyl)pyridine on rat hepatic microsomal cytochrome P450 and drug-metabolizing enzymes in vivo and in vitro.

The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12), and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.

[Pharmacological and physiological effects of ginseng on actions induced by opioids and psychostimulants].

Pharmacological and physiological effects of ginseng on actions induced by opioids and psychostimulants were summarized. Analgesic effects of opioids, such as morphine and U-50,488H, were blocked by ginseng in a non-opioid dependent manner. Furthermore, ginseng inhibited the tolerance to and dependence on morphine, and prevented the suppressive effect on the development of morphine tolerance caused by co-exposure to foot-shock stress, but not psychological stress. On the other hand, behavioral sensitization (reverse tolerance to ambulation-accelerating effect) to morphine, methamphetamine (MAP) and cocaine was also inhibited by ginseng. Interestingly, ginseng also inhibited the appearance of the recurrent phenomenon (reappearance of the sensitized state was observed at the time of readministration of MAP and cocaine even after a 30-day discontinuation of drug administration) of the effect of MAP and cocaine. The conditioned place preference of MAP and cocaine was completely blocked by ginseng. These findings provide evidence that ginseng may be useful clinically for the prevention of abuse and dependence of opioids and psychostimulants.

Cloning and high expression of catalase gene from bacillus sp. TE124.

An efficient expression system for producing catalase in Bacillus was developed. A catalase was purified from Bacillus sp. TE124 and the catalase gene was cloned by plaque hybridization with a probe constructed from the N-terminal amino acid sequence of the enzyme. The gene, containing an open reading frame of 1452 bp, was subcloned into pHY300PLK for self-cloning into the organism. As a result, the production of catalase increased 20-fold over that of the parent strain.

Sex-related effect of hemin on cytochrome P450 and drug-metabolizing enzymes in rat liver.

We examined the effect of hemin on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (P450 2E1, 3A2, 2C11 and 2C12) under the induction of heme oxygenase activity in male and female rats. Hemin produced an inverse relationship between the induction of heme oxygenase activity and the decrease of P450 content in a dose-dependent manner. A time course study revealed that hemin produced a significant decrease in total P450 content in male rats to about 37% that of the controls at 24 hr after its administration. Western and Northern blot analyses revealed that the increase in both heme oxygenase-1 (HO-1) protein and HO-1 mRNA reached a maximum at 24 hr and returned to control levels within 120 hr in both sexes. With respect to P450-dependent monooxygenase activities, we found that there was a significant decrease of aniline p-hydroxylase activity to about 30% of the control animals, but not in erythromycin N-demethylase activity at various time intervals. Immunoblot analysis revealed that P450 2E1 in male rat liver was dramatically decreased at 24 hr to about 20% of the controls, but not P450 3A2. Parallel to the decrease of androstenedione 16 alpha-hydroxylase activity, there was a marked decrease of P450 2C11 or 2C12 in male or female rats, respectively, at 72 hr after the treatment; however, hemin did not decrease androstenedione 16 alpha-hydroxylase activity in phenobarbital-pretreated rat liver. These findings suggest that hemin predominantly affects constitutively expressed isozymes rather than inducible P450 species in rat liver.

Concurrent induction of rat hepatic microsomal cytochrome P450 and haem oxygenase by 2,2'-dipyridyl ketone: comparison with the effect of 2,2'-dipyridyl amine.

1. The effect of 2,2'-dipyridyl ketone and 2,2'-dipyridyl amine on the induction of hepatic microsomal cytochrome P450 (P450) and heme oxygenase was compared, and their effects on five different P450 isoforms (P4501A1, 3A2, 2B1, 2E1 and 2C11) in rat were examined. 2. Treatment of rat with 2,2'-dipyridyL amine resulted in the marked induction of haem oxygenase to about seven-fold of the controls with a decrease in p450 content. 2,2'-Dipyridyl ketone produced concomitant induction of both P450 and haem oxygenase activity in a dose- and time-dependent manner without showing any sex differences. 3. Immunoblot analysis revealed that 2,2'-dipyridyl ketone slightly increased CYP2E1 and CYP3A2 at low doses, but not at high dose levels. There was no effect on P4502C11. P4502B1 was induced by the treatment with 2,2'-dipyridyl ketone in a dose-dependent manner. 4. These results indicate that dipyridyl compounds having different bridges between two aromatic moieties act as differential inducers of hepatic microsomal P450s and haem oxygenase.

Opioid analgesic-induced apoptosis and caspase-independent cell death in human lung carcinoma A549 cells.

We characterized anticancer effects of opioid analgesics that are clinically used for cancer patients for pain relief. Treatment with 100 microM buprenorphine, a representative analgesic, induced cell death of human carcinomas, such as A549 (squamous epithelial cell of lung cancer), MCF-7 (breast cancer) and N417 (small cell of lung cancer), but not in KATO III (gastric cancer) cells as evaluated by alamar blue assay. Among 18 clinically utilized and related analgesics, buprenorphine and loperamide showed potent inhibition of cell viability. However, these anti-cancer effects were not affected by opioid receptor antagonists nor by pertussis toxin. Buprenorphine-induced cell death occurred as early as 1 h after the addition, and its T1/2 of cell viability inhibition was 3 h. The cell death manifested the characteristics of apoptosis, such as DNA-laddering and nuclear fragmentation, which were sensitive to a caspase inhibitor, Z-Asp-CH2-DCB. The nuclear fragmentation was independent of cell cycle phase specificity. The activity of caspase-3-like protease which is known to be closely related to apoptotic DNA laddering was markedly enhanced by buprenorphine. However, the inhibition of cell viability by buprenorphine was not affected by the caspase inhibitor. These findings suggest that some opioid analgesics induce typical apoptotic features sensitive to the caspase inhibitor, while also inhibition of cell viability insensitive to the inhibitor.

A protein kinase inhibitor, H-7, blocks naloxone-precipitated changes in dopamine and its metabolites in the brains of opioid-dependent rats.

The influence of an inhibitor of cAMP-dependent protein kinase and protein kinase C, H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], on naloxone (an opioid receptor antagonist)-precipitated withdrawal signs and changes in levels of dopamine (DA) and its metabolites in morphine- or butorphanol-dependent rats was investigated. Animals were infused continuously with morphine (a mu-opioid receptor agonist) or butorphanol (a mu/delta/kappa mixed opioid receptor agonist) for 3 days. Naloxone precipitated withdrawal syndrome and decreased the levels of DA in the cortex, striatum, and midbrain; 3, 4-dihydroxyphenylacetic acid (DOPAC) in the cortex, striatum, limbic areas, and midbrain; and homovanilic acid (HVA) in the striatum, limbic areas, and midbrain regions. In animals rendered dependent on butorphanol, the results obtained were similar to those of morphine-dependent rats except for the changes in DOPAC levels. Concomitant infusion of H-7 and opioid blocked both the expression of withdrawal signs and the decreases in DA, DOPAC, and HVA levels in a dose-dependent manner. These results suggest that the enhancement of cAMP-dependent protein kinase and/or protein kinase C activity accompanying the increase of DA neuron activity during continuous infusion of opioids leads to an abrupt reduction in levels of DA and its metabolites precipitated by naloxone, which is intimately involved in the expression of physical dependence on opioids.

A novel effect of polymorphonuclear leukocytes in the facilitation of angiogenesis.

The purpose of this study was to examine whether the adhesion of polymorphonuclear leukocytes (PMNs) to endothelial cells and/or reactive oxygen species (ROS) released from PMNs are responsible for inducing angiogenesis. Angiogenesis was assessed by tube formation using endothelial cells obtained from bovine thoracic aorta (BAECs) grown on a layer of collagen type I. Addition of PMNs to BAECs weakly induced angiogenesis. The angiogenesis induced by PMNs alone was further enhanced by treatment of the PMNs with N-formyl-methionyl-leucyl-phenylalanine (FMLP), a selective activator of PMN. The involvement of PMN adhesion to BAECs via adhesion molecules in angiogenesis was investigated by using monoclonal antibodies against E-selectin and intercellular adhesion molecule-1 (ICAM-1). These antibodies blocked both the PMN adhesion to BAECs and the enhancement of angiogenesis induced by FMLP-treated PMNs. Furthermore, the enhancement of angiogenesis by FMLP-treated PMNs was blocked by catalase, a scavenging enzyme of H2O2, but not by superoxide dismutase (SOD). These results suggest that PMNs induce angiogenesis in vitro, and that the mechanism of stimulation of angiogenesis by PMNs may involve the adherence of PMNs to endothelial cells via E-selectin and ICAM-1, and H2O2, but not superoxide. Thus, activated PMNs in pathological states may not only induce tissue injury, but may also function as regulators of angiogenesis.