Pretreatment with protein kinase C activator phorbol 12,13-dibutyrate attenuates the antinociception induced by mu- but not epsilon-opioid receptor agonist in the mouse

The effects of pretreatment with a protein kinase C activator, phorbol 12,13-dibutyrate, on antinociception induced by i.c.v.-administered mu-opioid receptor agonist (D-Ala2, NMePhe4, Gly(ol)5) enkephalin (DAMGO) or morphine and epsilon-opioid receptor agonist beta-endorphin were studied in male ICR mice. The tail-flick responses were used for antinociceptive tests. I.c.v. pretreatment with phorbol 12,13-dibutyrate (50 pmol) for 30 or 60 but not 10 min attenuated antinociception induced by i.c.v.-administered DAMGO. I.c.v. pretreatment with phorbol 12,13-dibutyrate (10 and 50 pmol) for 60 min caused a dose-dependent attenuation of DAMGO (19.5 pmol)- or morphine (6.0 nmol)-induced antinociception. The dose-response curve for DAMGO-induced antinociception was shifted to the right by 7.3-fold by i.c.v. pretreatment with phorbol 12,13-dibutyrate (50 pmol) for 60 min. However, the i.c.v.-administered beta-endorphin-induced antinociception was not affected by the same pretreatment with phorbol 12,13-dibutyrate. The attenuation of i.c.v.-administered DAMGO- and morphine-induced antinociception by phorbol 12,13-dibutyrate was reversed by concomitant i.c.v. pretreatment with a selective protein kinase C inhibitor calphostin C. These results suggest that activation of protein kinase C by phorbol 12,13-dibutyrate leads to the desensitization of mu-, but not epsilon-opioid receptor-mediated antinociception. These findings also provide additional evidence for differential intracellular modulation on antinociceptive action of mu- and epsilon-opioid receptor agonists.     

Role of intracellular calcium in modification of mu and delta opioid receptor-mediated antinociception by diabetes in mice

We examined the effects of calcium modulators on mu and delta opioid receptor agonist-induced antinociception in both diabetic and nondiabetic mice. In nondiabetic mice, intracerebroventricular (i.c. v.) pretreatment with calcium and thapsigargin, which increase intracellular calcium, reduced [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO)-induced antinociception by shifting its dose-response curve to the right. However, in diabetic mice i.c.v. pretreatment with calcium and thapsigargin did not affect DAMGO-induced antinociception. In contrast i.c.v. administration of agents that decrease intracellular calcium, such as EGTA and ryanodine, enhanced DAMGO-induced antinociception in both diabetic and nondiabetic mice. In contrast with DAMGO i.c.v. pretreatment with calcium and thapsigargin enhanced (-)-TAN67-induced antinociception in nondiabetic mice by shifting its dose-response curve to the left. However, (-)-TAN67-induced antinociception in diabetic mice was not affected by pretreatment with calcium or thapsigargin. Moreover i.c. v. pretreatment with EGTA, but not with ryanodine, reduced (-)-TAN67-induced antinociception in nondiabetic mice. In diabetic mice i.c.v. pretreatment with both EGTA and ryanodine reduced (-)-TAN67-induced antinociception. These results suggest that cytosolic calcium has different effects on mu and delta opioid receptor agonist-induced antinociception. Further, these results suggest that the modification of mu and delta opioid receptor agonist-induced antinociception by diabetes in mice may be due to increased levels of intracellular calcium.     

CD86 (B7-2), but not CD80 (B7-1), expression in the epidermis of transgenic mice enhances the immunogenicity of primary cutaneous Candida albicans infections

Transgenic (Tg) mice whose epidermal keratinocytes constitutively overexpress either B7-1 (CD80) or B7-2 (CD86) exhibited exaggerated cutaneous delayed type hypersensitivity (DTH) to haptens compared to non-Tg mice. To determine whether enhanced DTH in these Tg mice is seen in response to cutaneous fungal infections, a primary infection with Candida albicans was established by inoculating this organism on the occluded skin of Tg and non-Tg mice. These infections resolved 7 days after removal of occlusive dressing in all three groups of mice, without evidence of exaggerated inflammation in either the Tg or non-Tg mice. Only B7-2 Tg mice developed enhanced Th1-lymphocyte-mediated immune responses to C. albicans antigens after resolving this infection: enhanced footpad swelling in response to intradermal C. albicans antigens, enhanced production of mRNA encoding Th1 lymphokines in draining lymph nodes, and increased gamma interferon secreted into culture supernatants by lymph node T lymphocytes stimulated with Candida antigens in vitro. Lastly, Western blotting of sera from mice that had resolved this fungal infection indicated that only B7-2 Tg mice recognized a wide range of Candida-associated antigens. These data suggest that these two costimulatory molecules, when expressed by keratinocytes, do not deliver identical signals to C. albicans antigen-reactive Th1 lymphocytes. The enhanced immune response in B7-2 Tg mice to a cutaneous C. albicans infection demonstrates the importance of antigen presentation and costimulation in immune reactivity to fungi. Furthermore, B7-2 Tg mice may be useful in identification of protective Candida antigens.     

Streptozotocin-induced diabetes selectively enhances antinociception mediated by delta 1- but not delta 2-opioid receptors

We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of delta-opioid receptor agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective delta 1-opioid receptor antagonist, but not with naltriben (NTB), a selective delta 2-opioid receptor antagonist. There were no significant differences in the antinociceptive effect of [D-Ala2]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala2]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal delta 1-opioid receptor-mediated antinociception, but are normally responsive to activation of delta 2-opioid receptors.     

PKC and tyrosine kinase involvement in amyloid beta (25-35)-induced chemotaxis of microglia

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.     

7-D-ALA]-angiotensin 1-7 blocks renal actions of angiotensin 1-7 in the anesthetized rat

Exogenous angiotensin (Ang) 1-7 affects renal function, but the receptor(s) involved in this response remain(s) to be determined. In an in vitro preparation of proximal tubules, Ang 1-7 was shown to act on Ang II AT1 receptors (minor component), but also on a non-AT1, non-AT2 Ang receptor (major component) to inhibit reabsorption. In brain, Ang 1-7 also exerts effects mediated by a non-AT1, non-AT2 binding site; these effects are inhibited, however, by the angiotensin analog [7-D-Ala]-Ang 1-7. Therefore we tested the effect of Ang II AT1-receptor antagonist losartan and [7-D-Ala]-Ang 1-7 on the renal response to exogenous Ang 1-7 in standard renal-clearance experiments in the anesthetized rat. We found that Ang 1-7 (100 pmol/kg/min, i.a.) increased glomerular filtration rate (GFR), urinary flow rate (UV), and urinary sodium excretion (UNaV) without affecting mean arterial blood pressure (MAP) or urinary potassium excretion (UKV), confirming previous reports. Losartan (10 mg/kg, i.v.) blocked the pressor effect of exogenous Ang II (100 pmol/kg/min, i.a.), but did not significantly affect the renal response to Ang 1-7. Conversely, pretreatment with [7-D-Ala]-Ang 1-7 (5 nmol/kg/min) did not affect the pressor effect of Ang II, but abolished the renal response to Ang 1-7. Application of [7-D-Ala]-Ang 1-7 in the absence of exogenous Ang 1-7 did not alter MAP or GFR, but increased UNaV (by 52%). Our data indicate that similar to the response in brain, the renal response to exogenous Ang 1-7 may be mediated predominantly by a distinct non-AT1 binding site, which is sensitive to blockade by [7-D-Ala]-Ang 1-7. Furthermore, ambient endogenous Ang 1-7 acting on this distinct binding site may not contribute significantly to control of MAP or GFR, but exerts an antinatriuretic influence in the anesthetized rat.     

Cigarette smoke-induced bronchoconstriction: causative agents and role of thromboxane receptors

Inhalation of cigarette smoke induces a biphasic bronchoconstriction in guinea pigs: the first phase is induced by a combination of cholinergic reflex and tachykinins, whereas the second phase involves cyclooxygenase metabolites (J.-L. Hong, I. W. Rodger, and L.-Y. Lee. J. Appl. Physiol. 78: 2260-2266, 1995). This study was carried out to further determine the causative agents in the smoke and the types of prostanoid receptors and endogenous prostanoids mediating the bronchoconstriction. Inhalation of 10 ml of high-nicotine cigarette smoke consistently elicited the biphasic bronchoconstriction in anesthetized and artificially ventilated guinea pigs. Pretreatment with hexamethonium (10 mg/kg iv) significantly reduced the first-phase bronchoconstriction but did not have any measurable effect on the second-phase response. In sharp contrast, gas-phase smoke did not elicit any bronchoconstrictive effect. Furthermore, when the animals were challenged with low-nicotine cigarette smoke, only a single second-phase response was evoked, accompanied by increases in thromboxane (Tx) B2 (a stable metabolite of TxA2), prostaglandin (PG) D2, PGF2 alpha in the bronchoalveolar lavage fluid. The bronchoconstrictive response induced by low-nicotine smoke was completely prevented by pretreatment with SQ-29548 (0.3 mg/kg iv), a TxA2-receptor antagonist. These results indicate that 1) nicotine is the primary causative agent responsible for the first-phase bronchoconstriction and 2) nonnicotine smoke particulates evoke the release of TxA2, PGD2, and PGF2 alpha, which act on TxA2 receptors on airway smooth muscles and induce the second-phase response to cigarette smoke.     

Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions

The activation of protein kinase C (PKC) found in diabetic glomeruli and glomerular mesangial cells cultured under high glucose conditions has been proposed to contribute to the development of diabetic nephropathy. However, the abnormalities distal to PKC have not been fully elucidated yet. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) cascade, an important kinase cascade downstream to PKC and an activator of cytosolic phospholipase A2 (cPLA2) by direct phosphorylation, is activated in glomeruli isolated from streptozotocin-induced diabetic rats. MAPK cascade was also activated in glomerular mesangial cells cultured under high glucose (27.8 mmol/l) conditions for 5 days, and the activation of MAPK cascade was inhibited by treating the cells with calphostin C, an inhibitor of PKC. Furthermore, the activities of cPLA2 also increased in cells cultured under the same conditions and this activation was inhibited by both calphostin C and PD 098059, an inhibitor of MEK (MAPK or extracellular signal-regulated kinase [ERK] kinase). These results indicate that MAPK cascade is activated in glomeruli and mesangial cells under the diabetic state possibly through the activation of PKC. Activated MAPK, in turn, may induce various functional changes of mesangial cells at least through the activation of cPLA2 and contribute to the development of diabetic nephropathy.     

Calphostin C induces expression of amphiregulin mRNA via reactive oxygen species in IEC-6 cells

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is generally used as a specific inhibitor of protein kinase C. It is known that 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, induces expression of mRNA for amphiregulin (AR), a member of EGF-related polypeptides, in mammalian epithelial cells. In this work, we determined the effect of calphostin C on AR mRNA expression in IEC-6 cells, a rat intestinal epithelial cell line, and unexpectedly found that this compound enhanced the TPA-induced expression of AR mRNA. Moreover, calphostin C alone induced expression of AR mRNA in a light-dependent manner, and this effect was abrogated by pretreatment with N-acetylcysteine. These results suggest that calphostin C can upregulate expression of AR mRNA via reactive oxygen species.     

A hepatocyte-targeted conjugate capable of delivering biologically active colchicine in vitro

The regulatory mechanism of Bcl-2 protein expression was investigated in SH-SY5Y cells, the human neuroblastoma cell line that expresses natively Bcl-2 proteins. WHen the cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or retinoic acid, the level of Bcl-2 protein was increased compared with the control. These effects were inhibited by pretreatment with a protein kinase C (PKC) inhibitor, staurosporine or calphostin C. The level of Bcl-2 protein was also increased by treatment with carbachol, a muscarinic acetylcholine receptor (mAChR) agonist, and the effect were also inhibited by pretreatment with staurosporine or calphostin C. An addition, a carbachol-induced increase in Bcl-2 protein levels and a transient elevation of [Ca2+]i were inhibited by pretreatment with 4-DAMP (4-diphenylacetoxy-N-methylpiperidine), an m3 mAChR antagonist. In contrast, the level of Bcl-2 protein was decreased by treatment with dibutyryl cAMP (diBu-cAMP), forskolin, or cholera toxin, and the effects of diBu-cAMP were inhibited by pretreatment with a protein kinase A (PKA) inhibitor, H-89. From these results, we suggest that the expression of Bcl-2 proteins is regulated by PKC and PKA in positive and negative manners, respectively, in SH-SY5Y cells. Furthermore, the nucleosomal DNA fragmentation induced by serum depletion for 4 h was observed in SH-SY5Y cells when the level of Bcl-2 protein was down-regulated by treatment with 1 mM diBu-cAMP for 3 days, although the DNA fragmentation by serum depletion for 4 h was not observed in nontreatment cells, indicating that Bcl-2 proteins whose expression is regulated by PKC and PKA play important roles in serum depletion-induced apoptosis.     

Lipopolysaccharide and the glycoside ring of staurosporine induce CD14 expression on bone marrow granulocytes by different mechanisms

We established previously that lipopolysaccharide (LPS) can induce the expression of LPS-binding sites on bone marrow cells (BMC). We now report that staurosporine (STP), a glycosylated indolocarbazole alkaloid with potent inhibitory activity for various protein kinases, can induce the same effect. With both agents, the newly expressed LPS receptor was found to be CD14. The STP-induced effect was independent of its protein kinase inhibitory activity because several other protein kinase inhibitors, such as the indolocarbazole K-252a, the bisindolylmaleimide RO-31-8220, the perylenequinone calphostin C, and the isoquinolinesulfonamide H7, did not induce CD14 expression. The observation that the STP analog K-252a with an identical polyaromatic aglycon moiety was inactive yet the analog UCN-01 with an identical glycoside ring was active suggests that the induction of CD14 expression is triggered by the sugar moiety of STP. Three lines of evidence show that the mechanism of CD14 expression induced by STP differs from that induced by LPS: (i) unlike LPS, STP can stimulate BMC from LPS-unresponsive C3H/HeJ mice, (ii) LPS and STP effects are additive at a saturating dose of LPS, and (iii) the protein kinase inhibitor K-252a inhibits the LPS-induced but not STP-induced stimulation. Therefore, our findings show that both a protein kinase-dependent (LPS-induced) and a protein kinase-independent (STP-induced) mechanism can lead to the expression of the LPS receptor CD14 on BMC. We also found that the STP-induced stimulation of BMC is modulated by cyclosporin A, vinblastine, and verapamil. This observation may suggest that the inducible effect of STP could be initiated by its interaction with P-glycoprotein, a membrane pump with drug efflux function that plays a critical role in the multidrug resistance of cancer cells.     

Acute tolerance to spinally administered morphine compares mechanistically with chronically induced morphine tolerance

The mechanistic similarity between acutely and chronically induced morphine tolerance has been previously proposed but remains largely unexplored. Our experiments examined the modulation of acutely induced tolerance to spinally administered morphine by agonists that affect the N-methyl-D-aspartate receptor and nitric oxide synthase systems. Antinociception was detected via the hot water (52.5 degrees C) tail flick test in mice. Intrathecal pretreatment with morphine (40 nmol) produced a 9.6-fold rightward shift in the morphine dose-response curve. This shift confirmed the induction of acute spinal morphine tolerance. Intrathecal copretreatment with the receptor antagonists (competitive and noncompetitive, respectively) dizolcipine (MK801, 3 nmol) or LY235959 (4 pmol) and morphine [40 nmol, intrathecally (i.t.)] attenuated acute tolerance to morphine measured 8 hr later. A 60-min pretreatment of 7-nitroindazole (6 nmol, i.t.), a selective neuronal NOS inhibitor, followed by administration of morphine (40 nmol, i.t.) blocked the induction of morphine tolerance. Intrathecal copretreatment with morphine (40 nmol, i.t.) and agmatine (4 nmol, i.t.), an imidazoline, receptor agonist and putative nitric oxide synthase inhibitor, almost completely abolished acute spinal morphine tolerance. The results of these experiments agree with previous reports using models of chronically induced morphine tolerance. This evidence supports the proposal that the mechanisms responsible for acute morphine tolerance parallel those underlying chronic morphine tolerance. This study attests to the powerful predictive value of acute induction as a model for morphine tolerance.     

Endogenous benzodiazepine-like compounds and diazepam binding inhibitor in serum of patients with liver cirrhosis with and without overt encephalopathy

PURPOSE: The purpose of our work was to determine the usefulness of double-phase helical CT during arterial portography (CTAP) for the detection of hypervascular hepatocellular carcinoma (HCC). METHOD: Eighty-four patients with 176 hypervascular HCC nodules underwent double-phase CTAP. Hypervascular HCCs were diagnosed by iodized oil CT after transcatheter arterial chemoembolization (TACE). The first-phase images were obtained 30 s after the initiation of injection of a nonionic iodinated contrast medium into the superior mesenteric artery or splenic artery, and the second-phase images were obtained after 70 s. These images were interpreted separately for detection of HCC. RESULTS: The double-phase CTAP detected two nodules and six nodules that were missed by the first- and second-phase images, respectively. The sensitivity for hypervascular HCC nodules was 89% for the first phase, 91% for the second phase, and 93% for the first phase and second phase combined. The double-phase CTAP showed significantly superior sensitivity to the first-phase CTAP for detecting HCC nodules (p < 0.05). However, there was no statistically significant difference between the sensitivities of the double-phase CTAP and the second-phase CTAP. The positive predictive values of the double-phase images were inferior to those of either the first-phase or the second-phase images alone. CONCLUSION: Double-phase helical CTAP was found to be no better than second-phase CTAP alone for the detection of hypervascular HCC nodules.     

Ca(2+)-dependent and Ca(2+)-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells

Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.     

The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.     

Role of intracellular calcium on the modulation of naloxone-precipitated withdrawal jumping in morphine-dependent mice by diabetes

The role of intracellular calcium in the modifications of naloxone-precipitated withdrawal jumping in morphine-dependent mice by diabetes was examined. Naloxone-precipitated withdrawal jumping was significantly less in morphine-dependent diabetic mice than in morphine-dependent non-diabetic mice. Intracerebroventricular (i.c.v. ) pretreatment with ryanodine attenuated naloxone-precipitated withdrawal jumping in morphine-dependent non-diabetic mice. However, naloxone-precipitated withdrawal jumping in morphine-dependent diabetic mice was not affected by i.c.v. pretreatment with ryanodine. Moreover, i.c.v. pretreatment with thapsigargin, a Ca2+-ATPase inhibitor, enhanced naloxone-precipitated withdrawal jumping in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice. The noradrenaline (NA) turnover in the frontal cortex in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice, was significantly increased by naloxone injection. Naloxone-induced enhancement of NA turnover in morphine-dependent non-diabetic mice, but not in morphine-dependent diabetic mice, was blocked by i.c.v. pretreatment with ryanodine. In contrast to ryanodine, thapsigargin enhanced naloxone-induced enhancement of NA turnover in morphine-dependent non-diabetic mice. These results suggest that increased intracellular calcium augmented naloxone-precipitated withdrawal jumping and the turnover rate of NA in the frontal cortex in morphine-dependent non-diabetic mice. Furthermore, it seems likely that the attenuation of naloxone-precipitated withdrawal jumping in morphine-dependent diabetic mice may be due, in part, to the dysfunction of intracellular calcium store.     

Reduction of voltage-dependent Mg2+ blockade of NMDA current in mechanically injured neurons

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors is implicated in the pathophysiology of traumatic brain injury. Here, the effects of mechanical injury on the voltage-dependent magnesium (Mg2+) block of NMDA currents in cultured rat cortical neurons were examined. Stretch-induced injury was found to reduce the Mg2+ blockade, resulting in significantly larger ionic currents and increases in intracellular free calcium (Ca2+) concentration after NMDA stimulation of injured neurons. The Mg2+ blockade was partially restored by increased extracellular Mg2+ concentration or by pretreatment with the protein kinase C inhibitor calphostin C. These findings could account for the secondary pathological changes associated with traumatic brain injury.     

Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation

Proliferation of airway smooth muscle results from persistent inflammatory cytokine and growth factor stimulation and is a critical component of airway luminal narrowing in chronic asthma. Using primary cultures of bovine tracheal smooth muscle (BTSM) cells to examine the signaling basis of cell proliferation, platelet-derived growth factor (PDGF)-BB and thrombin (which act through distinct receptor types) were found to induce DNA synthesis in BTSM cells. Mitogen-induced DNA synthesis could be completely inhibited by LY294002, a selective phosphoinositide 3-kinase (PtdIns 3-kinase) inhibitor. Exposure of BTSM cells to PDGF-BB or thrombin resulted in rapid activation of PtdIns 3-kinase and accumulation of phosphoinositide-3,4,5-trisphosphate. Protein kinase B, a novel signaling protein kinase, was identified in BTSM cells and was activated by PDGF-BB and thrombin in a PtdIns 3-kinase-dependent manner; this may underlie mitogen-stimulated activation of p70(s6k). PD98059, a mitogen-activated protein kinase kinase 1 inhibitor, also partially inhibited PDGF-BB- and thrombin-stimulated DNA synthesis, indicating a modulatory role for mitogen-activated protein kinase in proliferation. GF109203X, Ro 31-8220, calphostin C, and chelerythrine (selective protein kinase C inhibitors) had no effect on PDGF-BB- or thrombin-stimulated DNA synthesis, suggesting that, despite abolishment of mitogen-stimulated protein kinase C activity, cell proliferation stimulated by PDGF-BB and thrombin is protein kinase C-independent. These data demonstrate that the PtdIns 3-kinase/protein kinase B pathway represents a key signaling route in airway smooth muscle proliferation, with the mitogen-activated protein kinase kinase 1/mitogen-activated protein kinase cascade providing a complementary signal required for the full mitogenic response.     

Continuous assay of proteases using a microtiter plate fluorescence reader

The present study was designed to determine if spinal calcium channels, calmodulin, and calcium/calmodulin-dependent protein kinase II were involved in the production of antinociception induced by cold water swimming stress (CWSS). The effects of intrathecal (i.t.) injection of nimodipine, omega-conotoxin GVIA, calmidazolium, or (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperaz inyl)-propyl]phenyl ester (KN-62) on CWSS-induced antinociception were studied in ICR mice. The antinociception was assessed by the tail-flick test. CWSS produced inhibition of the tail-flick response. Various doses of nimodipine (10-40 ng), omega-conotoxin GVIA (5-40 ng), calmidazolium (10-40 ng), or KN-62 (5-40 ng) injected i.t. alone did not show any antinociceptive effect in the tail-flick test. I.t. pretreatment with omega-conotoxin GVIA, calmidazolium, or KN-62 dose dependently attenuated the CWSS-induced inhibition of the tail-flick response. However, i.t. pretreatment with nimodipine did not affect the inhibition of the tail-flick response induced by CWSS. Our results suggest that spinal N-type calcium channel, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the production of antinociception induced by CWSS. On the other hand, CWSS-induced antinociception appears not to be mediated via the spinal L-type calcium channel.