Ethanol increases the activity of rat excitatory amino acid transporter type 4 expressed in Xenopus oocytes: role of protein kinase C and phosphatidylinositol 3-kinase

Background: Glutamate is the major excitatory neurotransmitter in the central nervous system and is critical for essentially all physiological processes, such as learning, memory, central pain transduction, and control of motor function. Excitatory amino acid transporters (EAATs) play a key role in regulating glutamate neurotransmission by uptake of glutamate into cells. EAAT4 is the major EAAT in the cerebellar Purkinje cells. The authors investigated the effects of ethanol on EAAT4 and the mediatory effects of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in this context. Methods: Excitatory amino acid transporter 4 was expressed in Xenopus oocytes by injecting EAAT4 mRNA. l ‐aspartate‐induced membrane currents were measured using a two‐electrode voltage clamp. Responses were quantified by integrating current traces and are represented in microCoulombs (μC). Results: Ethanol increased EAAT4 activity in a dose‐dependent manner. At ethanol concentrations of 25, 50, 100, and 200 mM, the responses were significantly higher than untreated control values. Ethanol (25 mM) significantly increased the V_max (1.5 ± 0.1 μC for control vs. 2.0 ± 0.1 μC for ethanol, p < 0.05), but did not affect K_m (2.3 ± 0.6 μM for control vs. 1.7 ± 0.7 μM for ethanol, p > 0.05) of EAAT4 for l ‐aspartate. Preincubation of oocytes with phorbol‐12‐myristate‐13‐acetate (PMA, a PKC activator) significantly increased EAAT4 activity. However, combinations of PMA and ethanol versus PMA or ethanol alone did not increase responses further. Two PKC inhibitors, chelerythrine and staurosporine did not reduce basal EAAT4 activity but abolished ethanol‐enhanced EAAT4 activity. Pretreatment with wortmannin (a PI3K inhibitor) also abolished ethanol‐enhanced EAAT4 activity. Conclusions: These results demonstrate that acute ethanol exposure increases EAAT4 activity at clinically relevant concentrations and that PKC and PI3K may mediate this. The effects of ethanol on EAAT4 may play a role in the cerebellar dysfunction caused by ethanol intoxication.     

Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement

Abstract : Background: Glutamate transporters (excitatory amino acid transporters, EAAT) regulate extracellular concentrations of glutamate, a major excitatory neurotransmitter. We reported that acute ethanol exposure increases the activity of a major neuronal EAAT, EAAT3. This effect is consistent with the general inhibitory effect of acute alcohol toxicity in the central nervous system (CNS). However, chronic ethanol exposure has CNS presentations different from acute alcohol toxicity. We hypothesize that chronic ethanol exposure will affect the EAAT3 activity differently from acute ethanol exposure. Methods: EAAT3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Oocytes were incubated with diluted ethanol for 24–96 hr. Using two‐electrode voltage clamp, membrane currents were recorded after the application of L‐glutamate. Responses were quantified by integration of the current trace and reported as microCoulombs (μC). Results: Ethanol (10–100 mM) reduced EAAT3 activity in a time‐dependent and reversible manner. After a 96 hr‐incubation, the activity was significantly decreased compared to the control values at any concentrations tested in this study. Kinetic study demonstrated that a 96 hr‐exposure to 50 mM ethanol significantly decreased Vmax (3.6 ± 0.3 for control versus 2.6 ± 0.3 μC for ethanol, n = 20, p < 0.05) but had no effect on Km (57.6 ± 12.8 for control versus 51.8 ± 16.3 μM for ethanol, n = 20, p > 0.05) of EAAT3 for glutamate. When ethanol (50 mM for 96 hr)‐treated oocytes were incubated with phorbol‐12‐myrisate‐13‐acetate (50 or 100 nM for 10 min), ethanol‐induced decrease in EAAT3 activity was abolished. Preincubation of the oocytes with 100 μM chelerythrine significantly decreased EAAT3 activity (1.00 ± 0.08 for control versus 0.51 ± 0.09 μC for chelerythrine group, n = 18–20, p < 0.05). However, there was no statistical difference among the chelerythrine, ethanol, or chelerythrine plus ethanol groups. Likewise, staurosporine (2 μM for 1 hr) significantly decreased EAAT3 activity and there was no statistical difference among the staurosporine, ethanol, or staurosporine plus ethanol groups. Conclusions: Our results show that chronic ethanol exposure decreases EAAT3 activity at clinically relevant concentrations and that this effect may be protein kinase C‐dependent. Such an effect could be a neuroadaptive mechanism to overcome the inhibitory effect of ethanol on the excitatory neurotransmission.     

Inhibition of the activity of excitatory amino acid transporter 4 expressed in Xenopus oocytes after chronic exposure to ethanol

Background: The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) in these effects. Methods: EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol‐containing solution for 24 to 96 hours. Membrane currents induced by l ‐aspartate were recorded using 2‐electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (μC). Results: Ethanol dose‐ and time‐dependently reduced EAAT4 activity. EAAT4 activity after a 96‐hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 μC for control vs. 1.6 ± 0.2 μC for ethanol, n = 18, p < 0.05) of EAAT4 for l ‐aspartate. Preincubation of ethanol‐treated (50 mM for 96 hours) oocytes with phorbol‐12‐myrisate‐13‐acetate (100 nM for 10 minutes) abolished the ethanol‐induced decrease in EAAT4 activity. While staurosporine (2 μM for 1 hour) or chelerythrine (100 μM for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 μM for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions: The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri‐Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion.     

Microsomal Acetaldehyde Oxidation is Negligible in the Presence of Ethanol

The microsomal ethanol oxidizing system (MEOS), inducible by ethanol and acetone, oxidizes ethanol to acetaldehyde, which causes many toxic effects associated with excess ethanol. Recent studies reported that rat liver microsomes also oxidize acetaldehyde, thereby challenging the validity of the assessment of MEOS activity by measuring acetaldehyde production and suggesting that MEOS activity results in the accumulation not of acetaldehyde but, rather, of its less toxic metabolite, acetate. To address these issues, we compared both metabolic rates of ethanol and acetaldehyde and the effect of ethanol on the acetaldehyde metabolism. Liver microsomes were prepared from Sprague-Dawley rats induced either with acetone for 3 days or ethanol for 3 weeks. NADPH-dependent acetaldehyde (300 /μM) metabolism was measured in two ways: (1) by detection of acetaldehyde disappearance by headspace gas chromatography, and (2) by assessment of acetaldehyde oxidation by liquid scintillation counting of acetate formed from [1,2-¹⁴C]acetaldehyde. Ethanol (50 mM) oxidation was measured by gas chromatography. In acetone- and ethanol-induced rat liver microsomes, the acetaldehyde disappearance (p < 0.0001) and oxidation (p < 0.0001) rates were both significantly increased. The rates of acetaldehyde oxidation paralleled those of p-nitrophenol hydroxylation (r= 0.974, p < 0.0001), with a K_m of 82 ± 14 μM and a V_max of 4.8 ± 0.5 nmol/min/mg protein in acetone-induced microsomes. Acetaldehyde disappearance in acetone-induced microsomes and acetaldehyde oxidation in acetone-induced and ethanol-induced microsomes were significantly lower than the corresponding ethanol oxidation, with rates (nmol/min/mg protein) of 4.6 ± 0.6 versus 9.0 ± 0.8 (p < 0.005), 4.4 ± 0.3 versus 9.1 ± 0.5 (p < 0.0005), and 14.0 ± 0.9 versus 19.5 ± 1.8 (p < 0.05), respectively. The presence of 50 mM ethanol decreased this metabolism to 0.9 ± 0.3 (p < 0.005), 0.5 ± 0.1 (p < 0.001), and 1.8 ± 0.3 (p < 0.001), resulting in rates of acetaldehyde metabolism of only 9.8 ± 3.2%, 6.0 ± 0.5%, and 9.5 ± 1.2% (respectively) of those of ethanol oxidation. In conclusion, rat liver microsomes oxidize acetaldehyde at much lower rates than ethanol, and this acetaldehyde metabolism is strikingly inhibited by ethanol. Accordingly, acetaldehyde formation provides an accurate assessment of MEOS activity. Furthermore, because acetaldehyde production vastly exceeds its oxidation, the net result of MEOS activity is the accumulation of this toxic metabolite.     

Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes

Phorbol myristate acetate (PM A) increased the formation of |³²P | PI 4,5-P, in ³²P-prelabeled human platelet. In saponin-permeabilized platelets, in which ³²P from exogenous |γ-³²P| ATP was incorporated into PI 4-P and PI 4,5-P₂, addition of 10 nM PMA resulted in increased formation of |³²P|PI 4,5-P₂ and |³²P|PI 4-P. In order to distinguish whether increased [³²P]PI 4,5-P₂ formation by PMA reflected merely an increase of [³²P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC.

However, 100 nM PMA did not stimulate [³²P]PI 4,s-P₂ formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P₂ inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca²⁺ -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca²'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca²⁺ -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p₂ and Ca²⁺ -independent PKC(nPKC).     

Febrile temperatures control antineutrophil cytoplasmic autoantibody–induced neutrophil activation via inhibition of phosphatidylinositol 3‐kinase/Akt

Objective

Neutrophil activation by antineutrophil cytoplasmic autoantibodies (ANCAs) is central to the pathogenesis of the ANCA‐associated vasculitides. Febrile infections occur frequently during these diseases, often in the context of immunosuppressive treatment. Heat exposure may affect the underlying pathophysiologic processes of the vasculitis. In this study we tested the hypothesis that short‐term exposure to heat inhibits ANCA‐induced neutrophil activation.

Methods

After exposure to temperatures from 37°C to 42°C, human neutrophils were primed with either tumor necrosis factor α (TNFα) or granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and stimulated with monoclonal antibodies to myeloperoxidase or to proteinase 3. Respiratory burst activity was assayed using rhodamine and a nitroblue tetrazolium reduction assay. Specific inhibition experiments against p38 MAPK, ERK, and phosphatidylinositol 3‐kinase (PI 3‐kinase)/Akt, and Western blotting with phospho‐specific antibodies were used to identify key components in the antibody‐induced respiratory burst.

Results

A temperature‐dependent reduction in ANCA‐induced respiratory burst was observed over a range of heat exposures from 37°C to 42°C. Inhibition of human ANCA–induced neutrophil stimulation was significant at 40°C (after priming with 2 ng/ml TNFα, mean [± SEM] fluorescence intensity [MFI] 114 ± 12 at 37°C versus 53 ± 6 at 40°C; after priming with 20 ng/ml GM‐CSF, MFI 92 ± 16 at 37°C versus 35 ± 6 at 40°C; both P < 0.01). In the priming phase, the transient activation of the p38 MAPK, ERK, and PI 3‐kinase/Akt pathways by TNFα was blocked by prior exposure of the neutrophils to heat, but GM‐CSF–induced activation was unaltered by heat. However, in the second, antibody‐induced wave of kinase activation, exposure to heat inhibited only the PI 3‐kinase/Akt pathway, and these effects were independent of the priming agent used.

Conclusion

Short‐term spikes of modest heat abrogate ANCA‐induced activation of neutrophils via inhibition of PI 3‐kinase/Akt signaling. Febrile responses in ANCA‐mediated diseases may therefore have a physiologic purpose.

     

Characterization of a [3H]glutamate binding site in rat pineal gland: Enhanced affinity following superior cervical ganglionectomy

Abstract: Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (K_d= 612 ± 23 nM, B_max= 3.17 ± 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [³H]glutamate binding displayed a higher apparent affinity (K_d= 412 ± 28 nM) (P < 0.05) without a change in binding site number (B_max= 3.60 ± 0.24 pmol/mg protein). No difference in [³H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.     

p38丝裂素活化蛋白激酶在高糖损伤人脐静脉内皮细胞中的作用

目的探讨p38丝裂素活化蛋白激酶在高糖导致人脐静脉内皮细胞损伤过程中的作用及p38丝裂素活化蛋白激酶激活是否为蛋白激酶C依赖途径。方法体外分离培养人脐静脉内皮细胞,加入22 mmol/L葡萄糖及PMA、GF109203X(蛋白激酶C特异抑制剂)、SB203580(p38丝裂素活化蛋白激酶特异抑制剂)培养72 h。采用Western-blot检测磷酸化p38丝裂素活化蛋白激酶蛋白的表达,用逆转录聚合酶链反应检测p38丝裂素活化蛋白激酶mRNA的表达,流式细胞仪检测细胞凋亡率。结果高糖及PMA使磷酸化p38丝裂素活化蛋白激酶蛋白表达量与mRNA水平明显升高,人脐静脉内皮细胞凋亡率明显升高。高糖培养人脐静脉内皮细胞72 h后磷酸化p38丝裂素活化蛋白激酶蛋白表达量、mRNA水平、人脐静脉内皮细胞凋亡率分别由0.189±0.0103、0.313±0.0153和5.15%上升至0.605±0.0407、0.447±0.0252和16.8%(P<0.05)。SB203580和GF109203X预处理后使p38丝裂素活化蛋白激酶蛋白磷酸化的表达量与mRNA水平明显下降,人脐静脉内皮细胞凋亡率下降(P<0.05)。结论p38丝裂素活化蛋白激酶激活在高糖导致人脐静脉内皮细胞损伤过程中起促进作用,p38丝裂素活化蛋白激酶激活可能为蛋白激酶C依赖途径。p38丝裂素活化蛋白激酶特异阻断剂对高糖损伤内皮细胞有保护作用。     

ENT1 Regulates Ethanol‐Sensitive EAAT2 Expression and Function in Astrocytes

Background: Equilibrative nucleoside transporter 1 (ENT1) and excitatory amino acid transporter 2 (EAAT2) are predominantly expressed in astrocytes where they are thought to regulate synaptic adenosine and glutamate levels. Because mice lacking ENT1 display increased glutamate levels in the ventral striatum, we investigated whether ENT1 regulates the expression and function of EAAT2 in astrocytes, which could contribute to altered glutamate levels in the striatum. Methods: We examined the effect of ENT1 inhibition and overexpression on the expression of EAAT2 using quantitative real‐time PCR and measured glutamate uptake activity in cultured astrocytes. We also examined the effect of 0 to 200 mM ethanol doses for 0 to 24 hours of ethanol exposure on EAAT2 expression and glutamate uptake activity. We further examined the effect of ENT1 knockdown by a specific siRNA on ethanol‐induced EAAT2 expression. Results: An ENT1‐specific antagonist and siRNA treatments significantly reduced both EAAT2 expression and glutamate uptake activity while ENT1 overexpression up‐regulated EAAT2 mRNA expression. Interestingly, 100 or 200 mM ethanol exposure increased EAAT2 mRNA expression as well as glutamate uptake activity. Moreover, we found that ENT1 knockdown inhibited the ethanol‐induced EAAT2 up‐regulation. Conclusions: Our results suggest that ENT1 regulates glutamate uptake activity by altering EAAT2 expression and function, which might be implicated in ethanol intoxication and preference.     

Polyamines contribute to ethanol withdrawal-induced neurotoxicity in rat hippocampal slice cultures through interactions with the NMDA receptor

Background: Several reports demonstrate that withdrawal from long‐term ethanol exposure is associated with significant central nervous system neurotoxicity, produced at least in part by increased activity of N‐methyl‐d‐aspartate receptors (NMDARs). Recent evidence suggests that elevations in the synthesis and release of the polyamines spermidine and spermine, which are known modulators of NMDARs, contribute to the increased activity of the receptor during ethanol withdrawal. Therefore, the goal of this investigation was to examine what role, if any, spermidine and spermine have in the generation of ethanol withdrawal‐induced neurotoxicity. Methods: Neurotoxicity (measured as fluorescence of the cell death indicator propidium iodide, PI), glutamate release (measured by high‐performance liquid chromatography analysis), and polyamine concentrations (by high‐performance liquid chromatography) were measured in rat hippocampal slice cultures undergoing withdrawal from chronic (10 day) ethanol exposure (100 mM). In addition, the effects of the polyamine synthesis inhibitor di‐fluoro‐methyl‐ornithine (DFMO, 0.1–100 nM) and NMDAR polyamine‐site antagonists ifenprodil, arcaine, and agmatine (1 nM‐100 μM) on ethanol withdrawal‐ and NMDA‐induced neurotoxicity were measured. Results: Ethanol withdrawal significantly increased glutamate release (peaking at 18 hr with a 53% increase), increased concentrations of putrescine and spermidine (136% and 139% increases, respectively, at 18 hr), and produced significant cytotoxicity in the CA1 hippocampal region (56% increase in PI staining relative to controls) of the cultures. The cell death produced by ethanol withdrawal was significantly inhibited by ifenprodil (IC₅₀= 14.9 nM), arcaine (IC₅₀= 37.9 nM), agmatine (IC₅₀= 41.5 nM), and DFMO (IC₅₀= 0.6 nM). NMDA (5 μM) significantly increased PI staining in the CA1 region of the hippocampal cultures (365% relative to controls), but ifenprodil, arcaine, agmatine, and DFMO all failed to significantly affect this type of toxicity. Conclusions: These data implicate a role for polyamines in ethanol withdrawal‐induced neurotoxicity and suggest that inhibiting the actions of polyamines on NMDARs may be neuroprotective under these conditions.     

Elevated concentrations of 15‐deoxy‐Δ12,14‐prostaglandin J2 in chronic liver disease propose therapeutic trials with peroxisome proliferator activated receptor γ‐inducing drugs

Background/Aims: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF‐β. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor γ (PPARγ) expression in hepatocytes, thus sensitizing these cells to the well‐known inhibitory effect of 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐d‐PGJ₂) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15‐d‐PGJ₂ is required to exert an inhibitory effect on transforming growth factor‐β (TGF‐β) target genes such as CTGF. Methods: This study compared serum concentrations of 15‐d‐PGJ₂ in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non‐liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARγ‐inducing (i.e. CTGF inhibitory) drugs such as caffeine. Results: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2±5.9 μg/L) displayed strikingly higher serum concentrations of 15‐d‐PGJ₂ than healthy probands (mean 2.3±1.0) and Caucasian patients with NLD (mean 2.7±1.4 μg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3±0.7 μg/L) compared with Chinese healthy controls (mean 0.4±0.2 μg/L). Conclusions: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARγ expression, such as methylxanthine derivates.     

Effects of ivabradine on the pulmonary vein electrical activity and modulation of pacemaker currents and calcium homeostasis

Effects of Ivabradine on Pulmonary Veins . Introduction: Ivabradine is a novel heart rate decreasing agent with selective and specific antagonist effects on the pacemaker current (I_f). The aim of this study was to investigate the pharmacological effects of ivabradine on the pulmonary vein (PV) cardiomyocytes. Methods and Results: Whole‐cell patch‐clamp techniques and the indo‐1 fluorimetric ratio technique were used to investigate the characteristics of the I_f and intracellular calcium (Ca ²⁺_i ) in single isolated rabbit PV cardiomyocytes with pacemaker activity before and after an ivabradine administration (0.3, 3, 10, and 30 μM). Ivabradine (0.3, 3, 10, and 30 μM) concentration dependently decreased the spontaneous activity by 6 ± 3%, 32 ± 6%, 49 ± 5%, and 85 ± 4%, and decreased the I_f by 35 ± 8%, 47 ± 9%, 62 ± 5%, and 65 ± 7%, respectively, in PV cardiomyocytes. The decreased extent of the PV beating rate or I_f by the different concentrations of ivabradine correlated well with the baseline PV beating rates. The IC₅₀ of the spontaneous activity and I_f induced by ivabradine were 9.5 and 3.5 μM, respectively. Moreover, ivabradine (30 μM, but not 3 μM) decreased the Ca ²⁺_i transient in the PV cardiomyocytes and ivabradine (30 μM) decreased the L‐type calcium current in the PV cardiomyocytes. Conclusion: Ivabradine decreased the I_fs and Ca ²⁺_i transient in the PV cardiomyocytes, which may contribute to its inhibitory effects on the PV spontaneous activity. (J Cardiovasc Electrophysiol, Vol. 23, pp. 200‐206, February 2012)     

Melatonin potentiates running wheel‐induced neurogenesis in the dentate gyrus of adult C3H/HeN mice hippocampus

This study assessed the role of melatonin in modulating running wheel(RW)‐induced hippocampal neurogenesis in adult C3H/HeN mice. Chronic melatonin (0.02 mg/mL, oral for 12 days) treatment did not affect cell proliferation or cell survival determined by the number of BrdU‐positive cells in dentate gyrus of mice with access to fixed wheel (FW). RW activity significantly increased cell proliferation [RW (n = 8) versus FW (n = 6): dorsal, 199 ± 18 versus 125 ± 12, P < 0.01; ventral, 211 ± 15 versus 123 ± 13, P < 0.01] and newborn cell survival [RW (n = 7) versus FW (n = 8): dorsal, 45 ± 8.5 versus 15 ± 1.8, P < 0.01; ventral, 48 ± 8.1 versus 15 ± 1.4)] in the dorsal and ventral dentate gyrus. Oral melatonin treatment further potentiated RW activity‐induced cell survival in both areas of the dentate gyrus [melatonin (n = 10) versus vehicle (n = 7): dorsal, 63 ± 5.4 versus 45 ± 8.5 P < 0.05; ventral, 75 ± 7.9 versus 48 ± 8.1, P < 0.01] and neurogenesis [melatonin (n = 8) versus vehicle (n = 8): dorsal, 46 ± 3.4, versus 34 ± 4.5, P < 0.05; ventral, 41 ± 3.4 versus 25 ± 2.4, P < 0.01]. We conclude that melatonin potentiates RW‐induced hippocampal neurogenesis by enhancing neuronal survival suggesting that the combination of physical exercise and melatonin may be an effective treatment for diseases affecting the hippocampus neurogenesis.     

Pathogenic mechanisms of postinfectious functional gastrointestinal disorders: Results 3 years after gastroenteritis

Objective. Functional gastrointestinal disorders (FGID) may appear after acute gastroenteritis. The aim of this study was to evaluate the possible mechanisms (inflammation, visceral hypersensitivity, psychological and immunogenetic factors) related to the development of postinfectious (PI) FGID 3 years after a Salmonella outbreak. Material andmethods. Biopsies of the antrum, and right- and left colon from 16 PI-FGID patients, 8 PI control patients, and 18 healthy controls (H-controls) were processed for immunohistochemistry, cytokines, and mast-cell electron microscopy. DNA was typed for cytokine gene polymorphisms. Visceral sensitivity (satiety test and rectal barostat) and psychological factors (SCL-90 and vital events) were assessed. Results. The number of mast cells and T lymphocytes was similar among the groups in all locations. Mast cells within 5 µm of nerve fibers of both PI groups were increased compared to H-controls: (stomach: 5.6±1.2 versus 6.6±1.5 versus 2.5±1.1; right colon: 9.7±1.3 versus 8.0±1.3 versus 4.1±1.7; left colon: 8.9±0.9 versus 8.5±1.8 versus 2.2±2.0 per field) (p<0.05). No differences in the production of IL-1β, IL-1ra, IL-6, and IL-10 or in their genotypes were found. PI-FGID patients showed a lower pain threshold to rectal distention (29±2 versus 37± 2 mmHg; p<0.05). Scores for anxiety (0.63±0.11 versus 0.28±0.14) and somatization (1.01±0.15 versus 0.45±0.15) were higher in PI-FGID patients than in PI controls (p<0.05). The number of stressful life events was not significantly different between both PI groups. Conclusions. Three years after salmonellosis, PI-FGID patients showed no evidence of inflammation in the gastric or colonic mucosa, but visceral sensitivity and anxiety/somatization levels were increased. The close anatomical mast cell-nerve fibers relation does not seem to be related to the FGID but to the infection itself.     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short‐term ethanol perfusion in the rat

Abstract: Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O₂^·?) release into the hepatic sinusoids during short‐term exposure to ethanol. Methods: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert‐buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O₂^·? production, MCLA (2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one), a Cypridina luciferin analogue, was simultaneously infused and MCLA‐enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL₃) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine–threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4‐methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol‐induced chemiluminescence were also evaluated. Sites where O₂^·? could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Results: Both ethanol and tert‐buthanol rapidly caused O₂^·? release. GdCL₃ suppressed the ethanol‐induced O₂^·? release by 61%. Staurosporine and DPI, but neither IB nor 4‐MP, also significantly inhibited the ethanol‐induced O₂^·? release. In the histochemical examination, ethanol‐stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl₃‐pretreated liver had the precipitate only on SECs. Conclusions: This study shows that ethanol itself stimulates both SECs and KCs to release O₂^·? via activation of NADPH oxidase probably involving protein kinase C (PKC).     

Melatonin‐mediated inhibition of Purkinje neuron P‐type Ca2+ channels in vitro induces neuronal hyperexcitability through the phosphatidylinositol 3‐kinase‐dependent protein kinase C delta pathway

Although melatonin receptors are widely expressed in the mammalian central nervous system and peripheral tissues, there are limited data regarding the functions of melatonin in cerebellar Purkinje cells. Here, we identified a novel functional role of melatonin in modulating P‐type Ca²⁺ channels and action‐potential firing in rat Purkinje neurons. Melatonin at 0.1 μm reversibly decreased peak currents (I_Ba) by 32.9%. This effect was melatonin receptor 1 (MT_R1) dependent and was associated with a hyperpolarizing shift in the voltage dependence of inactivation. Pertussis toxin pretreatment, intracellular application of QEHA peptide, and a selective antibody raised against the G_β subunit prevented the inhibitory effects of melatonin. Pretreatment with phosphatidylinositol 3‐kinase (PI3K) inhibitors abolished the melatonin‐induced decrease in I_Ba. Surprisingly, melatonin responses were not regulated by Akt, a common downstream target of PI3K. Melatonin treatment significantly increased protein kinase C (PKC) activity 2.1‐fold. Antagonists of PKC, but not of protein kinase A, abolished the melatonin‐induced decrease in I_Ba. Melatonin application increased the membrane abundance of PKC_δ, and PKC_δ inhibition (either pharmacologically or genetically) abolished the melatonin‐induced I_Ba response. Functionally, melatonin increased spontaneous action‐potential firing by 53.0%; knockdown of MT_R1 and blockade of P‐type channels abolished this effect. Thus, our results suggest that melatonin inhibits P‐type channels through MT_R1 activation, which is coupled sequentially to the βγ subunits of G_i/o‐protein and to downstream PI3K‐dependent PKC_δ signaling. This likely contributes to its physiological functions, including spontaneous firing of cerebellar Purkinje neurons.     

Antibodies to high‐density lipoprotein and β2‐glycoprotein I are inversely correlated with paraoxonase activity in systemic lupus erythematosus and primary antiphospholipid syndrome

Objective

To determine the prevalence of anti–high‐density lipoprotein (anti‐HDL) antibodies and to establish a possible relationship between anti‐HDL, anticardiolipin antibodies (aCL), anti–β₂‐glycoprotein I (anti‐β₂GPI), and paraoxonase (PON) activity in patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS).

Methods

Thirty‐two patients with SLE and 36 with primary APS were enrolled in a cross‐sectional study. Twenty age‐ and sex‐matched healthy subjects were used as controls. Serum levels of IgG and IgM aCL, anti‐β₂GPI, and antiprothrombin antibodies and IgG anti‐HDL were measured by enzyme‐linked immunosorbent assay. Total cholesterol, HDL cholesterol, HDL₂, and HDL₃ were determined by standard enzymatic techniques. PON activity was assessed by quantification of nitrophenol formation, and total antioxidant capacity (TAC) by chemiluminescence.

Results

Levels of total HDL, HDL₂, and HDL₃ were reduced in patients with SLE compared with controls (mean ± SD 0.51 ± 0.3, 0.37 ± 0.3, and 0.14 ± 0.1 mmoles/liter, respectively, versus 1.42 ± 0.9, 1.01 ± 0.7, and 0.40 ± 0.2). Patients with SLE and primary APS had higher titers of anti‐HDL antibodies and lower PON activity than controls. In the SLE population, PON activity was inversely correlated with IgG anti‐HDL titers (r = −0.48, P = 0.005) whereas in the primary APS population, IgG anti‐β₂GPI was the only independent predictor of PON activity (r = −0.483, P = 0.003). In the SLE group, anti‐HDL was inversely correlated with TAC (r = −0.40, P < 0.02), and PON activity was positively correlated with TAC (r = 0.43, P < 0.02).

Conclusion

IgG anti‐HDL and IgG anti‐β₂GPI antibodies are associated with reduced PON activity in patients with SLE and primary APS. Since the physiologic role of PON is to prevent low‐density lipoprotein oxidation with its attendant atherogenic effects, the reported interactions may be relevant to the development of atherosclerosis in SLE and primary APS.

     

High glucose level inhibits capacitative Ca2 + influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism

Summary In cultured mesangial cells (MC), capacitative Ca^{2 +} influx via store-operated channels (SOC) is potentiated by agents that release Ca^{2 +} from intracellular stores, and inhibited by protein kinase C (PKC). Cells grown under high glucose conditions, as a model of the diabetic microenvironment, display reduced Ca^{2 +} signalling in response to vasoconstrictors, probably due to downregulation by elevated PKC activity. Since SOC might be relevant to this phenomenon, we assessed Ca^{2 +} influx by microfluorometry of fura-2-loaded rat MC cultured for 5 days in normal (5.5 mmol/l, NG) or high glucose (30 mmol/l, HG). The addition of 1–10 mmol/l Ca^{2 +} to NG cells equilibrated in Ca^{2 +} -free media induced an immediate Ca^{2 +} influx with a free cytosolic Ca^{2 +} ([Ca^{2 +} ]_i) plateau of 155 ± 50 and 318 ± 114 nmol/l, respectively. Basal influx was reduced to 88 ± 8 and 145 ± 17 nmol/l [Ca^{2 +} ]i (1–10 mmol/l Ca^{2 +} , p < 0.01) by 30 mmol/l d-glucose. This effect of HG was confirmed by Mn^{2 +} quenching of fura-2, indicating reduced entry of divalent cations via the capacitative pathway. Equimolar l-glucose had no effect on Ca^{2 +} influx, consistent with a non-osmotic mechanism. Arginine vasopressin (10 μmol/l) elicited weaker release of stored Ca^{2 +} and subsequent influx in HG cells (191 ± 33 vs 153 ± 24 nmol/l, 400 ± 76 vs 260 ± 33 nmol/l, 1–10 mmol/l Ca^{2 +} , NG/HG, p < 0.05). To examine the involvement of PKC in the effect of HG on capacitative Ca^{2 +} influx, the enzyme was activated or downregulated by treatment with 0.1 μmol/l phorbol myristate acetate (PMA) for 3 min or 24 h, respectively. PMA acutely inhibited Ca^{2 +} influx in NG cells, while PKC downregulation restored it in HG cells. Similarly, the PKC inhibitors staurosporin or H-7 normalized SOC activity in HG cells. In summary, impairment of Ca^{2 +} influx via SOC by HG is one mechanism of the reduced MC [Ca^{2 +} ]_i responsiveness to vasoconstrictors. This event is mediated by PKC and may contribute to the glomerular haemodynamic changes in the initial stages of diabetes mellitus. [Diabetologia (1997) 40: 521–527] Received: 25 November 1996 and in revised form: 30 January 1997     

ATP modulates sulfobromophthalein uptake in rat liver plasma membrane vesicles

The hepatic uptake of the bilirubin-bilirubin-sulfobromophthalein (BSP) group of organic anions is a carrier-mediated process and is accounted for by at least four distinct plasma membrane proteins (bilitranslocase, BSP/bilirubin-binding protein, organic anion-binding protein and the organic anion transport protein). In order to investigate the regulation of basolateral organic anion uptake, BSP transport was measured in rat basolateral liver plasma membrane vesicles in the presence of ATP. ATP significantly stimulated the electroneutral uptake of BSP with an increment in V_max compared with control (1.57±0.14 vs 0.73±0.06 nmol BSP/mg protein per 15 s, respectively; P< 0.001) while the apparent K_m was not changed significantly (12±1 vs 12±2 μmol/L). The stimulatory effect was dose-dependent for ATP (K_m 1.01±0.37 mmol/L). ATP had no detectable effect on the electrogenic component of BSP transport. Other nucleotides (ADP, AMP, GTP) and non-hydrolysable ATP did not enhance BSP uptake, suggesting that ATP hydrolysis was necessary for the effect. This was supported by the lack of effect on BSP uptake when ATP was added in the presence of vanadate. The addition of phorbol-12-myristate 13-acetate, an activator of protein kinase C (PKC), increased BSP uptake in a dose-dependent manner in the presence, but not in the absence, of ATP. Incubation of vesicles with staurosporine, an inhibitor of PKC activity, resulted in a dose-dependent inhibition of ATP-sensitive BSP transport. These data indicate that electroneutral BSP hepatic uptake is modulated by ATP. The effect is related to ATP hydrolysis and involves the activity of PKC.     

Investigation of the effect of oral metformin on dipeptidylpeptidase‐4 (DPP‐4) activity in Type 2 diabetes1

Aims Glucagon‐like peptide‐1 (GLP‐1) is an insulinotropic hormone and major component of the enteroinsular axis. Its therapeutic potential in human diabetes is limited by rapid degradation and inactivation by the enzyme dipeptidylpeptidase‐4 (DPP‐4). We investigated the acute effects of metformin with and without food on DPP‐4 activity in Type 2 diabetes. Methods Ten subjects with Type 2 diabetes (6 male/4 female, age 65.8 ± 2.6 years, body mass index 30.0 ± 1.2 kg/m², glycated haemoglobin (HbA_1c) 6.3 ± 0.2%, mean ± sem ) received metformin 1 g orally or placebo together with a standard mixed meal (SMM) in a random crossover design. Six subjects re‐attended fasting and received metformin 1 g without a SMM. Results Following SMM (n = 10), DPP‐4 activity was not suppressed by metformin compared with placebo [area under curve (AUC)_{0–4 h} 1574 ± 4 vs. 1581 ± 8 μmol/ml/min, respectively]. Plasma glucose, insulin and active GLP‐1 were not different. However, DPP‐4 activity was suppressed with metformin following fasting compared with a SMM (n = 6) (AUC_{0–4 h} 1578 ± 4 vs. 1494 ± 9 μmol/min, P < 0.02). Metformin serum levels were significantly lower (P < 0.001) after SMM than fasting (AUC_{0–4 h} 350 ± 66 vs. 457 ± 55 mg/ml/min). Conclusion Metformin inhibits DPP‐4 activity in Type 2 diabetic patients in the fasting state but not when taken with a standard mixed meal. Metformin serum concentrations are lower if the drug is taken with food. These findings should be taken into account in establishing how to maximize efficacy of the drug.