Avian influenza H5N1 virus induces cytopathy and proinflammatory cytokine responses in human astrocytic and neuronal cell lines.

It has previously been reported that the avian H5N1 type of influenza A virus can be detected in neurons and astrocytes of human brains in autopsy cases. However, the underlying neuropathogenicity remains unexplored. In this study, we used differentiated human astrocytic and neuronal cell lines as models to examine the effect of H5N1 influenza A viral infection on the viral growth kinetics and immune responses of the infected cells. We found that the influenza virus receptors, sialic acid-alpha2,3-galactose and sialic acid-alpha2,6-galactose, were expressed on differentiated human astrocytic and neuronal cells. Both types of cells could be infected with H5N1 influenza A viruses, but progeny viruses were only produced from infected astrocytic cells but not neuronal cells. Moreover, increased expression of interleukin (IL)-6 and/or tumor necrosis factor alpha (TNF-alpha) mRNA was detected in both astrocytic and neuronal cells at 6 and 24 h post-infection. To examine the biological consequences of such enhanced cytokine expression, differentiated astrocytic and neuronal cells were directly treated with these two cytokines. TNF-alpha treatment induced apoptosis, as well as proinflammatory cytokine, chemokine and inflammatory responses in differentiated astrocytic and neuronal cells. Taken together, our findings reveal that avian influenza H5N1 viruses can infect human astrocytic and neuronal cells, resulting in the induction of direct cellular damage and proinflammatory cytokine cascades. Our observations suggest that avian influenza H5N1 infection can trigger profound CNS injury, which may play an important role in the influenza viral pathogenesis.

Novel hypoglycemic effects of Ganoderma lucidum water-extract in obese/diabetic (+db/+db) mice.

In this study, we evaluated the pharmacological effects of Ganoderma lucidum (G. lucidum) (water-extract) (0.003, 0.03 and 0.3g/kg, 4-week oral gavage) consumption using the lean (+db/+m) and the obese/diabetic (+db/+db) mice. Different physiological parameters (plasma glucose and insulin levels, lipoproteins-cholesterol levels, phosphoenolpyruvate carboxykinase (PEPCK), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and isolated aorta relaxation of both species were measured and compared. G. lucidum (0.03 and 0.3g/kg) lowered the serum glucose level in +db/+db mice after the first week of treatment whereas a reduction was observed in +db/+m mice only fed with 0.3g/kg of G. lucidum at the fourth week. A higher hepatic PEPCK gene expression was found in +db/+db mice. G. lucidum (0.03 and 0.3g/kg) markedly reduced the PEPCK expression in +db/+db mice whereas the expression of PEPCK was attenuated in +db/+m mice (0.3g/kg G. lucidum). HMG CoA reductase protein expression (in both hepatic and extra-hepatic organs) and the serum insulin level were not altered by G. lucidum. These data demonstrate that G. lucidum consumption can provide beneficial effects in treating type 2 diabetes mellitus (T2DM) by lowering the serum glucose levels through the suppression of the hepatic PEPCK gene expression.

Modulation by simvastatin of iberiotoxin-sensitive, Ca2+-activated K+ channels of porcine coronary artery smooth muscle cells.

BACKGROUND AND PURPOSE: Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors) have been demonstrated to reduce cardiovascular mortality. It is unclear how the expression level of HMG CoA reductase in cardiovascular tissues compares with that in cells derived from the liver. We hypothesized that this enzyme exists in different cardiovascular tissues, and simvastatin modulates the vascular iberiotoxin-sensitive Ca2+-activated K(+) (BK(Ca)) channels. EXPERIMENTAL APPROACHES: Expression of HMG CoA reductase in different cardiovascular preparations was measured. Effects of simvastatin on BK(Ca) channel gatings of porcine coronary artery smooth muscle cells were evaluated. KEY RESULTS: Western immunoblots revealed the biochemical existence of HMG CoA reductase in human cardiovascular tissues and porcine coronary artery. In porcine coronary artery smooth muscle cells, extracellular simvastatin (1, 3 and 10 microM) (hydrophobic), but not simvastatin Na+ (hydrophilic), inhibited the BK(Ca) channels with a minimal recovery upon washout. Isopimaric acid (10 microM)-mediated enhancement of the BK(Ca) amplitude was reversed by external simvastatin. Simvastatin Na+ (10 microM, applied internally), markedly attenuated isopimaric acid (10 microM)-induced enhancement of the BK(Ca) amplitude. Reduced glutathione (5 mM; in the pipette solution) abolished simvastatin -elicited inhibition. Mevalonolactone (500 microM) and geranylgeranyl pyrophosphate (20 microM) only prevented simvastatin (1 and 3 microM)-induced responses. simvastatin (10 microM ) caused a rottlerin (1 microM)-sensitive (cycloheximide (10 microM)-insensitive) increase of PKC-delta protein expression. CONCLUSIONS AND IMPLICATIONS: Our results demonstrated the biochemical presence of HMG CoA reductase in different cardiovascular tissues, and that simvastatin inhibited the BK(Ca) channels of the arterial smooth muscle cells through multiple intracellular pathways.

Endogenous release and multiple actions of secretin in the rat cerebellum.

Previous studies demonstrated that secretin could modulate synaptic transmission in the rat cerebellum. In the present report, we provide evidence for the endogenous release of secretin in the cerebellum and further characterize the actions of secretin in this brain area. First, to show that secretin is released endogenously, blocks of freshly dissected cerebella were challenged with a high concentration of KCl. Incubation with KCl almost doubled the rate of secretin release. This KCl-induced release was sensitive to tetrodotoxin and cadmium suggesting the involvement of voltage-gated sodium and calcium channels. The use of specific channel blockers further revealed that L-type and P/Q-type calcium channels underlie both basal and KCl-evoked secretin release. In support of this, depolarization of Purkinje neurons in the presence of NMDA, group II mGluR and cannabinoid CB1 receptor blockers resulted in increased inhibitory postsynaptic current frequency. Second, we found that the previously reported facilitatory action of secretin on GABAergic inputs to Purkinje neurons is partly dependent on the release of endogenous glutamate. In the presence of CNQX, an AMPA/kainate receptor antagonist, the facilitatory effect of secretin on GABA release was significantly reduced. In support of this idea, application of AMPA, but not kainate receptor agonist, facilitated GABA release from inhibitory terminals, an action that was sensitive to AMPA receptor antagonists. These data indicate that a direct and an indirect pathway mediate the action of secretin in the basket cell-Purkinje neuron synapse. The results provide further and more solid evidence for the role of secretin as a neuropeptide in the mammalian CNS.

Expression and spatial distribution of secretin and secretin receptor in human cerebellum.

The expression and spatial distribution of secretin and its receptor in human cerebellum were investigated by in situ hybridization and immunohistochemical techniques. Secretin mRNAs are found in Purkinje cells whereas secretin receptor transcripts are present in Purkinje cells and basket cells in the molecular cell layer. In addition, secretin-immunoreactivities are localized in both the soma and dendrites of Purkinje cells. These data are the first demonstration of the spatial distribution of secretin and its receptor in distinct neurons within the human cerebellum. The cellular localizations of this ligand-receptor pair are consistent with the proposed actions of secretin in the cerebellum of rodents and hence suggest that secretin also serves specific neural functions in the human cerebellum.

Secretin controls anion secretion in the rat epididymis in an autocrine/paracrine fashion.

There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.

Gubernacular development in Müllerian inhibiting substance receptor-deficient mice.

OBJECTIVE: To determine, in mice with disrupted Müllerian inhibiting substance (MIS) receptor genes, whether MIS affects gubernacular development; MIS causes Müllerian duct regression and is proposed to be involved in the first stage of testicular descent, because gubernacular development is abnormal in humans with persistent Müllerian duct syndrome. MATERIALS AND METHODS: Ten wild-type, 11 heterozygotic and 12 homozygotic mice for MIS receptor mutations were killed at 17.5 or 18.5 days after conception or at birth, to provide serial sagittal sections of the pelvis. The amount of cremaster muscle, mitotic bodies in the gubernacular bulb, and gubernacular size were quantified by computer analysis (four mice/group). RESULTS: Müllerian ducts were present in the homozygous mutants, partially present in the heterozygotes and absent in the wild-type controls. All mice had descended testes. The cremaster muscle was significantly less developed in homozygous mutants than in wild-type controls (P < 0.001) and heterozygotes (P < 0.01) at birth. The mitotic index between the gubernacula of all groups was indistinguishable. There was no statistical difference in gubernacular area amongst the groups. Poor cremaster muscle development in homozygous mutants gave the muscle a loose mesenchymal appearance. CONCLUSIONS: Although there was an observable effect on cremaster muscle development in these mutant mice, gubernacular development and testicular descent were otherwise normal, and thus there must be other reasons for the observed differences in humans with persistent Müllerian duct syndrome.