Activation of protein kinase C induces internalization of the GABA(C) receptors expressed in Xenopus oocytes

In a previous study, we showed that the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) inhibited the gamma-aminobutyric acid (GABA)-gated currents in Xenopus oocytes expressing human rho1 GABA(C) receptors. To investigate whether the inhibition of currents was due to a decrease in efficacy or in the potency of rho1 GABA(C) receptor, concentration-response curves for GABA were compared before and after PMA treatment. The EC50 concentrations of GABA obtained during the maximally inhibited period were not statistically different from the concentrations obtained before PMA treatment (1.74 +/- 0.33 and 1.45 +/- 0.28 microM, respectively). These results indicate that the inhibition depends on a change in number or conductance of active receptor channels, but not on a change in affinity for GABA. To allow histochemical detection of rho1 GABA(C) receptors, we constructed a receptor tagged at the C-terminal position with human c-myc epitope. Electrophysiologically, the tagged receptors showed almost the same sensitivities for GABA and PMA as those of wild-type rho1 GABA(C) receptors. Immunohistochemistry with anti-myc antibody detected a dense concentration of tagged receptors at the surface area of Xenopus oocytes. Transient exposure to PMA reduced the density of immunofluorescence at the surface area and increased it in the subsurface area. These results suggest that the stimulation of protein kinase C leads to internalization of rho1 GABA(C) receptors expressed in Xenopus oocytes.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.     

The stimuli releasing histamine from murine bone marrow-derived mast cells. 2. Mechanisms involved in histamine release induced by extracellular ATP and its metabolites.

Extracellular ATP stimulated histamine release and generation of leukotrience C4 (LTC4) accompanied with the formation of inositol phosphates and a rapid increase in intracellular Ca2+ ([Ca2+]i) in mouse bone marrow-derived cultured mast cells (BMMC). The rank order of histamine-releasing potency of ATP and its metabolites is ATP greater than ADP greater than AMP greater than adenosine. Nonhydrolyzable ATP analog, adenosine-5'-O-[2-thiotriphosphate] (ATP-S) released more histamine from the cells than ATP. On the other hand, simultaneous addition of adenosine analogues at micromolar concentrations potentiated histamine release from the cells induced by ATP (50 microM) or DNP-HSA antigen (0.1 ng/ml) in the following rank order: adenosine greater than AMP much greater than ADP = ATP. Histamine release potentiated by adenosine was blocked by the treatment with pertussis toxin, whereas histamine release induced by ATP was not affected by the toxin, suggesting that extracellular ATP stimulate histamine release from BMMC probably via mechanisms independent of the potentiation of histamine release induced by adenosine.     

In vivo expression of CD69 on lung eosinophils in eosinophilic pneumonia: CD69 as a possible activation marker for eosinophils.

The antigen, CD69, has been demonstrated to be expressed on activated T cells and natural killer cells. There have been no studies concerning the expression of CD69 on eosinophils. In this article, we demonstrate that lung eosinophils obtained from the bronchoalveolar lavage fluid of patients with eosinophilic pneumonia expressed significant levels of CD69, whereas peripheral blood (PB) eosinophils did not express CD69. We also activated PB eosinophils in vitro using phorbol myristate acetate and cytokines to determine whether CD69 was expressed. PB eosinophils expressed CD69 after short-term culture with phorbol myristate acetate and eosinophil hemopoietic cytokines (interleukin-3, granulocyte-macrophage--colony-stimulating factor, and interleukin-5). These findings suggest that CD69 may be a useful marker for activated eosinophils at inflammatory sites.     

Two Autopsy Cases of Primary Leiomyosarcoma of the Liver Superiority of Muscle-specific Actin Immunoreactivity in Diagnosis

Two autopsy cases of leiomyosarcoma of the liver in a 49-year old female and 63-year-old male are reported. Both of the liver tumors showed electron microscopically dense patches in the cytoplasm and intermediate junctions between the tumor cells, suggesting a smooth muscle cell origin, irrespective of their different histological features. The nature of both tumors was confirmed by positive immunoreactivity for muscle-specific actin in the tumor cells, whereas desmin immunoreactivity was labile in both cases, showing a higher diagnostic value of the former compared with the latter in these leiomyosarcomas. Both cases, showed extensive distant metastases in spite of an evident difference in their mitotic indices, indicating that this index is not reliable for judging the metastatic potential of these tumors. Acta Pathol Jpn 41: 461–465, 1991.     

Repeated administration of CGP 46381, a gamma-aminobutyric acidB antagonist, and ethosuximide suppresses seizure-associated cyclic adenosine 3'5' monophosphate response element- and activator protein-1 DNA-binding activities in lethargic (lh/lh) mice

To characterize seizure-associated increases in cerebral cortical and thalamic cyclic AMP responsive element (CRE)- and activator protein 1 (AP-1) DNA-binding activities in lethargic (lh/lh) mice, a genetic model of absence seizures, we examined the effects of ethosuximide and CGP 46381 on these DNA-binding activities. Repeated administration (twice a day for 5 days) of ethosuximide (200 mg/kg) or CGP 46381 (60 mg/kg) attenuated both seizure behavior and the increased DNA-binding activities, and was more effective than a single administration of these drugs. These treatments did not affect either normal behavior or basal DNA-binding activities in non-epileptic control (+/+) mice. Gel supershift assays revealed that the increased CRE-binding activity was attributable to activation of the binding activity of CREB, and that the c-Fos-c-Jun complex was a component of the increased AP-1 DNA-binding activity.     

A light and electron microscopical study of membranocystic lesions in a case of lupus erythematosus profundus

A light and electron microscopical study on membranocystic lesions (MCL) in a case of lupus erythematosus profundus (LEP) is reported. The patient was a 16-year-old female who presented with subcutaneous nodules on both upper arms. The light microscopic features were consistent with LEP, and the result of an immunofluorescence band test supported this diagnosis. A peculiar finding in this case was MCL in the subcutaneous tissue. Ultrastructurally, these were thin membranes without a tubular structure and tortuous thick membranes composed of minute tubules. The lesions were very similar to the fatty tissue changes in membranous lipodystrophy. On the other hand, the basement membranes of the blood vessels were thickened and multilayered, and the lumina were narrowed by endothelial swelling and thickening of the vessel wall. Our findings suggest that the MCL in LEP result from circulatory disturbance of the fat tissue.     

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.