The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics ; Animal ; Cell Differentiation ; Cyclic AMP/*metabolism ; Forskolin/*pharmacology ; Isoenzymes/genetics ; Melanoma, Experimental/*enzymology/*pathology ; Mice ; Signal Transduction

Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.
Animals ; Antigens, Differentiation/metabolism ; Cell Adhesion ; *Cell Differentiation ; Cell Lineage ; Cell Separation ; Cells, Cultured ; Forskolin/pharmacology ; Mice ; Mice, Inbred ICR ; Muscle, Skeletal/*cytology/metabolism ; Neurons/*cytology/metabolism ; Stem Cells/*cytology/metabolism ; Tretinoin/pharmacology

We investigated the mechanism of Cl- secretion by fluoroaluminate(AlF4-) and sodium orthovanadate(vanadate) using the human colonic T84 cell line. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure short circuit current(ISC). Serosal addition of AlF4- or vanadate to T84 monolayers produced a sustained increase in ISC. Removal of Ca2+ from the serosal bathing solution partially inhibited AlF4-(-)and vanadate-induced ISC, and readministration of Ca2+ restored AlF4-(-)and vanadate-induced ISC. Carbachol application in the presence of forskolin, AlF4- or vanadate induced a synergistic increase of ISC. Forskolin and vanadate significantly increased cellular cAMP level, while carbachol and AlF4- did not. Carbachol, AlF4- and vanadate significantly increased [Ca2+]i. After Na+ in mucosal bathing solution was replaced with K+, and the mucosal membrane of T84 cell was permeabilized with amphotericin B, AlF4-, vanadate, and carbachol increased K+ conductance, but forskolin did not. After sodium chloride in serosal bathing solution was replaced with sodium gluconate and the serosal membrane was permeabilized with nystatin, forskolin, AlF4-, and vanadate increased Cl- conductance, but carbachol did not. AlF4-(-)induced ISC was remarkably inhibited by the pretreatment of pertussis toxin(2 micrograms/ml) for 2 hours. These results indicate that AlF4- and vanadate can increase Cl- secretion via simultaneous stimulation of Cl- channel and K+ channel in T84 cells. However, the AlF4- action is mostly attributed to stimulation of pertussis toxin-sensitive G-proteins, whereas the vanadate action mostly results from G protein-independent mechanisms.
Aluminum/*pharmacology ; Amphotericin B/pharmacology ; Carbachol/pharmacology ; Cell Polarity ; Cells, Cultured/drug effects ; Chloride Channels/drug effects/*physiology ; Chlorides/*physiology ; Colon ; Electrophysiology ; Fluorine/*pharmacology ; Forskolin/pharmacology ; GTP-Binding Proteins/physiology ; Human ; Pertussis Toxin ; Potassium/pharmacology ; Potassium Channels/drug effects/physiology ; Second Messenger Systems ; Signal Transduction ; Support, Non-U.S. Gov't ; Vanadates/*pharmacology ; Virulence Factors, Bordetella/pharmacology

No abstract available.
1-Methyl-3-isobutylxanthine/pharmacology ; Ammonium Compounds/pharmacology ; Animal ; Biological Transport/physiology ; Biological Transport/drug effects ; Cell Membrane/metabolism ; Cyclic AMP/metabolism* ; Forskolin/pharmacology ; Guanidines/pharmacology ; Mice ; Mice, Knockout ; Pancreatic Ducts/metabolism* ; Phosphodiesterase Inhibitors/pharmacology ; Protons ; Sodium-Hydrogen Antiporter/metabolism* ; Sodium-Hydrogen Antiporter/genetics ; Sulfones/pharmacology

1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.
Adrenergic beta-Agonists/pharmacology* ; Animal ; Biological Transport/physiology ; Biological Transport/drug effects ; Cell Size/physiology ; Chlorides/metabolism* ; Cyclic AMP/metabolism ; Cytosol/metabolism ; Enzyme Inhibitors/pharmacology ; Female ; Fetus/cytology ; Forskolin/pharmacology ; Nitrobenzoates/pharmacology ; Pregnancy ; Protein-Tyrosine Kinase/metabolism* ; Rats ; Rats, Wistar ; Respiratory Mucosa/enzymology* ; Respiratory Mucosa/embryology ; Respiratory Mucosa/cytology ; Sodium/metabolism* ; Tyrphostins/pharmacology

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.
Animal ; Blotting, Western ; Bucladesine/pharmacology ; Cholera Toxin/pharmacology* ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Forskolin/pharmacology ; GTP-Binding Proteins/genetics* ; GTP-Binding Proteins/biosynthesis ; Gene Expression Regulation* ; Glioma ; Membrane Proteins/analysis ; RNA, Messenger/metabolism ; RNA, Messenger/genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (DeltaIsc,amil), cAMP-dependent Cl- secretion (DeltaIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (DeltaIsc,ATP). In mice exposed to 98% PO2 for 36 hr, DeltaIsc,forsk decreased, DeltaIsc,amil and DeltaIsc,ATP was not affected. In gulo(-/-) mice, both DeltaIsc,forsk and DeltaIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid.
Animals ; Ascorbic Acid Deficiency/*metabolism ; Biological Transport/drug effects ; Chlorides/*metabolism ; Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors/drug ; Forskolin/pharmacology ; Hyperbaric Oxygenation ; Hyperoxia/*physiopathology ; Ion Transport/drug effects ; Mice ; Mice, Inbred C57BL ; Mice, Inbred ICR ; Mice, Knockout/metabolism ; Mice, Transgenic ; Microscopy, Fluorescence ; Oxidative Stress ; Oxygen/adverse effects/pharmacology ; Potassium Channels/metabolism ; Respiratory Mucosa/drug effects/*metabolism/secretion ; Sodium ; Sugar Acids/metabolism

Human central neurocytoma is a kind of the brain tumors that are usually found in anterior part of the lateral ventricles. In this study, we established conditions that allowed proliferation of neurocytoma cells culture and analyzed characteristics of neurocytoma cells in vitro. For in vitro, a condition that used for culturing neural stem cells and contained basic fibroblast growth factor (bFGF) provided high proliferation. RT-PCR analaysis showed that nestin was found in neurocytoma cells, indicating that the neurocytomas possess neural stem cell properties. Interestingly, treatment of neurocytoma cells with forskolin increased expression of glial fibrillary acidic protein with a concomitant decrease in the nestin expression. Forskolin also induced morphological changes of neurocytoma cells to adopt an astrocyte-like phenotype. The results suggest that neurocyotma cells may have properties of multipotent neural stem cells.
Animals ; Astrocytes/cytology/*physiology ; Cell Differentiation/*drug effects ; Cell Proliferation ; Cell Shape ; Fibroblast Growth Factor 2/pharmacology ; Forskolin/*pharmacology/*therapeutic use ; Humans ; Intermediate Filament Proteins/metabolism ; Nerve Tissue Proteins/metabolism ; Neurocytoma/*drug therapy/pathology ; Research Support, Non-U.S. Gov't ; Tumor Cells, Cultured

Pigmentation may result from melanocyte proliferation, melanogenesis, migration or increases in dendricity. Recently, it has been reported that secreted phospholipase A2(sPLA2) known as a component of bee venom (BV), stimulates melanocyte dendricity and pigmentation. BV has been used clinically to control rheumatoid arthritis and to ameliorate pain via its anti-inflammatory and antinociceptive properties. Moreover, after treatment with BV, pigmentation around the injection sites was occasionally observed and the pigmentation lasted a few months. However, no study has been done about the effect of BV on melanocytes. Thus, in the present study, we examined the effect of BV on the proliferation, melanogenesis, dendricity and migration in normal human melanocytes and its signal transduction. BV increased the number of melanocytes dose and time dependently through PKA, ERK, and PI3K/Akt activation. The level of cAMP was also increased by BV treatment. Moreover, BV induced melanogenesis through increased tyrosinase expression. Furthermore, BV induced melanocyte dendricity and migration through PLA2activation. Overall, in this study, we demonstrated that BV may have an effect on the melanocyte proliferation, melanogenesis, dendricity and migration through complex signaling pathways in vitro, responsible for the pigmentation. Thus, our study suggests a possibility that BV may be developed as a therapeutic drug for inducing repigmentation in vitiligo skin.
Animals ; Base Sequence ; Bee Venoms/*pharmacology ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Cyclic AMP/metabolism ; DNA Primers/genetics ; Forskolin/pharmacology ; Gene Expression/drug effects ; Humans ; Melanins/biosynthesis ; Melanocytes/cytology/*drug effects/physiology ; Microphthalmia-Associated Transcription Factor/biosynthesis/genetics ; Monophenol Monooxygenase/biosynthesis/genetics ; Signal Transduction/drug effects

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.
1-Methyl-3-isobutylxanthine/pharmacology ; Animals ; Cell Line ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Antagonism ; Forskolin/pharmacology ; *Humulus ; Intramolecular Oxidoreductases/antagonists & inhibitors/biosynthesis ; Melanins/antagonists & inhibitors/*biosynthesis ; Melanocytes/*drug effects/*metabolism ; Melanoma, Experimental ; Membrane Glycoproteins/antagonists & inhibitors/biosynthesis ; Mice ; Microphthalmia-Associated Transcription Factor/antagonists & inhibitors ; Monophenol Monooxygenase/antagonists & inhibitors/biosynthesis/genetics ; Oxidoreductases/antagonists & inhibitors/biosynthesis ; Propiophenones/*pharmacology ; Signal Transduction/drug effects ; alpha-MSH/metabolism