Neutrophils play a major role in host defense against microbial infection. There are some clues indicate that neutrophils may also play a role in the pathophysiology of the airway obstruction in chronic asthma. We studied the roles of intracellular calcium and GTP gamma S in the regulation of neutrophils exocytosis using pipette perfusion and membrane capacitance measurement technique in whole cell patch clamp configuration. The results showed that the membrane capacitance increase induced by calcium revealed a biphasic process. The first phase occurred when the calcium level was between 0.2-14 micromol/L with a plateau amplitude of 1.23 pF and a calcium EC50 of 1.1 micromol/L. This phase might correspond to the release of the tertiary granules. The second phase occurred when the calcium concentration was between 20-70 micromol/L with a plateau increment of 6.36 pF, the calcium EC50 being about 33 micromol/L. This phase might represent the release of the primary and secondary granules. Intracellular calcium also simultaneously increased the exocytotic rate and the eventual extent in neutrophils. On the other hand, GTP gamma S can increase the exocytotic rate in a dose-dependent manner but had no effect on the eventual extent of membrane capacitance increment (>6 pF) if the cell was stimulated for a long period (>20 min). GTP gamma S (ranging from 20 to 100 micromol/L) induced the neutrophils to release all four types of the granules at very low intracellular calcium level.
Calcium ; metabolism ; Cell Degranulation ; drug effects ; Exocytosis ; drug effects ; GTP-Binding Proteins ; metabolism ; physiology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Humans ; Neutrophils ; metabolism ; physiology ; Patch-Clamp Techniques

Methylation events play a critical role in various cellular processes including regulation of gene transcription and proliferation. We observed that methyltransferase activity underwent time-dependent changes in the cytosol of the rat hepatocytes upon partial hepatectomy. However, any change in the methylation of nuclear proteins is not clear during hepatocyte proliferation. The nuclear fraction possesses basal level of methyltransferase to catalyze methylation of several proteins ranging from 7 to 70 kD prior to any hepatecmony. The specific p16 (16 kD) band was transiently and heavily methylated post 1 day hepatectomy, and then became non- detectable, but not in the control liver. Methylation of p16 band was completely inhibited by exogenously added histones, particularly 2AS, 1, 2A and 2B subtypes. The methylated p16 protein remains stable in either acid or alkali- induced demethylation conditions, indicating that methylation is not likely to occur on isoaspartyl or C-terminal cysteinyl residues. Exogenous addition of non-hydrolyzable GTP caused a dose- dependent suppression of a p16 methylation suggesting that G-proteins might play a role as an endogenous methylation inhibitor in vivo. Taken together, we have identified the proliferation event associated-methylation of the nuclear p16 protein in the hepatocytes undergoing liver regeneration.
Alkalies/pharmacology ; Animals ; Cell Proliferation ; Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology ; Hepatectomy ; Hepatocytes/drug effects/*metabolism ; Histones/pharmacology ; Liver Regeneration/drug effects/*physiology ; Methylation/drug effects ; Nuclear Proteins/*metabolism ; Rats ; Research Support, Non-U.S. Gov't ; Sodium Chloride/pharmacology

Although the apoptosis of chondrocytes plays an important role in endochondral ossification, its mechanism has not been elucidated. In this study, we show that guanosine induces chondrocyte apoptosis based on the results of acridine orange/ ethidium bromide staining, caspase-3 activation, and sub-G1 fraction analysis. The potent inhibitory effect of dipyridamole, a nucleoside transporter blocker, indicates that extracellular guanosine must enter the chondrocytes to induce apoptosis. We found that guanosine promotes Fas-Fas ligand interaction which, in turn, leads to chondrocyte apoptosis. These findings indicate a novel mechanism for endochondral ossification via metabolic regulation.
Tumor Necrosis Factors/metabolism ; Signal Transduction/drug effects ; Receptors, Tumor Necrosis Factor/*metabolism ; Rats, Sprague-Dawley ; Rats ; Nucleoside Transport Proteins/metabolism ; Membrane Glycoproteins/metabolism ; Guanosine/*pharmacology/physiology ; Fas Ligand Protein ; Chondrocytes/*drug effects/metabolism ; Apoptosis/*drug effects ; Antigens, CD95 ; Animals

OBJECTIVETo observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.

METHODSUsing whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.

RESULTS(1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.

CONCLUSIONSDual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.

Animals ; Enkephalin, D-Penicillamine (2,5)- ; pharmacology ; GTP-Binding Proteins ; physiology ; Glioma ; metabolism ; pathology ; Guanosine Monophosphate ; analogs & derivatives ; pharmacokinetics ; Hybrid Cells ; metabolism ; pathology ; Mice ; Neuroblastoma ; metabolism ; pathology ; Patch-Clamp Techniques ; Pertussis Toxin ; pharmacology ; Potassium Channels, Inwardly Rectifying ; metabolism ; Rats ; Receptors, Opioid ; agonists ; Thionucleotides ; pharmacokinetics