guanosine-triphosphate and 9-(tetrahydro-2-furyl)-adenine

The proliferation of human lymphoblastoma cell line (H9) was differently stimulated by Peptide Histidine Methionine (PHM) and Vasoactive Intestinal Peptide (VIP). PHM induced a cyclic AMP (cAMP) accumulation, abolished by Adenylate Cyclase (AC) inhibitors leading to a loss of proliferative effect. VIP mitogenic activity was Pertussis toxin (PTX) sensitive and AC inhibitors insensitive. Pharmacological experiments performed on H9 membranes with or without a GTP analogue indicated expression of both GTP-insensitive and -sensitive PHM/VIP high-affinity binding sites (HA). H9 cells expressed only the VPAC1 receptor. VIP(10-28), known as a VPAC1 antagonist, bond to all GTP-insensitive PHM sites and inhibited evenly the PHM and VIP mitogenic actions. These data strongly suggested different mechanisms initiated by VIP and PHM and highlighted the key role of GTP-insensitive binding sites in the control of cell proliferation.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Blotting, Southern; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression; Guanosine Triphosphate; Guanylyl Imidodiphosphate; Humans; Imines; Iodine Isotopes; Lymphoma; Peptide Fragments; Peptide PHI; Pertussis Toxin; Protein Binding; Radioligand Assay; Receptors, Cell Surface; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Vasoactive Intestinal Peptide

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

Topics: 8,11,14-Eicosatrienoic Acid; Adenine; ADP Ribose Transferases; Antibodies; Carbazoles; Cells, Cultured; Drug Interactions; Electrophysiology; Enzyme Inhibitors; Gene Expression Regulation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Indoles; Kidney; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channel beta Subunits; Large-Conductance Calcium-Activated Potassium Channels; Potassium Channels; Potassium Channels, Calcium-Activated; Pyrroles

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells, where they regulate cardiac function during metabolic impairment. The present study analyzes the effects of extracellular ATP (ATPc), a P2-purinergic agonist that can be released under various conditions in the myocardial cell bed, on KATP current (IK-ATP) in rat ventricular myocytes. Under the whole-cell patch-clamp configuration at a physiological level of intracellular ATP, applying ATPc in the micromolar range did not activate IK-ATP. However, dialyzing the cell with a low-ATP (100 mumol/L) pipette solution elicited a slowly, quasilinearly increasing IK-ATP that was markedly enhanced by applying ATPe in the presence of a Purinergic antagonist. The effect was reversible on washing out the agonist. The IK-ATP enhancement was inhibited by cholera toxin treatment of the myocytes, suggesting that a Gs protein was involved to mediate the effect. Experiments on excised patches allowed us to exclude a membrane-delimited G protein-dependent pathway. Rather, the results suggested that ATPe activates the adenylyl cyclase, since its inhibition by 2'-deoxyadenosine 3'-monophosphate and SQ-22536, which respectively interact with the purine and catalytic site of the cyclase, strongly reduced the ATPe-induced IK-ATP enhancement, whereas neither compound affected IK-ATP in inside-out patches. Inhibition of cAMP-dependent protein kinase by protein kinase inhibitor peptide 5-24 did not alter the purinergic effect. The findings suggests that ATPe triggers the activation of adenylyl cyclase, which causes a subsarcolemmal ATP depletion sufficient to enhance IK-ATP as it develops during low-ATP dialysis of rat ventricular myocytes.

Topics: Adenine; Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Benzopyrans; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Colforsin; Cromakalim; Deoxyadenine Nucleotides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; GTP-Binding Proteins; Guanidines; Guanosine Triphosphate; Heart Ventricles; Male; Patch-Clamp Techniques; Peptide Fragments; Pinacidil; Potassium Channels; Pyrroles; Rats; Rats, Wistar; Uncoupling Agents

Extracellular guanosine, guanosine triphosphate (GTP), and 5'-N'-ethylcarboxamidoadenosine (NECA), each significantly enhanced the proportion of nerve growth factor (NGF)-treated rat pheochromocytoma (PC12) cells which had neurites, greater than that in cultures exposed to NGF alone. Guanosine and NECA, but not GTP, increased intracellular cAMP concentrations. An adenylate cyclase inhibitor, SQ22536, completely blocked the cAMP increase induced by both guanosine and 0.1 microM NECA. However, SQ22536 only partially blocked guanosine enhanced neurite outgrowth, although it completely blocked the neuritogenic effect of NECA. Therefore guanosine-enhanced neurite outgrowth through both cAMP-dependent and -independent mechanisms, while the effect of GTP was cAMP-independent.

Topics: Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Antineoplastic Agents; Colforsin; Cyclic AMP; Enzyme Inhibitors; Guanosine; Guanosine Triphosphate; Nerve Growth Factors; Neurites; PC12 Cells; Rats

The adenosine analogue 9-(Tetrahydro-2-furyl)adenine, SQ 22536, inhibited adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity of crude membrane preparations from catfish (Ictalurus melas) and rat isolated hepatocytes in a non-competitive manner. The IC50s were reduced in the presence of NaF. SQ 22536 reduced the activity of adenylate cyclase also in the presence of increasing concentrations of GTP, as well as Mg++ and Mn++. In the presence of catecholamines (epinephrine, norepinephrine, isoproterenol, phenylephrine) SQ 22536 reduced their activating effect on adenylate cyclase in both catfish and rat membranes. SQ 22536 also inhibited the effect of glucagon (0.1 microM) on rat membrane cyclase activity.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Cell Membrane; Cells, Cultured; Epinephrine; Female; Glucagon; Guanosine Triphosphate; Ictaluridae; Isoproterenol; Kinetics; Liver; Male; Norepinephrine; Phenylephrine; Rats; Sodium Fluoride; Species Specificity

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclase Inhibitors; Animals; Animals, Newborn; Brain; Cells, Cultured; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Membrane Potentials; Models, Neurological; Neurons; Pertussis Toxin; Potassium Channels; Rats; Signal Transduction; Substance P; Virulence Factors, Bordetella

Substance P excites neurons by suppressing inward rectification channels. We have investigated whether the substance P receptor interacts with the inward rectification channels through a guanine nucleotide-binding protein (G protein) by using dissociated cultured neurons from the nucleus basalis of newborn rats. During intracellular application of guanosine 5'-[gamma-thio]triphosphate and 5'-guanylyl imidodiphosphate, hydrolysis-resistant GTP analogues that irreversibly stimulate G proteins, substance P application almost irreversibly suppressed the inward rectification channels. Pretreatment with pertussis toxin did not significantly influence substance P action. Intracellular application of cAMP and 3-isobutyl-1-methylxanthine or of 9-(tetrahydro-2-furyl)adenine (SQ 22,536), an inhibitor of adenylate cyclase, did not alter the substance P-induced response. We conclude that the inhibition of inward rectification channels by substance P is mediated through a G protein. However, the effect is not mediated through adenylate cyclase or the cAMP system. This G protein, which is insensitive to pertussis toxin, could be an unidentified G protein.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylate Cyclase Toxin; Adenylyl Cyclase Inhibitors; Animals; Animals, Newborn; Brain; Cells, Cultured; Cyclic AMP; GTP-Binding Proteins; Guanosine Triphosphate; Ion Channels; Neurons; Pertussis Toxin; Potassium; Rats; Somatostatin; Substance P; Virulence Factors, Bordetella