endothelin-1 and 9-(tetrahydro-2-furyl)-adenine

Endothelin-1 (ET-1) affects glucose uptake in adipocytes and may play an important role in adipose physiology. One of the principal functions of adipose tissue is the provision of energy substrate through lipolysis. In the present study, we investigated the effects of ET-1 on lipolysis in 3T3-L1 adipocytes. When glycerol release in the culture medium was measured as an index of lipolysis, the results showed that ET-1 caused a significant increase that was time and dose dependent. With a concentration of 10 nM ET-1, stimulation of glycerol release plateaued after 4 h of exposure. This effect was inhibited by the ETA receptor antagonist BQ-610 (10 microM) but not by the ETB receptor antagonist BQ-788 (10 microM). To further explore the underlying mechanisms of ET-1 action, we examined the involvement of the cAMP-dependent protein kinase A-mediated, phospholipase A2 (PLA2)-mediated, protein kinase C (PKC)-mediated, phosphatidylinositol 3 (PI 3)-kinase-mediated, and the mitogen-activated protein kinase (MAPK)-mediated pathways. Inhibition of adenylyl cyclase activation by SQ-22536 (100 microM) did not block ET-1-induced lipolysis. Pretreatment of adipocytes with the PLA2 inhibitor dexamethasone (100 nM), the PKC inhibitor H-7 (6 microM), or the PI 3-kinase inhibitor wortmannin (100 nM) also had no effect. ET-1-induced lipolysis was blocked by inhibition of extracellular signal-regulated kinase (ERK) activation using PD-98059 (75 microM), whereas a p38 MAPK inhibitor (SB-203580; 20 microM) had no effect. Results of Western blot further demonstrated that ET-1 induced ERK phosphorylation. These data show that ET-1 induces lipolysis in 3T3-L1 adipocytes via a pathway that is different from the conventional cAMP-dependent pathway used by isoproterenol and that involves ERK activation.

Topics: 3T3-L1 Cells; Adenine; Adipocytes; Adipose Tissue; Animals; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Glycerol; Lipolysis; Mice; Mitogen-Activated Protein Kinases; Oligopeptides; Phosphatidylinositol 3-Kinases; Phospholipases A; Phospholipases A2; Piperidines; Protein Kinase C; Receptor, Endothelin A

PTHrP has important roles in lung development and function. Here we determined the vasomotor responses of isolated pulmonary arteries and veins of newborn and adult sheep to PTHrP. In vessels constricted with endothelin-1, PTHrP (PTHrP 1-34) caused greater relaxation of veins than of arteries. In both vessel types, relaxation to the peptide was less in adult than in newborn vessels. In newborn lambs, PTHrP-induced relaxation was not affected by endothelium removal, inhibition of eNOS, or inhibition of adenylyl cyclases by SQ-22536. However, relaxation was attenuated by 4-aminopyridine, inhibitor of voltage-dependent potassium channels, in both arteries and veins, and by charybdotoxin, inhibitor of calcium-activated potassium channels, in veins. When vessels were saturated with 8-BrcAMP (3 x 10(-4) M), to eliminate relaxation mediated by endogenous cAMP, PTHrP-induced relaxation was partially attenuated. In vessels treated with 8-BrcAMP (3 x 10(-4) M), 4-aminopyridine but not charybdotoxin inhibited relaxation induced by PTHrP 1-34 in both arteries and veins. Radioimmunoassay showed that, in the presence of a general phosphodiesterase inhibitor, PTHrP caused a concentration-dependent increase in intracellular cAMP content in arteries and veins, which was largely abolished by SQ-22536. Our results demonstrate that PTHrP is a potent vasodilator of pulmonary vessels, with a greater effect in veins than in arteries. Relaxation induced by the peptide contains both cAMP-dependent and -independent components. In both arteries and veins, voltage-dependent potassium channels mediate the response to PTHrP, at least in part, in a cAMP-independent fashion; and in veins, calcium-activated potassium channels may be stimulated by elevated cAMP levels.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Animals; Animals, Newborn; Charybdotoxin; Cyclic AMP; Endothelin-1; Endothelium, Vascular; Enzyme Inhibitors; Lung; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Organ Culture Techniques; Parathyroid Hormone-Related Protein; Potassium Channels, Calcium-Activated; Potassium Channels, Voltage-Gated; Pulmonary Artery; Pulmonary Veins; Sheep, Domestic; Vasodilation

The role of cAMP in forskolin-induced relaxation was studied in isolated pulmonary veins of newborn lambs (7-12 days). In vessels preconstricted with endothelin-1, forskolin at concentrations < or =10(-7) M had no effect on cAMP content and adenylyl cyclase activity but caused up to 50% relaxation. At higher concentrations, forskolin markedly elevated cAMP content and adenylyl cyclase activity and caused a further relaxation. SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine; an adenylyl cyclase inhibitor] and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide; a calmodulin-dependent adenylyl cyclase inhibitor] had no significant effect on forskolin-induced relaxation but markedly inhibited the elevation of cAMP content and adenylyl cyclase activity caused by forskolin. Rp-8-CPT-cAMPS [8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate; an inhibitor of cAMP-dependent protein kinases] and Rp-8-Br-PET-cGMPS (beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate; an inhibitor of cGMP-dependent protein kinases) attenuated the relaxation caused by a cAMP analog but not that caused by forskolin. These results suggest that cAMP may not play a major role in forskolin-induced relaxation of pulmonary veins of newborn lambs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenine; Adenylyl Cyclases; Animals; Animals, Newborn; Cell Membrane; Colforsin; Cyclic AMP; Cyclic GMP; Endothelin-1; Enzyme Induction; Female; Male; Muscle, Smooth, Vascular; Pulmonary Veins; Sheep; Sulfonamides; Thionucleotides; Time Factors; Vasodilation

Tyrphostin-23 is commonly used as inhibitor of tyrosine kinase (TK). We found that tyrphostin-23 concentration-dependently increased basal steroid-hormone secretion from dispersed human and rat adrenocortical cells, the maximal effective concentration being 10(-5) M. Tyrphostin-23 (10(-5) M) enhanced 10(-9) M angiotensin-II- and endothelin-1-stimulated secretion of human and rat adrenocortical cells, but not the secretory response to 10(-9) M ACTH However, it increased the response to lower concentrations (10(-12) or 10(-11) M) of ACTH. The secretagogue effect of tyrphostin-23 on dispersed rat adrenocortical cells was abolished by either the adenylate cyclase inhibitor SQ-22536 (10(-4) M) or the protein kinase A (PKA) inhibitor H-89 (10(-5) M). Tyrphostin-23 (10(-5) M) raised basal cyclic-AMP release by dispersed rat adrenocortical cells, but in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 10(-3) M) it was ineffective. Both tyrphostin-23 and IBMX increased cyclic-AMP release by rat adrenocortical cells in response to 10(-10) M ACTH, and their effects were not additive. Taken together, our findings suggest that tyrphostin-23, acting as an inhibitor of phosphodiesterases in adrenocortical cells, increases the intracellular concentration of cyclic-AMP available for PKA activation thereby stimulating steroid-hormone secretion. They also stress that caution must be used in interpreting the results of studies aimed at investigating the possible cross-talk between adenylate cyclase- and TK-dependent signaling cascades.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylyl Cyclase Inhibitors; Adrenal Cortex; Adrenal Cortex Hormones; Adrenocorticotropic Hormone; Aldosterone; Angiotensin II; Animals; Corticosterone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Enzyme Inhibitors; Humans; Hydrocortisone; Isoquinolines; Male; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Sulfonamides; Tyrphostins

The role of cAMP and cAMP-dependent protein kinase A (PKA) in endothelin (ET)-stimulated migration of human neutrophils was studied. Endothelins caused an increase in neutrophil migration when they were applied in low (nanomolar) concentrations; stimulation of migration was either predominantly chemokinetic (ET-1) or chemotactic (ET-2, ET-3). All endothelins, at concentrations which gave maximal stimulation of migration, caused an increase of cAMP level. Two inhibitors of adenylate cyclase, MDL-12330A and SQ-22536, completely inhibited migration activated by ET-1, ET-2 or ET-3, indicating that cAMP generation played a decisive role in endothelin-activated migration. The role of PKA in endothelin-activated migration was considered. Two specific antagonists of PKA strongly inhibited endothelin-activated migration. KT-5720, an inhibitor of PKA, also inhibited ET-activated migration, but only when electroporated cells were used. The results suggest that the effect of cAMP on endothelin-activated migration was mediated by PKA.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Carbazoles; Cell Movement; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Endothelin-2; Endothelin-3; Endothelins; Enzyme Inhibitors; Humans; Imines; Indoles; Neutrophils; Pyrroles; Thionucleotides