h-89 and Pheochromocytoma

Cyclooxygenase-2 (COX-2) plays crucial roles in the development and invasion of tumors. Celecoxib, a selective COX-2 inhibitor, has been shown to be chemopreventive against cancer. However, to date, the mechanisms of these effects remain unclear. In this study, we investigate the effects of celecoxib on voltage-gated calcium channel (VGCC) currents in undifferentiated pheochromocytoma (PC12) cells using whole-cell patch clamp. Our results showed that celecoxib, instead of rofecoxib or NS-398, another selective COX-2 inhibitor, reversibly inhibited the current density of VGCC in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 microM), an L-type calcium channel blocker. Upon pre-incubation of PC12 cells with omega-conotoxia GVIA (1 microM), an N-type calcium channel blocker, omega-agatoxin IVA (1microM), a P/Q-type calcium channel blocker, or SNX-482 (1microM), a R-type calcium channel blocker, celecoxib (1microM) inhibited the currents by 36%, 28%, and 25%, respectively. Celecoxib up-shifted the current-voltage (I-V), and hyperpolarizedly shifted the inactivation curve, but did not markedly affect the activation curve. Intracellular application of H89, a protein kinase A inhibitor, failed to affect the celecoxib's VGCC currents inhibition. Taken together, our present results suggested that celecoxib inhibited L-type calcium channels in PC12 cells via a COX-2 independent pathway, which might be responsible for its clinical effects including anti-tumor.

Topics: Adrenal Gland Neoplasms; Animals; Antineoplastic Agents; Barium; Calcium Channel Blockers; Calcium Channels, L-Type; Celecoxib; Cell Proliferation; Cyclooxygenase 2 Inhibitors; Dose-Response Relationship, Drug; Isoquinolines; Lactones; Membrane Potentials; Nifedipine; Nitrobenzenes; omega-Agatoxin IVA; omega-Conotoxin GVIA; PC12 Cells; Pheochromocytoma; Protein Kinase Inhibitors; Pyrazoles; Rats; Spider Venoms; Sulfonamides; Sulfones

We have previously shown that prolactin-releasing peptide (PrRP) stimulates catecholamine release from PC12 cells (rat pheochromocytoma cell line). However, it is not known whether PrRP also affects catecholamine biosynthesis. Thus, we examined the effect of PrRP on catecholamine biosynthesis in PC12 cells. PrRP31 (>10 nM) and PrRP20 (>100 nM) significantly increased the activity and expression level of tyrosine hydroxylase (TH), a rate-limiting enzyme, in catecholamine biosynthesis. However, the PrRP20-stimulated TH activity was markedly weaker than that of PrRP31. PrRP31 (>1 nM) and PrRP20 (>10 nM) significantly induced an increase in the level of PKC activity. Both Ro 32-0432 (a protein kinase C inhibitor) and H89 (a protein kinase A inhibitor) effectively suppressed the PrRP31 (100 nM)-induced TH mRNA level. Next, we examined the effect of PrRP on mitogen-activated protein kinases (MAPKs). PrRP31 (1 microM) significantly induced an increase in the activity of extracellular signal-related kinases (ERKs) and the stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK). In contrast to ERKs and JNK, PrRP31 did not affect P38 MAPK activity. Consistent with these findings, pretreatment of cells with the MEK-1-inhibitor, PD-98059 (50 microM), significantly inhibited the PrRP31 (100 nM)-induced increase in TH mRNA. These results indicate that PrRP stimulates catecholamine synthesis through both the PKC and PKA pathways in PC12 cells.

Topics: Animals; Catecholamines; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Flavonoids; Hypothalamic Hormones; Indoles; Isoquinolines; MAP Kinase Signaling System; Neuropeptides; Pheochromocytoma; Prolactin-Releasing Hormone; Protein Kinase C; Pyrroles; Rats; RNA, Messenger; Signal Transduction; Stimulation, Chemical; Sulfonamides; Tyrosine 3-Monooxygenase

We examined the effect of prolactin-releasing peptide (PrRP) on catecholamine secretion and DNA synthesis in rat pheochromocytoma PC12 cells. We initially confirmed the expression of both PrRP and its receptor in PC12 cells. PrRP31 and PrRP20 (> or =10 nM) significantly increased dopamine secretion from PC12 cells. However, PrRP20-stimulated dopamine secretion was markedly weaker than that of PrRP31. Both EDTA (extracellular Ca2+ chelator) and BAPTA-AM (intracellular Ca2+ chelator) effectively suppressed PrRP31 (100 nM)-induced dopamine secretion. PrRP31and PrRP20 (> or =1 nM) significantly induced an increase in the level of cAMP. The PKA inhibitor H89 (at 10 microM) impeded PrRP31- and PrRP20-induced dopamine secretion. Finally, we confirmed that PrRP did not affect DNA synthesis. These results indicate that PrRP may regulate catecholamine secretion but not the mitogenic effects in chromaffin cells.

Topics: Animals; Blotting, Northern; Catecholamines; Chelating Agents; Cyclic AMP; Dose-Response Relationship, Drug; Drug Interactions; Edetic Acid; Egtazic Acid; Enzyme Inhibitors; Hypothalamic Hormones; Isoquinolines; Neuropeptides; Nicotine; Nicotinic Agonists; PC12 Cells; Pheochromocytoma; Prolactin-Releasing Hormone; Rats; Receptors, Neuropeptide; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sulfonamides

Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A2A receptor.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Adenosine; Adrenal Gland Neoplasms; Animals; Blotting, Western; Caffeine; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Enzyme Activation; Enzyme Inhibitors; Isoquinolines; Kinetics; PC12 Cells; Phenethylamines; Pheochromocytoma; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Polymerase Chain Reaction; Purinergic P1 Receptor Agonists; Rats; Receptor, Adenosine A2A; Receptors, Purinergic P1; Recombinant Proteins; Sulfonamides

Topics: Adenylyl Cyclases; Adrenal Gland Neoplasms; Animals; Chloramphenicol O-Acetyltransferase; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Gene Expression; Isoquinolines; Kinetics; Naphthalenes; Neuropeptide Y; PC12 Cells; Pheochromocytoma; Polycyclic Compounds; Promoter Regions, Genetic; Protein Kinase C; Rats; Sulfonamides; Transcription, Genetic

A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.

Topics: Adrenal Gland Neoplasms; Animals; Axons; Bucladesine; Cell Line; Colforsin; Isoquinolines; Kinetics; Neoplasm Proteins; Nerve Growth Factors; Pheochromocytoma; Phosphorylation; Protein Kinase Inhibitors; Sulfonamides

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adrenal Gland Neoplasms; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin-Binding Proteins; Cell Differentiation; Isoquinolines; Neoplasm Proteins; Pheochromocytoma; Phosphorylation; Piperazines; Protein Binding; Protein Kinase Inhibitors; Protein Kinases; Protein Processing, Post-Translational; Rats; Second Messenger Systems; Substrate Specificity; Sulfonamides; Tumor Cells, Cultured