In this study, we investigated which subtype of GTP-binding protein (G protein) is related to muscarinic activation of nonselective cation (NSC) channels in gastric smooth muscle. Inward cationic current was activated by the application of 50 microM carbachol (ICCh) at a holding potential of -60 mV with the same CsCl-rich solution in both pipette and bath. The same cationic current as ICCh was slowly activated by the dialysis of guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) through the pipette. Since it is known that pertussis toxin pretreatment can block ICCh, antibodies (Abs) against Galpha,i (anti-Galpha,i) or Galpha,o (anti-Galpha,o) were tested. Activation of ICCh was blocked by the addition of anti-Galpha,o. However, anti-Galpha,i Abs had no significant effect on ICCh. The expression of Galpha,o in guinea-pig gastric smooth muscle was confirmed by Western immunoblot analysis. These results suggest that Go-type protein may mediate signals from the muscarinic receptor to NSC channel in guinea-pig gastric myocytes.
The beta and gamma subunits of heterotrimeric GTP-binding proteins (Gbetagamma) were found to bi-directionally regulate the UV-induced activation of p38 and c-Jun NH(2)-terminal kinase, and the UV-induced activation of p38 was reported to enhance the resistance of normal keratinocytes to apoptosis. However, the signaling pathway downstream of Gbetagamma for this UV-induced p38 activation is not known. Thus, we examined the role of the Rho GTPase family in the regulation of UV-induced p38 activation by Gbetagamma. We found that overexpression of Gbetagamma increased the UV-induced activation of Cdc42 and that overexpression of constitutively active V12 Cdc42 increased the UV-induced p38 activation. Transfection of dominant negative N17 Cdc42 or small interfering RNA for Cdc42 blocked UV-induced p38 activation mediated by Gbetagamma in COS-1 and HaCaT cells. UV-induced p38 activation by Gbetagamma was blocked by overexpression of dominant negative p21-activated kinase (PAK)-interacting exchange factor beta (betaPix), and wild type betaPix stimulated the UV-induced p38 activation, which was blocked by N17 Cdc42. Gbetagamma increased the UV-induced activation of Ras, and the overexpression of V12 Ras increased UV-induced p38 activation, which was blocked by dominant negative betaPix. UV-induced p38 activation was inhibited by N17 Ras and a farnesyltransferase inhibitor, manumycin A. Gbetagamma also increased the UV-induced phosphorylation of the epidermal growth factor receptor (EGFR), and the UV-induced p38 activation was blocked by an EGFR kinase inhibitor, AG1478. From these results, we conclude that Gbetagamma mediates UV-induced activation of p38 in a Cdc42-dependent way and that EGFR, Ras, and betaPix act sequentially upstream of Cdc42 in COS-1 and HaCaT cells.
Ultraviolet (UV) irradiation induces various cellular responses by activating many UV-responsive enzymes including mitogen-activated protein kinases (MAPKs). Various G protein-coupled receptor agonists also activate MAPKs, but it is not known whether or not G proteins also mediate the UV-induced activation of MAPKs. Therefore, this study was undertaken to determine whether the G protein ␥-subunit (G␥) mediates the UV-induced activation of p38 and JNK. G␥ overexpression in COS-1 cells amplified the UV-induced activation of p38 but reduced JNK activation. The overexpression of the C-terminal region of -adrenergic receptor kinase (ARKct) decreased the UV-induced activation of p38 but increased JNK activation. G 1 ␥ 2 expression increased MKK3/6 phosphorylation with a concomitant decrease in MKK4 phosphorylation, which contrasts with ARKct expression. G 1 ␥ 2 or ARKct expression resulted in corresponding changes in the transcriptional activity of CHOP and c-Jun. Treatment with a p38 inhibitor, SB203580, or the expression of a kinase-inactive p38 increased the UV-induced JNK activation. Expression of the constitutively active MKK6 decreased the UV-induced JNK activation. In summary, although the endogenous G␥ was found to mediate about half of the UV-induced activation of p38, it was found that exogenous G␥ mediates the bi-directional regulation of UV-induced p38 and JNK activation, and that this bidirectional regulation results from the inhibition of JNK activation by the p38 activated via G␥ in the COS-1 cells.
Heart disease is one of the major cause of death in diabetic patients, but the pathogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to assess the significance of G protein signaling pathways in the pathogenesis of diabetic cardiomyopathy, we analyzed the expression of G proteins and the activities of second messenger dependent protein kinases: cAMP-dependent protein kinase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent protein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat heart. The expression of Gα α α αq was increased by slightly over 10% (P<0.05) in diabetic rat heart, while Gα α α αs, Gα α α αi, and Gβ β β β remained unchanged. The PKA activity in the heart did not change significantly but increased by 27% (P<0.01) in the liver. Insulin treatment did not restore the increased activity in the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and insulin treatment did not restore such increase. The CaM kinase II activity in the heart remained at the same level but was slightly increased in the liver (14% increase, P<0.05). These findings of increased expression of Gα α α αq in the streptozotocin-diabetic rat heart that are reflected by the increased level of PKC activity and insensitivity to insulin demonstrate that alteration of Gα α α αq may underlie, at least partly, the cardiac dysfunction that is associated with diabetes.
Electroconvulsive shock (ECS) has been suggested to affect cAMP signaling pathways to exert therapeutic effects. ECS was recently reported to increase the expression of PDE4 isoforms in rat brain, however, these studies were limited to PDE4 family in the cerebral cortex and hippocampus. Thus, for comprehensive understanding of how ECS regulates PDE activity, the present study was performed to determine whether chronic ECS treatment induces differential changes in the expression of all the PDE isoforms in rat brains. We analyzed the mRNA expression of PDE isoforms in the rat hippocampus and striatum using reverse transcription polymerase chain reaction. We found chronic ECS treatment induced differential changes in the expression of PDE isoform 1, 2, 3, 4, 5 and 7 at the rat hippocampus and striatum. In the hippocampus, the expression of PDE1A/B (694%), PDE4A (158%), PDE4B (323 %), and PDE4D (181%) isoforms was increased from the controls, but the expression of PDE2 (62.8%) and PDE7 (37.8%) decreased by chronic ECS treatment. In the striatum, the expression of PDE1A/B (179%), PDE4A (223%), PDE4B (171%), and PDE4D (327%) was increased by chronic ECS treatment with the concomitant decrease in the expression of PDE2 (78.4%) and PDE3A (67.1%). In conclusion, chronic ECS treatment induces differential changes in the expression of most PDE isoforms including PDE1, PDE2, PDE3, PDE4, PDE5, and PDE7 in the rat hippocampus and striatum in an isoform-and brain region-specific manner. Such differential change is suggested to play an important role in regulation of the activity of PDE and cAMP system by ECS.
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