Modulation of the inhibitory action of ATP on acetylcholine-activated current by protein phosphorylation in rat sympathetic neurons

Modulation by protein phosphorylation of the relation between acetylcholine (ACh)-activated current (I _ACh) and adenosine triphosphate-(ATP)-activated current (I _ATP) was investigated with the whole-cell voltage-clamp technique in rat sympathetic neurons. During simultaneous activation by 100 M ATP of an inward current, the current evoked by 100 M ACh was reduced to 60–70% of that in the absence of ATP. Effects of compounds that are known to modulate protein phosphorylation were tested by including them in the intracellular solution. The reduction ofI _ACh by ATP was not observed when K252a (1 M), a non-selective protein kinase inhibitor, adenosine 5-O-(3-thiotriphosphate) (ATP[S], 1 mM) or,-methylene ATP (1 mM) were included in the intracellular solution. Activators of protein kinases, adenosine 3,5-cyclic monophosphate (cAMP, 100 M), guanosine 3,5-cyclic monophosphate (cGMP, 100 M), phorbol 12-myristate 13-acetate (PMA, 1 M), also abolished the reduction by ATP ofI _ACh. The effects of okadaic acid, a protein phosphatase inhibitor, were paradoxical: okadaic acid (2 M) itself abolished the reduction by ATP ofI _ACh but it antagonized the abolishment by cAMP or cGMP of the reduction ofI _ACh. Okadaic acid did not affect the disappearance of the reduction ofI _ACh by ATP in the presence of intracellular PMA. The results suggest that the interaction betweenI _ACh andI _ATP is regulated by protein phosphorylation/dephosphorylation. Possible mechanisms underlying the effects of these modulators of protein phosphorylation are discussed.     

Skinned smooth muscle: calcium-calmodulin activation independent of myosin phosphorylation

In chemically skinned chicken gizzard smooth muscle fibers investigated shortly after preparation, a contraction may be induced by calcium and calmodulin which is independent of myosin phosphorylation at intermediate Ca²⁺-concentrations. However, fibers stored for a prolonged period also contract in the absence of exogenous calmodulin and exhibit a close relationship between force development and myosin phosphorylation.     

失血性休克对大鼠血管平滑肌BKCa通道酪氨酸磷酸化水平的影响

目的:探讨失血性休克对大鼠肠系膜上动脉血管平滑肌BK_Ca通道酪氨酸磷酸化的影响以及NO对BK_Ca通道酪氨酸磷酸化的调控。方法:建立大鼠失血性休克模型[(35±5)mmHg]并提取肠系膜上动脉总裂解蛋白,采用免疫沉淀及免疫印迹技术,观察休克血管平滑肌BK_Ca通道酪氨酸磷酸化的变化情况;采用原代培养的大鼠肠系膜上动脉血管平滑肌细胞,观察NO对BK_Ca通道酪氨酸磷酸化的调控及其时-效、量-效关系。结果:失血性休克2 h及4 h后大鼠肠系膜上动脉血管平滑肌BK_Ca通道α亚基酪氨酸磷酸化明显增强 (P＜0.01 );L-精氨酸(5×10^-5-5×10^-4 mol/L)孵育30 min即可诱导培养血管平滑肌细胞BK_Ca通道α亚基酪氨酸磷酸化增强,2 h内无明显下降;且L-精氨酸诱导BK_Ca通道α亚基酪氨酸磷酸化具有剂量依赖性。结论:重症失血性休克可增强血管平滑肌BK_Ca 通道酪氨酸磷酸化,且NO参与了该调控过程。     

Inhibitory effect of ketamine on phosphorylation of the extracellular signal-regulated kinase1/2 following brain ischemia and reperfusion in rats with hyperglycemia

To determine if the inhibitory effects of ketamine on the extracellular signal-regulated kinase (ERK) 1/2 are involved in reduction of the hyperglycemia-exaggerated cerebral ischemic lesion, rats with normoglycemia, hyperglycemia, or hyperglycemia supplemented with ketamine were subjected to 15 min of forebrain ischemia, and then, reperfusion for 0.5, 1, and 3h. Phosphorylation of ERK1/2 in the brain tissues was assessed by immunohistochemistry and Western blot analysis. In rats with normoglycemia, we demonstrated a moderate increase of the ERK1/2 phosphorylation in the cingulum cortex and hippocampus CA3 following an ischemic intervention. It quickly dropped to control levels after reperfusion for 0.5h. In rats with hyperglycemia, however, the increase of the ERK1/2 phosphorylation in these areas was significantly higher in all animals reperfused. The neuronal death, detected by the TdT-mediated-dUTP nick end labeling assays, was found in the cingulum cortex (5.23+/-2.34, per high power feild) and hippocampus CA3 areas (6.29+/-3.68, per 1mm(2)) in hyperglycemic group after reperfusion for 3h. With ketamine treatment, the ERK1/2 phosphorylation in cingulum cortex and hippocampus CA1 and CA3 areas was found to be the same as that in normoglycemia rats. Our results suggest that hyperglycemia may increase the ischemic insult through modulation of the signal transduction pathways involving ERK1/2. The inhibitory effects of ketamine on the hyperglycemia-activated ERK1/2 phosphorylation are probably through inhibition of the N-methyl d-aspartate-mediated calcium influx, which subsequently reduce the hyperglycemia-exaggerated cerebral damage.     

Expression of early activation antigen (CD69) during human thymic development.

The novel early activation antigen, EA1, has been shown to be induced by mitogens, antigens and the tumour promoter, phorbol myristate acetate (PMA), on human lymphocytes. This antigen has been designated to be CD69. EA1 has also been shown to be expressed on thymocytes without exogenous activation stimuli. In order to characterize further the expression of EA1 on thymocytes, the ontogeny of its expression was studied. EA1 appeared between 7 and 9.5 weeks of gestation, after colonization of the thymic rudiment with CD7+ T cell precursors, but before the onset of compartmentalization of the thymus into cortical and medullary zones. After cortico-medullary differentiation, the majority of medullary thymocytes expressed EA1 while only a fraction of the cortical thymocytes expressed this antigen. In the fetal and post-natal cortex, EA1 expression appeared to cluster in the subcapsular cortex. EA1+ cells were also scattered throughout the inner cortex. By two-colour fluorocytometric analysis of post-natal thymocytes, it was shown that EA1 was expressed on 30 to 65% of thymocytes. EA1 was expressed on CD4+ CD8+ as well as on the more immature CD4- CD8- thymocytes. In contrast to circulating T cells, thymocytes were much less responsive to PMA stimulation for the expression of EA1. Molecular characterization showed that EA1 on thymocytes had the same structure as that of activated peripheral T cells. In addition, thymic EA1 was constitutively phosphorylated. Thus, EA1 expression is acquired early during thymic development after colonization of the thymic rudiment by CD7+ T cell precursors. However, the specific role that EA1 may play in the activation and function of developing thymocytes remains to be determined.     

Reduced protein tyrosine phosphatase (PTPase) activity of CD45 on peripheral blood lymphocytes in patients with systemic lupus erythematosus (SLE)

To disclose the mechanism of aberrant function of peripheral blood lymphocytes (PBL) in SLE, we focused on the catalytic function of CD45, and determined the CD45 PTPase activity in SLE patients. The sample population consisted of 32 SLE patients with different disease activity. PTPase activity of cell lysates immunoprecipitated by anti-CD45 MoAb was assayed against phosphotyrosine analogue PNPP, followed by measuring the release of para-nitro phenol at 410 nm. CD45 PTPase activity of PBL was significantly decreased in SLE patients, compared with that of normal controls and patients with systemic sclerosis (964 ± 265, 1202 ± 172, 1210 ± 125, respectively; SLE versus normal, P < 0.05). It was correlated with SLE Disease Activity Index (SLEDAI) score (r = 0.597, P = 0.0006), but not with the dose of prednisolone (r = 0.214, P = 0.2657), indicating that CD45 PTPase activity became reduced when the disease was active, but it was not affected by prednisolone. Moreover, it was not corrected by in vitro culture with or without stimulation. The expression of CD45 on PBL was comparable between normal and SLE, raising a possibility that it may be due to aberrant regulation of catalytic function of CD45 in SLE. Given the evidence that tyrosine phosphorylation of cellular proteins by tyrosine kinases and phosphatases is one of the key biochemical events in the signal transduction pathway, the decreased CD45 PTPase activity in SLE may account for the defective signal transduction via TCR/CD3, leading to dysregulated effector function of the lymphocytes.     

Rapid tyrosine phosphorylation of Grb2 and Shc in T cells exposed to anti-CD3, anti-CD4, and anti-CD45 stimuli: differential effects of aging

Two adapter proteins, Grb2 and Shc, have recently been implicated in the transmission of activation signals from the stimulated T cell receptor to Ras. We show here that in vitro stimulation of mouse splenic T cells with crosslinked anti-CD3 antibody leads within 30 s to phosphorylation of both Grb2 and Shc. Treatment with crosslinked anti-CD45 antibody leads to phosphorylation of Grb2 and also to a slight retardation in the mobility of this protein in an SDS polyacrylamide gel; both changes are seen within 30 s of crosslinking. Crosslinked anti-CD4 antibody leads to phosphorylation of Shc and to the phosphorylation of a 30-kDa protein that cross-reacts with anti-Grb2 antibodies. Aging leads to a decline in CD3-stimulated phosphorylation of Shc (but not Grb2), and to an increase in CD4-stimulated phosphorylation of Grb2, Shc, and the 30-kDa Grb2-like protein. Increased tyrosinephosphorylation of Grb2 after exposure to either anti-CD3 or anti-CD45 suggests that Grb2 may be a common substrate for both CD3-linked kinases and the CD45 phosphatase. The differences between T cells from young and old mice suggest that aging may lead to a set of alterations in kinase/substrate coupling that contribute to immune dysfunction in the elderly, and that activation of the Ras pathway might be impaired by aging in T lymphocytes.     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.