Tingchung Wang

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

Abstract The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of 32 P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.

The shift of an increase in phosphofructokinase activity from protein synthesis-dependent to -independent mode during concanavalin A induced lymphocyte proliferation

Abstract Phosphofructokinase activity increased dramatically in cultured mouse spleen lymphocytes 8 hours after concanavalin A stimulation and preceded the onset of DNA synthesis by 8 hours. The increase in enzyme activity and [ 3 H]-thymidine incorporation were mitogen-concentration dependent. Enzyme activity increased 12-fold over control level at 48 hours when DNA synthesis peaked. The protein synthesis inhibitor, cycloheximide, blocked the rise in phosphofructokinase only when given prior to the increase in enzyme activity. Once the increase began, later addition of cycloheximide became progressively less inhibitory. These observations suggest that the period of increase in phosphofructokinase activity involves the activation of preexisting enzyme molecules.

Increased phosphorylation of a specific protein in mitogen-stimulated lymphocytes☆

The phosphorylation of proteins from murine splenic lymphocytes was studied. When the phosphorylation of proteins in extracts from Concanavalin A (ConA)-treated lymphocytes was compared with that of resting lymphocytes, there was only one detectable difference between them. A protein of 135,000 mol. wt was highly phosphorylatable in extracts from ConA-treated cells while phosphate incorporation into this protein was slight in extracts from untreated cells. This effect could be observed 12 h after ConA treatment and was maximal during S phase. This soluble protein was also phosphorylated in intact lymphocytes.

Inhibition of spermine biosynthesis and thymidine incorporation in concanavalin a transformed lymphocytes by S-adenosyl-(±)-2-methylmethionine

Summary S-Adenosyl-(±)-2-methylmethionine, an inhibitor of mammalian S-adenosyl-L-methionine decarboxylase, inhibited the increase in [ 3 H]-thymidine incorporation but did not interfere with the rise in [ 3 H]-uridine and [ 3 H]-leucine incorporation induced by concanavalin A in cultured mouse lymphocytes. Polyamine synthesis which increases during lymphocyte proliferation, was also selectively inhibited. Cellular levels of putrescine and spermidine increased despite the presence of the inhibitor but the rise in spermine concentration was blocked. Moreover, 24 hours after the addition of S-adenosyl-(±)-2-methylmethionine and concanavalian A to lymphocyte cultures, a concentration-dependent decrease in both [ 3 H]-thymidine incorporation and cellular spermine concentration was observed with dose-dependent increases in the other two polyamines. This is the first study in which selective inhibition of spermine synthesis also blocked DNA synthesis suggesting that this polyamine may participate in the regulation of proliferation in lymphocytes.

Enhanced cardiac efficiency with dobutamine after global ischemia

Dopamine and dobutamine are used in low output states following cardiopulmonary bypass but the consequences of increased inotropic activity on myocardium recovering from ischemia is unknown. Dogs on cardiopulmonary bypass were subjected to 20 min of normothermic global ischemia followed by 20 min of reperfusion. Dopamine or dobutamine (both at 10 μg/kg/min) or normal saline infusion was begun and 10 min later the dogs weaned from cardiopulmonary bypass while the infusions continued. Serial measurements were made of regional myocardial and systemic blood flow (15 μm radiolabeled spheres), myocardial oxygen consumption, creatine phosphate, and ATP levels. On bypass mean aortic pressure was decreased and heart rate, oxygen consumption, and left ventricular blood flow were increased by both catecholamine infusions (P < 0.01), but neither drug lowered ATP or creatine phosphate levels. Renal blood flow was decreased in dobutamine-treated dogs (P < 0.01). Off bypass, heart rate and mean aortic pressure were similar in all groups. While both drugs increased left ventricular blood flow to a similar extent (P < 0.01), dopamine treatment raised cardiac output by only 30% (P < 0.05) and dobutamine treatment increased cardiac output by 85% (P < 0.01). In addition, myocardial oxygen consumption was increased in dopamine-treated dogs (P < 0.05) while values in dobutamine animals were similar to controls. Therefore, dobutamine seems advantageous to dopamine following bypass because it increases cardiac output (by increasing stroke volume) but does not increase myocardial oxygen consumption. Both drugs are potentially detrimental on bypass because they greatly increase heart rate and oxygen consumption and, in addition, dobutamine causes an unexplained fall in renal blood flow.

Effects of dopamine and dobutamine on the myocardial and systemic circulation during and following cardiopulmonary bypass in dogs

SummaryThe effect of dopamine (Dp) and dobutamine (Db) on myocardial and systemic blood flow (BF) distribution was compared in dogs being weaned from cardiopulmonary bypass (CPB) after 20 min of normothermic global myocardial ischemia. Drug infusions (10 mcg/kg/min) were begun and BF was measured (radiolabeled microspheres) prior to weaning and 60 min off CPB.On CPB: Pump flows, by design, were similar (100 ml/kg/min) in Dp, Db, and saline control (NS) dogs. Dp and Db significantly (p<0.05) increased myocardial BF. Dp did not alter renal, visceral, and skeletal muscle BF and only increased BF to the cervical spinal cord and medulla. Db, however, significantly reduced renal (−49%), splenci (−58%), pancreatic (−27%), and colonic (−47%) BF but increased perfusion in essentially all of the central nervous system.Off CPB: Cardiac output during Dp and Db infusions was significantly greater than NS dogs (107±7 vs 152±12 vs 82±10 ml/kg/min resp.); the greater increase for Db resulting from a larger stroke volume. Dp and Db significantly increased myocardial BF. Dp increased splenic (+77%), gastric (+139%), and gallbladder (+125%) BF but had no effect on renal, hepatic, skeletal muscle, and intestinal BF. Db infusion maintained renal BF similar to NS and elevated BF to most visceral organs. The results of this study show that the myocardium responded to inotropic stimulation despite the previous ischemic insult but the BF changes varied among regional vascular beds with Dp and Db infusions during and following CPB. Of the 2 drugs, Db showed the greater inotropic response off CPB, a similar increase in myocardial perfusion and greater visceral organ bloodflow.