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308 Tyrosine phosphorylation of particulate proteins in lectin stimulated human T lymphocytes
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EFFECT OF TWO BETA ANTAGONISTS, BUCINODOLOL AND METOPROLOL. ON PULMONARY FUNCTION IN SUBJECTS WITH ASTHMA. P.W. Welsh, B.A.,-C.E. Reed, M.D., L.J. Jackson, R.N., and R.J. Seidehamel, Ph.D., Rochester. Minnesota. and Evansville. Indiana. Many patients with asthma who could benefit from treatment of cardiovascular disorders with beta adrenergic blocking drugs cannot tolerate the resulting increased airway obstruction. Bucindolol, 2-[2-hydroxy-3((%-(2H indol-3 yl) I,l-dimethyl ethyl) amino) propoxy] benzonitrile hydrochloride, is a beta adrenergic antagonist with partial beta agonist, and alpha antagonist activity. It is a weak bronchodilator. In a parallel double blind study, 36 subjects with mild stable asthma were randomly assigned to one of 3 groups, receiving either placebo, bucindolol or metoprolol for three weeks. The first week the dose of beta antagonist was 50 mg, the second 100 mg, and the third 150 mg, all t.i.d. Spirometry was performed before and 90 minutes after the first dose of each treatment and again after the last dose. Symptoms and peak flow were recorded Five patients withdrew from the study daily. because of increased respiratory symptoms, two each in the placebo and metoprolol groups and one in the bucindolol group. Both drugs were well tolerated. Neither had a great effect on pulmonary function though bucindolol increased air flow and metoprolol decreased it. The difference between the drugs was statistically For example, bucinodolol, 150 mg, significant. increased mean FEVI by 0.10 1 and FEF25-7 by 0.22 l/s while metoprolol decreased mean 8 EVI by 0.06 1 and FEF25-75 by 0.16 l/s.
CALCIUM; ESSENTIAL MESSENGER IN EARLY AND LATE T CELL ACTIVATION. D.L. Birx, MD, R.J. Summers, MD and T.A. Fleisher, MD, Wash, D.C., Bethesda, MD. We have utilized nassive channel calcium anand diltiazem nifedipine, tagonists: verapamii, to study the role of this channel and calcium in Addition of the the activation of T cells. agents resulted in similar dose related noncytotoxic suppression of mitogen and tetanus induced lymphocyte proliferation. The blocking agents inhibited the normally observed 45Ca influx folTo examine the effect of lowing PHA activation. the blocking agents on earlier events in mitogen induced T cell activation, we studied the appearance of the IL-2 and transferrin receptors. Both surface receptor expressions were inhibited in a dose dependent manner by the drugs. We then used these agents to evaluate the role of extracellular Ca and the passive channel in late cell activation phenomena (IL-2 induced Addition of the drugs to +TAC proliferation). human T cells inhibited proliferation in a dose dependent noncytotoxic manner. The agents did not inhibit IL-2 independent T cell tumor lines. We were able to specifically demonstrate that the binding of pure recombinant IL-Z to its receptor (TAC), resulted in 45Ca influx. The specificity of the process was confirmed by experiments in which preincubstion of the +TAC T cell lines with * TAC blocked IL-2 induced 45Ca influx and the proliferation of these cells. In summary, we have shown that extracellular Ca influx by means of the passive calcium channel plays a pivotal role in both the early activation signal following mitogen or antigen stimulation and the later proliferation signal generated by IL-Z binding to its receptor.
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K CHANNEL REQUIREMENT FOR HUMAN T-CELL ----II_-- MEDIATED CYTOTOXICITY. K.G. Chandy, B. Sharma-L-A...: T E DeCoursey, M.D. Cahalan, S. wta, Irvine, CA. A voltage-gated potassium (K)/Ibannel js required for T-cell activation. i\ si-:i.iar K channel has been identified in a mouse c);t..tt?x ii: T-lymphocyte (CTLj clone. The lethal hi* :‘ji:~se is accompanied by increased K conductan:,e .-ind “*Rb efflux, suggesting a role for the 1, c.h:innel. Here we have determined whether func.tioca: K \,h;innels are required during human CTL-med’i;;tcd target cell (TC) lysis. T4fT8- and TgrT4- I’:‘!‘l. were generated in a one-way mixed lynphoc:;;c ~~uiture and tested for their ability to ki.lj i $1~ iabeled allogeneic-PHA stimulated T-cell binsts during a five hour assay. We used the gipa--r>:m! seal patch clamp technique to study K channels in individual CTL. Both phenotypes expressed sim:lJa~ numbers of K channels with properties like th<,r+c in human peripheral blood T-cells. 5OZ bl.och ct K currents was produced by 0. l%nM &aminopyritiine (hAP) and 14mM tetraethylammonium (TEA). Both blockers reversibly inhibited CTL-mediated ‘Ti‘ 1ysis by both in a dose-dependent manner anti at phenotypes, roughly the same concentrations that block K channels. Tetramethylammonium, an analog to TEA, neither blocked the channel nor inhibited 7C lysis. CTJ, in the presence of blockiari were viable as assessed by trypan blue dye Gxciusion. 4AP and TEA did not increase release rcf “Cr, from CTL or TC labeled with ‘;‘CI-, r,irnpare;l with cells not exposed to the bloc-ke;-s. “‘hi].;., inhihition of TC Jysis by 4AP and TEA j-6 “oL &I+ to non-specific toxicity. These studie.5 dernonsfratc for functianal K chan9els during a requirement CTL-mediated TC lysis though th<, irs-,~‘t ro!+ r(imains to be determined.
TYROSINE PHOSPHORYLATION OF PARIICULA’~“; PROTEINS IN LECTIN STIMULATED HUMAN T LYMPHOCYTES. L Cort, G. Bass, and H.J. Wedner, St. Louis, MO Tyrosine phosphorylation ha5 been associated with the induction of cell growth in many cells. We have recently described the rapid phosphorylation on tyrosine of a 66kD soluble protein in lectin stimulated T lymphocytes. To examine tyrosine phosphorylation in the particulate fraction, human peripheral blood T lymphocytes were isolated by dextran sedimentation, isopycnic centrifugation and nylon wool filtration and subsequently were exposed to maximal mitogenic concentrations of Concanavalin A or The lymphocytes were then disrupted control. and particulate fraction was isolated by centrifugation at 16,000 x G. Aliquots containing 1Opg of crude protein were incubated with 20 NCi 32P-ATP in the presence of 10 nM Zn. The proteins were then separated by single dimension SDS-PAGE and the tyrosine phosphoproteins were identified by autoradiography following in$ubation of the gels in 1N NaOH for 2h at 55 C. Fifteen alkali stable bands were seen in both the lectin stimulated and control cells. Of these, there was a marked increase in 32P incorporation in three bands with M- 63, 53, and 33kD in the lectin stimulated cells, most prcminently in the 63kD band (stimulation ratio This data confirms the activation of 3.06). tyrosine kinase activity assocjoted with the activation of human T lymphocytes. These newly tyrosine phosphorylated particulate proteins may represent either the tyrosine kinase itself or membrane associated substrates which are important in the induction of lymphocyte growth.
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EFFECT OF TWO BETA ANTAGONISTS, BUCINODOLOL AND METOPROLOL. ON PULMONARY FUNCTION IN SUBJECTS WITH ASTHMA. P.W. Welsh, B.A.,-C.E. Reed, M.D., L.J. Jackson, R.N., and R.J. Seidehamel, Ph.D., Rochester. Minnesota. and Evansville. Indiana. Many patients with asthma who could benefit from treatment of cardiovascular disorders with beta adrenergic blocking drugs cannot tolerate the resulting increased airway obstruction. Bucindolol, 2-[2-hydroxy-3((%-(2H indol-3 yl) I,l-dimethyl ethyl) amino) propoxy] benzonitrile hydrochloride, is a beta adrenergic antagonist with partial beta agonist, and alpha antagonist activity. It is a weak bronchodilator. In a parallel double blind study, 36 subjects with mild stable asthma were randomly assigned to one of 3 groups, receiving either placebo, bucindolol or metoprolol for three weeks. The first week the dose of beta antagonist was 50 mg, the second 100 mg, and the third 150 mg, all t.i.d. Spirometry was performed before and 90 minutes after the first dose of each treatment and again after the last dose. Symptoms and peak flow were recorded Five patients withdrew from the study daily. because of increased respiratory symptoms, two each in the placebo and metoprolol groups and one in the bucindolol group. Both drugs were well tolerated. Neither had a great effect on pulmonary function though bucindolol increased air flow and metoprolol decreased it. The difference between the drugs was statistically For example, bucinodolol, 150 mg, significant. increased mean FEVI by 0.10 1 and FEF25-7 by 0.22 l/s while metoprolol decreased mean 8 EVI by 0.06 1 and FEF25-75 by 0.16 l/s.
CALCIUM; ESSENTIAL MESSENGER IN EARLY AND LATE T CELL ACTIVATION. D.L. Birx, MD, R.J. Summers, MD and T.A. Fleisher, MD, Wash, D.C., Bethesda, MD. We have utilized nassive channel calcium anand diltiazem nifedipine, tagonists: verapamii, to study the role of this channel and calcium in Addition of the the activation of T cells. agents resulted in similar dose related noncytotoxic suppression of mitogen and tetanus induced lymphocyte proliferation. The blocking agents inhibited the normally observed 45Ca influx folTo examine the effect of lowing PHA activation. the blocking agents on earlier events in mitogen induced T cell activation, we studied the appearance of the IL-2 and transferrin receptors. Both surface receptor expressions were inhibited in a dose dependent manner by the drugs. We then used these agents to evaluate the role of extracellular Ca and the passive channel in late cell activation phenomena (IL-2 induced Addition of the drugs to +TAC proliferation). human T cells inhibited proliferation in a dose dependent noncytotoxic manner. The agents did not inhibit IL-2 independent T cell tumor lines. We were able to specifically demonstrate that the binding of pure recombinant IL-Z to its receptor (TAC), resulted in 45Ca influx. The specificity of the process was confirmed by experiments in which preincubstion of the +TAC T cell lines with * TAC blocked IL-2 induced 45Ca influx and the proliferation of these cells. In summary, we have shown that extracellular Ca influx by means of the passive calcium channel plays a pivotal role in both the early activation signal following mitogen or antigen stimulation and the later proliferation signal generated by IL-Z binding to its receptor.
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K CHANNEL REQUIREMENT FOR HUMAN T-CELL ----II_-- MEDIATED CYTOTOXICITY. K.G. Chandy, B. Sharma-L-A...: T E DeCoursey, M.D. Cahalan, S. wta, Irvine, CA. A voltage-gated potassium (K)/Ibannel js required for T-cell activation. i\ si-:i.iar K channel has been identified in a mouse c);t..tt?x ii: T-lymphocyte (CTLj clone. The lethal hi* :‘ji:~se is accompanied by increased K conductan:,e .-ind “*Rb efflux, suggesting a role for the 1, c.h:innel. Here we have determined whether func.tioca: K \,h;innels are required during human CTL-med’i;;tcd target cell (TC) lysis. T4fT8- and TgrT4- I’:‘!‘l. were generated in a one-way mixed lynphoc:;;c ~~uiture and tested for their ability to ki.lj i $1~ iabeled allogeneic-PHA stimulated T-cell binsts during a five hour assay. We used the gipa--r>:m! seal patch clamp technique to study K channels in individual CTL. Both phenotypes expressed sim:lJa~ numbers of K channels with properties like th<,r+c in human peripheral blood T-cells. 5OZ bl.och ct K currents was produced by 0. l%nM &aminopyritiine (hAP) and 14mM tetraethylammonium (TEA). Both blockers reversibly inhibited CTL-mediated ‘Ti‘ 1ysis by both in a dose-dependent manner anti at phenotypes, roughly the same concentrations that block K channels. Tetramethylammonium, an analog to TEA, neither blocked the channel nor inhibited 7C lysis. CTJ, in the presence of blockiari were viable as assessed by trypan blue dye Gxciusion. 4AP and TEA did not increase release rcf “Cr, from CTL or TC labeled with ‘;‘CI-, r,irnpare;l with cells not exposed to the bloc-ke;-s. “‘hi].;., inhihition of TC Jysis by 4AP and TEA j-6 “oL &I+ to non-specific toxicity. These studie.5 dernonsfratc for functianal K chan9els during a requirement CTL-mediated TC lysis though th<, irs-,~‘t ro!+ r(imains to be determined.
TYROSINE PHOSPHORYLATION OF PARIICULA’~“; PROTEINS IN LECTIN STIMULATED HUMAN T LYMPHOCYTES. L Cort, G. Bass, and H.J. Wedner, St. Louis, MO Tyrosine phosphorylation ha5 been associated with the induction of cell growth in many cells. We have recently described the rapid phosphorylation on tyrosine of a 66kD soluble protein in lectin stimulated T lymphocytes. To examine tyrosine phosphorylation in the particulate fraction, human peripheral blood T lymphocytes were isolated by dextran sedimentation, isopycnic centrifugation and nylon wool filtration and subsequently were exposed to maximal mitogenic concentrations of Concanavalin A or The lymphocytes were then disrupted control. and particulate fraction was isolated by centrifugation at 16,000 x G. Aliquots containing 1Opg of crude protein were incubated with 20 NCi 32P-ATP in the presence of 10 nM Zn. The proteins were then separated by single dimension SDS-PAGE and the tyrosine phosphoproteins were identified by autoradiography following in$ubation of the gels in 1N NaOH for 2h at 55 C. Fifteen alkali stable bands were seen in both the lectin stimulated and control cells. Of these, there was a marked increase in 32P incorporation in three bands with M- 63, 53, and 33kD in the lectin stimulated cells, most prcminently in the 63kD band (stimulation ratio This data confirms the activation of 3.06). tyrosine kinase activity assocjoted with the activation of human T lymphocytes. These newly tyrosine phosphorylated particulate proteins may represent either the tyrosine kinase itself or membrane associated substrates which are important in the induction of lymphocyte growth.