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Lysosomal involvement in cellular internalization of clostridium difficile toxin b
8th World C o n l ~ s on ~mhnal, ~ and bfi=obial ToWns HALOTHANE INDUCES A CARDIOTOXIN-LIKE CONTRACTURE IN HUMAN SKELETAL MUSCLE PRETREATED WITH BEE VENOM PHOSPHOLIPASE A~ By J . E . FLETCHER, H. ROSENBERG AND M. H I L F . D e p t . o f Anesthesfo-q'~y--~--,
Hahnemann University, Philadelphia, PA 19102, U.S.A. Malignant hyperthermia (MH) susceptible humans and porcine stress syndrome (PSS) pigs are characterized by a low contracture threshold of skeletal muscle strips when challenged in vitro with halothane (4). Phospholipase A ~ ( P L A ~ | activity is ele~te~--i-n skeletal muscle from PSS (2) and MH (unp~blish&d observations) susceptibles. SCh stimulates endogenous PLA~ activity (5} and acts synergistically with halothane to induce contrac~ures {3). Cardiotoxin (CTX] acts synergistically with PLA~ to induce contractures in skeletal muscle (1}. Therefore, to test if ~levated PLA~ activity might account for the increased sensitivity to halothane, we eRamined the effects of bee venom PLA 9 on muscle biopsied from humans diagnosed as normal by the halothane co~tracture test. Bee venom PLA. (~uM) was added to the bath 2 min before halothane or SCh. The value~ in the Table are the maximum contracture (X+SE| within 5 min of halothane or SCh addition for six patients. Bee venom PLA~ caused these preparations to exhibit contractures to halothane, but-not to SCh. These studies suggest that CTX and Agent -PLA 2 +PLA 2 halothane may act through a similar mechanism. PLA~ activity enhances Contracture (g) the activity of both of these Halothane 3% 0.17+0.08 0.77+0.19, agents, presumedly through the SCh 50 mM 0.00~0.00 0.03~0.02 liberation of unsaturated fatty * P<.01 (paired t-test) acids.
.REFERENCES 1)Chang, C.C. (1979) In Snake Venoms (ed C.Y. Lee) p309. Springer-Verlag 2)Cheah, K.S. and Cheah A.M. (l--§-~Biochem biophys Acta 638, 40 3)Fletcher, J.E. and Rosenberg, H. (1985} Anesthesiology,--i-n press 4)Gronert, G.A. (1980| Anesthesiology 53, 395 5)Olthoff, D., Kunze, D. and Kries, H.-~1973) Acta Biol Med Ger 3_~I, 317 KEY WORDS phospholipase A21 contracture~ skeletal muscle
~
IN ~erzJw~R ~rl'~inL_7'ZRPION
elf I ~ B ~ H I ~ D I !
I H ] ~ ' n ' ~ r z 'LCIXIN B
B~ I. F l o r i n and M. 'Rmleetam. Dept o f Bncter-lo!ogy, Karo]/rmka I n s t i t u t e ,
S-I04 01 ~ I m , ~ diffi=kLo toxin s 4,. a potent ~ Im~l~ Int~e etiology of ~ I c - a s s o c l a t e d ~litis (Borlello, 1984). The toxin is internalized aultured ~ 1 1 s by e , ~ t o s i 8 (Fiarin and T,e l m ~ m , 1963), ~ t~o microfilamant system is disorganized and a c y ~ t h o g e n i o effect (CPE) is developed. Recently diphtheria toxin was shown to enter the aytoeol from a of the endosomes with lysooome8 (Sanavig et al., 1984). ~ aim of this study w u to clarify whether ~ ~ I n B enter8 the cytoool c U r e c t l y ~-om a prelysoscmal compartment or not. ~l~w~ of t~e CPB in tcxln-treated ~ l l s ~ Inhihlted at om~iw~ere fusion ~ e t w N n ~ and lynoocme8 is prever~a- such = ~ i are m ° C ( ~ n et al., 1980), ~ of ~2 M ~Cl (m--~i~er and Fiate, 19e2) ~ 10 ~M ~ r ~ o l {To1~ and Berg, I~82). A~ait~n of L ~ of l y ~ o m ~ ~ { c ~ . m x . ~ . , ~m~e~in, m ~ t ~ i n ) ~ o inhi~Ite~ the appearance of the CP~ results ~
t~.
~
tc~in B oann~ ~ter
the cFto~ol f r u m a
prelyso~omal compartment, but proceeds to the lysooomes and is activated by I y ~ o ~ a l proteuee ~ f ~ ~fer ~ t~e c~=~ol. Baenziger, J.U. and Fiete, ~ (1982) J. Biol. Chem. 257, 6007-6009. B~riel~o, ~P. (1984) ~ ~ and a u ~ 4 ~ e ~ t e d diarrhoea and ~litis. Martlnu8 NiJhoff P u b l ~ Dunn, W.A. et al. (1980) J. Biol. C~m. 255, 5971-5978. Florin, I. and ~nelesta~, M. (1983) _n!_-w~d~ B i o ~ Rcta 763, 383-392. Sanavig, K. at al. (1984) J. C e l l Biol. 98, 963-970. Tolled, F, and Berg, T. (1982) B i o c h ~ R~a-~_1~ 31, 593-595. ~ l e
toxin B~ cellular ~
~
lym~me~
Hahnemann University, Philadelphia, PA 19102, U.S.A. Malignant hyperthermia (MH) susceptible humans and porcine stress syndrome (PSS) pigs are characterized by a low contracture threshold of skeletal muscle strips when challenged in vitro with halothane (4). Phospholipase A ~ ( P L A ~ | activity is ele~te~--i-n skeletal muscle from PSS (2) and MH (unp~blish&d observations) susceptibles. SCh stimulates endogenous PLA~ activity (5} and acts synergistically with halothane to induce contrac~ures {3). Cardiotoxin (CTX] acts synergistically with PLA~ to induce contractures in skeletal muscle (1}. Therefore, to test if ~levated PLA~ activity might account for the increased sensitivity to halothane, we eRamined the effects of bee venom PLA 9 on muscle biopsied from humans diagnosed as normal by the halothane co~tracture test. Bee venom PLA. (~uM) was added to the bath 2 min before halothane or SCh. The value~ in the Table are the maximum contracture (X+SE| within 5 min of halothane or SCh addition for six patients. Bee venom PLA~ caused these preparations to exhibit contractures to halothane, but-not to SCh. These studies suggest that CTX and Agent -PLA 2 +PLA 2 halothane may act through a similar mechanism. PLA~ activity enhances Contracture (g) the activity of both of these Halothane 3% 0.17+0.08 0.77+0.19, agents, presumedly through the SCh 50 mM 0.00~0.00 0.03~0.02 liberation of unsaturated fatty * P<.01 (paired t-test) acids.
.REFERENCES 1)Chang, C.C. (1979) In Snake Venoms (ed C.Y. Lee) p309. Springer-Verlag 2)Cheah, K.S. and Cheah A.M. (l--§-~Biochem biophys Acta 638, 40 3)Fletcher, J.E. and Rosenberg, H. (1985} Anesthesiology,--i-n press 4)Gronert, G.A. (1980| Anesthesiology 53, 395 5)Olthoff, D., Kunze, D. and Kries, H.-~1973) Acta Biol Med Ger 3_~I, 317 KEY WORDS phospholipase A21 contracture~ skeletal muscle
~
IN ~erzJw~R ~rl'~inL_7'ZRPION
elf I ~ B ~ H I ~ D I !
I H ] ~ ' n ' ~ r z 'LCIXIN B
B~ I. F l o r i n and M. 'Rmleetam. Dept o f Bncter-lo!ogy, Karo]/rmka I n s t i t u t e ,
S-I04 01 ~ I m , ~ diffi=kLo toxin s 4,. a potent ~ Im~l~ Int~e etiology of ~ I c - a s s o c l a t e d ~litis (Borlello, 1984). The toxin is internalized aultured ~ 1 1 s by e , ~ t o s i 8 (Fiarin and T,e l m ~ m , 1963), ~ t~o microfilamant system is disorganized and a c y ~ t h o g e n i o effect (CPE) is developed. Recently diphtheria toxin was shown to enter the aytoeol from a of the endosomes with lysooome8 (Sanavig et al., 1984). ~ aim of this study w u to clarify whether ~ ~ I n B enter8 the cytoool c U r e c t l y ~-om a prelysoscmal compartment or not. ~l~w~ of t~e CPB in tcxln-treated ~ l l s ~ Inhihlted at om~iw~ere fusion ~ e t w N n ~ and lynoocme8 is prever~a- such = ~ i are m ° C ( ~ n et al., 1980), ~ of ~2 M ~Cl (m--~i~er and Fiate, 19e2) ~ 10 ~M ~ r ~ o l {To1~ and Berg, I~82). A~ait~n of L ~ of l y ~ o m ~ ~ { c ~ . m x . ~ . , ~m~e~in, m ~ t ~ i n ) ~ o inhi~Ite~ the appearance of the CP~ results ~
t~.
~
tc~in B oann~ ~ter
the cFto~ol f r u m a
prelyso~omal compartment, but proceeds to the lysooomes and is activated by I y ~ o ~ a l proteuee ~ f ~ ~fer ~ t~e c~=~ol. Baenziger, J.U. and Fiete, ~ (1982) J. Biol. Chem. 257, 6007-6009. B~riel~o, ~P. (1984) ~ ~ and a u ~ 4 ~ e ~ t e d diarrhoea and ~litis. Martlnu8 NiJhoff P u b l ~ Dunn, W.A. et al. (1980) J. Biol. C~m. 255, 5971-5978. Florin, I. and ~nelesta~, M. (1983) _n!_-w~d~ B i o ~ Rcta 763, 383-392. Sanavig, K. at al. (1984) J. C e l l Biol. 98, 963-970. Tolled, F, and Berg, T. (1982) B i o c h ~ R~a-~_1~ 31, 593-595. ~ l e
toxin B~ cellular ~
~
lym~me~