ate cyclase because this is not attained with amounts of LH which would fully saturate the surface receptors. It is poss- ible therefore that LH receptors are ...
28
BlOCH EM ICAL SOCl ETY TRANSACTIONS
ate cyclase because this is not attained with amounts of LH which would fully saturate the surface receptors. It is possible therefore that LH receptors are recycled and are able to bind fresh hormone and thus further activate the adenylate cyclase. The possible recovery of the cells to desensitization was determined by incubating the cells for 1 h with 1.0, 10 and 100 ng of LH/ml and then, after washing, incubating the cells for various times up to 120min. I t was found that the cells desensitized with 1 .O ng of LH/ml gave nearly a 100% response in terms of cyclic AMP production when rechallenged with LH after 2 h , but with IOOng of LH/ml no increase in cyclic AMP production was obtained. These experiments demonstrate that desensitization is reversible after incubation with physiological amounts of LH. E8ki.t of‘ phorhol esters und calcium The ability of tumour-promoting phorbol esters to cause desensitization of adenylate cyclase has been reported with several systems. The phorbol ester 4[&phorbol- 12P-myristate-13a-acetate (PMA) was found to cause a dose- and time-dependent desensitization of rat Leydig cells (Habberfield et ul., 1986d). After 30min incubation with different amounts of PMA the cells were challenged with LH (100 ng/ ml); it was found that maximum PMA-induced desensitization (approx. 40%) occurred with 50nM. With longer incubation times and with 50 nM-PMA the desensitization reached a maximum after 90min. The amount of surface binding of I” I-hCG also decreased by incubation with PMA: a loss of approx. 40% occurred within 30min. When the calcium ionophore A23187 was added 10min before 5nM- and IOOnM-PMA it was found that the degree of I” I-hCG surface binding was markedly decreased in the presence of the low but not the high concentration of PMA. These experiments indicate that a phorbol ester can mimic LH in causing desensitization and loss of cell-surface LH receptors and furthermore that its effects may be modulated by Ca’+. I t is possible therefore that LH-induced desensitization and LH-receptor internalization are caused by activation by LH of Ca’+ -dependent protein kinase Cmediated phosphorylation. We are grateful to the M.R.C. and the Peter Samuel Royal Free Fund for financial assistance. Call, K. J.. Harwood. J. P.. Agiulera. G . R. & Dufau. M . L. (1979) Nu/urc, (London) 280. 109 I I6
Cooke. B. A.. Janszen, F. A.. van Drricl. M. J. A. & van dcr Molen. H. J. (1979) Bioc,hin?.Biop/rjx. Ac,/u 583, 320 331. Cooke, B. A.. Dix. C. J.. Maggee-Brown. R . . Janszcn. F. H. A. & van der Molen. H. J. (1981) Adr. C~clic. Nuc.Imick, RCS.14. 593 609 Dehejia. A., Nozu, K . . Catt. K . J. & Dufau. M. L. (1982) J . B i l J c ’ / l ( ~ . 257. 7x1 7x6 Dix. C. J.. Schumacher. M. & Cooke. B. A. (1982) B i ~ ~ c hJ ~. 202, i. 739 745 Dufau. M. L. & Catt. K. J. (1978) Viruni. Hornr. ( N . Y . ) 36, 461 592 Habberfield, A. D.. Dix. C. J. & Cooke. B. A. ( 1 9 8 6 ~ )B i ~ c h ~J~. i . 233, 369-376 Habberfield, A. D.. Dix. C. J. & Cooke. B. A. (19X6h) J . Enr/oc,rinol. 107. abstract 126 Habberfield. A. D., Dix, C. J. & Cooke, B. A. ( 1 9 8 6 ~J) . Endocrinol. 108 (Suppl.). abstract 329 Habberfield. A. D., Dix. C. J. & Cooke, B. A. (l986d) in 4th Europeun Workshop of Molecular and Cellulur Endocrinolo~y of /he Tesris, p. 78. Elsevier. Amsterdam. in the press Haour, F. & Saez. J. M. (1977) M o l . Cell. Enc/ocrino/. 7, 17 24 Hseueh, A. J. W.. Dufau, M. L. & Catt. K. J. (1977) Proc,. Null. Acucl. Sci. U . S . A . 74. 592 595 Johnsen, T., Purvis, K.. Torjesesn. P. A. & Hansson. V. (1981) Arch. Anrlrol. 6, 155 162 Knecht. M., Darbon. J. M.. Ranta. T.. Bankel. A. J. & Catt. K. J. (1984) Endocrinolugj, 1 15. 4 1-49 Luborsky. J. L.. Dorfinger, L. J.. Wright. K. & Behrman. H. R . (1984) Endocrinology 115. 2210 2216 Mather, J. P.. Saez, J. M. & Haour. F. (19x2) Endoc~rino/og~, 110, 933 940 Munson, P. J. & Rodbard. D. (1980) A n d . Bioc.hcw. 107. 220 239 Purvis, K. & Hansson. V. (l97X) Arch. Anrlrol. 2. X9 91 Purvis. K.. Torjesen. P. A.. Haug, E. & Hansson. V. (1977) M o l . C’c~ll Enr/oc,rinol. 8. 73 80 Rosenthal. H. E. (1967) Anal. Biocheni. 20. 525 532 Saez. J. M.. Haour, F. & Cathiard. A . M. (1978) Bioc~licvn.B ~ c J ~ ~ ~ s . Rex. Coniniun. 81, 552 558 Scatchard. G . (1949) Ann. N . Y . A c ~ d .%I. . 51 I . 660 672 Sharpe, R. M. (1977) Bioc~hcw.B i o p h j ~ .Rcs. Conitnun. 75, 7 I I 71 7 Sibley, D. R. & Lefkowitz. R. J. (1985) Ntr/urc,(Lo,rr/o,i) 317, 124 129 J . 236. 45 51 Sullivan. M. H. F. & Cooke. B. A. (19x6) BilJ~’/ifwl. Tsien, R. Y . . Pozzan. T. & Rink, T. J. (19X2)J.Cell. BilJl. 94, 325 334 Tsitouros. P. D.. Kowatch. M. A. & Harman. S. M . (1979) Elidllocrinologj. 105. 1400 1405 Tsuruhara. T.. Dufau. M . L.. Cigorraga. S. & Catt. K. J. (1977) J . Biol. Chew. 252. 9002 9009 Wu, F. C . W.. Zhang. G . Y . . Williams. B. C . & de Kretser. D. M. ( 1985) Mid. Cdl. Enrkoc~rinol.40. 45 46
Received 15 July 1986
Inositol trisphosphate and tetrakisphosphate phosphomonoesterases of rat liver that the primary consequence of receptor occupation is the phosphodiesteric hydrolysis of PtdIns(4,5)P, leading to the generation of Ins( l.4.5)P3 and diacylglycerol (Kirk ct d . , 1981: Michell ct a/., 1981; Berridge & Irvine, 1984). Both these products can act as second messengers within the Receptor-mediated inositol lipid degradation is a widespread stimulated cell. Ins( I,4,5)P1 provokes Ca’+ release from an mechanism of stimulus-response coupling in those eukary- intracellular source (Streb c ~ tul., 1983; Berridge & Irvine, ote cells which utilize the Ca’+ ion as an intracellular mess- 1984), whilst diacylglycerol activates the Ca” - and phosenger. Work from a number of laboratories has established pholipid-dependent ‘protein kinase C’ (Nishizuka, 1984). In view of the role of Ins(1,4.5)P3 as an intracellular second messenger, the metabolic pathways which remove this comAbbreviations used: Ptdlns(4.5)P2. phosphatidylinositol 4.5-bisphospound from the cell may have a regulatory function. phatc: InsP. inositol monophosphate: Ins/‘?. inositol bisphosphate; We have previously reported on the hepatic enzymes InhP,. inositol trisphosphatc; InsP,, inositol tetrakisphosphate: lnsl P. inositol I-phosphate: Ins4P. inositol 4-phosphate; Ins( 1 . 3 ) P 2 . inositol which catalyse the dephosphorylation of Ins( I ,4.5)P3and its I .3-bisphosphate: Ins( l.4)P,. inositol 1,4-bisphosphate; lns(3.4)P2. initial hydrolysis product, Ins( l,4)P, (Storey r t ul., 1984; inositol 3.4-bisphosphate: Ins( I ,4,5)P inositol I ,4.5-trisphosphate; Shears (’I ul.. 1985). Recent work has identified additional Ins( I ,3.4)P1. inositol I ,3,4-trisphosphate; Ins( 1.3.4.5)P4. inositol I J.4.5inositol phosphates that accumulate in stimulated cells. tctrakisphosphate. Theseare Ins(l,3,4)P,(lrvine~,ta/.. 1984)and Ins(1.3,4.5)P4 CHRISTOPHER J. KIRK, ROBERT H. MICHELL, JOCELYN B. PARRY and STEPHEN B. SHEARS Ikptrrttiicnt of Bioc,lict,ii.str~.,Universitj. of’ Birmingham, PO BO.Y363, Birtninghuni B15 2TT, U . K .
1987
29
619th M E E T I N G , C A M B R I D G E (Batty ct al.. 1985). The latter compound can be formed by the direct phosphorylation of Ins( 1,4,5)P, in various tissues including rat liver (Irvine et d., 1986; S. B. Shears, J . B. Parry, R. H. Michell & C . J . Kirk, unpublished work). lrvine (it al. (1985) have suggested that the phosphorylation of Ins( 1,4.5)P3may represent a major route for its removal from the cell and several workers have speculated that the lns(1,3.4,5)P4 so formed may also act as an intracellular messenger. With these ideas in mind, we have investigated the pathways by which lns(1,3,4,5)P,, and lns(1,3,4)P, are further metabolized in rat liver. Ins ( I , 3 , 4 , 5 )P , phosphomonot~stt~rase We have prepared [' HIlnsP, by the ATP-dependent phosphorylation of ['H]lns( 1,4.5)P (Amersham International, U . K . ) in rat liver 100000 g supernatants (the Ins(1,4,5)P3 kinase activity of rat liver is largely soluble; J . B. Parry & S. B. Shears, unpublished work). The reaction was performed in 0.25 M-SUCTOSC. 50 mM-Tes (pH 7.5), 20 mMMgCI?, 10 mM-ATP, 25 m~-2,3-disphosphoglycerate(sodium salt) and 0.2mg of saponin/ml. Ins( 1,3,4,5)P, was purified by ion-exchange chromatography (Irvine et al., 1986). The material so produced co-elutes o n h.p.1.c. (Palmer et al., 1986) with authentic Ins( 1,3,4,5)P, derived from rat cortex as described by Batty et al. (1985). Hence, we believe that this compound is Ins( 1,3,4,5)P,. We have incubated approx. 3 n ~ - [ ' H ] h s P , with diluted rat liver homogenate containing 100 mM-KCI, 10 mM-NaCI, 2 0 m ~ - H e p e s( p H 7.2), 1 mM-EGTA, 4 m free ~ Mg" and 0.1 M free Ca" . Reactions were terminated with HCIO, (final concn. 3%, v/v), and the acid extract was neutralized with 1.5 ~ - K o H / 5 mM-Hepes 0 or freon/octylamine ( 1 : 1, v/v; Downes et al., 1986) before analysis by ion-exchange chromatography or h.p.1.c. respectively (Batty e t al., 1985; Palmer c't d.. 1986). In these experiments, the hydrolysis of ['H]lns( I ,3.4,5)P4 followed first-order kinetics. When the results were extrapolated to the concentration of enzyme present in the original tissue, the first-order rate constant for the reaction was found to be 0.31 f 0.03s I . This corresponds to a half-life for InsP, in situ of about 2.2 s. When the hydrolysis of Ins( l,4,5)P, was studied under similar conditions, the corresponding rate constant and half-life were 0.49 f 0.05s ' and 1.4s respectively. I t is clear that both Ins( I .4.5)P, and Ins( 1,3,4,5)P, are subject to rapid hydrolysis within the cell. This observation is compatible with the notion that the latter compound may also fulfil a second messenger role. Ins( 1 ,3,4,5)P4 phosphomonoesterase activity in rat liver homogenate was insensitive to 50 mM-Li' , which largely inhibits the activities of Ins( 1) P phosphomonoesterase and Ins( 1 ,4)Pz 4-phosphomonoesterase (Sherman et al., 198 I ; Berridge e t al., 1982; Storey et al., 1984). However, the activity of Ins( 1 ,3,4,5)P, phosphomonoesterase was inhibited by 10 m~-2.3-diphosphoglycerate. (The calculated first-order rate constant was 0.038 s ' in the presence of 2,3diphosphoglycerate and 0.31 s I in its absence.) The InsP, fraction, arising from the hydrolysis of ['H]lns( 1 ,3.4,5)P4 in liver homogenate, was separated by anion-exchange chromatography and analysed by h.p.1.c. The product eluted with the expected mobility of Ins( l,3.4)P7 and was separated from a [32P]lns(1,4.5)P7 standard (Fig. I). Unfortunately, standard samples of other InsP, isomers were not available to us, so we cannot exclude the possibility that this peak includes some Ins( 1 , 3 3 4 and/or Ins(3,4,5)P,. We have further investigated the identity of the hydrolysis product of Ins( 1.3,4,5)P4 by studying the simultaneous metabolism of [4,5-"P]Ins( l,3,4,5)P4 and ['H]lns( 1,3,4,5)P, in liver homogenate. The "P-labelled tetrakisphosphate was prepared by the ATP-dependent phosphorylation of [4,5Vol. 15
20
21
22
23
Elution time (min)
Fig. 1. H.p.1.c. profile of thc InsP, product of ['H]Ins( 1 , 3 , 4 , 5 ) P ,hydrolysis bji liver homogenate ['H]lns( 1,3,4,5)P, was prepared and incubated with diluted liver homogenate as described in the text. Incubations were terminated after 10 min and inositol phosphates were separated by anion-exchange chromatography. The 'Ins P7 fraction' (-) was mixed with a standard sample of [4,5-'*P]lns( l.4,5)P7 (---) prepared from human erythrocytes (Downes i't ul.. 1982) and analysed by h.p.1.c.
"P]lns( 1,4,5)P derived from human erythrocytes as described by Downes et al. ( 1 982). The preparation used in this study had 71% of incorporated "P in the 5-phosphate and 29% in the 4-phosphate (see also Hawkins et al.. 1984). A mixture of the two Ins( 1,3,4,5)P, preparations was incubated with a diluted homogenate of rat liver in the incubation medium described above. After lOmin the reaction was terminated with HCIO, (final concn. 3%, v/v), and the mM-Hepes acid extract neutralized with 1.5 M - K O H / ~ O before the separation of inositol phosphates by anionexchange chromatography (Batty ct al., 1985). The ratio of ['*PI/[' HI was determined in fractions corresponding to InsP,, InsP, and InsPz (Table I). As a result of the uneven distribution of "P between the 4- and 5-phosphates of [4,5-"P]Ins( 1,3,4,5)P, (see above), the change in the ["PI/ ['HI ratio of the InsP, hydrolysis product(s) as compared with that of the InsP, precursor should help identify the InsP, isomer(s) which accumulate in these conditions. Thus if Ins( 1,3,4)P7was the sole product of InsP, hydrolysis, then
Table 1. Hydrolysis o/[4,5--"P]In.s(1,3.4.5)P4und ['H]lns( 1 , 3 , 4 . 5 )P, by liver honiogcnarr [4,5-"P]Ins( l,3,4,5)P4 and ['H]lns( I,3.4.5)P4 were prepared and incubated with diluted liver homogenate as described in the text. Incubations were terminated after 10 min and inositol phosphates separated by anion-exchange chromatography. The [" P]/['H] ratio in individual inositol phosphate fractions was determined by liquid scintillation spectrometry. Asterisks denote the monoester phosphate groups which are labelled with "P. Results are means ~ s . I . M . from three separate determinations. Fraction InsP,
['ZP]l[iH] ratio
Interpretation
0.247
Ins( 1 . 3 . 4 . 3 ) ~ ~
0.027
/w* InsP,
0.064 & 0.007
Ins( I - 3 . 4 ) ~ '
InsP,
0.056 & 0.005
Ins( I ,4)P2or
I
lns(J.4)P2
30
BIOCHEMICAL SOCIETY TRANSACTIONS
its ["P]/['H] ratio would be predicted to be 29% of that of its precursor. In fact, the ['ZP]/[3H]ratio of the InsP, fraction generated in this experiment was 26% of that of the starting material and did not differ significantly from the value predicted above (Table I). This indicates that Ins( 1,3,4)P, was the major InsP, product to accumulate in these conditions. We have investigated the distribution of InsP, phosphomonoesterase activity between a IOOOOOg supernatant and a particulate fraction prepared from rat liver. The first-order rate constants, extrapolated to the concentration of enzyme present in the original tissue, were 0.027 f 0.007s-' and 0.36 f 0.03s for supernatant and particulate fractions respectively, showing that hepatic InsP, phosphomonoesterase is almost entirely membrane-associated. In order to further investigate the subcellular distribution of this enzyme we have subjected liver homogenates to the Percoll density gradient fractionation which we previously utilized to investigate the distribution of hepatic Ins( l,4,5)P, 5-phosphomonoesterase (Storey et a/., 1984). Our preliminary results indicate that InsP, phosphomonoesterase is confined to a single peak on the Percoll gradient, which co-separates with alkaline phosphodiesterase, a plasma membrane enzyme. However, this region of the gradient also contains substantial activity of the Golgi marker, galactosyltransferase (see Storey et a/., 1984). Further experiments will be necessary to determine the distribution of Insf, phosphatase between plasma and Golgi membranes. N o InsP, phosphomonoesterase activity was found at the top of the gradient where the cytosol markers were located.
'
Table 2. Ejfi1c.t (?/'Lit rind I n s ( l , 4 ) P ,upon InsP., metabolism in liver liomogenatc~ [4,5-"P]Ins( 1,4,5)P, and [3H]Ins(1 ,3.4)P, were prepared and incubated with liver homogenate as described in the text. The final dilution of the liver homogenatc was 1 : 60 with respect to intact liver. Incubations with [4,5-"P]Ins( I.4,5)P3 were terminated after 5min and those with ['H]lns( I,3,4)P3 after 20 min. Inositol phosphates were analysed by anion-exchange chromatography. Results are means f S.E.M. of the number of observations in parentheses. P vs addition-free controls: *
