3 14s Biochemical Society Transactions ( 1 994) 22
Characterisation and Caz+-dependency of the soluble and
particulate Ins(1,4,5)P3 5-phosphatase in bovine tracheal
smooth muscle
(ii) Particulate 5-Phosphatrw
(i) Soluble EPhosohaIase
.
0
ace+
lWC.I+
BARBARA J. LYNCH, R.A. JOHN CHALLISS and EDWIN R.
CHILVERS.
0
ImMC.Z+
LO
Respiratory Medicine Unit, University of Edinburgh, Medical
School, Teviot Place, EH8 9AG, UK and *Department of Cell
Physiology and Pharmacolgy, MSB, University Road, University
of Leicester, LEI 9HN, UK.
10
0
Receptor-mediated hydrolysis of PtdIns(4,5)P2 is thought to
play a pivotal role in mediating pharmacomechanical coupling
in ASM [l]. In contrast to many other tissues, (eg. rat cerebral
cortex [2]), the activation of muscarinic cholinoceptors in ASM
results in a transient accumulation of Ins(1,4,5)P3 despite
sustained PtdIns(4,5)P2 hydrolysis 131. These data imply that
agonist-stimulated metabolism of Ins(1,4,5)P3 in ASM is an
important factor regulating its accumulation. Although previous
reports have indicated that the Ins(1,4,5)P3 3-kinase is an
important route for Ins(1,4,5)P3 metabolism in many cell types
141, our studies have shown that the Ins(1,4,5)P3 5-phosphatase
is the dominant pathway involved in the early metabolism of
Ins(1,4,5)P3 in BTSM [5]. In view of previous studies
highli hting the potential for the 5-phosphatase to be stimulated
by C 3 + [6,7], we have sought to examine the effect of W7, a
calmodulin antagonist, on Ins(1,4,5)P3 accumulation in intact
tissue and the modulation of the Ins(1,4,5)P3 5-phosphatase by
Ca2+.
Aliquots (50 pl) of gravity-packed BTSM slices 181 were
incubated in the presence or absence of 100 pM W7 for 20min
prior to addition of carbachol (100 1M) or buffer for 0-300s.
Ins(1,4,5)P3 was assayed in neutralised trichloroacetic acid
extracts according to Challiss et a/. [9]. Figure 1 demonstrates
that W7 had no effect on the basal or initial (<1Os) Ins(1,4,5)P3
accumulation but caused a small, yet significant, attenuation of
the rate at which Ins(1,4,5)P3 accumulation declined (10-60s)
after carbachol challenge.
BTSM was homogenised in a Tris-maleate/sucrose buffer (pH
7.5) containing 1 mM PMSF and 0.1% (v/v) mercaptoethanol
and centrifuged at 48,000 xg for 90min to generate particulate
and soluble fractions. After extensive washing, the particulate
fraction was solubilised using 1% Nonidet P40 and re-spun at
48,000 xg to remove remaining insoluble material. The activities
of the soluble and particulate 5-phosphatase enzymes were
determined by measuring the conversion of
a
Vo/[SI (ml min-1 mg protein-1)
I600
2600
Jdoo
Vo/[SI (ml min-1 mg protein-1)
Each value represents the mean f SEM of 3 separate expts.
Figure 2. Effects of [Ca2+I on the Km (pM) and Vmax (nmol
m i n - l mg protein-l) of the soluble and particulate
Ins(1,4,5)P~5-phosphatase in BTSM.
[3H]Ins(1,4,5)P3 to [3H]Ins(1,4)P2 under linear kinetic
conditions. The [3H]Ins(l,4,5)P3 and [3H]Ins(1,4)P2 were
separated by Dowex AGI-X8 anion exchange chromatography
and the Km and Vmax values calculated from Vo versus Vo/[S]
(EadieHofstee) plots (figure 2 and table).
These data demonstrate very similar Km values for the
soluble and particulate Ins(1,4,5)P3 5-phosphatase enzymes,
but higher Vmax values for the membrane-associated 5phosphatase. These Km values, whilst in close agreement with
previously reported values in bovine aortic smooth muscle [lo],
are some 5-fold lower than reported in rabbit ASM [ll]. The
above data also indicate that, while at supraphysiological
[Ca2+l Ins(1,4,5)P3 5-phosphatase enzyme activity clearly
deviates from standard Michaelis-Menten kinetics, the activity
of this enzyme is not influenced by changes in [Ca2+]across the
physiological range. This would suggest that the attenuation of
agonist-stimulated Ins(1,4,5)P3 metabolism observed in the
presence of W7 is more likely to reflect inhibition of the
Ca2+/calmodulin-stimulated3-kinase activity, and indicates
that an alternative, non-Ca2+-dependent, mechanism mediates
the agonist-stimulated removal of Ins(1,4,5)P3 in ASM.
We thank the Wellcome Trust and Chest, Heart and Stroke
Association (UK) for financial support.
Hall, I.P. & Chilvers, E.R. (1989) Pulm. Pharmac. 2, 113120
2. Challiss, R.A.J., Chilvers, E.R., Willcocks, A.L. &
Nahorski, S.R. (1990) Biochem. J. 265, 421-427
3. Chilvers, E.R., Batty, I.H., Challiss, R.A.J., Barnes, PJ. &
Nahorski, S.R. (1991) Biochem. J. 275, 373-379
4. Shears, S.B. (1991). Pharmac. Ther. 49, 79-104
5. Lynch, B.L. & Chilvers, E.R. (1992) Eur. Resp. J. 5, 65-66s
6. Sasaguri, T., Hirata, M. & Kuriyama, H. (1985) Biochem. J.
231, 497-503
7. Kukita, M., Hirata, M. & Koga, T. (1986) Biochim.et
Biophys. Acta 885, 121-128
8. Chilvers, E.R., Barnes, P.J. & Nahorski, S.R. (1989)
Biochem. J. 262, 739-746
9. Challiss, R.A.J., Batty, I.H. & Nahorski, S.R. (1988)
Biochem. Biophys. Res. Commun. 157,684-691
10. Polokoff, M.A., Bencen, G.H., Vacca, J.P., deSolms, S.J.,
Young, S.D. & Hoff, J.R. (1988)J. Biol. Chem. 263, 1192211927
11. Rosenburg, S.M., Berry, G.T., Yandrasitz, J.R. &
Grunstein, M.M. (1991)J. Biol. Chem. 88, 2032-2038
1.
4
0
100
200
300
Time ( s )
Figure 1. Effect of W7 on carbachol-stimulated Ins(1,4,5)P3
accumulation in BTSM slices. Data represent mean f SEM of
3 expts each performed in triplicate.
indicates p<0.05.
Control (01, control+W7 (o),Carbachol (o),Carbachol+W7 (8)
Abbreviations used: Ptdlns(4,5)P2, phosphatidylinositol 4,5bisphosphate; ASM/BTSM airways/bovine tracheal smooth
muscle; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; W7, N-(6aminohexyl)-5-chloro-1-napthalenesulphonamide.