Vol. 73, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RELEASE OF UNSATURATED VITAMIN B BINDING CAPACITY FROM HUMAN GRANULOCYTE&*BY THE CALCIUM IONOPHORE A23187 James D. Simon, Hospital,
Received
William
E. Houck and Maurice
M. Albala
Division of Clinical Hematology, Rhode Island and Brown University, Providence, R.I. 02902
September
21,
1976
SUMMARY: The ionophore A23187, in the presence of calcium ions, was capable of eliciting the release of granule-associated unsaturated vitamin B12 binding capacity from human granulocytes. The ionophoreinduced extrusion of unsaturated vitamin Bl2 binding capacity was concentration, time and temperature-dependent. The release was blocked by 2-deoxyglucose and was unaccompanied by cytotoxicity (trypan-blue uptake and lactate dehydrogenase release). The unsaturated vitamin B12 binding capacity release was accompanied by the release of lysozyme, a specific granule marker enzyme. INTRODUCTION There
is
considerable
capacity
(UBBC)"
UBBC is
a measure
released
of the
Corcino
proteins. were
is
et al
particle-bound
and Peters
(5)
specific
granules
lithium,
inhibited
inhibitors,
degree (1)
reported
that
unsaturated
vitamin
B12 binding
human granulocytes
(PMN's)
(l-4).
suggested
primarily
granule-associated is no evidence
granulocytes.
is well
that
azide, (6)
in the
the
from human PMN's is
that
the
by other
by
metabolic
2,4-dinitrophenol concluded
changes
substances
establishes
localized
Kane
of UBBC was stimulated
and Herbert
known
proteins
Recently,
exclusively
sodium
through
B12-binding
B12-binding
and unaffected
cyanide,
was not mediated it
vitamin
The release
fluoride,
Stebbins
Although
that
UBBC was almost
of human PMN's. by sodium
of vitamin
neutrophilic
(3).
*Abbreviations: polymorphonuclear buffered saline;
that
of unsaturation
the
such as potassium
AMP levels.
there
from
within
and 2-deoxyglucose release
evidence
that
UBBC
in intracellular
secretion
of a number
Ca++-dependent
involvement
cyclic of
(7-l*),
of Ca++ in the
UBBC, unsaturated vitamin B12 binding capacity; PMN's, leukocytes; ACD, acid citrate dextrose; PBS, phosphate LDH, lactate dehydrogenase.
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
444
Vol. 73, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL
A23187
Fig. 1. incubated
Ionophore A23187 in PBS complete
RESEARCH COMMUNICATIONS
(,uM)
dose response curve with human PMN's medium for 30 minutes at 37OC,
CALCIUM
Fig.
(mM)
2. Ca"+ -induced release of UBBC from PMN's incubated for minutes at 37OC. Ionophore concentration was 1.0 PM. Ca++ and ?4g++ -free medium was supplemented with Ca++ at the indicated concentrations. 30
445
BIOCHEMICAL
Vol. 73, No. 2, 1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
release of UBBC. Wenow report that the ionophore A23187-induced release of UBBCfrom humanPMN's is a Ca++-dependent process. MATERIALSANDMETHODS Preparation of granulocyte suspensions, HumanPMN's from normal donors were isolated from buffy coats of freshly drawn ACDblood using Ficoll Isopaque according to the method of Boyum (13). Red blood cells were lysed with ammoniumchloride by the procedure of Cells were washed Dioguardi et al (14) with minor modifications. twice with saline and suspended in Dulbecco's phosphate buffered saline (PBS), pH 7.2 (Associated Biomedic Systems, Inc., Buffalo, N.Y.) with or without Ca++ and Mg++ containing 0.1% bovine serum albumin. PBS complete medium contained Ca++ and Mg++ concentraCa++ and Mg++-free tions of 0.9 mMand 0.4 mM, respectively. mediumwas supplemented with CaC12and MgC12 salts, as indicated in the individual experiments. Measurement of UBBC, lysozyme and lactate dehydrogenase (~LDH)release, Approximately 1 x 107 PMN's were incubated with shaking in 3 ml of buffer in 17 x 100 mmcotton-stoppered plastic tubes, Cells were preincubated 5 minutes in the buffer before addition of the Ca++ ionophore A23187 (generously supplied by Dr. Robert L. Hamill, Eli Lilly Co,, Indianapolis, Ind.) Following various periods of incubation, the PMN suspensions were placed in an ice bath, centrifuged at 1120 x g for 10 minutes at hoc, and the cell-free supernatants decanted for assay. Th.e UBBCwas determined by the method of Gottleib et al (15), 1ySOZynle (EC 3.2.1.17) according to the procedure of Litwack (16) using Micrococcus lysodeikticus as substrate and LDH (EC 1.1.1.27) by the method of Wacker et al (17). Release of UBBCwas expressed as picograms released per 1 x 107 cells after correction for untreated controls. Lysozyme and LDHrelease Fre expressed as a percentage of the total enzyme released by 1 x 10 PMN's after incubation for 30 minutes in mediumcontaining 0.2% Triton X-100. RESULTS The release of UBBCfrom humanPMN's was ionophore A23187 and Ca++ Maximumrelease of UBBCfrom PMN's
dose-dependent (Figs. 1 and 2).
suspended in complete PBS mediumoccurred at an ionophore concentration of 1.0 uM (Fig.
1).
Cells in Ca++ and Mg++-free mediumcontaining
1.0 uM ~23187 released UBBCwith the addition CaC12(Fig.
2).
Maximumrelease, without
occurred with Ca++ concentrations
observed by light
microscopy.
These alterations
of 5.0 mMCa++. 446
as 0.1 mM damage,
1.0 mM. Concentra-
in macroscopic changes in
and distorted
damageand could account for slightly concentration
apparent cellular
of approximately
tions of Ca++ higher than 2.0 mMresulted the appearance of the cell pellet
of as little
Wright-stained
cells
suggested cellular
higher release observed at a
Vol. 73, No. 2, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
70006000-
5
0
IO
IS
TIME
Fig. 3. Time and temperature PMN'S. Ionophore concentration
20
25
30
(Minuted
dependence was 1.0
of UBBC release from uM in complete PBS medium.
500(
4ooc
2 z 0 1c 0 c : x5
3ooc
2000
1000
(0.4mM)
(0.9mM)
+ Mg**(0.4mM)
Fig. 4. Requirement of Ca++ for the ionophore-induced release of UBBC from PMN's incubated for 30 minutes at 37oC. Ionophore concentration was 1.0 uM. Ca++ and Mg++-free medium was supplemented with CaCl and MgClp salts to the indicated concentrations. The open and shade 2 bars represent reaction mixtures without and with ionophore, respectively. Each bar represents the mean of three experiments +- S.E.M.
447
Vol. 73, No. 2,1976
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
40.0-
!iQ
30.0-
W ii Y & B a E+
20.0
lO.O-
0
0
r 5
I IO
_1 IS TIME
LDH
I 20
1 2s
1 30
(Minutes)
5. Time dependence of lysozyme and LDH release from PMN's incubated at 37OCin complete PBSmedium containing 1.0 uM ionophore, Fig.
The release of UBBCwas temperature and time-dependent (Fig. Maximumrelease occurred after
incubation
3).
for 30 minutes at 37°C in
complete PBSmedium. Although not shown, UBBCrelease at 60 minutes was 14%less than that seen at 30 minutes.
The ionophore-induced ++ release of UBBCrequired Ca++, but not Mg++ (Fig. 4). Mg alone, in
the presence of ionophore, did not induce release of UBBC. Furthermore, it did not enhance the release observed with Ca++ plus ionophore. at higher concentrations
(up to 1.8 mM), in the presence of 1.0 nM ionophore
and 0.9 mMCa++, gave the sameresults Addition
Mg+'
as those seen with 0.4 mMMg++.
of glucose (1.0 mg/ml) to the complete PBS mediumcontaining
ionophore (1.0 uM) yielded the sameamount of UBBCreleased in unsupplemente medium. However, UDBCrelease,
induced by ionophore (1.0 PM) in complete
PBS medium, was reduced about 87%by 1 mM2-deoxyglucose.
The mean+
standard error of the mean (n=3) for UBBCrelease per 1 x lo7 PMN's incubated for 30 minutes at 37'C, was 547 2133 and 3570 + 383 picograms for reaction
mixtures with or without
2-deoxyglucose,
respectively.
The release of UDBCwas accompanied by the release of lysozyme,
448
BIOCHEMICAL
Vol. 73, No. 2, 1976
a specific were
granule
similar
to those
enzyme activity compared
marker
absence
seen for
changes
The kinetics
30 minutes
of release
RESEARCH COMMUNICATIONS
Approximately
UHBC release.
lack
5).
UBBC.
78% for
by the
trypan
(Fig.
during
of significant
excluded
enzyme
was released
to about
as indicated
AND BIOPHYSICAL
of release
37% of the
total
of incubation.
Cell
viability
of cytoplasmic
This
was not
altered,
LDH and by the
in the percentage
(>98%)
of cells
which
blue. DISCUSSION
The role PMN's the
is well
ionophore
of Ca++ in the established A23187,
Mg++ (181,
g reatly
(7-10,12),
eosinophil
of anaphylaxis basophils release
which
that
the
the UBBC (5), in the
release
This UBBC from
it
the
of granule-associated
factor
investigation
specific
from
the
to examine
substances
Since
that
the
granules,
is
it
was recently
nearly
demonstrated
human PMN's.
The phenomenon
similar
granule-associated
substances
to that
no requirement
inhibition
of Ca++ ionophore-induced et al
(7),
for
we did
that
of
of Ca ++ and A23187
observed
Mgi'
not
Ca++ induced
required
(8,9,20).
dependent,
for
Although
was observed. lysozyme
the
the
presence
the
release
the
see any inhibition
glycolytic
of the
process
system
for
UBBC release
by 2-deoxyglucose.
of A23187
the
Mg'+
reported
by
449
was shown by the these
PMN
was Ca++-
of UBBC release
However,
of
of other
release
Unlike release
release
Mg++.
inhibition
all
of UHBC.
investigation
An intact
and
from human
PMN specific
the role
++
enzymes
of human PMN's contain
was of interest
of Ca
observation
(7,12).
Ca++,
reacting
and histamine
A23187
granules
fluxes
and slow
in the
ionophore
at a concentration
Goldstein
(11)
stems
an enzyme located
by Ca++ and the
reported
transmembrane
release
from human
of extracellular
facilitates
from human leukocytes
This
of substances
In the presence
chemotactic
of lysozyme,
stimulated
of a number
(7-12).
enhanced
(19)
(20).
release
results
by
Vol. 73, No. 2, 1976
are in direct
BIOCHEMICAL
contrast
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
to Li+-stimulated
unaffected by 2-deoxyglucose (3).
release of UBBCwhich was
A further
difference
between Cat+
ionophore and Li.+ -induced release of UBBCis evident in the time required for maximumrelease. maximal effect
Whereas the ionophore produced its
in minutes, Li+ required greater than 8 hours.
This report describes the first
direct
evidence of Cat+-dependent
ionophore release of UBBCfrom humanPMN's. The mechanismof release and its physiological
significance
are under investigation.
ACKNOWLEDGEMENTS: The authors wish to thank Dr. Horace Martin for performing the lactate dehydrogenase assays. This investigation was supported by the American Cancer Society Grant No. IN-45-P. REFERENCES
1. 2. 3. 4. 2: 7. a.
9. 10.
Corcino, J., Krauss, S., Waxman, S., and Herbert, V. (1970) J.Clin. Invest. 9, 2250-2255. Rachmilewite, B., Rachmilewitz, M,, and Gross, J. (1974) Br,J, Haematol. 26, 557-567. Scott, J-M., Bloomfield, F.J., Stebbins, R., and Herbert, V. (1974) J.Clin.Invest. 53, 228-239. Simon, J.D., Albala, M-M,, and Rauth, B.J. (1975) Clin. Res. 23 4C6A+ 49, 171d2. Kane, S.P., and Peters, T.J. (1975) Clin.Sci.Mol,Med, Stebbins, R., and Herbert, V. (1974) Proc. Soc.Exp.BiocMed. l& 734-736. Goldstein, I.M., Horn, J.K., Kaplan, H.B., and Weissman, G. (1974) Biochem. Biophys.Res.Commun. 60, 807-812. Koza, E.P., Wright, T.E., and Becker, E.L. (1975) Proc.Soc.Exp.Biol.Med. 149, 476-479. Smith, R.J.,and Ignarro, L.J. (1975)Proc.Nat.Acad.Sci.U.S.A. 72,108-112. Zabucci, G., Soranzo, M.R., Rossi, F.,and Romeo, D. (1975) FEE Letters 2,
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Czarntzki, B.M.,Konig, W., and Lictenstein, L.M. (1976)J.Imm~n01.~,229-234 Estensen,R.D., Reusch, M.E.,Epstein, M.L., and Hill, H.R. (1976) Infect. Immunol. 1.3, 146-151. Boyum, A. (1968) Stand. J. Clin. Lab. Invest. (Suppl.) 97, 77-89. Dioguardi, N., Agostoni, A., Fiorelli, G., and Lomanto, B. (1963)J.Lab.Clin. Med. 6l, 713-723. Gottleib,C., Lau,K.S., Wasserman, L.R., and Herbert, V. (1965) J.Hematol. z,a75-884.
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19. 20.
Litwack,G. (1955) Proc.Soc.Exp.Biol.Med. &, 401-403. Wacker,W.E.C., Ulmer,D.D., and Vallee, B.L. (1956)N.Eng.J.Med. =,449-456. Reed,P.W., and Lardy, H.A. (1972) J.Biol.Chem. 247, 6970-6977. Conroy, M.C., Orange, R.P., and Lichtenstein, L.M. (1976) J.Immunol. uA, 1677-1681. Lichenstein, L.M. (1975) J. Immunol. ll4, 1692-1699.
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