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,

11. 12. 13. 14. 15.

44-48.

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.

16. 17. 18.

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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Release of unsaturated vitamin B12 binding capacity from human granulocytes by the calcium ionophore A23187.

Vol. 73, No. 2, 1976 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS RELEASE OF UNSATURATED VITAMIN B BINDING CAPACITY FROM HUMAN GRANULOCYTE&*...
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