Elevation of intracellular Ca2+ concentration in rabbit nonpigmented ciliary epithelial cells by allicin

Comp. Riochem. Physiol. Vol. 115C, No. 1, pp. 89-94, Copyrighr 0 1996 Elsevittr Science Inc.

ISSN 0742-8413/96/$15.00 PII SO742-8413(96)00115-6

1996

ELSEVIER

Elevation of Intracellular Ca2+ Concentration in Rabbit Nonpigmented Ciliary Epithelial Cells by Allicin Teh-Ching Chu, * Jarrett L. Burch, t Marco A. de Paula Brow, j Tony L. Creagao, t Joan Hun,# Grace Y. Hunf and David E. Potter *DEPARTMENTOF PHARMAC~LOCY AND TOXICOLOGY,MVREH~USESCHOOLOF MEDICINE, ATLANTA, GEORGIA,USA; ~DEPARTMENT OF CELLULARBIOLOGYAND ANATOMY, MEDICALCOLLEGEOF GEORGIA;AND $DEPARTMENT OF CHEMISTRY,MOREHOUSECOLLEGE

ABSTRACT. A previous study has shown that allicin produces changes in aqueous humor dynamics, and this study was conducted to examine possible cellular mechanisms. In rabbit nonpigmented ciliary epithelial cells, basal levels of [Ca”], were determined to be 164 t 34 nM. Allicin, a sulfhydryl-reactive agent, induced Ca’+ transients at 0.01 mM and at 0.2 mM, the Cal+ transienr peaked at 732 -t 35 nM. Allicin-induced Ca’+ transients were prevented a slight,

by pretreatment

insignificant,

extracellular

Ca’+-free

of allicin’s

effect.

with dithiothreitol

effect on L/type conditions.

These

Pretreatment

allicin-induced

Ca”

Cal+ transients

are most likely mediated

Science

Else&r

KEY WORDS. current,

rabbit,

with through

allicin,

intracellular

sulfhydryl

group,

Ca”

garlic (Allium

organosulfur

sativum

merous biological bacterial

effects

(1,2),

reduction

modification

of sulfhydryl

administration in rabbits

can be lowered secretion

derived

L.), has been reported

(3), inhibition

pressure

including

anti-

of serum

cholesterol

and

aggregation (6,7).

has been shown

(8). It is known

effectively

(4,5) and

Recently,

topical

to lower intraocular

that intraocular

by inhibition

from the nonpigmented

to possess nu-

activities

of platelet enzymes

from

pressure

of aqueous

ciliary epithelium

humor (9) and

that changes in intracellular Ca2+ may play an important role in the modulation of aqueous humor secretion by drugs (10,ll).

Therefore,

fects by examining tion. In this report, rabbit

an inhibitor

of Cal+-induced-Ca’+-release,

nonpigmented

it is important mechanisms experiments ciliary

to determine involving

allicin’s

intracellular

Ca!’

Thus, stores.

inhibited allicin-induced

Copyright

o 1996

1990. ryanodine,

nonpigmented

ciliary

epithelial

cells, Ca’+

cells

Ca*+ transients

dine, an inhibitor cellular stores.

MATERIALS Drugs

scribed

AND

(diallyl

thesized,

from intra-

C,H&X(O)-C1Ht)

and assayed

(12). Tetrodotoxin,

purchased

with ryano-

METHODS

thiosulfinate,

purified

obtained

pretreatment

Chemicals

and

Allicin

following

of Ca*+-induced-Ca*+-release

ionomycin,

from Calbiochem from Molecular

was syn-

by the method

recently

and ryanodine

(La Jolla, CA). Fura-2/AM Probes,

Inc. (Eugene,

dewere was

OR).

ef-

Ca2+ mobiliza-

were performed epithelial

by ryanodine.

under source

Cal+-induced-Ca’+-release

compound

and biochemical

triglycerides allicin

Ca’+ stores are the tnost probable

ryanodine-sensitive

concentration,

had only

were also present

intracellular

induced

the primary

the basal Ca’+ levels. Allicin Cal+ transients

levels were unaffected

Inc. COMPBIOCHEMPHYSIOL115C;1:89-94,

INTRODUCTION Allicin,

that

ryanodine,

but the basal Cal+

did not affect

and allicin-induced

data suggest

of cells

transients,

which

Ca’+ currents,

in cultured

to determine:

1) the effects of allicin on Ca” transients and Ca*+ currents, 2) the possible antagonism of allicin’s effect by dithiothreitol, 3) allicin’s effect in Ca *+-free conditions, and 4) allicin-

Address reprint requests TV: T. Chu, Department of Pharmacology and Taxic&>gy, Morehouse School of Medicine, 720 Westview Dr. SW, Atlanta, Georgia, 30310-1495, USA. E-mail: TC’@link.msm.edu.

Cell CuIture The rabbit derived

nonpigmented

ciliary epithelial

after transformation

cells, a cell line

of a semiconfluent

primary

cul-

ture (13), were grown to confluence in culture medium containing Dulbecco’s modified Eagle medium/Ham’s F12 mixture (l:l),

15 mM NaHCOj,

1 mM

ascorbic

acid,

100 U/

ml penicillin, 100 pg/ml streptomycin, and 10% fetal bovine serum at pH 7.4. The second passage of nonpigmented ciliary

epithelial

cells,

used for this study.

obtained

from

a frozen

stock,

was

90

T. Chu et al.

Fura-

Loading

Semiconfluent tomed

stored cells

dishes

to attach

of the following

5.6 at pH 7.4. The

Ringer’s

at room

deesterification

solution

temperature

1.8; HEPES, the cells

in a rotating

for 30 min. The cells were washed

the above-mentioned stabilized

composition

dish containing

was placed

bath

for analysis

using

a microcomputer.

at

three

times

with

without

fura-

and

for 25 min to allow for the

cals (mM): HEPES,

TEA.Cl,

TEA.OH.

The

taining

CsCl,

Na+-

CsOH.

to 300 mosm/kg.

were performed

Briefly, the concentration was measured

were

[Ca’+], were length

excitation

fluorescence

performed

measured

were obtained

described

of free intracellular

by utilizing

manipulations

as previously

with

fura-

ratio technique according

Ca*+ ([Ca”],)

microscopy

in the

dark.

using

the

for fura-

with high Cal’

and low Ca”,

At the end of experiments, by exposure

of cells with

mM EGTA.

Free fura-

mented

ciliary epithelial

scribed

previously

Ringers.

Additional

/3

nm.

and with

(30 PM)

cells was measured

25

in nonpig-

by methods

de-

Th’ is concentration is expected buffering effect. The effect of allicin by bathing

experiments

mm) of dithiothreitol

the cells with

or Ca’+-free

were also determined

within

in response

either

of allicin-induced following

Ca”

pretreatment

(2 mM) or ryanodine

licin and other drugs were introduced

diameters

(10

(0.1 mM). Al-

through

a bath perfu-

Statistical

clamped

using the perforated

viously described

epithelial

cells

patch-clamp

(14). Patch

electrodes

were

voltage-

technique

as pre-

were fabricated

on

a micropipette puller (M P-97, Flaming/Brown Sutter Instrument Co., San Rafael, CA) from Corning 7052 capillary tubing ( 1.65 mm OD, 1.2 mm ID; Garner Glass Co., Claremont,

CA)

and had resistances

of 1.0-2.0

MG when

with internal solution. Pipettes were coated (Dow Corning, Midland, MI) and fire-polished forge tered filter, data

capaci-

following

seal

forming.

After

the unfiltered

tran-

began

step from

-80

4094 digital

within

conTo de-

to -70

oscilloscope

the cell capacitance.

mV, at 2 The

with an ocular

the microscope.

Analysis

The data are given number

as means

of measurements

-C SE, where

n refers to the

made for each condition.

cal significance

(p < 0.05) was evaluated t test for paired or non-paired data.

RESULTS Akin-Induced In cultured

filled

with Sylgard on a micro-

immediately prior to use. Membrane currents were filat 2-5 kHz (- 3dB) using a 8-pole low-pass Bessel digitized with a I2-bit 330-kHz A/D converter (Digi1200, Axon Instruments, Inc., Foster City, CA) and

Ca”

Statisti-

by the Student’s

rabbit

Transients

nonpigmented

of [Ca’+], were

experiment,

transient

Recordings of Cu’+ Currents ciliary

MG).

cells were determined

contained

basal levels

nonpigmented

good voltage

clamp,

to obtain

of patched

micrometer

typical

The

on a Nicolet

with

(0.1 mM EGTA)

sion system.

Whole-cell

to a voltage

nystatin-

the electrode

pores

voltage

solusolu-

Data were usually

immediately

nystatin

and integrated

with

was 510

capacitance,

and before

MHZ

Instruments.

compensated

the whole-cell

were

recording

was controlled

10 min after obtaining

achieving

con-

4; HEPES,

of solutions

back-filled

the series resistance

formation

solution

was added to the pipette was dip-filled with internal

Llata acquisition

cell membrane was

Osmolalities

from Axon

trol (i.e., when termine

K+-free

130; MgCl:,

and then,

20;

and R,,,,, were determined

10 ,uM ionomycin

(1.8 mM Cal’)

transients

(14,17),

(14,181.

on [Ca’+], was examined

collected

was captured

at 380 nm of a solution

concentration

to exert no appreciable Ca’+-rich

values

R is the ratio at 340/380 R,,,

software

sient,

where

signal

pClamp

tance

(16):

- R)

constant

in

dual-wave-

(15). Quantitative

W’l, + & X P X (R - LAL, is the ratio of fluorescence

and all

Changes

to the Ca*+ equation

KJ IS the dissociation

(14).

nystatin solution.

1.8; CsCl,

For perforated-patch

(0.05 mg/ml)

without

containing

and

10; Cs:S04,

10 at pH 7.35 with adjusted

tion Measurements

Experiments

Cal+ cur-

0.003 at pH 7.4 with

5.6; tetrodotoxin,

internal

(mM):

(19), nystatin

of dye.

110; CaC12, 1.8; MgCl?,

10; glucose,

tion. The tip of the pipette [Cu”],

The

rents were recorded at room temperature using extracellular Nat- and K’-free solution containing the following chemi-

to glass-bot-

140; KCl, 4.0; MgClz, 1.8; CaClz,

and 2.0 PM fura-Z/AM 37°C

allowed

in the Ringer’s

(mM): NaCI, 10; glucose,

were

which

allicin

peaked allicin,

ciliary

epithelial

(0.2 mM)

induced

out

promptly

to the basal level. The allicin-induced

sient was inhibited which

the

fluorescence

by pretreatment reacts

a large Caz’

lasted at least 1 min (Fig. 1A). After

washing

an agent

cells,

164 + 34 nM (n = 65). In a

with allicin

ratio

returned Ca’+ tran-

of 2 mM dithiothreitol, forming

disulfide

bonds

(Fig. 1B). The

basal Cal+ level was unaffected by dithiothreitol pretreatment ([Ca’+], = 141 2 31 nM, n = 15). Dose-related changes of Ca” transients induced by allicin

(0.01, 0.1,0.2 mM) were 221 ? 40, 254 + 36, and 732 ? 35 and allicin-induced Cal ’ transients were

nM respectively, antagonized

by dithiothreitol

([Ca’+],

=

167 C 46 nM).

These data are summarized in Fig. 2. Allicin (1 PM) did not produce any changes in [W-l,. The maximal response was induced by a concentration of 0.2 mM. It is hypothesized that increased [Cal’], may result from activation of

Allicin and Intracellular

A

91

Ca”

Traces

600

were elicited

(L-types

1

a

blocker

between

zero current effect

of allicin

tionships tivated

-80

currents

currents

A and B. The Ca’+ curincreases

by dithiothreitol(O.1 current

density

tol, at a test potential

was increased

the by al-

2 0.1 (n = 10) and recov-

of 10 mV. The mean

was also increased

of dithiothreiT-type

by allicin

current

from -0.13

? 0.09 (n = 10) but was reduced

to -0.25

(0.01

mM). For example,

(PA/cm’)

% 0.1 to -0.50

by allicin

in Ca’+ currents

-’ 0.08 (n = 5) in the presence

by di-

? 0.02 (n = 5) at test potential

of

mv.

Allicin-Induced To determine

Cu” whether

tribute

significantly

iments

were performed

Transient other

in Cu2+-Free Conditions

sources

of external

to the allicin-induced

Ca’* con-

transients,

under CaL+-free external

exper-

conditions.

The basal [Ca’*], levels were 116 ? 20 nM (n = 6). Follow-

0-l

I

25seconds

DTT + Allicin

FIG. 1. The effect elicited by ahicin on [Ca’+]i in cultured rabbit nonpigmented Gary epithelial cells and its antagonism by dithiothreitol (DTT) treatment. The Ca2+ transient induced by 200 PM allicin (A) and its inhibition by pre.treatment with 2 mM DTT (B).

of plasma

from intracellular

membrane

or/and

Ca’+-release

stores.

Allicin’s Effect on Voltage-Dependent whether

Ca’+ currents

Ca’+ transient,

might

Ca’+ Currents play a role in the

Cal+ currents

arate T- and L-type Cal+ currents

in embryonic

cardiac

my-

ocytes (20). The Ca*+ current traces from the nonpigmented ciliary epithelial cells are shown in Fig. 3. The membrane

capacitance

was

Effect of dithiothreitol pretreatment on allicin-evoked increase in calcium concentration. 800 s5 6 600 ._ 5L E g 6 400 0

2 mM DlT+

200 pM Allicin

E .z ; 200 0

were measured

under various conditions. The voltage clamp protocols were as previously described and have been used routinely to sep-

cell

ac-

the T-type

allicin-induced

(PA/cm’)

currents

mV; Fig. 4C depicts

quite small, were increased

ered to -0.34

relato cell currents

and the

? 0.05 to -0.32

average

@A/

the L-type

although

-30

density

normalized

mM),

thiothreitol

allicin-induced

currents

rents,

density

To study

Ca”

between

licin from -0.38

1

The

mV; Fig. 4B shows

from -40

mean L-type

600

on Ca’+ current

from difference

were inhibited

Allicin

and

subtracted.

Fig. 4A shows both L- and T-type

from

activated

inward current

in Fig. 4 with the mean current-voltage

for L- and T-type

capacitance.

mV. It

was increased by 0.01 mM

Cal+ channel was measured as the

the peak of transient

(0.01 mM)

-40 mV

of

of +lO

at 400 ms with the leak current

cm’) is shown

25seconds

dihydropyridine-sensitive The Cal’ current

(Fig. 3C).

difference

C%+ channels

potential

to a test potential

was found that the control (Fig. 3A) current by 0.01 mM allicin (Fig. 3B) and inhibited nifedipine,

B

from a holding

Ca’+ currents)

8.4

?

3.4 pF.

0

*

p
FIG. 2. Summary of dose-related changes of Ca” transients induced by allicin ( 10, 100,200 FM) and antagonism of al. licin’s effect by dithiothreitol (DTT, 2 mM). Data are expressed as mean f SE, n 2 12. Asterisk denotes significance (p < 0.05) from the basal.

92

T. Chu et al.

sensitive tissues

intracellular and cells

Ca”

(21).

cells, the Ca2+ transient

Control

blocked ment

([Ca”],

stores has been shown

In nonpigmented induced

Ca’+-release.

by allicin

= 165 ? 35 nM)

of 0.1 mM ryanodine, The resting

ciliary

epithelial

(0.2 mM) was

following

an inhibitor

in various

the pretreat-

of Ca’+-induced-

Cal+ levels were unaffected

by ry-

-0.6 -80

B

-60 -40

I

-20

0

20

40

60

I

Nifedipine __

_____ I ’

’ “/.‘I

I

I

(

I(

+I0 mV

I.

-40

Allicin

-o-

-0.6 1 I.

-20

! 0

I

.I.

20

40

60

//

400 msec

-40 mV _/

c,

FIG. 3. Effects of ahicin and nifedipine on L.type Ca” current (PA) traces from the cultured nonpigmented ciiiary epithelial cells. A: Control; B: Allicin ( 10 PM); C: Nifedipine (10 ,uM). Letype Ca2+ current was elicited by first inactivating T-type Ca2+ current with a 500 msec prep&e to -40 mV before stepping to the test potential of 10 mV. Peak Ca’+ current was measured as difference between peak of transient inward current and current at the end of the 400 ms test pulse. Dotted lines indicate 0 current. A&in enhanced both types of Ca*+ currents whereas nifedipine inhibited the magnitude of the L-type Ca2+ current only.

t

-Om6// -80

-60

-40

-26

0

Potential (mV) ing addition were increased extracellular

of 0.2 mM allicin

to the bath,

[Ca’+], levels

to 772 -+ 40 nM (n = 6). Thus, Ca*+ had virtually

removing

no effect on the allicin

tran-

sients.

Allicin’s Dependence Because

on Intracellular

Stores

Ca*+ transient was not depenCa2+ source extracellular Cal+, an intracellular from ryanodinewas postulated. Ca ‘+-induced-Ca’+-release dent

the allicin-induced

Cd+

upon

FIG. 4. Current-voltage relationships showing the effects of allicin ( 10 FM) on La and T+type (panel A), L-type (panel B) and T-type (panel C) Ca”+ current density (PA/cm’) in rabbit nonpigmented ciliary epithelial cells (n 2 10). Voltage steps to various test potentials from a holding potential of -80 mV were used to elicit both T- and L-type currents. The L-type Ca*+ current was elicited by tirst inactivating T. type Ca’+ current with a 500 msec prepulse to -40 mV before stepping to the test potential. T-type current was determined as the difference between the currents elicited from test pulses from -80 and -40 mV. The Ca’+ currents were normalized by cell surface area.

Allicin

and Intracellular

93

Ca’+

Effect of ryanodine pretreatment on allicin-evoked increaee in calcium concentration #

800

nels exist in rabbit ciliary epithelium

E 200 pMAlllcin q Alllcin+ 100 pt.!Ryanodine

600

threshold

T-type

epithelial

cells has been

ted-patch

recordings,

duced

s ‘C 2 f 400 e s E ; 200

small

However,

Ca”

transients

Cal+

induced

extracellular Therefore,

0

x

were

Ca’+.

In contrast,

creasing

p
Ca”

stores.

that

pretreatment

of these

= 168 2 40 nM, n = 6) and

([Ca”],

treatment

experiments

are summarized

in Fig. 5.

of cells

posed that allicin

increases

intracellular

Although

the

therapeutic

value

of various

drugs has been established,

few studies

exact

whereby

cellular

traocular

mechanisms

pressure.

ous humor determining

intraocular

cellular

mechanism(s)

aqueous

humor

The limited

by which

understanding epithelial

complicated

interactions

neurotransmitters,

The current zation

hormones

Ca’+ release from internal

fold increase

of

is primarhumor

by allicin,

that allicin

of [Ca’+], in nonpigmented Ca’+ transient

pretreatment

of sulfhydryl

groups

(23).

3.

could affect

which

in the action

out that allicin

produces

ciliary

a 4-

epithelial

was inhibited

by

of allicin.

It should

reacts rapidly with sulfhydryl

In order to determine sients induced by allicin, were also studied.

the mechanism(s) voltage-dependent

Previously,

be

groups

of Ca’+ tranCa” currents

it has been shown

dihydropyridine-sensitive

5.

suggests the involvement

of cysteine (12), thus, allicin may exert its effect by modifying essential cysteinyl residues of proteins located either intracellularly and/or at cell membrane.

threshold,

2.

4.

allicin-induced

1,4,5

messen-

Cal+ stores. Future the possible

for the changes

involve-

in [Ca”],

in-

by allicin.

(L-type)

that highCa’+

chan-

C.J.; Bailey, J.H. Allicin, the antibacterial principle 1. Isolation, physical properties and antibacterial action. J. Am. Chem. Sot. 66:1950-1951;1944. Deshpande, R.G.; Khan, M.B.; Bhat, D.A.; Nalvalker, R.G. Inhibition of mycobacterium avium complex isolates from AIDS patients by garlic (A&m satiuum). J. Antimicrob. Agents Chemother. 32:623-626;1993. Augusti, K.T.; Mathew, P.T. Lipid lowering effect of allicin (diallyl disulphide oxide) on long term feeding tn normal rats. Experientia 30:468-470;1974. Lawson, L.D.; Ransom, D.K.; Hughes, B.G. Inhibition of whole blood platelet-aggregation by compounds in garlic clove extracts and commercial garlic products. Thromb. Res. 65:141-156;1992. Maveux, P.R.; Agrawal, K.C.; Tou, J-SH.; King, B.T.; Lippton. H.L.; Hyman, A.L.; Kadowitz, P.J.; McNamara, D.B. The pharmacological effects of allicin, a constituent of garlic oil. Agents Actions 25:182-190;1988. Willis, E.D. Enzyme inhibition by allicin, the active principle of garlic. B&hem. J. 63:514-519;1956. Han, J.; Lawson, L.; Chu, T.C.; Potter, D.E.; Han, G.; Han, P. Modification of catalytic properties of chicken live fructose 1,6-bisphophatase by allicin. Biochem. Mol. Biol. Int. 31:1007-1015;1993. Chu, T.C.; Ogidigben, M.; Han, J.C.; Potter, D.E. Allicin induced hypotension in rabhit eyes. J. Ocul. Pharmacol. 9:201209;1993.

of Al&m s&rum.

body and to

ciliary epithelial

showing

to determine

to the

inositol

as a major intracellular

trisphosphate

References 1. Cavallito,

is due to

a number

of the aqueous

as induced

dithiothreitol pointed

of

elusive.

mechanisms

cells with

in

secretion.

This is the first report cells. The

factor

study would suggest that effects on Ca’+ mobili-

humor

that

This work was supported, in part, by NIH Grants EY 06338 (DEP) , S06GM 45199 (GYH) and HL 36059 (TLC). Rabbit non&ymmted ciliary epithelial cells were generously provided by Dr. M. Coca&ados, Yale University.

and drugs (22). It is generally

for the secretion

and Ca’+ currents,

aqueous

in-

secretion

of iris-ciliary

of these

that the nonpigmented

ily responsible

decreased

of cellular

shown

on ryanodine-

contribute

the specific

by drugs remains

structure

the

lower

contributing

(9); however,

can be produced

the complex

accepted

pressure

drugs

that the rate of aque-

is an important

which

diminit is pro-

anti-glaucoma

have elucidated these

It is well-accepted

formation

greatly Thus,

ger regulating

duced

of intracellular

by allicin.

is considered

of inositol

inare

ryanodine

trisphosphate

ment

that

of allicin

by the evidence

[Cal+], by acting

Ca2+ stores

of [Cal+],. It has been

studies will be conducted DISCUSSION

with

extracellular

action

is supported

induced

channels. Ca’+ tran-

suggest

on mobilization

probability

to Ca’+

to result from

upon

this study by the

the

ion

allicin-induced

from

CaL+ level

transient

that

through

dependent

results

This

ished the Ca”

regulation results

primarily

cytosolic

sensitive anodine

not

pro-

significantly

are not possibly

that

perfora-

allicin

of Ca’+ currents.

indicating

of Ca’+

most apt to be dependent

FIG. 5. Effect of ryanodine (0.1 mM, 10 min) pre-treatment on allicin-evoked increase in intracellular Ca’+ concentrae tion. Data are expressed as mean + SE, n 2 12. Asterisk denotes significance (p < 0.05) from the response to 200 PM allicin.

magnitude

by allicin

it is proposed

sients

that

that they contribute

movement

ciliary

Utilizing

showed

transients

a low-

pigmented

(25).

data

in the

it is unlikely

(24). Moreover,

in bovine

identified

these

change

allicin-induced

d

channel

6. 7.

8.

94

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