Shinshu M ed J, 59 ⑴ :13〜26, 2011

Thrombin Activates Ca ‑permeating Nonselective Cation Channels
through Protein Kinase C in Human Umbilical Vein Endothelial Cells
Natsuko TSUJINO , Tsutomu NAKADA , Kiyotaka TSUBOUCHI , Masaaki KOBAYASHI
Yoshiko KAWAI , Shiharu YANO , Tamihide MATSUNAGA , Masamichi HIROSE
Shigeru OHMORI , Toshio OHHASHI and Mitsuhiko YAMADA ＊1） Department of Molecular Pharmacology, Shinshu University School of Medicine
2） Department of Pharmacy, Shinshu University Hospital
3） New Product Development Department, Technology Center, HIOKI E.E. CORPORATION
4） Department of Physiology, Shinshu University School of Medicine

We analyzed Ca ‑permeating nonselective cation channels (NSCs)mediating thrombin‑induced contractionof human umbilical vein endothelial cells (HUVECs). A Ca

chelater, BAPTA‑AM (10μM), signiﬁcantly

inhibited the thrombin‑induced contraction of HUVECs.Thrombin induced inward currents at ‑60 mV in thepresence of intracellular M gATP. Removal of extracellular Ca

signiﬁcantly decreased the currents. A

selective phospholipase C inhibitor, U73122 (1μM) but not its inactive analogue, U73343 (1μM) almost
completely inhibited the currents. Neither a selective inhibitor of Ca ‑ATPase of endoplasmic reticulum,
thapsigargin (1μM )nor a diacylglycerol analogue,1‑oleoyl‑2‑acetyl‑glycerol (30μM)activated the currents.
However, a selective protein kinase C inhibitor, bisindolylmaleimide I (500 nM) signiﬁcantly inhibited thecurrents.The thrombin‑induced currents were signiﬁcantly inhibited by SKF96365 (50μM )but not by La(1mM), ruthenium red (10μM) or ﬂufenamic acid (100μM ). As assessed with RT‑PCR, HUVECs expressedtransient receptor potential(TRP)M4,7,TRPV1,2,4,TRPC1,4 and 6 subunits of NSCs.These results indicatethat thrombin activates Ca ‑permeating NSCs containing TRPC4 through protein kinase C in HUVECs.
Thus,drugs speciﬁcally inhibiting TRPC4‑containing channels might be eﬀective to control fatal diseases suchas sepsis where thrombin mediates the vicious cycle between inﬂammation and coagulation. Shinshu Med J
59 : 13 ―26, 2011
(Received for publication June 21, 2010:accepted in revised form September 8, 2010)
Key words:human umbilical vein endothelial cells, thrombin, nonselective cation channels,
transient receptor potential C4, protein kinase C

Introduction

create gaps between ECs and increase vascularpermeability . Plasma proteins leaking from theEndothelial cells (ECs) form a barrier between

blood into tissues assemble into a provisional

intra‑ and extra‑vascular spaces and regulate thematrix that supports the attachment, survival andvessel wall permeability for ﬂuid, electrolytes, pro-migration of invading neutrophils. Inﬂammation

teins and blood cells . In the initial phase ofleads to activation of coagulation,whereas coagula-inﬂammation, ligands for G protein‑coupled rece-tion also markedly aﬀects inﬂammation . In thisptors such as histamine induce contraction of ECs,

crosstalk, thrombin plays a pivotal role. Thrombin
potently induces the contraction of ECs throughCorresponding author:M itsuhiko Yamada
Department of M olecular Pharmacology,Shinshu Univer-protease‑activated receptors (PAR) 1 which acti-

sity School of Medicine, 3‑1‑1, Asahi, M atsumoto‑city,

vate G

Nagano‑prefecture, 390‑8621, Japan

phoinositide (PI) hydrolysis and thereby increases

No. 1, 2011

and G

proteins . G

induces phos-

13

Tsujino.Nakada.Tsubouchi et al.

concentration ［( Ca ］) . This

form SOCs activated by thrombin. Freichel et al.

results in the phosphorylation of myosin light chain

found that TRPC4 knockout mice lacked SOCs in(M LC) by activating M LC kinase and induces theECs , and Tiruppathi et al. showed that in thesecontraction of ECs. G

activates Rho and Rho

mice, thrombin failed to induce the contraction ofKinase . Rho kinase suppresses a type I phos-lung vascular ECs . On the other hand, it was

phatase, thereby increasing the phosphorylation ofshown that thrombin activates SOCs formed by

MLC and augmenting the contraction of ECs.

TRPC1 through Rho‑induced interaction of TRPC1

intracellular Ca

PI hydrolysis increases［Ca ］ through two dis-with IP

receptors

or through activation of

tinct mechanisms .Inositol 1,4,5‑trisphosphate(IP )PKC , thereby inducing contraction of human pul-induces rapid and transient Ca

release from themonary ECs and human umbilical ECs (HUVECs),

endoplasmic reticulum (ER). Subsequently, PI

respectively. Thus, TRPC1 and/or TRPC4 mayhydrolysis causes sustained Ca inﬂux through Ca

form SOCs mediating the thrombin‑induced con-channels in the plasma membrane.For the contrac-

traction of ECs. However, the whole‑cell current

tion of ECs, the Ca

inﬂux plays a critical role .

density of SOCs in ECs is known to be extremely

These channels are conceptually classiﬁed into two

small in amplitude .In this study on HUVECs,wetypes depending on the way in which the channels

found that thrombin induces the contraction in a

are activated .The “
store‑operated”Cachannels

Ca ‑dependent manner and activates Ca ‑per-(SOCs)are activated by the IP ‑mediated depletion

meating nonselective cation channels that are likelyof Ca

to contain TRPC4 through PKC but not store deple-

in ER. On the other hand, the “
secondmessenger‑operated”Cachannels (SMOCs) aretion.These results suggest that selective blockers ofactivated by second messengers/kinases recruited

TRPC4 channels may be utilized for the treatment

by PI hydrolysis such as diacylglycerol (DAG),

of fatal diseases such as sepsis where thrombin

arachidonic acid metabolites, protein kinase C

mediates the vicious cycle between inﬂammation(PKC), etc. Two major classes of molecules haveand coagulation.

been known to form these Ca

channels. “Tran-

M aterials and M ethods

sient receptor potential”(TRP) channels are thehomo‑ or hetero‑tetramers of putative six‑trans-

Solutions and Chemicals

membrane polypeptide subunits . The TRP super-

Modiﬁed Tyrode solution contained (in mM):family comprises six related proteins: TRPC

NaCl 136.5;KCl 5.4;CaCl 1.8;M gCl 0.53;HEPES(canonical), TRPV (vallinoid), TRPM (melastatin),

10;and glucose 10 (pH adjusted to 7.4 with NaOH).

TRPP (polycystin),TRPM L (mucolipin)and TRPA

Hypertonic extracellular solution contained (in(ankyrin).Among them,TRPC,TRPV,TRPM,andmM ):mannitol 220;Ca‑gluconate 20;cesium meth-

TRPA channels can serve as SMOCs regulated byanesulfonate 5.4; HEPES 10; and glucose 10 (pHPI hydrolysis, whereas most of the TRPC channels

adjusted to 7.4 with CsOH). Pipette solution

can also operate as SOCs. Another class of Ca

contained (in mM): cesium methanesulfonate 140;

channels regulated by PI hydrolysis is Orai/

Ca‑gluconate 1.74;MgSO 3;HEPES 10;EGTA 5;

CRACM1 channels

BAPTA 2;ATP 3;and GTP 0.1(pH adjusted to 7.2. Orai channels are oligomersof putative four‑transmembrane polypeptide
subunits and correspond to a special entity of SOCs

Primary human umbilical vein endothelial cells

channels. Orai(HUVECs), endothelial growth medium (EBM ‑2),level in ER via single‑trans-

and a supplement for EBM ‑2 (EGM ‑2) were pur-membrane polypeptides termed stromal interaction

chased from Cambrex Corporation (East Rutherfor-

molecules 1 .

d, NJ, USA); Penicillin/streptomycin, Trypsin‑termed Ca

release‑activated Cawith CsOH).

channels sense a Ca

In ECs, TRPC channels have been reported to
14

EDTA, BAPTA‑AM , and SKF96365, from Sigma‑
Shinshu M ed J Vol. 59

Cachannels in endothelial cells

Aldrich,Inc.(St Louis,M O,USA);Cellsmatrix typeand 0.02% saponin in PBS) for 15 min, phalloidin

I‑P, from Nitta Gertin Inc. (Osaka, Japan);FITC‑

solution (4 U/ml FITC ‑phalloidin and 0.02%

phalloidin, from Invitrogen Corporation (Carlsbad,

saponin in PBS) for 30 min and with propidium

CA, USA); normal goat serum, from Dako A/S

iodide solution (PI in PBS)for 15 min. After wash-(Glostrup, Denmark); Isogen, from Nippon Geneing with PBS three times, the coverslips were

(Tokyo, Japan); and GoTaq M aster Mix, frommounted onto glass slides with the mounting

Promega (Madison, WI, USA). All other chemicals

medium. The morphology of HUVECs visualized

were purchased from Wako Pure Chemical Indus-

with FITC ﬂuorescence was pictured with a ﬂuores-

tries, Ltd (Osaka, Japan).

cence microscopy (AX80, Olympus, Tokyo, Japan).

Stock solutions of BAPTA‑AM ,U73122,U73343,

The percentage of surface area of HUVECs per unit

thapsigargin, bisindolylmaleimide I, 1‑oleoyl‑2‑

area was measured by Photoshop (Adobe Systems

acetyl‑glycerol, SKF96365, ruthenium red, andIncorporated, San Jose, CA, USA). Images were

ﬂufenamic acid were prepared in 100% DMSO atacquired at x200 magniﬁcation, and 6 images on

50, 2, 2, 1, 1, 100, 50, 10 and 100 mM , respectively.

each sample were analyzed.

The maximum ﬁnal concentration of DM SO (0.1%)
did not aﬀect membrane currents.
Cell Culture

For the electrical assessment of the contractionof HUVECs,the impedance of a HUVEC monolayer
was measured with a Culture Cell Analyzer (HIOKIHUVECs were cultured in the EBM ‑2 medium

E.E. Corp., Ueda, Nagano, Japan). This system

supplemented with EGM ‑2 and 10% fetal bovine

comprises three components: an electronic sensor

serum. Cells were maintained at 37 ℃ in aanalyzer,a device station and 4×Indium Tin Oxide

humidiﬁed atmosphere of 5% CO and 95% air(ITO) electrode sensor wells. The ITO electrodeuntil they formed subconﬂuent layers. Cells were

was fabricated by sputtering ITO on a glass plate.

then detached with trypsin‑EDTA, resuspended in

HUVECs were grown on the surfaces of ITO elec-

fresh culture medium and passaged. HUVECs

trodes. Sensor electrical impedance quantitatively

between passages 1 and 7 were used for experi-

reﬂects changes in the number, viability, morphol-ments.

ogy and adherence of cells.

Measurement of Contraction of Endothelial

To quantify cell status, a parameter termed

Cells

reactance ratio was derived from the following

HUVECs were seeded at a density of 20,000 cell/

equation :cm on glass coverslips coated with 0.01% collagen,

Reactance ratio＝X(t)/X(0),Eq. 1grown to conﬂuence and incubated in serum‑freewhere X(0)and X(t)are the electrode reactances (i.

medium for 2 h before experiments.HUVECs weree. a component of impedance) before or at time tthen incubated with the indicated concentrations ofafter drug addition, respectively. When the number

thrombin in serum‑free medium. In some experi-of cells in the well is constant, a change in thements,HUVECs were pretreated with BAPTA‑AM

reactance ratio reﬂects a change in the cell status(10μM)for 1 hr before addition of thrombin.

such as morphology.For instance,a decrease in cell

Contraction of HUVECs in response to thrombin

adhesion or spreading reduces the cell‑electrode

was assessed by morphological and electricalcontact area, leading to a decrease in X(t) and themethods. For the morphological assessment, thereactance ratio. To monitor the thrombin‑induced

cells were treated with thrombin for the indicatedcontraction of HUVECs, the cell‑electrodeintervals and then washed twice with PBS, ﬁxed

impedance was continuously measured,and a corre-

with 20% formalin for 15 min and washed threesponding time‑dependent change in the reactancetimes with PBS. The cells were then treated withratio was derived and recorded.

blocking and permeation solution (1% goat serum
No. 1, 2011

Electrophysiological Analyses
15

Tsujino.Nakada.Tsubouchi et al.

HUVECs were seeded at a density of 450 cell/cm

reading provided by the patch‑clamp ampliﬁer

on glass coverslips coated with 0.01% collagen andwhen the cells were perfused with the hypertonic

grown in the EBM ‑2 medium supplemented withextracellular solution and was thus disregarded in

EGM ‑2 and 10% fetal bovine serum for 1‑3 days

this study.

until the days of experiments.The cells were treat-

The holding potential was ‑60 mV.At the timings

ed with the serum‑free medium for 2 hr before

indicated in Figures,a ramp pulse from ‑100 to ＋60

experiments.

mV for 1 sec was applied.In some experiments,theThe whole‑cell membrane currents of single

500 msec rectangular pulses ranging from ‑100 to ＋

HUVECs were measured with the patch‑clamp

60 mV with a 10 mV increment were applied every3

method . In all experiments, data acquisition wassec.

done with an in‑house computer program. Patch

Recorded cell currents were low‑pass ﬁltered at

pipettes were fabricated from borosilicate glass

10 kHz (‑3 dB), digitized at 47.2 kHz with a PCM

capillaries (Kimax‑51,Kimble Glass Inc.,Vineland,

converter system (VR‑10B, Instrutech Corp., New

NJ, USA) and had a resistance of 2‑4 M Ω when

York, NY, USA) and recorded on videocassetteﬁlled with the pipette solution. The pipette oﬀset

tapes. For oﬀ‑line analysis, data were reproduced,

potential was adjusted with a patch clamp ampliﬁer

low‑pass ﬁltered at 2 kHz (‑3 dB), digitized at 5

(Axopatch 200B, Axon Instruments, Foster City,

kHz with an AD converter (ITC16I, InstrutechCA)when the tip of a pipette was immersed in theCorp., New York, NY, USA) and analyzed with

modiﬁed Tyrode solution (see below). The series

Patch Analyst Pro (MT Corp., Hyogo, Japan).

resistance and cell capacitance compensated with

RT‑PCR

the ampliﬁer were 8 ± 1.0 M Ω and 73 ± 6.9 pF,

Total RNA of HUVECs was isolated with Isogen

respectively. All experiments were performed at(Nippon Gene, Tokyo, Japan) according to theroom temperature (25‑26 ℃).

manufactureʼs instructions. For cDNA synthesis, 5

Before whole‑cell recordings, cells were super-

μg of total RNA was reverse‑transcribed withfused with the modiﬁed Tyrode solution. Afteroligo(dT) primer and SuperScript III First‑Strand

establishment of the whole cell conﬁguration, cells

Synthesis System for RT‑PCR (Invitrogen, Carls-

were superfused with the hypertonic extracellular

bad,CA,USA).To assess the expression of the TRP

solution at a rate of 1.5 ml/min with a peristaltic

channels of the HUVEC, PCR was performed withpump (Dynamax, Rainin,Oakland,CA,USA).Boththe above cDNA and GoTaq M aster M ix (Promega,

pipette and hypertonic extracellular solutions were

Madison, WI, USA). All primer sets used are listed

devoid of Na , K and Cl to eliminate these cur-in Table 1.

rents . Drugs were dissolved in the hypertonic
extracellular solution and applied to the cells.

Data analysis
Data values are shown as mean ± SE.StatisticalAn agar‑bridge was used for a reference elec-

diﬀerences were evaluated with Studentʼ
s paired ortrode to reduce the liquid junction potential.In thisunpaired t‑test. A value of P＜0.05 was consideredelectrode, agar containing the hypertonic extracel-signiﬁcant.

lular solution was connected to silver chloride via
the pipette solution. The liquid junction potentials

Results

were directly measured with the patch‑clamp

Thrombin‑induced contraction of HUVECs

ampliﬁer and a 3 M KCl reference electrode .The

We ﬁrst examined the eﬀect of thrombin on thepotentials of the modiﬁed Tyrode solution in refer-shape of cultured HUVECs (Fig.1).In Fig.1A,actin

ence to the pipette and the hypertonic extracellularstress ﬁbers and nuclei were visualized with FITC‑

solution were ＋11 and ＋10 mV,respectively.Thus,

phalloidin (green) and propidium iodide (red),the membrane potential diﬀered by ‑1 mV from therespectively. Addition of thrombin (0.5 U/ml) to16

Shinshu M ed J Vol. 59

Cachannels in endothelial cells

Table 1 Primer sets used for RT‑PCR
Gene
TRPM 1

TRPM 2

TRPM 3

TRPM 4

TRPM 5

TRPM 6

TRPM 7

TRPM 8

TRPV1

TRPV2

TRPV3

Sequence
actatttcctatcccctggaagaaacc

StrandSense

cagacacaattactaagctgcttacactac Antisense
tcaccatggaccccatccgt

Sense

gtcacacgccacagcccatt

Antisense

ttctgtcggaatgatccttcgt

Sense

tgaccagcaagatgtttgcc

Antisense

tggaaccagtgcgacctagtg

Sense

tgccctgtactttgccgaatg

Antisense

agcgcatgatgaaggacgtctt

Sense

ggaagttctccttctggactgtctc

Antisense

gcctccgtaaagctatgagagtcgt

Sense

gctctccccgagtgtactcatagg

Antisense

tctccatttaagccagctatggatac

Sense

acattcttccacagcaaaccact

Antisense

cgctggaggtggatattccgtt

Sense

tgcctcatttcctctgaggtgt

Antisense

tggcctatgtaattctcacctacatcc

Sense

cttgaagacctcagcgtcctct

Antisense

cgcttccttctgatctacttagtcttc

Sense

aggacagggttctcgagagt

Antisense

agcagaactccaagtatcccattSense

tttccgggaattcctcgactt

Antisense

Gene
TRPV4

TRPV5

TRPV6

TRPC1

TRPC3

TRPC4

TRPC5

TRPC6

TRPC7

TRPA1

Sequence

Strandaacccgtgtgccaacatgaaggtgt

Sense

ctcattcttgcccgggtcctcgtt

Antisense

cttcgccatcattgccacact

Sense

cccctcactaaggttcagtccaa

Antisense

tcatcgatggcccagccaact

Sense

tgttgattatcccacgcaggtctctc

Antisense

aaggattgggatgcattccatcctac

Sense

cgtacatttgtcatcaaagtagctaagcc

Antisense

tctgggttcttggaatgatgtggt

Sense

agcattaactttagccccaaggtagt

Antisense

acgaggtccgctgtaactgtg

Sense

gaggctgtgtggcagataaac

Antisense

acagccaactcccacttaggac

Sense

gcatgatcggcaataagctgatagg

Antisense

gagttgtggaacatgcttga

Sense

tttgcaccaatgtagtaggag

Antisense

gaccacaaattcatcgagaacattgg

Sense

gagccgtttcatgattttctggtatc

Antisense

cagctcatctgcagtggcaa

Sense

aagttccacctgcatagctatcc

Antisense

extracellular medium induced contraction of

HUVECs (Fig. 1B, closed circles), indicating that

HUVECs. Fig. 1B shows quantiﬁcation of the sur-thrombin induced the contraction at least partiallyface area of each HUVEC,indicating that thrombin

by increasing intracellular Ca

concentrationinduced the contraction of HUVECs in a time‑［( Ca ］)as previously reported.

dependent manner (open circles).Thrombin induced

Thrombin‑induced Ca ‑permeable channels

the contraction also in a concentration‑dependent

in HUVECs

manner with a half‑maximum eﬀective concentra-

It is known that thrombin causes the contraction

tion (EC )of 〜0.05 U/ml (Fig. 1C). We also asses-of HUVECs mainly via protease‑activated rece-

sed the shape change of HUVECs by electricallyptors (PAR)1. G

measuring the reactance ratio of their monolayer

vates phospholipase C (PLC), which produces(Fig. 1D). Thrombin changed the ratio in a time‑

inositol 1,4,5‑trisphosphate (IP )and diacylglyceroland concentration‑dependent manner similar to(DAG) from phosphatidylinositol 4,5‑bisphosphate.

that estimated morphologically (Fig. 1B, C). Prein-

This pathway causes an initial transient increase incubation of HUVECs with a cell‑permeable［Ca ］ by IP ‑induced Ca

proteins coupled to PAR1 acti-

release from the ER

chelator, BAPTA‑AM (10μM ) signiﬁcantly

and a subsequent sustained increase in［Ca ］ byattenuated the thrombin‑induced contraction ofactivating Ca ‑permeable channels in the plasmaCaNo. 1, 201117

Tsujino.Nakada.Tsubouchi et al.

Ａ

Ｂ

Ｃ

Ｄ

Fig. 1 Contraction of HUVECs induced by thrombin
A, Thrombin induced contraction of HUVEC in a time‑dependent manner. Actin stress ﬁbers and nuclei werevisualized with FITC‑phalloidin (green)and propidium iodide (red), respectively. Fluorescence images were taken at
indicated timings after addition of thrombin (0.5 U/ml). B, Quantitative analysis of a change in cell surface area in
response to thrombin (0.5 U/ml). Cell surface area at the indicated timings after application of thrombin is expressed
as a percentage of that before application of thrombin. Data were obtained either under the control condition (opencircles,n =4)or after pretreatment of HUVECs with BAPTA‑AM (10μM)for 1 hr (ﬁlled circles,n =4). p＜0.05 versus
control. C, Thrombin induced contraction of HUVECs in a concentration‑dependent manner. Cell surface area wasmeasured 60 min after application of diﬀerent concentrations of thrombin.EC of thrombin was 〜0.05 U/ml (n =4).
p＜0.05 versus the control. D, Thrombin‑induced change in reactance ratio of HUVEC monolayers. HUVECs weregrown to conﬂuence on electrodes and treated with indicated concentrations of thrombin (n =4‑9 for each concentration). p＜0.05 versus control, ♯p＜0.05 versus 0.5 U/ml.
18

Shinshu M ed J Vol. 59

Cachannels in endothelial cells

membrane.In the sustained contraction of HUVECs

dothelial cells .Thus,we added 1μM thapsigargin,

induced by thrombin,the latter mechanism has been

an inhibitor of Ca ‑ATPase in ER that eﬀectively

shown to play a more important role than the for-causes ER Ca

mer. Thus, we measured the eﬀect of thrombin

However, it did not induce Ca currents inon the whole‑cell membranecurrents with thepatch‑

HUVECs where thrombin (0.2 or 2 U/ml)clearlydid

clamp technique. Under the present experimentalso (Fig.3D,E).We also applied thapsigargin with 10

condition where Na , K , and Cl currents wereμM IP to the pipette solution or thapsigargin (1suppressed, virtually only CaμM)to the extracellular solution and again failed toand Cscarrieddepletion, to the pipette solution.

membrane currents (see Materials and Methods).

detect measurable Ca

Under this condition, thrombin (2 U/ml) gradually

Second, DAG generated by PLC may mediate theincreased inward currents at ‑60 mV (Fig. 2A).

activation of Ca

channels. However, we could

After reaching the maximum, thrombin‑induced

not activate Ca

channels with a DAG analogue,

currents gradually decreased in the continuous pres-1‑oleoyl‑2‑acetyl‑glycerol (OAG) (30μM ) as

ence of thrombin.The current‑voltage(IV)relation-eﬀectively as thrombin (0.2 U/ml) (Fig. 3F, G).

ship was linear or slightly outwardly rectiﬁed in the

Third, protein kinase C (PKC) activated by DAG

absence (a) and presence (b) of thrombin (2 U/ml)

may mediate the thrombin‑induced activation of(Fig. 2B). The thrombin‑induced currents reversedCain direction at ＋56 ± 10 mV (n =6). Fig. 2C showsbisindolylmaleimide I (BIM) (500 nM) signiﬁcantly

the membrane currents elicited by rectangular volt-

attenuated the activation of inward currents byage pulses between ‑100 and ＋60 mV in a 10 mV

thrombin (2 U/ml) (Fig. 3H, I), indicating that

increment (a)in the absence (b)and presence (c)ofthrombin induces Ca

thrombin (2 U/ml). The thrombin ‑induced

chidonic acid metabolites can also mediate activa-

diﬀerence currents (d) were time‑ and voltage‑in-tion of Ca

dependent and slightly outwardly rectiﬁed. Throm-

the addition of 10μM of arachidonic acids tobin increased the amplitude of the inward currents

HUVECs did not result in activation of Ca ‑perme-

at ‑60 mV in a concentration‑dependent mannerable channels (data not shown).

with EC of 0.22 U/ml.Substitution of extracellularwith equimolar N‑methyl‑D(‑)‑glucamineCasigniﬁcantly decreased the amplitude of the throm-currents (data not shown).

channels. In fact, a selective PKC inhibitor,

currents via PKC. Ara-

channels by PI hydrolysis . However,Analyses of the molecules forming thrombin‑
activated Cachannels

The thrombin‑activated Cachannels were volt-

bin‑induced inward currents (Fig. 2E, F). Thus,

age‑independent.These channels were activated bythrombin activated voltage‑independent Ca ‑per-

PKC but not by store depletion. Thus, candidate

meable channels in HUVECs.

genes responsible for these channels seem to be

Signal transduction pathways mediating theinduction of Ca

currents by thrombintransient‑receptor potential (TRP) C, TRPV,
TRPM,and TRPA channels .As assessed with the

A selective PLC inhibitor, U73122 (1μM )but not

RT‑PCR technique, transcripts of TRPM4, 7,

its inactive analogue, U73343 (1μM ) almost com-

TRPV1, 2, 4, TRPC1, 4 and 6 genes existed inpletely inhibited the thrombin‑induced Ca

cur-

HUVECs (Fig. 4A). To analyze the molecules

rents (Fig.3A‑C),indicating that PLC mediated the

responsible for the thrombin‑induced currents, wethrombin‑induced activation of Ca

examined the eﬀects of diﬀerent channel blockers

currents. Sev-

eral mechanisms have been proposed for the PLC‑

on thrombin‑induced Ca

currents (Fig. 4B‑F). In

mediated activation of Ca

channels . First, IP ‑Figs. 4B and C where blockers were applied after

mediated depletion of ER Ca

results in activation

the addition of thrombin, experiments were con-of Ca

currents (i.e. store‑operated Cachannels

ducted in the selected patches where the eﬀect of(SOCs)). It has been shown that SOCs exist in en-

thrombin was sustained rather than transient. A

No. 1, 201119

Tsujino.Nakada.Tsubouchi et al.

Ａ

Ｂ

Ｃ

Ｄ

Ｅ

Ｆ

Fig. 2 Ca currents of HUVECs induced by thrombin
A, Whole‑cell voltage‑clamp recording of a HUVEC showed that thrombin (2 U/ml)transiently induced an inward
current at a holding potential of ‑60 mV.A horizontal broken line indicates the zero current level.At timings indicatedby vertical broken lines (a and b), a ramp pulse from ‑100 to ＋60 mV for 1 s was applied. B, The current‑voltage
relationships of the whole cell currents measured with the ramp pulse protocol in A.The current was measured before(a)and after (b)application of thrombin. The two curves crossed at ＋56 ± 10 mV (n =6). C, The whole cell currents
measured with rectangular voltage pulses. a. The voltage protocol. The membrane potential was changed from ‑100mV to ＋60 mV for 500 ms with an increment of 10 mV every 3 s.The holding potential was ‑60 mV.The currents were
measured before (b)and after (c)application of thrombin (2 U/ml).d shows the thrombin‑induced diﬀerence currents.
D, The relationship between concentrations of thrombin and the amplitude of currents induced by thrombin. The
currents were measured at ‑60 mV.Symbols and bars indicate the mean and SE,respectively(n =9 ‑15 for each point).
The curve indicates a ﬁt of the data with the following Hill equation :［Thrombin］
D＝D /(1＋(K /) ),where D is a thrombin‑induced diﬀerence current density (‑ delta current density); D , the maximum thrombin‑
induced diﬀerence current density; K , the concentration of thrombin which gives the half‑maximum activation ;［Thrombin］,the concentration of thrombin ;and n,the Hill coeﬃcient.The ﬁtting yielded a D ,K and n of 3.69 pA/
pF, 0.22 U/ml and 1.42, respectively. E and F, The eﬀect of removal of extracellular Ca on the membrane current
induced by thrombin (0.2 U/ml). E shows a typical trace recorded at a holding potential at ‑60 mV. Substitution of
extracellular Ca with equimolar N‑methyl‑D(‑)‑glucamine decreased the amplitude of thrombin‑induced inward
currents. F, The amplitude of thrombin (0.2 U/ml)‑induced inward currents was signiﬁcantly smaller in the absence
than in the presence of extracellular Ca . Values are mean ± S.E. (n =4). p＜0.05.
20

Shinshu M ed J Vol. 59

CaＡchannels in endothelial cells

Ｃ

Ｂ
Ｅ

Ｄ

Ｇ

Ｆ

Ｉ
Ｈ

Fig. 3 The mechanism underlying the activation of inward currents by thrombin
A‑B,Thrombin was applied to HUVECs pretreated with a selective inhibitor of phospholipase C,U73122 (1μM )(B)or its inactive analogue, U73343 (1μM)(A). U73343 or U73122 was applied to HUVECs 〜3 min before application ofthrombin. C. The summary of pooled data. U73122 inhibited thrombin‑induced inward currents signiﬁcantly morestrongly than U73343. D, Application of an inhibitor of endoplasmic Ca ‑ATPase, thapsigargin (1μM )to the pipettesolution failed to induce inward currents in HUVECs where thrombin (2 U/ml)did so.E,Thrombin (0.2 U/ml)induced
inward currents,whereas intracellular thapsigargin (1μM )induced small outward rather than inward currents.F and
G, An analogue of diacylglycerol, 1‑oleoyl‑2‑acetyl‑glycerol (OAG) (30μM) induced signiﬁcantly smaller inward
currents than thrombin (0.2 U/ml).H and I,A selective inhibitor of protein kinase C,bisindolylmaleimide I (BIM)(500nM )signiﬁcantly attenuated the activation of inward currents by thrombin (2 U/ml).BM I was applied to HUVECs 〜1
hr before application of thrombin. Values are mean ± S.E. (n =4‑6 for each experiment). p＜0.05
No. 1, 201121

Tsujino.Nakada.Tsubouchi et al.

Ａ

Ｂ

Ｃ

Ｄ

Ｅ

Ｆ

Fig. 4 Identiﬁcation of the gene responsible for the thrombin‑induced inward currents in HUVECs
A,As assessed with the RT‑PCR technique,HUVECs expressed some members of transient receptor potential(TRP)
families:TRPV1, 2, 4, TRPM 4, 7, TRPC1, 4 and 6. B, SKF96365 (50μM ) (a blocker of nonselective cation channels(NSCs))applied to HUVECs in the presence of thrombin (0.2 U/ml)inhibited the thrombin‑induced inward currents.C,
La(1 mM) (a blocker of NSCs except for TRPC4 and TRPC5 channels) applied to HUVECs in the presence ofthrombin (0.2 U/ml)slightly inhibited the thrombin‑induced inward currents.D,ruthenium red (RR)(10μM)(a blocker
of TRPV channels) applied 〜3 min before application of thrombin did not prevent HUVECs from responding tothrombin (0.2 U/ml).E,Flufenamic acid (FFA)(100μM )(a blocker of TRPM2 and TRPM4 channels and an activatorof TRPC6 channels) applied 〜3 min before application of thrombin slightly inhibited thrombin (0.2 U/ml)‑induced
inward currents. F. The summary of pooled data. mean ± S.E. (n =4‑6 for each experiment). p＜0.05
22

Shinshu M ed J Vol. 59

Cachannels in endothelial cells

blocker of non‑selective cation channels (NSCs),

human vascular ECs under the same experimental

SKF96365 (50μM) signiﬁcantly inhibited the curcondition as ours . Thrombin induced the Ca

rents (Fig. 4B, F). However, La(1 mM), whichcurrents less potently than the contraction inalso blocks NSCs except for TRPC4‑ or TRPC5‑HUVECs (Fig. 1C, 2D). This is probably because

containing channels

thrombin induces contraction of ECs not only

, did not signiﬁcantly

inhibit the currents (Fig.4C,F).SKF96365 and La

through a rise in［Ca ］ but also through activation

exhibited a similar degree of blocking when appliedof Rho/Rho kinase .

before thrombin (data not shown),and these results

A PLC inhibitor, U73122 but not its inactive

are included in Fig. 4F. Pretreatment of HUVECs

analogue U73343 almost completely inhibited thewith a blocker of TRPV channels, ruthenium red

thrombin‑induced currents (Fig. 3A‑C), indicating(RR)(10μM)also failed to inhibit the currents (Fig.

that thrombin activated the currents through PLC.

4D, F). Flufenamic acid (FFA), a potent blocker of

PI hydrolysis activates Ca

channels through IP ‑TRPM2 and 4 channels and an activator of TRPC6

induced depletion of Ca

in ER (SOCs) and/or

channel tended to inhibit the currents (Fig.4E),but

second messengers/kinases (SM OCs) . Thapsigar-

this eﬀect did not reach statistical signiﬁcance (Fig.

gin, an inhibitor of Ca ‑ATPase in ER eﬀectively

4F). These results indicate that thrombin activatedcauses depletion of Ca in ER and activates SOCs .

TRPC4‑containing Ca ‑permeable channels in

Although Ahmmed et al. showed 〜1.5 pA/pF of

HUVECs (see Discussion).

thapsigargin ‑induced currents at ‑50 mV in
HUVECs , we could not detect such a current

Discussion

irrespective of whether thapsigargin was applied to

We have found that thrombin induced contraction

the pipette solution alone or in combination with IP

of HUVECs in a［Ca ］‑dependent manner,consis-

or whether thapsigargin was applied to the intra‑ortent with previous reports (Fig. 1B). Because

extra‑cellular solution (Fig.3D,E,data not shown).

inﬂux through the plasma

This discrepancy might have occurred because

thrombin causes a Ca

membrane by activating PAR1/G
sought to characterize Ca

/PLC

, we

currents induced bythrombin in detail.

Ahmmed et al. (2004) buﬀered intracellular Ca
more strongly than we did : their pipette solution
contained 10 mM of BAPTA without additional

Thrombin induced inward currents at ‑60 mV

Ca(i.e.virtual absence of free Ca )whereas ours

(Fig. 1). Under the present experimental condition,

had 5 mM of EGTA,2 mM of BAPTA and 1.74 mM

Na ,K ,and Cl currents were suppressed,and only

of Ca

Ca

Whatever the reason is, thrombin clearly induced

and Cs carried membrane currents (see Mate-rials and Methods). The calculated equilibration
potentials for Ca

and Cs were ＋167 and ‑86 mV,Ca(i.e. free Ca

currents in HUVECs in which thapsigargin

failed to do so (Fig.3D,E),indicating that thrombin

respectively, whereas thrombin‑induced currents

did not activate the Ca

reversed at ＋56 ± 10 mV,indicating that thrombin

depletion.

activated Ca ‑permeable NSCs and that the throm-

concentration of 44 nM).

channels through store

Although we examined whether second messen-

bin‑induced inward currents were mainly mediatedgers produced by PI hydrolysis other than IP

by Ca . This is consistent with the ﬁnding that

activated the currents ,a DAG analogue,OAG (30substitution of extracellular Ca with equimolar N‑μM)or arachidonic acid (10μM) failed to activate

methyl‑D(‑)‑glucamine suppressed the currents

Ca(Fig. 2E). Thrombin‑induced Ca

currents showedshown). On the other hand, a selective PKC in-time‑and voltage‑independent kinetics and slightly

hibitor, BIM (500 nM)signiﬁcantly inhibited throm-

outward rectiﬁcation (Fig.2).Groschner et al.foundbin‑induced Cathat histamine activated similar Ca

likely that thrombin activated the Ca

No. 1, 2011

currents incurrents in HUVECs (Fig.3F,G,and data notcurrents (Fig. 3H, I). Thus, it ischannels
23

Tsujino.Nakada.Tsubouchi et al.

through PKC in HUVECs.

(Fig.2B,E)

From these results, candidate genes for thethrombin‑activated Cachannels in HUVECs are.However,it has been reported that

TRPC4 forms SOCs in mouse aortic ECs ,and thatthrombin failed to induce a Ca

inﬂux in lung

TRPC,TRPV,TRPM,and TRPA .RT‑PCR anal-

vascular ECs of TRPC4 knockout mice . The

ysis indicated that TRPM 4,7,TRPV1,2,4,TRPC1,

discrepancy between these and the present studies

4 and 6 were expressed in HUVECs (Fig. 4A).

might have arisen from species diﬀerence or again

TRPM4 is unlikely to mediate thrombin‑induced

from diﬀerent［Ca ］. Indeed, the activation ofcurrents because TRPM 4 channels are voltage‑

TRPC4 ‑containing

dependent,Ca ‑impermeable and potently blocked［Ca ］

by FFA (Fig. 4E, F) . TRPM7 also cannot be thecontaining channels through store depletion in thecandidate gene for thrombin‑activated Ca ‑perme-virtual absence of intracellular Ca

able channels because TRPM7 channels are strong-through PKC in the presence of a certain amount of

ly inhibited by intracellular M gATP that amply

intracellular Ca

existed in the pipette solution used in this study .

are clearly needed to determine the subunit compo-

TRPV channels diﬀer from thrombin‑activatedsition of the thrombin‑activated Cachannels because a TRPV blocker, RR did not

HUVECs by using antisense oligonucleotides orinhibit the currents (Fig. 4D, F)

small interference RNA.

. It is alsochannels

dependson.Thus,thrombin might activate TRPC4‑
but directlyin ECs. However, further studies
channels in

unlikely that TRPC6 forms thrombin‑activatedTo summarize, thrombin induced the contraction

Ca ‑permeable channels because FFA activatesof HUVECs in a［Ca ］‑dependent manner.It was

rather than inhibits TRPC6 channels (Fig.4E,F) .

likely that thrombin induced this response byThus, the remaining candidate genes for thrombin‑

activating Ca ‑permeable SMOCs containing

activated Ca ‑permeable channels are TRPC1

TRPC4 through PKC. In future, drugs speciﬁcally

and/or TRPC4.It is known that TRPC1 and TRPC4

inhibiting TRPC4‑containing channels might beform either homomeric channels or heteromericdeveloped and used to control fatal diseases such as

TRPC1/TRPC4 channels

sepsis where thrombin mediates the vicious cycle.

The thrombin‑activated channels in HUVECs

between inﬂammation and coagulation.

were similar to TRPC4‑containing channels in theirrelative insensitivity to La

Acknowledgement(Fig. 4C, F), the tran-sient response to agonists (Fig.2A),the insensitivity
to OAG (Fig. 3F, G), and the permeability to Ca

We are grateful to M s.Reiko Sakai for secretarial assistance.

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