以奈米現場效應電晶體及光學感測器探討神經網路功能－1.奈米線場效電晶體及偵測生物系統之應用2.以量子點探討神經分泌膠囊的新陳代謝機制(總計畫暨子計畫一)(2/3)

行政院國家科學委員會專題研究計畫 期中進度報告

以奈米現場效應電晶體及光學感測器探討神經網路功能--1.奈米線場效電晶體及偵測生物系統之應用 2.以量子點探

討神經分泌膠囊的新陳代謝機制(總計畫暨子計畫一)(2/3)

期中進度報告(精簡版)

計 畫 類 別 ： 整合型

計 畫 編 號 ： NSC 95-2627-M-002-003-

執 行 期 間 ： 95 年 08 月 01 日至 96 年 10 月 31 日

執 行 單 位 ： 國立臺灣大學化學系暨研究所

計 畫 主 持 人 ： 陳逸聰

共 同 主 持 人 ： 潘建源

報 告 附 件 ： 出席國際會議研究心得報告及發表論文

處 理 方 式 ： 本計畫可公開查詢

中 華 民 國 96 年 07 月 16 日

行政院國家科學委員會補助專題研究計畫

成果報告

計畫名稱

：

以奈米線場效電晶體及光學感測器探討神經網路功能

計畫類別： □個別型計畫 █ 整合型計畫

計畫編號： NSC

95-2627-M-002-003

執行期間：

95 年 8 月 1 日至 96 年 7 月 31 日

計畫主持人：

陳逸聰 教授

共同主持人：潘建源 助理教授

計畫參與人員：

蔡佳璋（博士後研究員）

，廖國棠（博士後研究員）

，賴俊陽（博士後研究

員）

，王建惟（助理）

，吳智陽（助理）

，吳幸臻（博士生）

，李長琪（碩士

生）

，江佩玲（碩士生）

，于書翰（碩士生）

。

成果報告類型：█精簡報告 □完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

執行單位：國立臺灣大學化學系暨研究所

中 華 民 國 96 年 5 月 16 日

In the past year, we have accomplished two research studies in (a) In-situ detection

of chromogranin A released from living neurons with single-walled carbon nanotube

field-effect transistor

s and (b)

Lysophospholipids regulate the cellular excitability and

exocytosis in cultured bovine chromaffin cells

.

In the in-situ detection of cellular excitability and exocytosis with

nanotube/nanowire field-effect transistors

, we have demonstrated that

single-walled carbon nanotube field effect transistors (SWCNT-FETs) can be used to

monitor the synaptic transmissions among primary cultured embryonic cortical neurons.

Chromogranin A (CgA) is one of the molecules released from the secretory vesicles

when the vesicles are fused with plasma membrane. The expression of CgA has been

identified in neuron and neuroendocrine cells. It has been reported that the appearance

of CgA in plasma is an excellent marker for neuroendocrine tumors and

neurodegenerative diseases like Parkinson’s and Alzheimer’s diseases. Therefore, the

detection of CgA is an indicator of synaptic activity when synaptic vesicle at the

presynaptic terminal is triggered to undergo exocytosis. In this study, we have

demonstrated that CgA released from the synaptic terminal of living neurons can be

detected directly with high selectivity and sensitivity by the CgA-antibody modified

SWCNT-FETs. This novel sensory technique of SWCNT-FETs is promising in medical

examination and can further be applied to study the activity of an individual neuron cell

which should open a new window to enlighten the neurophysiology in neuronal

network.

This work has been accepted for publication: "In-situ detection of chromogranin A

released from living neurons with single-walled carbon nanotube field-effect transistor",

C.-W. Wang, C.-Y. Pan, H.-C. Wu, P.-Y. Shih, C.-C. Tsai, K.-T. Liao, L.-L. Lu, W.-H.

Hsieh, C.-D. Chen, and Y.-T. Chen; Small, 3, 000-000 (2007).

In the study of lysophospholipids regulating the cellular excitability and exocytosis

in cultured bovine chromaffin cells, bioactive lysophospholipids (LPLs) released by

blood cells can be used to modulate many cellular activities such as angiogenesis and

cell survival. In our study, the effects of sphingosine-1-phosphate (S1P) and

lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells

were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated

Ca

current was inhibited by S1P and LPA pretreatment in a concentration-dependent

manner with IC50s of 0.46 and 0.79 μM, respectively. Inhibition was mostly reversible

upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na

current was inhibited by S1P, but not by LPA. However, recovery of Na

channels from

inactivation was slowed by both LPLs. The outward K

current was also significantly

reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to

minimal injections of depolarizing current. Repetitive activity was dramatically reduced

by LPLs. Consistent with the reduction in Ca

current, exocytosis elicited by a train of

depolarizations and the ensuing endocytosis were both inhibited by LPL pretreatments.

These data demonstrate the interaction between immune and endocrine systems

mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin

cells.

This work has been accepted for publication: "Lysophospholipids regulate the

cellular excitability and exocytosis in cultured bovine chromaffin cells", C.-Y. Pan, A.-Z.

Wu, and Y.-T. Chen; Journal of Neurochemistry, 100, 000-000 (2007).

In-Situ Detection of Chromogranin A Released from Living Neurons

with Single-Walled Carbon Nanotube Field-Effect Transistor

1. Introduction

Nanotubes or nanowires based field effect transistors (NT/NW-FETs) as biosensors have

recently drawn more and more attention in biological research because of their selectivity,

sensitivity, and real-time detection capabilities. For biological studies, several one-dimensional

semiconducting materials have been applied as sensing elements to construct NT/NW-FETs,

such as silicon nanowires,

carbon nanotubes,

and indium oxide nanowires,

for a variety

of successful detections of proteins,

nucleic acids,

cancer markers,

and viruses.

In

particular, Lieber’s group recently applied silicon NW-FETs to monitor the electrical signals

from single mammalian neurons, where each nanoscale nanowire-axon junction was used for

spatially resolved, highly sensitive detection, stimulation, and/or inhibition of neuronal signal

propagation.

Their elegant non-invasive measurements of the rate, amplitude, and shape of

signals propagating along individual axons and dendrites from the use of silicon NW-FETs have

opened a new avenue to the study of neurosciences.

Chromogranin A (CgA), a protein with calculated molecular weight ~50 kDa (apparent

molecular weight is 74~80 kDa), is one of the molecules released from secretory vesicles when

fused with plasma membrane. CgA plays many important biofunctions, e.g. as an innate

immunity to fight against bacterial infection,

as a mediator for neuron inflammation,

and

as a Ca

chelator to modulate the secretion of other functional biomolecules

and to mediate

neuronal apoptosis.

It has also been reported that the appearance of CgA in plasma is an

excellent marker for the diagnosis of neuroendocrine tumors and neurodegenerative diseases

like Parkinson’s and Alzheimer’s diseases.

Therefore, the detection of CgA can be an

indicator of synaptic activity when exocytosis occurs at the presynaptic terminal and a

diagnostic tool for clininic examination. In the past year, we chose single-walled carbon

nanotubes (SWCNTs) to configure FETs on account of their high biocompatibility for neuron

cells

and their sensitive detection capability of the CgA released from living cells. The

experimental results show that the SWCNT-FETs are a promising biosensor to monitor the

in-situ release of CgA during synaptic transmission among primary cultured embryonic cortical

neurons and can be used as a new tool to examine the synaptic activity.

2. Results and Discussion

2.1. Preparation of SWCNT-FET biosensors

Figure 1. (a) The mask design for the photolithographic fabrications of SWCNT-FET device array.

(b) The device array on magnified scales. (upper) An optical image for the circuits in the yellow square area and (lower) the SEM image of an SWCNT-FET array with the source-drain separation of 2 μm. The scale bar is 50 μm (c) Schematic illustration of an SWCNT-FET sensor for the source (Cr, 50 nm in thickness), drain (Cr, 50 nm), and backgate (Ni/Au, 10/50 nm) electrodes on a SiO2

(400 nm)/Si substrate. The source and drain electrodes were further passivated with an insulating layer of SiO2 (50 nm) to avoid electric leakage to sample solution. The schematic also shows the

immobilization and molecular recognition procedures: 1. The adsorption of linkers onto the SWCNT through a π-π interaction. 2. The immobilization of antibody. 3. The detection of antigen by antibody.

(a)

(b)

(c)

devices were fabricated following a standard photolithographic procedure. After the fabrication

of outer electrodes (represented in white color in Figure 1a) with Au/Cr (50/50 nm in thickness),

SWCNTs of 2 nm in diameter (Thomas Swan Co. & Ltd.) were transferred to a SiO

/Si

and 0.125% sodium dodecylbenzene sulfonate (NaDDBS) as a surfactant following the method

developed by Islam et al.

After baking at 200

C for 12 hr to remove the NaDDBS,

the

as-dispersed SWCNTs in the central area (the reddish rectangles in Figures 1a~b) were

electrically contacted by 50 nm thick Cr leads (represented in yellow color in Figure 1a) which

were further passivated with a layer of 50 nm thick SiO

by thermal evaporation to avoid

electric leakage to sample solution. The original pattern design and photolithographic

fabrication for this kind of device array by Lieber’s group can be found in Refs. 10, 11, and 20.

Ni/Au (10/50 nm in thickness) layers coated to the bottom of the SiO

/Si substrate were used as

a backgate.

The as-fabricated SWCNT-FETs were then conversed to functional sensors by

immobilizing the complementary probes, which will later be used against target molecules, onto

the surfaces of SWCNTs (Figure 1c). In our experiment, a goat IgG antibody against CgA,

denoted as CgA-Ab (Santa Cruz Inc.), served as the probe and was immobilized to the sensing

Figure 2. (a) Representative SEM image of an SWCNT-FET device consisting of a pair of source

and drain electrodes connected by a small bundle of SWCNTs. (b) A typical

Isd

SWCNT-FET device measured in the ambient air. The inset presents the same data but in a semi-log plot.

Isd

devices of SWCNT-FETs with a literature-reported procedure developed by Dai’s group.

After 1-pyrenebutanoic acid succinimidyl ester was applied as a linker to connect antibody with

SWCNTs, tween 20 was further used to block nonspecific bindings.

Figure 2a shows the representative scanning electron microscopic (SEM) image for a pair

of source and drain electrodes connected by a bundle of SWCNTs. The electrical transporting

property of the SWCNT-FET was characterized in the ambient air by measuring the

source-drain current vs. backgate voltage (I

-V

) curves swept in a negative-positive-negative

voltage direction as illustrated in Figure 2b. The current decreases with increasing gate voltage,

indicating that the SWCNTs are of p-type. Avouris and co-workers have pointed out that the

CNT-FET operation mode is dominated by the Schottky barrier, and the hole-transporting

property is mainly due to the oxygen adsorption on the metal-SWCNT junction which causes

Figure 3. (a) The Isd-Vg curves measured in the ambient air before and after the immobilization of

CgA-Ab on an SWCNT-FET. Isd was measured at Vsd = 10 mV. The change of threshold voltages

(ΔVth) before and after the immobilization of CgA-Ab is indicated. (b) An optical image of the

SWCNTs located in the patterned spots fabricated photolithographically. The scale bar is 30 μm. (c) A fluorescence image of the SWCNTs modified with CgA which was further labeled with rhodamine-conjugated rabbit anti-goat IgG antibody. The procedures for the immobilization of CgA-Ab on SWCNTs are described in the context. The scale bar is 30 μm.

the Fermi level of metal to approach the valence band edge of SWCNTs.

Moreover, the

hysteresis in SWCNT-FETs is attributed to the adsorption of water molecules on SWCNTs as

discussed by Dai and co-workers.

The immobilization of CgA-Ab onto SWCNTs was demonstrated by two methods:

electrical characterization (Figure 3a) and fluorescence microscopy (Figures 3b~c). Figure 3a

depicts the I

-V

curves, measured in the ambient air, before and after the immobilization of

CgA-Ab onto SWCNTs. The shift of threshold-voltage (ΔV

) toward the more negative side

indicates an electron donation from the as-immobilized CgA-Ab to SWCNTs due to the

electron-donating amino groups of antibodies.

In addition, the decrease of current (I

) after

the immobilization of CgA-Ab is also attributed to the effect of potential scattering to reduce the

mobility of charge carriers.

These observations have indicated the functionalization of

CgA-Ab onto SWCNTs. In the fluorescence imaging experiment to characterize the

functionalization of CgA-Ab onto SWCNTs, densely matted SWCNTs were first stuffed by

repeatedly coating them into prefabricated 10 μm-deep photoresist wells made by a

photolithographic technique. After lifting off the photoresist, SWCNT films were left in the

patterned spots (Figure 3b). The CgA-Ab was then immobilized onto these SWCNTs with the

procedures described earlier. Subsequently, the CgA-Ab modified SWCNTs were incubated

with rhodamine-conjugated anti-goat IgG antibody (Santa Cruz Inc.). In Figure 3c, the

fluorescence image clearly displays the successful immobilization of CgA-Ab onto the surfaces

of SWCNTs.

2.2. Molecular recognition of CgAP by CgA-Ab

The SWCNT-FETs which have been modified with CgA-Ab on the surfaces of SWCNTs

will be represented as CgA-Ab/SWCNT-FETs hereafter. To examine the detection efficacy of

CgA-Ab/SWCNT-FETs, a peptide that encodes the amino acids 158~457 of CgA (denoted as

CgAP, Santa Cruz Inc.) was used. To deliver the CgAP solution onto the

CgA-Ab/SWCNT-FETs, a polydimethyl-siloxane (PDMS) microfluidic channel

(6.25×0.5×0.05 mm

) was designed to couple with the device arrays (the reddish rectangles in

Figures 1a~b) and the CgAP sample solution was driven into the channel by a syringe pump.

Figure 4a shows the current (I

) of a CgA-Ab/SWCNT-FET in response to different

concentrations of CgAP in PBS (phosphate buffer saline, 137 mM NaCl, 2.7 mM KCl, 10 mM

Na

HPO

, 2 mM KH

PO

, pH 7.4 with NaOH). After balanced with PBS, the I

was ~1.085 nA

in the beginning and showed no significant change in electric conductance when 60 μg/mL

bovine serum albumin (BSA) reached the CgA-Ab/SWCNT-FET sensor, manifesting the

binding specificity of CgA-Ab. The I

increased to ~1.093 nA, when 1 nM CgAP was

introduced. The I

moved further up to ~1.13 nA, when CgAP was increased to 10 nM. This

sensory device, however, did not respond in proportion to the addition of 100 nM CgAP,

indicating a saturation of the binding sites on the CgA-Ab/SWCNT-FET. The sensitive electric

responses have demonstrated the high affinity between CgAP and CgA-Ab/SWCNT-FET. For

CgA, a typical isoelectric point (pI) ranges 4.5 ~ 5,

and a pI value of 4.49 for the CgAP

sequence used in this experiment was provided by the manufacturer. In view of its low pI value,

CgAP should be negatively charged in PBS at pH = 7.4. Accordingly, the binding of CgAP onto

CgA-Ab/SWCNT-FET should have increased the carrier (hole) concentration, thus enhancing

the electric conductivity in the p-type semiconductor device due to a gating effect.

To further investigate the applicability of CgA-Ab/SWCNT-FETs in medical diagnosis,

CgAP was dissolved in FBS (fetal bovine serum, JRH Biosciences Inc.) which was aseptically

collected via cardiac puncture followed by centrifugation and filtration to remove most blood

corpuscles. Unlike PBS, composed of only simple ionic salts (e.g. K

, Na

, etc.), FBS contains a

variety of proteins and small biomolecules. Therefore, the detection of CgAP in FBS is a

Figure 4. Electric responses of a CgA-Ab/SWCNT-FET to CgAP of different concentrations

measured in ambient conditions of (a) PBS and (b) FBS. At the beginning, 60 μg/mL BSA was used as a negative control. Different concentrations of CgAP were then flowed into the PDMS microfluidic channel coupled with the device arrays. Isd was measured at Vsd = 10 mV with

stringent test for the diagnostic performance of CgA-Ab/SWCNT-FETs. Figure 4b shows the

current responses of a CgA-Ab/SWCNT-FET in the detection of CgAP in FBS with I

increasing from ~1.390 to ~1.397 nA as 100 pM CgAP reached the device, and further

increasing to ~1.41 nA when 1 nM CgAP was added. These results demonstrate that the

CgA-Ab/SWCNT-FETs are capable of detecting CgA selectively even at a very low

concentration level (≥1 nM) in the complex FBS environment (total protein content ranging

from 30 to 45 mg/mL). This capability ensures that the sensitive and selective

Figure 5. CgA can be detected in cultured cortical neurons. (a) A differential interference contrast

microscopic image of live cultured neurons grown on a coverslip. The scale bar is 10 μm. To label both CgA and nucleus in neurons, cells were fixed and stained with the antibody against CgA and DAPI, respectively. The (b) bright field, (c) CgA immunostaining, and (d) nucleus immunostaining images of a single neuronal cell are presented. The scale bars are 10 μm. (e) The cultured neurons were harvested and the total proteins were used for a Western blot experiment. The positions of molecular weight markers (left lane) and CgA from neuron lysate (right lane) are indicated.

CgA-Ab/SWCNT-FETs are suitable for neuronal and neuroendocrine cancer diagnosis.

More

importantly, these sensory devices have the merits of label-free and real-time detection

capabilities in medical applications.

2.3. In-situ detection of CgA released from living neuron

E14.5 cortical neurons were isolated from the embryos of Sprageu-Dawley rat and

cultured on a poly-L-lysine pretreated coverslip with the standard protocol as described

before.

Neurons were used after 7~11 days in culture for the following experiments. The

optical image of such cultured neurons is shown in Figure 5a. The cell bodies (somata) are ~10

μm in diameter and the silk-like neurites can be clearly identified. To verify the existence and

distribution of CgA in neurons, traditional immunochemistry was performed. Figures 5b~d

display the resultant immunostaining images, where single neuronal cells were first

immobilized by formalin (4%), and then treated with triton X-100 (0.5%) to permeabilize the

cells

and with BSA (1%) to avoid nonspecific binding. The endogenous CgA was then

stained by the same primary antibody as that was used in modifying the SWCNT-FET device.

After rinse, fluorescin isothiocyanate conjugated rabbit anti-goat IgG was used as the secondary

antibody (1:2000, diluted with PBS at pH 7.4) to obtain fluorescence image. The nucleus was

also stained by diamidino-2-phenylindole (DAPI) which binds selectively to the DNA double

helix (Figure 5c). The distribution of green dots indicates that the localization of CgA was in

both the cell body and neurites. To further examine the existence of CgA in neurons, the

proteins were extracted from cultured neurons and separated by an 8% SDS-PAGE. The

proteins on the gel were electrically transferred to a nitrocellulose paper and the antibody

against CgA was used to stain the position of CgA. The resultant Western blot examination is

depicted in Figure 5e, in which a single band at ~75 kDa was identified, referring to the

apparent existence of CgA. The shift of the molecular weight of CgA (~50 kDa) may be due to

the denaturing condition used in this experiment as reported by the antibody manufacturer

(Santa Cruz Inc.).

A schematic illustration of the in-situ detection of CgA released from neurons by a

CgA-Ab/SWCNT-FET is shown in Figure 6a. Glutamate, one of the most common

neurotransmitters in brain, was used to stimulate the neurons. The binding of glutamate to the

ionotropic glutamate receptors allows Na

fluxes into the cytosol and depolarizes the cell. The

subsequent opening of the voltage-gated Ca

channels at the synaptic terminal triggers

exocytosis to release CgA, which can then be detected by the CgA-Ab/SWCNT-FET. In the

following experiments, instead of using a PDMS microfluidic channel, a plastic wall (located on

the orange square in Figure 1a) of 3 mm in height was glued onto the SiO

/Si chip to hold the

normal saline buffer (145 mM NaCl, 5 mM glucose, 10 mM Na-HEPES, 1 mM MgCl

, 5 mM

KCl, and 2.2 mM CaCl

, pH 7.3 with NaOH) as illustrated in the cartoon of Figure 6c. The FET

protected by coating a layer of photoresist to prevent them from contacting ionic solution.

Before the neuron experiment, a negative control without neurons on the FET device was

performed by adding 50 μM glutamate to the CgA-Ab/SWCNT-FET which had been immersed

in 100 μL normal saline buffer. No I

change could be detected (Figure 6b), when glutamate

was added. To detect the CgA released from neurons, a piece of coverslip blanketed with

cortical neurons was pushed onto the FET chip and incubated in 100 μL normal saline buffer

(the cartoon inset of Figure 6c). Care was taken to let neurons face the CgA-Ab/SWCNT-FET.

Figure 6. The CgA released from neurons can be detected by CgA-Ab/SWCNT-FETs. (a) When

neurons are stimulated by glutamate, the voltage-gated Ca2+_{channels at the axon terminal will be}

opened and allow the entrance of Ca2+ _{into the cytosol. The Ca}2+ _{will induce the fusion of synaptic}

vesicles with plasma membrane to release CgA, which will then bind to the CgA-Ab immobilized on the SWCNT-FET. (b) Before the neuron experiment, a negative control without neurons on the FET device was performed by adding 50 μM glutamate to the CgA-Ab/SWCNT-FET. No Isd

change could be detected. (c) In-situ detection of the Isd changes elicited by glutamate. The

coverslip was positioned onto the FET device with neurons faced the circuit as illustrated by the inset cartoon and indicated by “cell” in the current trace. Glutamate (50 μM) was then added to stimulate the neurons to release CgA. The Isd was measured in the ambient solution at Vsd = 10 mV

After the addition of 50 μM glutamate to activate the glutamate receptors, a huge increase in the

I

of CgA-Ab/SWCNT-FET was detected as shown in Figure 6c.

CgA is widely detectable in central nervous system and is suggested to be co-released with

the neurotransmitters.

Although CgA can also be a marker for neuron labeling, there are only

very few reports measuring the release of CgA from neurons.

Some immunoassay kits for

detecting CgA in serum have been developed and the detection limit is at ~nM level.

However, these techniques could not be used to monitor the low amount of CgA released from

cultured neurons in real time. Moreover, after glutamate stimulation, the low amount of CgA

released in the bath buffer could not be detected by Western blot either (data not shown). In

contrast, our FET setup which directly measures the immediate vicinity release of CgA from

neurons provides a novel technique to monitor the neuron activities.

It is noted that the little increase in I

when neurons were mounted onto the

CgA-Ab/SWCNT-FET device (as marked by “cell” in Figure 6c) might be due to the basal

release of CgA. This small current change, however, did not affect the instant and prominent I

increase when neurons were stimulated by glutamate. In the neuron experiment, we also tested

the selectivity of bare SWCNT-FET devices (without the immobilization of CgA-Ab on the

SWCNTs) by adding 50 μM glutamate to the neurons in 100 μL normal saline buffer. Again, no

significant changes could be observed in this control experiment. These results suggest that the

surface functionalized CgA-Ab/SWCNT-FETs are of detection specificity and can be applied to

monitor the molecules released from living neurons.

3. Summary

Compared with the traditional time-consuming electrophoresis technique, e.g. Western

blot, our study has demonstrated that the CgA released from the synaptic terminal of neurons

can be detected in-situ by the CgA-Ab/SWCNT-FETs with high selectivity, sensitivity, and

real-time detection capabilities. This sensory technique is promising in medical diagnosis and

can further be applied to study the activity of individual neurons, which should open a new

window to enlighten the neurophysiology in neuronal network.

4. Experimental section

4.1 Electrical measurement and optical imaging

In the electric transport measurements, the conductance of SWCNT-FET was recorded by a

lock-in amplifier (Stanford Research 830) in an AC mode. To measure the I

-V

curves, a

power supply (Keithley 2400) was employed to apply the backgate voltage. The I

-V

curves

for CgA-Ab/SWCNT-FETs were measured after the CgA-Ab/SWCNT-FETs were fully rinsed

with deionized water and dried in the ambient air. In this study, while the I

-V

measurements

6) were carried out in the ambient aqueous solutions. The fluorescence image after the

immobilization of CgA-Ab onto SWCNTs (Figure 3c) was taken by an epifluoresce microscope

(Nikon TE2000) with 20× objective, and the images of the CgA immnostaings (Figures 5a~d)

were taken by a microscope (Leica DM-IRE) with 40× and 100× objectives.

4.2 Surface functionalization

In the processes of the surface functionalization of SWCNT-FETs, all sample solutions were

driven by a syringe pump at the flowing rate of 0.5~0.8 mL/hr into the PDMS microfluidic

channel coupled with the device arrays. The SWCNTs were first treated in 7.5 mM

1-pyrenebutanoic acid succinimidyl ester (Sigma Aldrich) for 1 hr and then flushed with pure

methanol for 15 min. Subsequently, the device was incubated for 2 hr in PBS (pH 7.4)

containing 0.2 μg/mL of goat IgG antibody against CgA. After the extra CgA-Ab was washed

off, the device was then incubated for 20 min in PBS containing 0.5% tween 20 (J. T. Baker).

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Lysophospholipids regulate excitability and exocytosis in cultured

bovine chromaffin cells

Chien-Yuan Pan,*

Adonis Z. Wu*

and Yit-Tsong Chenà

*Institute of Zoology, National Taiwan University, Taipei, Taiwan  Department of Life Science, National Taiwan University, Taipei, Taiwan àDepartment of Chemistry, National Taiwan University, Taipei, Taiwan

Abstract

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angi-ogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca2+ _{current was inhibited by}

S1P and LPA pre-treatment in a concentration-dependent manner with IC50s of 0.46 and 0.79 lmol/L, respectively.

Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na+ current was inhibited by S1P, but not by LPA. However, recovery of Na+channels from inactivation was slowed by both LPLs. The

outward K+ current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Con-sistent with the reduction in Ca2+_{current, exocytosis elicited}

by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.

Keywords: action potential, chromaffin cell, endocytosis, exocytosis, ion channels, lysophospholipids.

J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04584.x

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are two lysophospholipids (LPLs) secreted by platelets and macrophages during blood clotting and inflammation, respectively (Xiao et al. 2000; Okajima 2001). They have been reported to be involved in Ca2+ mobilization, cell survival and wound healing (Panetti 2002; Xu et al. 2003). These LPL-related responses are mainly mediated by cell surface G-protein coupled receptors (GPCRs) (Taha et al. 2004; Rosen and Goetzl 2005). At least five receptor subtypes have been linked to S1P signaling and three have been linked to signaling by LPA (Anliker and Chun 2004).

Several lines of evidence have demonstrated that S1P and LPA regulate the activities of ion channels. Most of these studies focused on endothelial, neuronal or fibroblast cells. S1P has been found to activate non-selective cation channels and large-conductance Ca2+-activated K+channels (BK) in human endothelial cells (Muraki and Imaizumi 2001; Kim et al. 2006). It has also been reported that S1P inhibits voltage-gated K+current (IK) in rat cerebral artery (Coussin et al. 2003) and LPA activates BK current in microglial cells

(Schilling et al. 2004). Both S1P and LPA have been found to activate Cl-current in cultured corneal keratinocytes (Wang et al. 2002) and myofibroblasts (Yin and Watsky 2005). In dorsal root ganglion neurons, LPA inhibits tetrodotoxin (TTX)-sensitive sodium current (INa) but enhances TTX-insensitive INa(Seung Lee et al. 2005). The exposure of rat

Received August 10, 2006; revised manuscript received February 21, 2007; accepted February 26, 2007.

Address correspondence and reprint requests to Dr C.-Y. Pan, Institute of Zoology, National Taiwan University, Rm730, Life Science Building, 1 Roosevelt Rd., Sec. 4, Taipei 106, Taiwan. E-mail: [email protected] or Dr Y.-T. Chen, Department of Chemistry, National Taiwan University, and Institute of Atomic and Molecular Sciences, Academia Sinica, PO Box 23-166, Taipei 106, Taiwan. E-mail: [email protected]

1_{C.-Y. Pan and A. Z. Wu contributed equally to this study.}

Abbreviations used: AHP, afterhyperpolarization potential; AP, action potential; BK, large-conductance Ca2+-activated K+channels; GPCR, G protein coupled receptor; HBSS, Hank’s balanced salt solution; ICa, Ca

2+

current; IK, K +

current; INa, Na +

current; LPA, lysophosphatidic acid; LPL, lysophospholipid; NMG, N-Methyl-D-glucamine; PLC, phospho-lipase C; PTX, Pertussis toxin; S1P, sphingosine-1-phosphate. Journal of Neurochemistry, 2007 doi:10.1111/j.1471-4159.2007.04584.x

 2007 The Authors

sensory neurons to S1P enhances the frequency of action potential (AP) firing (Zhang et al. 2006). However, there is little information concerning the regulatory roles of LPLs in neurotransmitter release and AP firing.

A variety of voltage-gated ion channels can be identified on the plasma membrane of adrenal chromaffin cells, which secrete catecholamines in response to splanchnic nerve stimulation under physiological conditions. Calcium influx through calcium channels is the main factor responsible for catecholamine release from chromaffin cells (Douglas and Rubin 1961; Douglas and Poisner 1962; Boarder et al. 1987). In addition, APs can be evoked in cultured chromaffin cells (Kidokoro and Ritchie 1980). Therefore, it is an excellent model for studying electrical excitability and associated exocytosis (Winkler 1993). In addition, modula-tion of stimulus-secremodula-tion coupling in chromaffin cells by LPLs may play an important role in the interaction between immune and endocrine systems.

This study was designed to determine: (a) whether S1P and LPA affect calcium currents (ICa), IK, and INain cultured bovine adrenal chromaffin cells; (b) the effects of LPLs on AP firing and (c) how LPLs modulate stimulus-secretion coupling. Our results suggest that LPLs attenuate the activities of voltage-gated cationic channels, reduce AP firing and play an important role in modulating the release of catecholamines from chromaffin cells.

Materials and methods

Chemicals

Oleoyl-L-a-lysophosphatidic acid (LPA, C18:1,

1-oleoyl-sn-gly-cerol-3- phosphate),D-erythro-sphingosine-1-phosphate (S1P) and

suramin sodium salt were purchased from Sigma (St. Louise, MO, USA). Pertussis toxin (PTX) and U73122 were obtained from CalBiochem (EMD Biosciences, San Diego, CA, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, and Hank’s balanced salt solution (HBSS) were purchased from Invitrogen Corp (Carlsbad, CA, USA). All other chemicals were commercially available and of reagent grade from Sigma. S1P and LPA were dissolved in chloroform : methanol/1:19 solution to a concentration of 1 mmol/L. It was then air-dried and redissolved in ethanol to make a stock of 1 mmol/L and stored at)20C.

Solutions

The composition of normal HBSS bath solution for recording was as follows (in mmol/L): 138 NaCl, 5.3 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4

MgSO4, 4 NaHCO3, 0.34 Na2HPO4, 0.44 KH2PO4, 10 Hepes, 5.6

glucose, pH 7.3. In some experiments, the concentration of CaCl2

was changed as indicated. To isolate INa, Ca2+-free HBSS solution

was used; to isolate ICa, cells were incubated in N-methyl-D

-glucamine (NMG) solution (in mmol/L): 130 NMG, 2 KCl, 5 CaCl2,

1 MgCl2, 5.6 glucose, 10 Hepes, pH 7.3. For measuring INaor ICa,

the patch pipette was filled with a Cs+_{-containing solution (in mmol/}

L): 130 Cs-aspartate, 20 KCl, 1 MgCl2, 0.1 EGTA, 3 Na2ATP, 0.1

Na2GTP and 20 Hepes, pH 7.3. To record membrane potential or IK,

cells were incubated in HBSS and the patch pipette was filled with a K+-containing solution (in mmol/L): 130 K-aspartate, 20 KCl, 1 MgCl2, 0.1 EGTA, 3 Na2ATP, 0.1 Na2GTP and 20 Hepes, pH 7.3.

To characterize the long-term effects of LPLs on inward currents, cells were incubated in HBSS containing LPL of different concentrations for 1 h before the start of recording. To identify the involvement of G-protein signaling pathway, cells were pre-treated with PTX (0.1 lg/mL) overnight; suramin (0.1 mmol/L) or U73122 (0.1 mmol/L) for 1 h. These chemicals were present before the establishment of the whole-cell configuration of patch-clamp technique and remained in the bath buffer during recording. For short-term treatment, S1P and LPA were added into the bath after a cell has already been whole-cell patched.

Cell preparation

Chromaffin cells were prepared by digestion of bovine adrenal gland obtained from local slaughterhouses with collagenase (0.5 mg/mL) and purified by density gradient centrifugation at 200 g as previously described (Pan et al. 2002). In brief, cells were plated at a density of 2· 105

cells per 35-mm culture dish on polyL -lysine-coated coverslips and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum. The medium was replaced every two days. All experiments were carried out between 5 and 10 days after cells were isolated. Electrophysiological measurements

Cells were transferred to a recording chamber mounted on the stage of an inverted microscope and bathed in HBSS at 25C. Patch pipettes were pulled from thin-wall capillaries with filament (Catalog 617000, A-M Systems Inc., Everett, WA, USA) using a two-stage microelectrode puller (P-97, Sutter Inc., Novato, CA, USA), and fire-polished with a microforge (MF-830, Narishige, Japan). When filled with pipette solution, the resistance ranged between 3–5 MW. To monitor the change in membrane capacitance, electrodes were coated with Sylgard (Catalog 184 Silicone Elastomer Kit, Dow Corning Co., Midland, MI, USA) to reduce nonspecific noise. Ionic currents, membrane capacitance, and APs were measured from whole-cell patch-clamped cells using an EPC10 patch-clamp amplifier (HEKA GmbH, Lambrecht, Germany) and controlled by Pulse software (HEKA GmbH).

For capacitance measurements, cells were whole-cell voltage clamped at )70 mV and depolarized with a train of 10 depolari-zations to + 10 mV for 150 ms with an interval of 200 ms between the start of two consecutive depolarizations. A 10-ms sinewave with a frequency of 1 kHz and amplitude of 20 mV was applied just before the start of each depolarization to monitor the membrane capacitance. After the end of this train of depolarizations, the same sinewave was applied continuously and the capacitance measured was averaged every 100 ms. The membrane capacitance was obtained by the Lock-in amplifier using sine + dc mode in the Pulse software program.

To monitor the whole-cell inward INaand ICa, cells were

voltage-clamped at a holding potential of)70 mVand depolarized to various potentials for 30 ms once every 15 s. The maximal inward current obtained during the first 5 ms was identified as INaand the current

recorded between 18 and 27 ms of depolarization as ICa. For outward

IK, cell was depolarized to various potentials for 0.4 s the current

during the last 100 ms of depolarization was averaged. To evoke

2 C.-Y. Pan et al.

Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04584.x  2007 The Authors

APs, cells were current clamped and adequate current was injected to bring the membrane potential slightly above threshold for 1.6 s.

To wash LPL out of the bath buffer, cell was placed in a perfusion chamber (JG-23 N/LP, Warner Instrument, US) containing 250 lL of NMG solution and patched with Cs+_{-containing pipette solution}

to isolate the ICa. The cell was depolarized with 30 ms pulses

to + 10 mV applied every 20 s from a holding potential of)70 mV. S1P or LPA was added directly to the bath to achieve a final concentration of 1 lmol/L. Five minutes later, the chamber was perfused continuously with NMG buffer containing no LPLs at a speed of 1 mL/min.

Data analysis

Signals were low-pass filtered at 3 kHz and stored in a Pentium III-based computer. Data are presented as mean ± SEM. For long-term LPL treatment, one-way analysis of variance with a least-signifi-cance difference method for multiple comparisons and unpaired Student’s t test were used for statistical evaluation of differences among means. For short-term treatment, paired Student’s t test was used to compare the results before and after the LPL addition. A value of p < 0.05 was considered to be statistically significant. The distributions of data were homogeneous as examined by Shapiro-Wilk Normality Test at 0.05 level.

The concentration-dependent inhibitory effects of LPLs on ICa

and INawere fitted to a Hill function where percent of remaining

current¼ 1  ðEmax½CnÞ=ðICn50þ ½C

n_{Þ; ½C represents the}

con-centration of LPL; IC50and n are the concentration of LPL required

for 50% inhibition and the Hill coefficient, respectively; Emaxis the

LPL-induced maximal percent inhibition of ICa, with a non-linear

least-squares fitting algorithm.

Normalized inactivation curves were fit to a Boltzmann function, using the least-squares method according to I¼ 1= ð1 þ exp ½V  a=bÞ; where V is the conditioning potential in mV, a is the membrane potential for half-maximal inactivation, and b is the slope factor of the inactivation curve.

Results

S1P inhibits bothINaandICa

To examine long-term effects of LPLs on inward currents, cells were incubated in HBSS containing different concentrations of S1P for 1 h before starting patch-clamp recording. Using this protocol, peak inward current (due largely to INa) and sustained inward current (due largely to ICa) from representative cells were both inhibited by S1P after 1 h pre-treatment (Fig. 1a). Concentration-response curves (Fig. 1b) obtained using depolarizations from a holding potential of )70 mV to + 10 mV gave concentrations for half-maximal inhibition (IC50) of INaand ICaof 0.57 and 0.46 lmol/L, respectively. However, even at 0.01 lmol/L, about 20% of the INa was inhibited (p < 0.05). Application of 1 lmol/L S1P reduced mean INafrom )1343.8 ± 204.7 to )684.1 ± 87.5 (Fig. 1c; n = 11 each; p < 0.05) and mean ICa from )265.4 ± 33.2 to )75.8 ± 13.5 pA (Fig. 1d; p < 0.05). Thus, long-term treatment with physiological concentrations of S1P in bovine chromaffin cells strongly reduces both INaand ICa.

LPA inhibitsICabut notINa

Unlike S1P, short-term LPA treatment inhibits ICabut not INa in chromaffin cells (Pan et al. 2006). A similar result was obtained for long-term treatment with LPA (Figs 2a–d). When depolarized to + 10 mV, the IC50 for the inhibitory effects of LPA on ICa was 0.79 lmol/L (Fig. 2b). At +10 mV, ICa was significantly decreased from )278.5 ± 44.7 to)149.8 ± 20.9 pA (n = 11 each, p < 0.05) by pre-treatment with 2.5 lmol/L LPA. Inhibition of mean INawas not insignificant ()1.34 ± 0.20 to )1.14 ± 0.16 nA). These results indicate that long-term treatment with LPA produces a concentration-dependent inhibition of ICa, but not INa, in bovine chromaffin cells.

S1P, not LPA, negatively shifts the steady-state inactivation ofINa

Changes in the steady-state inactivation of INa affect cell excitability (Fernandez et al. 2005). To isolate effects of LPLs on cell excitability that are INa-dependent, cells were bathed in Ca2+-free HBSS solution to avoid the influence of ICaand inactivation was studied using paired depolarizations. The first depolarization was a 100 ms conditioning pre-pulse to various potentials from the holding potential of)70 mV and was followed immediately by a second 20 ms test depolarization to + 10 mV. As the pre-pulse became more positive, INain response to the second depolarization became smaller (Fig. 3a). Normalized steady state inactivation curves obtained by plotting INaof the second depolarization versus pre-pulse potential were fit with a Boltzmann function as described in the Materials and methods (Figs 3b and c). For control cells (n = 7), the pre-pulse potential for half inacti-vation (a) was)34.5 ± 0.3 mV and with slope factor (b) of 7.4 ± 0.26 mV; in presence of S1P, a is negatively shifted to )41.9 ± 0.53 mV (n = 7, p < 0.01) but b was not signifi-cantly changed (7.8 ± 0.4 mV). This contrasts with LPA pre-treatment which produced no significant effects on steady-state inactivation (Control (n = 8): a =)34.6 ± 0.4, b = 8.8 ± 0.38 mV; 2.5 lmol/L LPA (n = 8): a =)38.1 ± 0.36 mV; b = 8.6 ± 0.32 mV). Thus, S1P, but not LPA, shifts the voltage-dependence of Na+ channels to more negative potentials.

Both S1P and lysophosphatidic acid prolong recovery of INafrom inactivation

The rate at which Na+ channels recover from inactivation determines the maximum frequency of AP firing (Lou et al. 2003). Recovery rate was measured using two depolariza-tions to + 10 mV separated by different time intervals (Fig. 4a). INa of the second pulse was normalized to that of the first pulse and plotted against the recovery interval (Figs 4b and c). For control cells, INa recovered almost completely after about 30 ms (Figs 4a–c). Pre-treatment with 1 lmol/L S1P prolonged recovery (Fig. 4b), as did pre-treatment with 2.5 lmol/L LPA (Fig. 4c). Fits of recovery

Lysophospholipids modulate chromaffin cells’ excitability 3

 2007 The Authors

times with a single-exponential function showed that S1P pre-treatment increased the time constant of the exponential from 8.1 ± 0.2 to 14.9 ± 0.6 ms (n = 6, p < 0.001) and LPA pre-treatment increased it from 7.8 ± 0.1 to 12.1 ± 0.3 ms (n = 6, p < 0.01). These results indicate that both LPA and S1P slow the recovery of INafrom inactivation.

Pre-pulse cannot rescue the inhibited Ca2+current Application of strong depolarizing pre-pulses often reverses inhibition of ICathat is due to G-protein bc subunits (Li et al. 2004). To determine whether ICainhibited by LPLs could be rescued by such a protocol, cells were incubated in NMG buffer to isolate ICa. In control cells, a strong pre-pulse increased ICarecorded during a test pulse to + 10 mV. When the same protocol was repeated 2 min after addition of S1P, ICa was inhibited as described in Fig. 1 and no facilitation was observed (Fig. 5a). The mean results show that the ICa was facilitated by a strong pre-pulse from)226.3 ± 22.3 to )266.3 ± 29.5 pA in control (n = 8, p < 0.05), but not after addition of S1P, where an insignificant decrease from )161.5 ± 15.9 to )152.6 ± 15.5 pA was observed (Fig. 5b).

Similarly, a conditioning pre-pulse increased ICa from )209.8 ± 21.9 to )258.2 ± 26.6 pA (n = 8, p < 0.05) before LPA treatment, but not after LPA treatment (from )161.7 ± 12.4 to )166.2 ± 13.7 pA) (Fig. 5c). These results indicate that the inhibitory effects of LPLs on ICa are not relieved by strong depolarizations, suggesting that inhibition is not mediated by the binding of Gbc subunits to Ca2+ channels in bovine chromaffin cells.

ICarecovers from inhibition after lysophospholipid washout

The concentration of LPLs in serum will eventually decrease to basal level after wounding. To ask whether the inhibition induced by both S1P and LPA is reversible, the LPLs in the recording chamber were washed out by continuous perfusion (Fig. 6). After beginning whole-cell recording in NMG buffer, ICain cells was monitored every 20 s with a depolarization from a holding potential of)70 mV to + 10 mV. There was little rundown during the first 3 min of recording. S1P (1 lmol/L) or LPA (1 lmol/L) were then added directly to the chamber. The magnitude of ICa decreased slowly and

(a) (b)

(c) (d)

Fig. 1 Inhibitory effects of S1P on inward currents. Cells were bathed in Hank’s balanced salt solution containing different concen-trations of S1P for 1 h before and during whole-cell voltage-clamp recording using a Cs+-containing pipette solution. Depolarizations (30 ms) to various potentials were applied once every 15 s from a holding potential of)70 mV. The inward maximal peak current was recorded as the Na+_{current (I}

Na); the current between the 18th and

27th ms of the depolarization was averaged and recorded as the Ca2+

current (ICa). The concentration of CaCl2in the bath solution was

6.8 mmol/L. (a) Representative current traces during depolarizations to + 10 mV from untreated cells (Control) or treated with S1P

(1 lmol/L) (S1P). (b) Concentration-dependent inhibitory effects of S1P on INaand ICa. The INa(j) and ICa(d) were acquired by a

step-depolarization from a holding of)70 mV to + 10 mV for 30 ms under different concentrations of S1P. The currents were normalized to the averages obtained from control cells without S1P treatment. The concentration response was fit as described in Materials and methods. Sample number was at least 10 for each concentration. (c and d) Average I–V relations of INaand ICa, respectively, acquired in the

absence (empty symbols) and presence (gray symbols) of 1 lmol/L S1P. Data are mean ± SEM from 11 cells each. *Student’s t-test p < 0.05 when comparing to cells without S1P treatment.

4 C.-Y. Pan et al.

Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04584.x  2007 The Authors

continuously. Five min after addition of S1P or LPA, currents had decreased to 61.5 ± 0.4 and 72.8 ± 1.2%, respectively, of their initial levels. Washout of the compounds resulted in recovery to 88.5 ± 5.2 and 93.0 ± 2.9% of their initial levels, after 9 min for S1P and LPA, respectively. These results suggest that inhibition of ICaby short-term treatment with LPL is reversible.

Suramin antagonizes the inhibitory effects of S1P and lysophosphatidic acid

To define the involvement of GPCR pathways in the inhibitory effects of S1P and LPA on ICa, cells were pre-treated with U73122, an inhibitor of phospholipase C (PLC) (Noh et al. 1998), PTX, an inhibitor of Gi/o (van Corven et al. 1989); or suramin, a general inhibitor of GPCR activation (Ancellin and Hla 1999) (Table 1). Though basal ICa was significantly inhibited by PTX and U73122 pre-treatments, application of S1P or LPA for 5 min further reduced the ICa. On the contrary, basal ICawas significantly and slightly elevated by suramin. In addition, in the presence

of suramin, application of S1P or LPA only marginally inhibited ICa. These results suggest that LPL may inhibit the ICathrough activation of GPCRs, but PLC and Gi/opathways may not be involved.

S1P and lysophosphatidic acid inhibit outward currents Action potential repolarization is mostly due to activation of K+ channels. To characterize the effects of LPLs on chromaffin cells, whole-cell outward IKwas monitored. After the incubation of chromaffin cells in S1P- or LPA-containing bath solution for one hour, IKwas inhibited when depolarized from a holding potential of )70 mV to various potentials (Fig. 7a). Averaged I–V relationships (Fig. 7b) show that IK was inhibited at all potentials. At + 50 mV, the magnitude of IKwas significantly reduced by S1P and LPA to 52.1 ± 6.6 (n = 6, p < 0.01) and 44.5 ± 5.7% (n = 6, p < 0.01) of the current measured from cells without LPL treatment (n = 8), respectively (Fig. 7c). Inhibition of IK was rapid, being decreased to 67.4 ± 12.1 or 57.6 ± 11.7% of the initial current by S1P (1 lmol/L) or LPA (2.5 lmol/L)

(a) (b)

(c) (d)

Fig. 2 Inhibitory effects of lysophosphatidic acid (LPA) on inward currents. Cells were bathed in Hank’s balanced salt solution containing different concentrations of LPA for 1 h before and during whole-cell voltage-clamp recording using a Cs+_{-containing pipette solution.}

Depolarizations (30 ms) to various potentials were applied once every 15 s from a holding potential of)70 mV. The inward maximal peak current was recorded as the Na+current (INa); the current between the

18th and 27th ms of the depolarization was averaged and recorded as the Ca2+current (ICa). The concentration of CaCl2in the bath solution

was 6.8 mmol/L. (a) Representative current traces from cells treated without (Control) or with 2.5 lmol/L LPA when depolarized

to + 10 mV. (b) Concentration-dependent inhibitory effects of LPA on INaand ICa. Patched cells were depolarized to + 10 mV; INa(j) and

ICa(d) acquired under different concentrations of LPA were

normal-ized to the averages of cells not treated with LPA. The concentration response was fit as described in Methods. Sample number is at least 12 for each concentration. (c and d) Average I–V relations of INaand

ICa, respectively, acquired in the absence (empty symbols) and

pres-ence (black symbols) of 2.5 lmol/L LPA. Data are mean ± SEM from 11 cells each. *Student’s t-test p < 0.05 when compared to cells without LPA treatment.

Lysophospholipids modulate chromaffin cells’ excitability 5

 2007 The Authors

(n = 10, p < 0.01 for both), respectively, just 2 minutes after their application. These results indicate that voltage-gated IKis also inhibited by LPL treatment.

(a)

(b)

(c)

Fig. 3 Effects of lysophospholipids on the steady-state inactivation of INa. Cells were bathed in Ca2+-free Hank’s balanced salt solution

and treated with S1P (1 lmol/L) or LPA (2.5 lmol/L) for 1 h before and during whole-cell voltage-clamp recording using a Cs+-containing pipette solution. Cells were depolarized with a conditioning pulse to various potentials for 100 ms followed by a 20 ms depolarization to + 10 mV. The peak inward current during the second depolarization was measured as a function of conditioning pulse potential. (a) Rep-resentative traces from a control cell with conditioning pulses to)80, )40, and + 10 mV as indicated. (b and c) Normalized amplitude of INa

(I/IMax) plotted against the conditioning pre-pulse for cells treated with

S1P and LPA, respectively. Data are mean ± SEM and fit to the Boltzmann equation as described in Materials and methods (n = 7–8 for each point).

(a)

(b)

(c)

Fig. 4 Effects of lysophospholipids on the recovery rate of INa. Cells

were bathed in Ca2+_{-free Hank’s balanced salt solution and}

pre-trea-ted with S1P (1 lmol/L) or LPA (2.5 lmol/L) for 1 h before and during whole-cell voltage-clamp recording using a pipette filled with a Cs-containing solution. Cells were first depolarized to)10 mV for 10 ms from a holding potential of)70 mV. After a variable interval, a second pulse to)10 mV for 20 ms was applied. (a) Overlapping representa-tive current traces from a control cell with different interpulse intervals from 1 to 46 ms. (b and c) INaratio (I2nd/I1st) plotted against interpulse

intervals for cells treated with S1P & LPA, respectively. Curves were fitted by a first-order exponential growth equation. Data are mean ± SEM (n = 6 for each treatment).

6 C.-Y. Pan et al.

Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04584.x  2007 The Authors

S1P and LPA decrease the firing frequency of action potentials

The above results showing that the both voltage-gated INa and IKare inhibited by LPLs suggested that AP firing will also be modulated by LPLs. To verify this, cells were recorded in whole-cell configuration under current clamp mode and APs were evoked by minimal current injection. Representative results (Fig. 8a) showed that multiple AP could be elicited from cells in control, but only one AP could be elicited from cells pre-treated with LPLs for 1 h. An average of 4.4 ± 0.4 spikes/s (n = 8) could be elicited by a 1.6-s suprathreshold depolarizing current injection in cells without LPL treatment (Fig. 8b). However, after being incubated in buffer containing S1P (1 lmol/L, n = 6) or LPA (2.5 lmol/L, n = 6) for 1 h., only a single AP could be evoked, no matter how much current was injected. A similar reduction in the frequency of AP firing was also observed

(a)

(b)

(c)

Fig. 5 Effect of LPLs on facilitated ICa. Cells were bathed in NMG

solution containing 5 mmol/L CaCl2and whole-cell voltage -clamped

with a Cs+_{-containing pipette solution. Cells were depolarized}

to + 10 mV for 20 ms with or without a 100 ms conditioning pre-pulse to + 100 mV that preceded the test pulse by 5 ms. After recording a pair of basal (without pre-pulse) and facilitated (with pre-pulse) currents, S1P (1 lmol/L) or lysophosphatidic acid (LPA) (2.5 lmol/L) was added to the recording chamber. Two minutes later another pair of basal and facili-tated currents was recorded. (a) Representative basal and facilifacili-tated current traces from a cell before (Control) and 2 min after (S1P) the addition of S1P. (b and c) Averaged baseline and facilitated ICatreated

with S1P and LPA, respectively. Data are Mean ± SEM (n = 8 for each treatment). *Student’s t-test p < 0.05; **p < 0.01.

(a)

(b)

Fig. 6 Inhibition of ICais reversible. Cell was bathed for 15 min in NMG

buffer and voltage-clamped with Cs+-containing buffer to isolate ICa. ICa

was measured by a 30 ms step depolarization from)70 to + 10 mV applied once every 15 s. Three min after beginning whole-cell recording, 1 lmol/L of S1P (a) or lysophosphatidic acid (b) was added to the bath (gray or black bar above each graph). Five min later the recording chamber was continuously perfused with NMG buffer to wash out LPLs. The measured ICawas plotted against time. The current traces on the

right were recordings before the addition of LPLs (i); before the perfu-sion (ii) and the last recording (iii) from a representative cell. Data pre-sented are mean ± SEM; sample numbers are 3 for each group.

Lysophospholipids modulate chromaffin cells’ excitability 7

 2007 The Authors

2 minutes after the addition of LPL. The firing frequency was significantly reduced by S1P (n = 7) and LPA (n = 6) from 4.6 ± 0.4 and 4.2 ± 0.3 to 2.4 ± 0.3 and 2.7 ± 0.2 spikes/s, respectively, in 2 min.

The peak membrane potential reached by the first evoked AP (approximatedly + 44 mV) was not significantly chan-ged by long- or short-term treatments with LPL. However, after being incubated in LPL-containing buffer for 1 h, the

(a) (b)

(c)

Fig. 7 Inhibitory effects of lysophospholipid (LPLs) on outward IK.

Cells were bathed in normal Hank’s balanced salt solution and whole-cell voltage-clamped with a K+-containing pipette solution. Cells were step depolarized to various potentials for 0.4 s and the outward current between 0.3 and 0.4 s during depolarization was averaged as IK. For

long-term treatment, cells were pre-treated with S1P (1 lmol/L, n = 6), LPA (2.5 lmol/L, n = 6) or without treatment (Control, n = 8) for 1 h before the establishment of whole-cell patch and same concentration of LPLs were used during the recording. For short-term treatment, S1P (1 lmol/L, n = 10) or LPA (2.5 lmol/L, n = 10) was added into the

bath after the cell was whole-cell patched; the voltage-dependent IK

was recorded before (Control) and 2 min again after the addition of LPLs. (a) Representative IKfrom cells pre-treated with LPLs as

indi-cated for 1 h and step-depolarized to various potentials from a holding potential of)70 mV. (b) Averaged voltage-dependent IKpre-treated

with S1P (d) or LPA (j) for 1 h. (c) Normalized IKacquired by step

depolarization to + 50 mV from cells treated with LPL for long-term (1 h) or short term (2 min). Data are mean ± SEM. **Student’s t-test p < 0.01 when comparing to control cells without (long-term) or before (short-term) LPL treatment.

Table 1 Effects of G protein coupled receptor signaling antagonists on Ca2+

currents

Pre-treatment )S1P +S1P )LPA +LPA

Control )226.3 ± 22.3 )161.5 ± 15.6** _{)209.7 ± 21.8 )161.7 ± 10.3}**

PTX )174.9 ± 23.9* _{)110.6 ± 19.8}** _{)179.9 ± 12.9}* _{)125.6 ± 19.2}**

U73122 )103.1 ± 10.3* )60.1 ± 7.5** )109.5 ± 13.5* )83.1 ± 10.7** Suramin )283.3 ± 20.9* _{)251.3 ± 20.6 )265.0 ± 23.8}* _{)215.8 ± 25.8}

Unit, pA.

Cells were bathed in HBSS and whole-cell voltage-clamped. ICawas obtained by step

depolar-ization form)70 to + 10 mV for 20 ms before ()) and 5 min after (+) the addition of LPLs. *Student’s t-test p < 0.05 when compared with the current without antagonist pre-treatment. **Paired Student’s t-test p < 0.01 when compared with the currents before the addition of S1P or LPA.

8 C.-Y. Pan et al.

Journal Compilation 2007 International Society for Neurochemistry, J. Neurochem. (2007) 10.1111/j.1471-4159.2007.04584.x  2007 The Authors

difference (Fig. 8d) between the baseline potential during current injection and the maximal hyperpolarization potential following the AP (afterhyperpolarization potential, AHP) was strongly decreased from 10.7 ± 0.4 of control cells to 2.9 ± 2.0 and 3.2 ± 1.3 mV by long-term treatments with S1P and LPA, respectively. The AHP was slightly but significantly decreased by short-term treatment with S1P and LPA to 9.3 ± 0.3 and 9.6 ± 0.7 from 10.9 ± 0.8 and 11.2 ± 1.1 mV, respectively. The half-width duration (Th) (Fig. 8e) of the first evoked AP was significantly increased from 3.0 ± 0.1 to 4.4 ± 0.4 and 3.9 ± 0.3 ms by long-term

S1P and LPA treatment, respectively. Similarly, the Thcould also be significantly increased to 3.8 ± 0.3 and 4.0 ± 0.4 from 2.9 ± 0.3 and 3.0 ± 0.2 ms by short-term S1P and LPA treatment, respectively. These results illustrate that LPLs reduce repetitive AP firing, decrease AHPs and increase the width of APs in chromaffin cells.

S1P and LPA inhibit both exocytosis and endocytosis To investigate whether the inhibition of ICaby LPLs leads to the modulation of exocytosis and endocytosis, the change in membrane capacitance evoked by a train of depolarizations

(a) (c)

(d)

(e) (b)

Fig. 8 Inhibitory effects of lysophospholipids (LPLs) on the firing frequency of action potentials (AP). Cells were bathed in normal hank’s balanced salt solution and whole-cell recorded in the current-clamp mode with a K+_{-containing pipette solution. For long-term}

treatment, cells were treated with S1P (1 lmol/L, n = 6), LPA (2.5 lmol/L, n = 6) for 1 h or without treatment (Control, n = 8) before the establishment of the whole-cell configuration and during record-ing. For short-term treatment, S1P (1 lmol/L, n = 7) or LPA (2.5 lmol/L, n = 6) was added into the bath after the cell was whole-cell clamped and the AP were evoked before (Control) and again 2 min after the application of LPLs. Cells were current clamped and injected with the minimal depolarizing current required to trigger AP for 1.6 s. (a) Representative membrane potential traces from

long-term LPL-treated cells. Cells were current clamped and injected with 1 pA of current for 1.6 s as indicated. (b) Averaged frequency of AP (firing from cells long- (1 h) or short-term (2 min) treated with LPLs. (c) The first APs from a control (solid line) and a S1P (dotted line) long-term treated cells. The magnitude of the AHP, the differ-ence between the lowest hyperpolarization potential and the nor-malized membrane potential (dashed line) during current injection; and half amplitude duration (Th, double-arrow) were analyzed. (d) The

averaged AHP from cells long- (1 h.) or short-term (2 min) LPL-treated cells. (e) The averaged Th from long- (1 h.) and

shorterm(2 min) LPL-treated cells. Data are mean ± SEM. *Student’s t-test p < 0.05 when comparing to that of control cells without (long-term) or before (short-(long-term) LPL treatments.

Lysophospholipids modulate chromaffin cells’ excitability 9

 2007 The Authors