bile acids are formed from cholesterol in the hepatocyte and stored in gallbladder , being released for transport lipids as mixed micells in the small intestine thereby promoting lipid absorption . in health ,
the enterohepatic circulation efficiently conserves bile acids , which results in the concentration of bile acids in plasma being extremely low . however , under pathological conditions as bile flow obstruction or bile duct disease , regurgitation of bile acids into the systemic blood stream occurs , resulting in an increased plasma level of bile acids as high as to 500~600 m .
increase in circulating level of bile acids may lead to a wide variety of pathophysiological conditions .
physiological role of bile acids besides emulsifying lipids have been recognized , for example , in glucose homeostasis [ 6 - 8 ] , thyroid function , and cardiovascular function .
bile acids also could produce pge2 via activation of cox-2 , and directly interact with muscarinic receptors .
these actions of bile acids appear to be mediated through their binding to specific receptors .
recently , an existence of cell surface receptors   besides nuclear receptors [ 15 - 17 ] has been proposed , which is thought to be coupled with g - protein .
bile acids also may directly activate ion channel protein such as large conductance ca activated k channel .
high concentration of bile acids free in circulation may affect the function of peripheral and/or central neurons .
in the present study , we explored this possibility by studying the effects of bile acids on neuronal ( n)-type ca channel that is known to be essential for neurotransmitter release at synapses of the peripheral and central nervous system .
biophysical properties of n - type ca channel at a single channel level have been extensively characterized in bullfrog sympathetic neuron , in which a major proportion of functionally expressed ca channel is n - type .
therefore , this system was adopted to assess the effects of low concentration of cholic acid ( ca ) that is relatively hydrophilic   thus less damaging to the cell membrane or least cytotoxic .
neurons were isolated from caudal paravertebral sympathetic ganglia of adult bull frogs ( rana catesbeiana ) and dissociated by a collagenase / dispase digestion and trituration .
cells were maintained in l-15 culture medium supplemented with 10% fetal bovine serum and penicillin / streptomycin .
n - type ca channel currents were recorded under a cell attached mode at room temperature using 100 mm ba as a charge carrier .
the pipette solution contained ( in mm ) : 100 bacl2 , 10 tetraethylammonium chloride , 5 4-aminopyridine and 10 n - methyl - d - glucamine ( nmg)-hepes . the extracellular solution was designed to zero the cell 's membrane potential and contained ( in mm ) 100 kcl , 10 khepes , and 5 nmg - egta , ph 7.2 .
the zeroing solution contained 10 m (  ) bay k8644 to reveal the presence of l - type ca channels in the patch .
these electrodes had resistances ranging from 15 to 40 m , which were coated with sylgard ( ge silicones rtv615 ; general electric co. ) .
the voltage clamp test pulse protocols were delivered , and single channel current responses were amplified and filtered ( at 2 khz ) by means of a patch clamp amplifier ( axopatch 200b , axon instruments , foster city , ca ) interfaced to a macadios ii analogue - digital converter ( gw instruments ) and a macintosh computer running s3 data acquisition software written by dr .
stephen ikeda ( nih , rockville , ma ) . currents were amplified an additional 10 times by a bessel filter ( warner , lpf-100b ) .
data were obtained in sets of 100 voltage steps of 100 ms duration delivered at a 2s interval , and analyzed using igorpro software ( wavemetrics ) .
chlolic acid was obtained from sigma ( st . louis , mo ) and dissolved in distilled water to make a stock of 10 m that were diluted in pipette solution immediately before each 
 use .
student t - test was performed and probabilities of less than 5% were considered statistically significant .
neurons were isolated from caudal paravertebral sympathetic ganglia of adult bull frogs ( rana catesbeiana ) and dissociated by a collagenase / dispase digestion and trituration .
cells were maintained in l-15 culture medium supplemented with 10% fetal bovine serum and penicillin / streptomycin .
n - type ca channel currents were recorded under a cell attached mode at room temperature using 100 mm ba as a charge carrier . the pipette solution contained ( in mm ) : 100 bacl2 , 10 tetraethylammonium chloride , 5 4-aminopyridine and 10 n - methyl - d - glucamine ( nmg)-hepes . the extracellular solution was designed to zero the cell 's membrane potential and contained ( in mm ) 100 kcl , 10 khepes , and 5 nmg - egta , ph 7.2 .
the zeroing solution contained 10 m (  ) bay k8644 to reveal the presence of l - type ca channels in the patch .
these electrodes had resistances ranging from 15 to 40 m , which were coated with sylgard ( ge silicones rtv615 ; general electric co. ) .
the voltage clamp test pulse protocols were delivered , and single channel current responses were amplified and filtered ( at 2 khz ) by means of a patch clamp amplifier ( axopatch 200b , axon instruments , foster city , ca ) interfaced to a macadios ii analogue - digital converter ( gw instruments ) and a macintosh computer running s3 data acquisition software written by dr .
currents were amplified an additional 10 times by a bessel filter ( warner , lpf-100b ) .
data were obtained in sets of 100 voltage steps of 100 ms duration delivered at a 2s interval , and analyzed using igorpro software ( wavemetrics ) .
chlolic acid was obtained from sigma ( st . louis , mo ) and dissolved in distilled water to make a stock of 10 m that were diluted in pipette solution immediately before each 
 use .
student t - test was performed and probabilities of less than 5% were considered statistically significant .
cholic acid ( ca ) at 10 m inhibited n - type ca channel currents .
ca increased the frequency of null ( no activity ) sweeps resulting a decrease in amplitude of pseudomacroscopic currents ( fig .
the proportion of null sweeps in the presence of ca in this specific patch was 71 , 46 and 40% at + 20 , + 30 and + 40 mv , respectively , whereas they were 15 , 12 and 8 in the control patch recorded without ca . when active sweeps are compared in diary plots ( bottom trace ) , the channels from the two patches were gating with similar open probability ( po ) .
an increase in the frequency of null by ca was manifested in a decreased po of the channel ( fig .
the po was calculated from a 100 sweep data set including null sweeps and averaged over several patches .
for example , at + 30 mv the po was 0.160.04 ( n=3 patches/3 frogs ) in the presence of ca ( closed circle ) , while that was 0.360.05 ( n=4 patches/3 frogs ) in the control without ca ( open circle ) .
the data were fit by a single boltzmann equation . in the presence of ca , the boltzmann half activation voltage ( v1/2 ) was 30 mv and a slope of e fold change was 8 mv with maximum po of 0.3 , while in the control without ca they were 30 mv , 6 mv and 0.6 , respectively .
the effects of ca on other biophysical properties of n - type ca channel was also determined ( fig .
( 10 m ) the single channel current amplitude appeared to be similar to that of control ( fig .
the instantaneous current - voltage relationship seemed to be also unaffected by ca ( fig .
the current elicited by a ramp voltage from -80 to + 120 mv ( ramp speed of 1 mv / ms ) was resolvable at potentials positive to ~+10 mv and the mean voltages generating the peak current was ~+35 mv ( n=5 patches/3 frogs , fig .
3b ) , which is similar to that of control patches ( data not shown ) .
single channel current amplitude measured at each patch potential was averaged from 5 patches and plotted against patch potential ( fig .
the slope conductance was calculated from 0 to + 50 mv by linear regression and was found to be 21.21.7 ps with a single channel current at 0 mv of -1.30.1 pa ( n=5 patches/3 frogs , fig .
3c ) , which were almost identical to those of controls and the properties of n - type ca channel previously reported .
mean open time and shut time histograms of n type ca channel in the presence of ca are illustrated in fig .
the mean open time ( o ) increased with voltage ( n=5 patches/3 frogs , containing one or two channels ) .
the shut time histograms were fit by two exponentials with the longer component ( sh2 ) decreased with voltage ( n=3 patches/3 frogs , containing one channel ) .
these properties are consistent with those of n - type ca channel . for example , at + 30 mv the mean open time was 1.6 ms and the mean shut times were 0.4 and 2.6 ms in the presence of ca , which were similar to those of controls ( 1.4 ms , and 0.4 and 2.5 ms , respectively ) .
thus , ca did not seem to affect the duration of n - type ca channel opening and closing .
the present study demonstrates that ca inhibits n - type ca channel function by altering channel gating .
other single channel properties such as current - voltage relationship , unitary slope conductance , single channel current amplitude , open times and shut times were unaffected by ca .
ca increased the frequency of null ( figs . 1 and 3 ) and consequently decreased po .
the null sweeps appeared to be clustered , suggesting that an open state is not attainable for a period of time during which the channel may not be responsive to excitatory signals or depolarization .
thus , under pathological conditions where circulating bile acids get elevated , n - type ca channel function may be suppressed leading to a synaptic depression .
it is not due to cytotoxicity , since ca is one of the least cytotoxic bile acids .
nonspecific membrane damage and cell lysis   is unlikely , since the concentration used in the present experiment is at least 1,000 times less than the concentration producing membrane toxicity   or critical micelle concentration [ 28 - 30 ] .
one possibility is that ca may modulate n - type ca channel function by directly interacting with channel protein .
however , one can argue that binding of bile acids to protein is correlated with their hydrophobicity and ca is relatively hydrophilic among bile acids .
dissociation constant of ca to albumin was 333 m [ 31 - 33 ] that is ~300 times greater than the concentration used in the present study .
thus , suppression of ca channel in the presence of 1 m ca may not be entirely attributable to ca binding directly to the channel protein .
another possibility is that ca may act through a g - protein coupled cell surface receptor protein .
g protein  subunit has been shown to modulate n - type ca channel gating in a membrane delimited manner . however , in the presence of norepinephrine known to inhibit n - type ca channel gating through g protein  subunit , no significant increase in null sweeps was observed , ruling out the possibility of involving membrane delimited pathway .
finally , ca may alter local lipid invironement where the channel protein is situated . synthetic detergent triton - x100 ( 10 m ) that decreases bilayer stiffness or promotes convex curvature of the membrane lipid shifted steady state voltage - dependent inactivation of n - type ca channel toward more negative potentials .
ca , due to its amphipatic nature , may also affect local lipid environment similarly and modify inactivation property of n - type ca channel .
one mechanism generating null sweeps may be inactivation from intermediate closed states of the channel on its way to opening .
if that is true , an increase in null sweeps and consequent rapid inactivation will lead to a left shift of the inactivation - voltage relationship .
however , we did not see any significant increase in null sweeps by triton - x100 at 10 m under the identical experimental condition ( data not shown ) .
therefore , the possibility of involving a specific protein effecting the interaction of ca on n - type ca channel gating still remains valid .
n - type ca channel is one of the main ca channels known to mediate neurotransmitter release at presynaptic terminals .
n - type ca channel is required for hippocampus - dependent learning and memory , sensory transmission , pain transduction in particular   and autonomic function . in mice deficient of n - type ca channel exhibited a substantial reduction in basal synaptic transmission , higher pain threshold   and decreased sympathetic nerve activity .
thus , it is reasonable to predict that a high concentration of circulating ca , by suppressing n - type ca channel function , could result in similar pathophysiological effects , but to different levels .
several studies have shown in various cell types that bile acids activated nonselective cation conductance ( insc ) .
taurodeoxycholic acid , dexoycholic acid or diluted fresh bile have been shown to activate insc   and/or na - dependent membrane depolarization . under some pathological conditions with increased concentrations of bile acids ,
the suppression of n - type ca channel function by ca may be beneficial in moderating excitation of the synapses .
 under some pathological conditions as bile flow obstruction or liver diseases with the enterohepatic circulation being disrupted , regurgitation of bile acids into the systemic circulation occurs and the plasma level of bile acids increases . 
 bile acids in circulation may affect the nervous system . 
 we examined this possibility by studying the effects of bile acids on gating of neuronal ( n)-type ca2 + channel that is essential for neurotransmitter release at synapses of the peripheral and central nervous system . 
 n - type ca2 + channel currents were recorded from bullfrog sympathetic neuron under a cell - attached mode using 100 mm ba2 + as a charge carrier . 
 cholic acid ( ca , 10 - 6 m ) that is relatively hydrophilic thus less cytotoxic was included in the pipette solution . 
 ca suppressed the open probability of n - type ca2 + channel , which appeared to be due to an increase in null ( no activity ) sweeps . 
 for example , the proportion of null sweep in the presence of ca was ~40% at + 40 mv as compared with ~8% in the control recorded without ca . 
 other single channel properties including slope conductance , single channel current amplitude , open and shut times were not significantly affected by ca being present . 
 the results suggest that ca could modulate n - type ca2 + channel gating at a concentration as low as 10 - 6 m. bile acids have been shown to activate nonselective cation conductance and depolarize the cell membrane . under pathological conditions with increased circulating bile acids , ca suppression of n - type ca2 + channel function 
 may be beneficial against overexcitation of the synapses . 
INTRODUCTION
METHODS
Neuronal cell preparation from bullfrog sympathetic neuron
Cell-attached single channel recording
Drugs
Statistics
RESULTS
DISCUSSION