Ceil Phjsiol
Bioph\s
(1994) 13,75 81
75
L-Type Calcium Channels May Fill Directly
t h e I P r s e n s i t i v e Calcium Store
H S G V G O \ l D B D I R I D Y N O W 1 K K BOL\
}
and E E D W I E I
2
1 Institute of Physiology Bulgaittin Academy of bcieni es
\cad C, Boncheff S'ti hi 2J 11 li Safin Bulqarm
2 McMastel Ihiueisiťy
hne nity of Health sciences
Depaitmt nt of Biomedical Matrices Division of Physiology and Pharmacology
1200 Main Sheet West Hamilton Ontario, Canada LHN JZ5
Abstract.
T h e íelationship between inositol-1 4 5-tiisphosphate-sensiti\e
stoies and C'a
2+
( n t n through potential-dependent L-t\pe C a
2+
Ca2+
c hannels was exa­
mined using whole-cell \oltage-clamp t< chmque m cells of longitudinal muscle la\ei
of guinea-pig ileum It was found t h a t hepaiin (10~ 1 { 1 mol/1) m the p i p e t t e l a p i d h
inhibited the c m i e n t t l n o u g h L-t\ pe C a 2 + channels Neithei an inhibitoi o f t h e s a i coplasmu l e t K u l u m C a _ + p u m p (( \ t lopia/omc acid) noi blockeis of C a - + - m d u ( e d
C a 2 + ieleas( ( n a n c x l m e oi l u t h e m u m led) affected t h e C a 2 + c m i e n t
T h e fa.il-
m e of hepai in to affect C a 2 + - c u n e n t s t l n o u g h L-t\pe C a 2 + (hannels m cells fiom
cnculai muscle of the same oigan suggested t h a t hepai m had no d u e t t effect on
L-t\pe C a " + channels
T h u s the inhibition of the lattei m hepai in-loaded cells
fiom the longitudinal la\ei is supposed to be Ca~ + -depeiident due to t h e o\eifilling
of the inositol-1,4 5-tiisphosphate-sensiti\e C a 2 + stoie
K e y w o r d s : Hepai in
L-t\pe C a ~
+
C\ clopiazonic acid
R u t h e n i u m led — C a 2 + stoies
channels — Smooth muscle
Introduction
It is w idel} accepted t h a t C a " + w Inch t u g g e i s contiaction m smooth muse le enteis
the cell fiom the outside oi is d e i n e d fiom the mositol-1 4 5-tnsphosphate-(IP3)sensitive C a " + - p o o l ( B e m d g e and I i \ m e 1989, Walsh 1991 foi ie\iew see Missiaeii
et al
1992)
But the feedback mechanisms b\ which cells íegulate t h e quantity
of depolanzation-e\oked calcium e n t n depending on the concent i at ions of Ca~ +
ions stored still l e m a m uncleai (Casteels et al
1992)
The existence of a close
contact between plasmaleimna and the membianes of I P j-sensiti\e stoie ( S o m h o
1985
\ a n B i e e m a n and Saida 1989
Stehno-Bittel and Stuiek 1992) indicate the
possibiht\ of d n e c t lefillmg of this pool b\ \oltage-dependent C a " + e n t n (Bouiieau
76
Gagov et al.
et al. 1991; Daniel et al. 1992). T h e latter could be provided by the L-type C a 2 +
channels, known to undergo, in a number of tissues, a very weak C a 2 + - d e p e n d e n t
inactivation, even at relatively high densities of Ca~ + currents (Ganitkevich et al.
1987; Yamamoto et al. 1989). We evaluated this possibility on guinea-pig ileal
smooth muscle cells freshly isolated from the longitudinal layer. The ionic currents
in membranes of these cells have been characterized previously (Droogmans and
Callewaert 1986; Duridanova et al. 1992), and the L-type C a 2 + channels were
found t o be the only voltage-dependent C a " + channels there. Recently Zholos et
al. (1991) reported t h a t procaine, which is known t o block the C a 2 + - i n d u c e d C a 2 +
release (Endo 1977), caused progressive decrease of inward C a 2 + current (ICA) m
the same cells, but did not discuss this finding. U y a m a et al. (1993) showed
that the cyclopiazonic acid, an inhibitor of sarcoplasmic C a 2 + p u m p , increases
the influx of calcium through voltage-gated calcium channels, and supposed "a
direct regulation of C a 2 + channels activity by the intracellular calcium storage
sites*'. In our experiments we tried to manipulate the functional availability of the
stored calcium by "locking" it separately: into IP3-sensitive stores located near t h e
sarcolemma (see Missiaeii et al. 1992), and the C a 2 + - i n d u c e d release sites which
may b e located deeper in the cells (Zholos et al. 1992; Low et al. 1992). T h e
d a t a presented suggest t h a t L-type C a 2 + channels conduct C a 2 + directly into the
IPs-sensitive pool, whose overloading leads to the C a 2 + - d e p e n d e n t inhibition of
Materials and M e t h o d s
Experiments were performed on single smooth muscle cells freshly isolated from the longitudinal or circular layer of the guinea-pig ileum. The animals weighed 300-400 g. Isolated
muscle strips were placed in physiological salt solution (PSS) and cut into small pieces.
The pieces were then transferred into the PSS without the addition of Ca 2 + at 37°C and
containing 0.5 g/1 collagenase (type 1A, Sigma), 1.5 g/1 soybean trypsin inhibitor and
2 g/1 bovine serum albumin. After 30-40 min of incubation at 37 °C the enzyme was
carefully washed from the pieces with 20 ml prewarmed Ca 2+ -free PSS. Single smooth
muscle cells were then obtained by gentle agitation of the pieces with two Pasteur pipettes
with different tip openings in 1 ml fresh modified "KB'" solution until the solution became
cloudy. In this solution cells were stored up to 12 h at 6°C for this study. For the purpose
of the study only fully relaxed cells were used. Cells which reduced their length by more
than 35% or did not contract at all aftei application of 10~G mol/1 acetylcholine were
rejected, as well as those that did not relax after acetylcholine-induced contraction. PSS
and drugs were perfused continuously to the bath chamber where cells adhered to the
glass bottom.
The whole-cell mode of the patch-clamp technique was used. The patch electrodes
were from borosilicate glass (Jencons) which, when filled with the internal solution, had
resistances of approximately 1.5 MOhms. Membrane currents were recorded using an
EPC-7 (List Electronics) amplifier. The capacitive and series resistance compensations
were made by the use of potentiometer furnished at the amplifier. Current signals were
L-Type C a 2 + Channels Fill Directly C a 2 + Store
77
recorded and further analysed on an AT 286 PC through a TL-1 DMA (AXOPATCH)
inteiface, and pCLAMP software, respectively, and simultaneously displayed on an oscilloscope monitor.
The physiological salt solution (PSS) in the experiments was of the following composition (in mmol/1): 110 NaCl, 12 KC1, 10 HEPES, 20 taurine, 20 glucose, 1.2 MgCl 2 ,
1.8 CaCl2, 5 Na-pyruvate, pH 7.4. 7ca were recorded in the presence of 5 mmol/1 tetraethvlammonium. The modified L'KB" medium, used in cell isolation consisted of 85 KC1,
30 KH2PO4, 5 MgCl2, 20 taurine, 5 Na2-ATP, 5 Na-pyruvate, 5 creatine, 5 oxalacetate,
I g/1 bovine serum albumin (pH 7.2). The internal solution into the recording pipette
contained: 105 KC1 (or CsCl instead of KC1 while recording inward currents), 10 HEPES,
II EGTA, 1 CaCl 2 , 2 MgCl 2 , 4 Na-pyruvate, 4 succinic acid, 4 oxalacetic acid, 1.5 Na 2 ATP, 0.001 cyclic AMP, pH 7.2. Ruthenium red or light heparin (Sigma) were diluted
into the internal solution. The cyclopiazonic acid (Sigma) and ryanodine (Calbiochem)
were added to the bath solution. All experiments weie carried out at room temperature
(25±2°C).
Results
Effects of heparin
on I(<A
Fig. 1J4 (inset) shows typical IQA waveforms, elicited by depolarization in cell from
the longitudinal layer, recorded by a caesium-loaded pipette to block potassium outward currents and t h e corresponding current-voltage relation curve (I /V-curve),
obtained under t h e holding potential (Vh) of —50 m V (Fig. 1A), which is close to
the resting potential of these cells (Droogmans and Callewaert 1986). In agreement
with earlier studies on the same cells (Duridanova et al. 1993), this inward current
is carried exclusively through the L-type (dihydropyridine-sensitive, long-lasting,
high-threshold) C a 2 + channels (Tsien 1983), because it can be strongly and reversibly inhibited by nifedipine (Fig. 1A), or activated by micromolar amounts of
the dihydropyridine BAY K 8644 (Droogmans and Callewaert 1986). On dialysing
cells with heparin-containing internal solution ( 1 0 ~ 1 0 mol/1 and above) a fast decrease of inward current amplitude developed (Fig. IB), and complete and irreversible block of i c a was reached in about 6 min after the beginning of the dialysis.
A subsequent change of bathing solution to Ca 2 + -free or Ba 2 + -containing (instead
of C a 2 + ) solutions (Fig. 1C), or changes in Vh t o various more negative values up
t o —120 mV did not recover from inactivation the inward current in cells from longitudinal layer (not shown). This led us to the suggestion t h a t the block of Tea w a s
caused by elevated intracellular .calcium concentration - [ C a 2 + ] , , which stopped
the ion entry into t h e channel, according to the model proposed for neurons and
heart (Hess et al. 1989; Rosenberg and Chen 1991).
Effect of blocking intracellular
Ca2+
stores
In control experiments (cells from longitudinal layer dialysed with heparin-free
solution), i c a w a s not affected by ryanodine (10~ 6 mol/1) or when C a 2 + uptake into
78
Gago\ et al
F i g u r e 1. C o m p a i isoii oi t In abiliU ol 111I1 dipinc and IK p u in to block ( a" e i u i e n t in
cells isolated from longitudinal la\ei ( 4) C u n e n t - \ o l t a g e (I/\ ) iclation c u i \ e of /r ,,
o b t a i n e d undei l i = —50 m \ eontiol (closed c u t les) m t h e p i e s e n t e of 0 0 3 mmol/1
nifedipine (open eucles) oi with 0 1 ninol/1 hepai m into t h e pipette 1 solution ( t n a n g l e s )
m e a s u i e d 10 ms afte i t h e \oltage s t e p
Cell with input i m p e d a n c e 1 2 G O h m s and
capacitance l i p ľ Inset o u g i n a l í e e o i d s of depolai l/ation-elu ited lc ( wavefoims of the
s a m e cell (photogiaplicd fiom t h e oscilloscope m o m t o i ) Depolai l/al ion steps applied t o
t h e p o t e n t i a l s signed fiom 11, — 50 m \ ( p o t e n t i a l applied heie and at t h e next figuies
a i e m a i k e d with digits) (13) Blocking effect of h e p a i n i (0 1 nmol/1) on / ( , m t h e same
tell as m ( 1 ) \ h = —50 in\ , test p o t e n t i a l t o + 1 0 m\ n u n aftei t h e beginning of t h e
d i a b s i s aic signed at t h e h g u i e ((') I he s a m e \oltage ])ioto<ol as m ( 1) was applied at
t he 12* ' n u n aftei t h e beginning of t he d i a b sis in t h e same c < 11 and at 1 he (>' ' m m aft CM
lepiac mg t h e C a 2 + - with B a " + c o n t a i n i n g (2 5 nmiol/1) e x t e r n a l solution
i n t e r n a l s t o i e s w a s i n h i b i t e d b\ 1 0 _ < > m o l / 1 c\ c l o p i a z o m e a c i d ( F i g
2). a selecti\e
m h i b i t o i of t h e C a 2 + p u m p of t h e s a i c o p l a s i n i c l e t i c u l u m in s t i i a t e d ( G o o g e i et
L-1\pe C a _ + Channels Fill Dneeth ( a " + Stoie
79
F i g u r e 2. 7/1 -ielation cuives of H , in cells fiom longitudinal la\ei \oltagt clamped
at \ h = —50 m\ in eontiol solution (idled cncles) in the piesence of c\e lopiazonie acid
f0~' mol/1 (tnangles) oi nanodme (10 ' mol/1) (open e ne les) Kc ctanguku depol uizing
pulse s of 400 ms dm at ion wei e ipphed at 10 m \ mciemcnts Data u e me ans foi 10 cells
withsmulai passne electiical piopeities Theie weie no significant chffeiences Dc\iation
bais u e not piesented foi clant\
-50
-30
10
30
50
70
V(mV)
F i g u r e 3 . Effect ol lutheniuin led (0 I mmol/1) on / t i m cells hoin longitudinal la'ser
Ij\ cur\e obtained as descnbed m legends of the pie\ious hguies Befoic (eontiol
closed circles) and aftei (open clicks) diabsis with luthenium led containing internal
solution Data aie means ± S T M foi 12 cells \ i, = —50 in\ Inset acceleiation of
whole cell mwaid cmient mactivation b\ liithemum led Oiigmal recoids fiom cell with
input impedance of 1 1 GOhms and capacitance 59 p i 11, = —50 m\ test potentid to
+ 10 m\ befoic and aftei (maiked with encle) 10 mm of ehahsis with liithemum led
containing internal solution Constants of mactnation estimated 102 b ms without and
73 9 ms m the piesence of luthenium led (104 i ± b 7 ms and 72 7 ± 7 2 ms íespectiveb
aie means ± S F \ I foi 7 cells)
al 1988 Seidlei et al 1989) and smooth muscle ( B o m i e a u et al 1991 Iniaiztiim
et al 1992 l h a m a et al 1992 Low et al 1992) Smiilai lack of effects on the
Gagov et al.
80
*
-SO
-30
100 ms
-»
X)
30
50
V(mV)
70
100 ms
Figure 4. Effect of heparin on /c» in cells from the circular layer of the ileum. (A)
7/V-curve of 7c a , elicited by depolarization from Vh = —50 mV (closed circles), in the
presence of 0.03 mmol/1 nifedipine (triangles) or after dialysis with heparin-containing (1
mmol/1) internal solution (open circles). Data are means ± S.E.M. for 6 cells. (Half-bars
are drawn in opposite directions for clarity). (73) Original records of depolarization-evoked
7ca in cell from circular layer with input impedance of f.9 GOhms and capacitance 62
pF. Depolarization steps were applied to potentials signed. (C) Current records in the
same cell after 10 min of dialysis with heparin-containing (f mmol/1) internal solution.
amplitudes, activation threshold and reversal potential of depolarization-elicited
Jca (Fig. 3) was obtained when 10~ 7 t o 1 0 - 4 mol/1 ruthenium red, a widely used
blocker of C a 2 + release channels of Ca 2 + -sensitive C a 2 + pool (Motitin et al. 1992)
was present in the pipette. Thus t h e inhibition of C a 2 + - i n d u c e d C a 2 + release or
emptying of C a 2 + stores did not inhibit ica- This implies t h a t only stores with
IP3-sensitive release sites are near plasmalemma (the inner m o u t h of the L-type
C a 2 + channels) and t h a t there is an overfilling of the stores rather t h a n general
elevation of [ C a 2 + ] , which influences Ica- However, an acceleration of whole-cell
L-Type C a 2 + Channels Fill Directly C a 2 + Store
81
/ca inactivation was observed to increase with time of dialysis (Fig. 3, inset). T h e
latter was taken as an evidence for C a 2 + - d e p e n d e n t inactivation of L-type channels
(Eckert and Chad 1984; Campbell et al. 1988), caused by the relative increase in
the concentration of C a 2 + in the IP3-sensitive C a 2 + pool, due to the block of the
Ca 2 + -induced C a 2 + release mechanism. It can be suggested t h a t b o t h pools may
have area of close contact where they exchange C a 2 + . Ruthenium red holds the
deeply situated Ca 2 + -sensitive C a 2 + pool (Zholos et al. 1991) full and "locked"
which hinders the entry of C a 2 + , released from the IP3-sensitive C a 2 + pool in t h a t
regions, when depolarization activates IcaDoes heparin
indirectly ?
bind the L-type
Ca"+ channels
or does it block the L-type
channels
We examined further the possibility for heparin to interact directly with the channel pore from the inside. For this purpose we isolated cells from the circular layer
of the same organ (guinea-pig ileum), using the procedure described above. Under
the same Vj, depolarization-evoked IQA in these cells had very similar 7/V-relation
properties and dihydropyridine sensitivity as the one observed in cells from longitudinal layer (Fig. 4/1, 73; the low-threshold dihydropyridine-resistant component of
IQA in cells, isolated from circular layer was negligible if Vj, = —50 m V was held for more details see Duridanova et al. 1993). But this current could not be affected
by heparin even at 1 mmol/1 concentration (Fig. AC). In some heparin-loaded cells
i c a tended to accelerate its inactivation course, but this effect was not significant.
It was concluded t h a t heparin is not able t o interact with L-type C a 2 + channels.
The lack of action of heparin in cells from circular layer may reflect the absence of
a close connection between IP3-sensitive stores and L-type Ca~ + channels.
Discussion
The above d a t a were taken as a proof t h a t effects of heparin in cells from longitudinal layer of guinea-pig ileum are specific for some cell types and do not include a
general ability of heparin to interact with the channel molecule. T h e latter conclusion is consistent with the experimental results on cardiac myocytes (Lacinova et
al. 1993) and is also in agreement with the high efficiency of the heparin-induced
Ica blockade observed in cells from longitudinal layer at concentrations of 100 times
lower t h a n expected (Kobayashi et al. 1988). As heparin is known to be the most
powerful and selective inhibitor of IP3-induced C a 2 + release in smooth muscle cells
(Ghosh et al. 1988; Kobayashi et al. 1989, Chadwick et al. 1990), where it prevents the interaction of IP3 with C a 2 + release channels (Kobayashi et al. 1989),
the most obvious explanation of the above d a t a is t h a t the inner m o u t h of L-type
C a 2 + channels opens directly into this pool. In this way, the C a 2 + ions stored and
"locked" into the pool by t h e heparin blockade, cannot be released by conventional
82
Gago\ et al
stimuli, so the pool l e m a m s h e a \ i h loaded with Ca-"1" which inhibits subsequent
movement s of ions t l n o u g h L-t\ pe channels fiom the outside This explanation is
also consistent with c\ tomoiphological d a t a , which h a \ c shown IP {-binding sites
on saieoplasimc leticultim to be placed close to the iiiiiei face of plasmalemma ( \ a n
B i e e m a n and Saida 1989 Missiaeii et al 1992)
Effectmmess of c\clopiazomc acid to íaise [Ca~ + ], m cells fiom longitudinal
la\ei mitialh was shown on K4" c m i e n t meastueiiients (Gagcn et al 19931)) It
was also found that the long-lastmg (10 m m and moie) exposme of ileal cells to
t \ c lopia/onu acid (10 (> mol/1) leads to a selective blockade ot the1 Ca 2 + -sensiti\e
K + condue t<uicc (Su/uki et al 1992, Gagcn et al 1993a) Oui e\])ciiments have
also shown that m h e p a i m loaded cells fiom longitudinal la\ei of the ileum evclo])ia7ome acid cause's almost a total inhibition of R4" cuiients m 5 mm (Gagcn
et al 1993b) The C a " + suppoitmg I \ + outwaid e m i e n t s m hepai m-loaded cells
entci tlnough L-t\ pe C,\' f channels e\en though 7c , could not be íecoided m
these conditions (Gagcn et al 19931)) These data showed t h i i when C a 2 + was
locked into IP {-sensitive stoies b\ h e p a i m dining the depenalization theie was an
ele\ation of [Ca 2 + ], neai the membiane which p i c s u m a b h o n g m a t e d fiom leakage out oi o\eihlled stoies tlnough the c \ c lopia/omc ac id-sensiti\e Ca 24 " p u m p
Such mechanism has been pioposed m ícgulation of Ca~ + stoie lehllmg m tiacheal
s m o o t h muscle (Janssen and Sims 1993) Lhus we supposed that this enoimotis
(due to the eneihllmg of the stoies) Ca~ + leak hoin the1 IPs sensitive Ca~ + -stoie
was eliieeted to the miiei mouths of the Ca~ + -sensitive R f channel poies It is also
consistent with e\ ideiice that empt\ mg the C<r + pool pioniotes C a " + enitiv into
the pool (Bomic an ct al 1991)
In e one lusion the d a t a obtained p i o \ i d c e\ ide ne e that m some smooth muscles
the I P ,-sc nsitne C a 2 + - p o o l undeigoes a v oltagc-ek pendent C a " + lehllmg t l n o u g h
L-t\ pe Ca 2 + -c hannels This niechainsm can be effee ti\el\ antagonized In nitiaeell u l a i h applied h e p a i m
A c k n o w l e d g e m e n t s . I his woik was suppoited b\ the Mmistn of Science ind Ilighei
Fducation of Bulgana giant \ o K-501
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Final version accepted April 7, 1994