455

Journal of Physiology (1992), 457, pp. 455-475 With 11 figures Printed in Great Britain

Ca2+ CURRENTS IN SINGLE MYOCYTES FROM HUMAN MESENTERIC ARTERIES: EVIDENCE FOR A PHYSIOLOGICAL ROLE OF L-TYPE CHANNELS BY SERGEY V. SMIRNOV* AND PHILIP I. AARONSONt From the Departments of Pharmacology and Medicine, United Medical and Dental Schools of Guy's and St Thomas's Hospitals, London SE1 7EH and *Department of Nerve-Muscle Physiology, A. A. Bogomolets Institute of Physiology, Ukrainian Academy of Sciences, Kiev, Ukraine

(Received 10 January 1992) SUMMARY

1. Voltage-gated Ca2+ currents (ICa) in isolated human mesenteric arterial cells were characterized in solutions containing normal (1P5 mm) Ca2+ and elevated concentrations of divalent cations using the conventional whole-cell patch clamp technique. 2. In normal Ca2+ solution, depolarization beyond -40 mV elicited a slowly decaying ICa which reached a maximum at + 10 mV and appeared to reverse between + 40 and + 50 mV. The amplitude of this current in a group of cells correlated with cell membrane capacitance. 3. In two of thirty-three cells a small transient component of inward current was detected in the voltage range between -40 and -10 mV when cells were held at -80 mV. This current was abolished at a holding potential of -40 mV, while the current at 10 mV was not affected. These currents were referred to as T- and L-type

Ca2+ respectively. 4. Elevation of the extracellular Ca2+ concentration to 20 mm shifted the voltage dependencies of Ca2+ current activation and inactivation by approximately + 20 mV; a small T-current component was then observed in seven of nine cells held at -60 mV. 5. Replacement of 1-5 mm Ca2+with 10 mm Ba2+ increased the amplitude of the current elicited at + 10 mV by a factor of 3-7 and a small barium current (IBa) through T-type Ca2+ channels was also observed in most cells studied. Activation and steady-state inactivation curves for L-type current were found to be almost identical in both solutions. The steady-state inactivation for the T-type IBa was, however, more than 30 mV more negative (half-inactivation potential of - 62-6 mV) of that for L-current in 1-5 mm Ca2+ and 10 mm Ba2+ solutions (-304 and - 24-9 mV respectively). 6. A sustained inward Ca2+ channel current was recorded in the presence of normal Ca2+ and high divalent cation concentrations during 30 s depolarizations. The

t To whom correspondence should be sent. MS 1030

456

S. V'. SMIRNOV AND P. I. AAROXSN0..

amplitude of this sustained current was found to be similar to the theoretical 'window current' predicted by the overlap of the activation and inactivation functions in these solutions. 7. Examination of the inactivation of the L-type current using a two-pulse protocol with a 240 ms prepulse revealed a U-shaped potential dependency for ICa' but not for IBa' suggesting the presence of a Ca2+-dependent component of the inactivation process. 8. These cells resemble other arterial smooth muscle cells previously studied in that they demonstrate both T- and L-components of 'Ca In normal-Ca21 solution, the T-current is quite small even when fully available, and likely to be mostly inactivated at the resting potential. The calculated window current predicts the presence of a small, sustained Ca21 influx through L-type Ca2' channels at negative voltages which would vary by an e-fold factor with both de- and hyperpolarizing deviations of less than 1O mV from -60 mV. Small changes in the membrane potential from its resting level would therefore have an important effect on Ca2± influx through these voltage-gated channels. INTRODUCTION

Excitation-contraction coupling in vascular smooth muscle involves a rise in the intracellular Ca2+ concentration which is in part dependent upon an influx of Ca21 through voltage-gated channels. Both single-channel and whole-cell currents mediated by these channels have been characterized in a number of types of isolated vascular smooth muscle cells (SMCs). It has in general been observed that there are two types of Ca21 channel, usually termed T-type and L-type, which underlie wholecell currents that are transient and sustained, respectively (Bean, Sturek, Puga & Hermsmeyer, 1986; Friedman, Suarez-Kurtz, Kaczorowski, Katz & Reuben, 1986; Benham, Hess & Tsien, 1987; Aaronson, Bolton, Lang & MacKenzie, 1988; Loirand, Mironneau, Mironneau & Pacaud, 1989; Ganitkevich & Isenberg, 1990). Dissection of these currents has usually been carried out in solutions containing elevated concentrations of Ca2+ or Ba2+; the presence of two components of current is not obvious when the external Ca2+ concentration ([Ca21].) is set at a physiological level (Aaronson et al. 1988; Ganitkevich, Smirnov & Shuba, 1989). Since arterial smooth muscles are usually non-excitable, and do not always depolarize markedly during excitation-contraction coupling (Droogmans, Raeymaekers & Casteels, 1977), it is clear that modulation of Ca2+ influx near the in vivo resting membrane potential may be of great physiological importance. The presence of a sustained, non-inactivating Ca2+ influx into SMCs which could be modulated to produce relaxation or contraction from the basal level of tone has previously been discussed (Bolton, 1979; Shuba, 1981; Nelson, Patlak, Worley & Standen, 1990), although it is becoming increasingly clear that intracellular effects on Ca2+ compartmentalization or sensitivity are also important in controlling tension. Hyperpolarization of the membrane potential is thought to relax cells by reducing Ca2+ influx through voltage-gated channels (reviewed by Nelson et al. 1990). Ca2+ currents have been detected near the resting potential of arterial cells at the wholecell level in normal [Ca2+]. (Aaronson et al. 1988). Single-channel openings have also been detected at very negative potentials in 10 mm Ba2 , which produce little surface

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY

457

potential-mediated shift in the voltage ranges of channel activation and inactivation (Ganitkevich & Isenberg, 1990; Nelson et al. 1990). The channels involved appear to be of the L-type. This report describes the voltage-gated Ca2+ current present in cells isolated from the human mesenteric artery in solutions containing normal [Ca2+]. and in 10 mM Ba2+. The results suggest that an L-type current provides a voltage-dependent steady-state Ca2+ influx in the physiological range of membrane potentials. A preliminary report of this work has been presented (Smirnov & Aaronson, 1992a). METHODS

Methods of cell isolation, current recording, and analysis and storage of data did not differ from those described in the previous paper (Smirnov & Aaronson, 1992b). Since cells demonstrated a high input resistance in the presence of Cs+ ions in both external and pipette solutions (11-5 + 1-5 GO, n = 65), compensation for ohmic leak currents was not deemed necessary. The main external solution contained (mM): 130 NaCl, 1 CsCl, 1-2 MgCl2, 1-5 CaCl2, 4 TEA-Cl, 10 HEPES; 10 glucose. High-Ba2+ solution was prepared from this solution, minus added CaCl2, by replacement of 10 mm NaCl with 10 mm BaCl2. The composition of the pipette solution was (mM): 135 CsCl, 2-5 MgCl2, 2 Na2ATP, 10 HEPES, 10 EGTA. The pH of all solutions was adjusted to 7-2 with NaOH. RESULTS

Ca2+ current in human mesenteric arterial cells at physiological [Ca2+]0 Calcium current (ICa) was recorded in single human mesenteric arterial cells in the presence of 1-5 mm Ca2+ in the external solution. The peak amplitude of Ica occurred between 0 and + 20 mV (usually at + 10 mV), and varied between 15 and 140 pA (mean 54-3 + 5-1 pA, n = 37). Results described in the previous paper showed that SMCs isolated from human mesenteric arteries vary widely in their sizes (Smirnov & Aaronson, 1992 b). An analysis of dependencies of the ICa peak amplitude on the cell membrane capacitance (Cm), determined as described in the previous paper, revealed a correlation (correlation coefficient of 0 74) between these parameters (Fig. 1), i.e. the density Of ICa is similar in small and large cells. Therefore the density of ICa rather than current amplitude in picoamps was used routinely in the following analysis. A family Of ICa activated by different depolarizing pulses from a holding potential of -60 mV is shown in Fig. 2A. ICa became apparent near -30 mV, reached its maximum at + 10 mV, and become outward at membrane voltages between +40 and + 50 mV. An average current-voltage (I-V) relationship for ICa in twenty-four cells (Cm and ICa peak ranged from 33 to 78 pF and from 14-8 to 108-1 pA respectively) is shown in Fig. 2B. The mean peak density Of ICa in human mesenteric arterial cells in the presence of 1P5 mm Ca2+ was 0-91 + 01 ,tA cm-2 (n = 24). In two cells (one of them presented in Fig. 3) of thirty-three cells studied at physiological [Ca2+]0 a rapidly inactivating component Of ICa was clearly observed in the voltage range between -40 and -10 mV when the holding potential was set to -80 mV. This current decayed almost completely within the first 100 ms of depolarization and was absent at a holding potential of -40 mV (Fig. 3A and B). The amplitude and time course of Ica activated at more positive potentials showed little dependency on the holding potential. These properties of Ica suggest the presence of two components of Ica, analogous to T- and L-type Ca2+ channels described in other tissues (e.g. Nowycky, Fox & Tsien, 1985; Bean, 1985; Fedulova,

S. V. SMIRNOV AND P. I. AARONSON

458

Kostyuk & Veselovsky, 1985; for review see Pelzer, Pelzer & McDonald, 1990), including SMCs (Benham et al. 1987; Aaronson et al. 1988; Akaike, Kanaide, Kuga, Nakamura, Sadoshima & Tomoike, 1989; Loirand et al. 1989; Ganitkevich & Isenberg, 1990). Cm (pF) 50

0

100

0~~~~~

-0 -

A

-100 -

-.00~~~ Q~~~~~ 0o

I0

\

0~~~~

0

cb 0

-50 -100

0 80t8

*

0 0 -.0

77-3pF

38-6pF

0

-150

120 pA

1 00 ms

Fig. 1. Dependence of 'Ca peak amplitude on the cell membrane capacitance (Cm). In several cells (@) 1Ca was estimated as the Cd2+-sensitive inward current. Continuous line is a regression line with a regression coefficient of 0 74. Inset shows ICa at + 10 mV in two different cells, marked by arrows in the graph, with Cm as indicated near each record.

The failure to observe the transient current component in most of the cells studied under these conditions may have been due to its small amplitude, or to its presence in only a fraction of the cells present, as was found for example in a primary cell culture from rat aorta (Akaike et al. 1989), or in freshly isolated SMCs from the guinea-pig coronary artery (Ganitkevich & Isenberg, 1990). We therefore examined ICa in solutions containing 20 mm Ca2+ or 10 mm Ba2 .

Effect of increasing the external Ca2+ concentration The dependency of the peak ICa amplitude (determined as described in the legend to Fig. 4A) on [Ca2+]o was fitted by the Langmuir equation with an apparent dissociation constant of 3-9 mm and a maximal saturated current ratio of 3-91 (Fig. 4A). In the presence of 20 mm Ca2+ (at which concentration ICa amplitude is close to its maximal value in these cells), the position of the peak ICa was shifted along the voltage axis by 20 mV from +10 to + 30 mV, and increased by 3-1+ 029 times (n = 11) in comparison with that at 1-5 mm Ca2+ (Fig. 4B and C). Moreover, this augmentation of the [Ca2+]0 revealed the rapidly inactivating component of ICa in seven of nine cells studied. This T-type Ca2+ channel current was observed at -30 and -20 mV in the cell presented even at a holding potential of -60 mV in contrast

^id*,>~ -1v0

459 Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY to ICa recorded in the presence of 1P5 mM Caa2+ (Fig. 4Ba and b). The amplitude of the T-type current, measured as the difference between the ICa peak and the current at the end of the 300 ms depolarizing pulse to -20 and -10 mV, was 3-9 + 0 3 pA at -20 mV and 6-2 + 1P8 pA at -10 mV for seven cells studied. This small amplitude A

30Uj 420 -10

r0

Of

r

-80

-40

0

20

10

30

20

40 pA

40

2

60 80

~~~~~V.(my)

001-

-02

-0.6

S-0-8-

50

60

1100 Ms

Fig. 2. ICa in normal [Ca2+]0. A, a family of ICa recorded at different test potentials indicated in mV near each current trace. Cm = 77 pF. B, the mean I-V relationship for IC. measured at the peak (0) and the end (0) of 300 ms pulse for twenty-four cells studied. Holding potential was -60 mV. Vertical bars in this and all following figures represent standard error of the mean value.

of the T-type current of the whole-cell ICa made a detailed investigation of its properties in human mesenteric arterial cells difficult. The shift of the I-V relationship for ICa towards positive potentials in high [Ca2+]o indicates the sensitivity of potential-dependent characteristics (activation and inactivation) ofICa to surface potential changes. This was seen from a comparison of the steady-state inactivation of Ica in solutions with normal (1P5 mM) and elevated (20 mM) Ca2+. Steady-state inactivation was measured at the peak of the I-V relationship in each solution (+ 10 and + 30 mV for 1P5 and 20 mm Ca2+, respectively) after 30 s at each conditioning potential studied (Fig. 5A and B). For a theoretical description of steady-state inactivation of Ica the Boltzmann function was used: (1) ho = [1+exp((V-Vo.5)/lk)]-l, where hco is the fraction of Ca2+ channels not inactivated, VJO.5 is the potential where

S. V. SMIRNOV AND P. I. AARONSON

460

50 % of these Ca2" channels are inactivated, and kh is a slope factor. The experimental data were fitted by eqn (1) with V^.5 and ih equal -30 4 and 8-8 mV at 1-5 mm and -7-1 and 7 mV at 20 mm Ca2" (Fig. 5C).

Ca2+ channel currents in the presence of 10 mM Ba2+ Since at elevated [Ca2+]. potential-dependent parameters of Ica are shifted significantly towards positive potentials, it was difficult to study directly the C

A

-30g;_

-20 _g

-10

0

10

20

t -

-90-60

r'-

r

|10 pA

100 ms

-30

0 30

Par~~~~~~~~~~~ V . I = V ) |20 pA S

lO 10msV

B

25

-25

0.11

-5 -50

Fig. 3. Two components of ICa at the normal Ca2+ concentration. A, a family of Icas recorded from the holding potential of -80 and -40 mV (lower and upper traces in each pair, respectively). Test potentials are indicated near each pair in mV. B, 'Ca recorded with 10 mV increment between -50 and -20 mV from a holding potential of -80 mV in the same cell. C, I-V relationships for the peak (open symbols) and current at the end of the 300 ms depolarizations (filled symbols) recorded from -40 and -80 mV (circles and triangles respectively). Cm = 47 9 pF.

properties and behaviour Of Ica over the range of potentials of physiological interest. It is known that Ba2+ is more permeable through the L-type Ca2+ channel, and counteracts negative surface charges less effectively, compared to Ca2+. This increases the current amplitude and elicits a shift of potential-dependent characteristics of Ca21 channels towards negative potentials when Ca21 is replaced by an equal concentration of Ba +. Due to these properties of Ba2+, it was possible to select a concentration of Ba21 which increased the amplitude of the inward current through Ca21 channels, but which caused very little shift in the voltage dependencies of

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY 461 activation and inactivation of the Ca2" channel current compared to those observed in physiological [Ca2"]. (e.g. see Ganitkevich et al. 1989). In human mesenteric arterial cells the replacement of 15 mm Ca2" by 10 mm Ba2+ did not shift the I-V relationship for 'Ba in comparison with that for ICam whereas the A 4-

(3)

32I-

(2) 1F

()

0 B

a

(11)

(5)

19

ll

5

10

15

20

[Ca 2+]. (MM) C

10

-60 (mV)

a) N

.c

z b 30

-60

-04 -0-6 -

-08 -

-1 0-

0-4-

b

-80 -40

02(mV)

-0*4-

Cu

_z

-0

-0 6 -

m

-0 8 -

z

-1-0-

Fig. 4. Effect of increasing external Ca2" concentration ([Ca2+].) on Ca2+ channel currents. A, dependence of relative ICa peak amplitude on the [Ca2+]0. ICa/'Ca 5 was determined as the ratio of ICa peak amplitude in a test solution to that in 1-5 mm da2+. Number of cells tested at each [Ca2+]0 indicated in parentheses. Continuous line is a Langmuir curve drawn with Kd = 3 9 mm and a maximal current ratio of 3-91. B, 'Ca recorded with 10 mV increments from the holding potential of -60 mV in the presence of 1-5 (a) and 20 (b) mM Ca2+. Cm = 55 pF. C, I-V relationships for the peak (open symbols) and current at the end of 300 ms pulse (filled symbols) recorded in each solution, a and b as in B. Current amplitude was normalized with respect to the peak of ICCa measured in 20 mm Ca2+ in each of nine cells studied.

S. V. SMIRNOV AND P. I. AARONSON

462

amplitude of inward current through Ca2+ channels was substantially increased over the entire voltage range studied. The ratio of the peaks of IBa and ICa was 3-7 at + 10 mV (data not shown). In the presence of 10 mm Ba2+ both T- and L-types Of ICa were activated by 300 ms depolarizing pulses from a holding potential of -80 mV A

C

10~~~~~~~1

06-

40 pA

100ms B

0*430

3~~~~~~~~~~~~~- -_

~~~02 -80 -60 -40 -20

0

20

40

Conditioning potential (mV) Fig. 5. Steady-state inactivation of Ica in normal and elevated Ca2+. A and B, Ic. recorded at the peak of the I-V curve in 1-5 and 20 mm Ca2+ at different conditioning potentials (test potentials were + 10 and + 30 mV respectively). Cm was 43-5 (A) and 34-5 pF (B). C, dependence of h. on the conditioning potential studied for six to nine cells in 1-5 mm (0) and for five cells in 20 mm (0) Ca2l. h. was determined as a ratio of the peak Ica measured after 30 s conditioning depolarization to the maximal current amplitude observed. Continuous lines are drawn according to eqn (1) as described in the text.

(Fig. 6B and C). The voltage range over which the T-type current was observed was qualitatively similar to that observed for the T-type current in the presence of 1.5 mM Ca2+ (See Fig. 3). This current was also completely blocked at a holding potential of -40 mV, while the L-type current amplitude showed very little change (Fig. 6A and B). As observed in high-Ca2+ solution, however, the amplitude of the Ttype component of the inward current, measured as the difference between the peak current and the current at the end of 300 ms pulse, was very small, totalling 2-3 + 0 5 pA at -40 mV, 6-0 + 1-3 pA at -30 mV and 8-6 + 2-3 pA at -20 mV for the seven cells shown in Fig. 6C. The steady-state inactivation of the L-type and T-type components of IBa was examined using test potentials of + 10 and -20 mV respectively, since the T-type component is quite prominent at the latter potential. As can be seen from Fig. 7, the steady-state inactivation for the T-type current was shifted towards negative

463 Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY potentials by about 40 mV in comparison to that for the L-type IBa. The voltage dependency of inactivation for IBa measured at + 10 mV was quite similar to that for ICa in the presence of 1'5 mm Ca2+ (Fig. 7 C, filled and open circles, correspondingly). The parameters of the Boltzmann function obtained from fitting the experimental A

1 30 pA

B

1 00 Ms

|10 pA

1 00 Ms

C

0-4-

a

-90 -60 -30

0

' *> ~~~~~~~0 ~ ~

E 0

Z

0-4

b

-90

30

COWL Em(mV) ~

~

N~~~~~~~~

-1-0Z

0

X

o

T4

V

Eli 10

Fig. 6. Ca2+ channel currents in the presence of 10 mm Ba2+. A, IB. recorded from holding potentials of -40 (shown by arrow in each pair) and -80 mV at the test potentials indicated in mV. B, T-type IB. in the same cell at -50, -40 and -30 mV; Vh = -80 mV. Cm = 47 9 pF. C, mean normalized I-V relationships for IB. in seven cells, recorded from holding potentials of -40 (a) and -80 (b) mV. Open and filled circles show IBa peak amplitude and current at the end of 300 ms pulses, respectively. Current amplitude was normalized with respect to the peak of IBa at Vh = -40 mV.

points in eqn (1) were VO.5 = -24-9 and -62-6 mV, and kh = 8-5 and 5.7 mV for the L- and T-type components of IBa, respectively. These results demonstrated the presence of T-type Ca2+ channels in the majority of the human mesenteric arterial cells studied at high Ca2+ or Ba2+ concentrations. The whole-cell current through these channels is, however, so small that this current

S. V. SMIRNOV AND P. I. AARONSON

464

is only poorly resolved at the normal physiological Ca2+ concentration. Due to the small amplitude of the T-type current and the negative voltage range over which it inactivates, these channels presumably play only a minor physiological role in human mesenteric arterial cells. We therefore carried out experiments to examine the A

B

10mV

°0

_4|-20mV -80

100 ms

C 1*00-80-60-4 0-2 00 __

-100

-80

-20 -60 -40 Conditioning potential (mV)

0

20

Fig. 7. Steady-state inactivation for L-type and T-type Caa2+ channel currents in 10 mM Ba2+. A and B, Ba2+ currents at test potentials of +10 and -20 mV, respectively, recorded after a cell was held for 30 s at conditioning potentials over the range shown in the voltage protocols. Cm was 49-4 pF. C, dependence of h., determined as described in the legend to Fig. 5 C, on conditioning depolarization for IBa at -20 (V1, average from six cells) and + 10 (@, nine cells studied) mV. The inactivation curve for ICa at 1-5 mm Ca2+ (0) which was described in Fig. 5 C is presented here for comparison. Continuous lines are drawn according to eqn (1) with parameters indicated in the text.

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY 465 role of the L-type ICa in contributing Ca2+ entry over the range of potentials likely to be of physiological importance. Comparison of the properties of L-type Ca21 channels in the normal-Ca2+ and high-Ba2+ solutions To study the possibility that L-type Ca2+ channels can produce a significant inward current near the resting potential, supposing this to be close to -60 mV, A

B

1-5 mm Ca2 1__

-.I.J

-

C

110mM m BBa2+

g

10-

Wm

M 30 pA

140 pA

20 ms

20 ms

0.8

2

I4

~~~-60-30

o -60 -30

30

0

8

0-0j0 -60 -30

0*

0 ~~~~

a-1

V (mV) D r -60

90

0

60

0

'60

Vm (mV) -40

-20

009

'V (mV)

P ~ ~ ~ ~

~~~~

E

~~-0.3

6~~~~~~~~~~~~ ~-0.6

E~~~~~~

U

-0*9 L

-0.8

-6

Fig. 8. Activation of L-type Ca2+ channel currents in normal-Ca2+ and high-Ba2+ solutions. A and B represent examples of Cd2+-sensitive 'Ca and IBa in the voltage range between -50 and + 10 mV with 10 mV increments from a holding potential of -60 mV (shown at the top, Cm was 90 9 and 78 pF for cells presented in A and B correspondingly). Mean I-V relationships (seven cells in each solution) for the peak of ICa (triangles) and IBa (squares) in the presence (filled symbols) and absence (open symbols) of 0 5 mm Cd2+ are shown below. The difference currents are shown for Ca2+ (0) and Ba2+ (@). Dashed lines are drawn according to eqn (5) (see text for detail). C, the activation dependencies for ICa (0) and IBa (@). Continuous and dashed lines are Boltzmann functions with parameters described in the text. D, Cd2+-sensitive experimental (0 and *) and theoretical (continuous and dashed lines) 'Ca and 'Ba, respectively, in the voltage range between -50 and -20 mV.

inward current elicited by 60 ms depolarizing pulses from the holding potential of -60 mV was compared in normal-Ca2+ (1-5 mM) and high-Ba2+ (10 mM) solutions before and after addition of 0-5 mm Cd2+ (Fig. 8A and B). Cd2+-sensitive ICa and IBa became apparent at a membrane potential of -40 mV and peaked at + 10 mV with a maximal current amplitude of 0-66 and 5-44 gA cm-2 respectively. Both ICa and IBa

S. V. SMIRNOV AND P. I. AARONSON revealed non-linear behaviour near the reversal potential, becoming outward at membrane voltages positive to +60 mV (the reversal potential was estimated as + 62-8 and + 72-9 mV for ICa and IBa, respectively). The fact that these reversal potentials are much more negative than would be predicted on the basis of permeation by Ca2+ alone, as well as the marked sensitivity of outward current to Cd2+, suggested that L-type Ca2+ channels in human mesenteric arterial cells are also permeable to monovalent cations as was shown in other tissues (e.g. Reuter & Scholz, 1977; Lee & Tsien, 1984). In addition, a contribution of outward caesium current flowing through K+ channels in the voltage range positive to the reversal potential cannot be excluded. In order, therefore, to describe the dependence of the Cd2+-sensitive inward current on the membrane potential in normal-Ca2+ and high-Ba2+ solutions, we used the Goldman-Hodgkin-Katz expression with the following assumptions (see Meves & Vogel, 1973; Hagiwara & Byerly, 1981; Sanchez & Stefani, 1983; Lee & Tsien, 1984). Firstly, the surface potential at the inner surface of the cell membrane was considered to be zero. Secondly, the Cd2+-sensitive inward current over the entire voltage range was assumed to be transferred only by divalent cations. Also, we neglected a possible contribution of the inactivation process to the peak inward current. The expression for the inward Ca2+ channel current used was: 466

-I4F2V=

[C2+]o(exp (2F(V-Erev)/RT) -1)

(2)

exp (2FVIRT) - I where P, is the permeability for Ca2+ or Ba2+ ions, I, is the Ca2+ or Ba2+ current, R, T and F have their usual meanings, and Erev, the reversal potential for ICa or IBa, was supposed to be 151 mV, assuming that the intracellular divalent cation concentration is 10-8 M. To account for the voltage dependence of Ca2+ channel activation, P, can be expressed as: C

RT

P" = P"M.,

(3)

where P, is the limiting divalent cation permeability. m., the Boltzman function for activation reflecting the fraction of Ca2+ channels being activated at each membrane potential, can be expressed as follows: (4) moo = [1 + exp ((V-VOT5)/km)]-, where VT.. and km are the mid-potential and the slope factor correspondingly. Combining the eqns (2) and (3) the final expression was obtained: IC

4VPMoo [C2+]O(exp (2F(V-Erev)/RT)- 1) RT

exp (2FV/RT) -1

(5)

where mc, is given by eqn (4). Equation (5) was used for a theoretical description of the I-V relationships for the Cd2+-sensitive inward current obtained in the normal-Ca2+ and high-Ba2+ solutions (Fig. 8A and B respectively), by varying the parameters P, VOT5 and km. The best coincidence between experimental and theoretical (shown by the dashed line in Fig. 8A and B) values was obtained atPca = 55 x 10-3 cm s-1, VoT5 = 5-2 mV and km = -7.79 mV for Ia and Ba = 6-2 x 10-3 cm s- , VoT5 = 4-2 mV and km = - 7-08 mV.

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY 467 It should be noted that the value of the reversal potential had a negligible effect on the parameters calculated. If the internal divalent cation concentration was assumed to be 10-6 or 10-10 M the parameters did not differ by more than 1-4% from those obtained above. Both sets of experimental data were well described by the theoretical function over the entire potential range, with the exception of potentials close to and positive to the reversal potential. This discrepancy is expected in the potential range where the outward flow of monovalent ions through Ca2+ and perhaps K+ channels becomes important. The activation curves for the Ca2+ channel current constructed using the fit of eqn (5) to the data in Fig. 8A and B were very similar, indicating only minor differences between the activation of whole-cell Ica and IBa at the external divalent concentrations used (Fig. 8 C). Figure 8D shows the experimental and theoretical ICa and IBa in the voltage range between -60 and -20 mV. The amplitude of inward current at -60 mV predicted on the basis of the constant field theory assuming the mean cell membrane capacitance of 46 pF (Smirnov & Aaronson, 1992 b) is about 0a 1 and 0-4 pA for Ica and IBa respectively. Currents with these amplitudes would be smaller then the cell membrane noise and therefore difficult to see under whole-cell conditions. Our data suggest, however, that a small fraction of L-type Ca21 channels are open even at -60 mV, as previously shown in patch clamp studies of IBa through single Ca2+ channels in guinea-pig coronary artery and rabbit basilar artery cells under similar conditions (Ganitkevich & Isenberg, 1990; Nelson et al. 1990).

Contribution of L-type Ca2+ channels to a sustained Ca2+ entry in human mesenteric arterial cells The results described in Figs 7 and 8 demonstrate that the activation and inactivation curves for the L-type Ca2+ current overlap over a wide potential range, suggesting the presence of a 'window current' which should persist for at least 30 s. In order to evaluate the potential dependence of such a window current we estimated the theoretical sustained current through the L-type Ca2+ channels in the normalCa2+ and high-Ba21 solutions, taking into account both the activation and inactivation processes. The expression used was similar to eqn (5) except that the permeability for Ca2+ or Ba2+ ions was represented as:

Pe = Pc mc, ha,

(6)

where hat and mao are described by eqns (1) and (4) respectively. The theoretical window current was calculated using the parameters for ICa and IBa described above. The fitted activation and inactivation curves for 1-5 mm Ca2+ (continuous lines) and 10 mM Ba2+ (dashed lines) are illustrated in Fig. 9A. The calculated window currents in these solutions are presented in Fig. 9B. Although the window current carried by 10 mM Ba2+ is much larger than that carried by 1-5 mm Ca2 , the potential dependency of current amplitude is similar for both situations. It is also apparent, especially for the current in 10 mm Ba2 , that a sustained inward current is predicted to exist at -60 mV. This current would increase by a factor of 3 with depolarization to -50 mV, and by a factor of 7-10 with depolarization to -40 mV. In order to determine whether these theoretical curves were likely to reflect actual sustained currents, 30 s depolarizations were applied from a holding potential of

468

S. V. SMIRNOV AND P. I. AARONSON

-60 mV. Such long-lasting depolarizations elicited inward current which decayed to a steady-state level which could be visualized by comparing these currents to those observed in the presence of 0-5 mm Cd2` (Fig. 10A). The amplitude of this sustained Cd2+-sensitive current measured at the end of a 30 s pulse was 1P2+ 07 and A

1.0

B

,.

-

-90

000

-

-031

-

60

30

0

30

.

60 Vm (mV)

0

a

O~ ~ ~ ~ ~ a N

E'

U

-042 -

-0*4 Fig. 9. 'Window current' in human mesenteric arterial cells. A, theoretical activation and inactivation curves for ICa (continuous line) and IBa (dashed line) reprinted from Figs 5, 7 and 8. B, predicted window currents generated as described in the text in normal Ca2+ (continuous line) and 10 mm Ba2+ (dashed line) solutions. The data points show the mean amplitudes of Cd2+-sensitive inward currents measured at the end of 30 s of depolarization in 1-5 mm Ca21 (0) and 10 mm Ba2+ (@ circles), in three cells.

1-6 + 0 9 pA at -20 and 0 mV, respectively, for three cells studied in 1-5 mm Ca2+. Increasing the external Ca2+ to 20 mm increased the amplitude of ICa (Fig. lOB). In this case test potentials were adjusted to -10 and 10 mV due to the 20 mV positive shift of the voltage characteristics of these channels, and the concentration of Cd2+ ions was increased to 1 mm to block ICa completely. The amplitude of Cd2+-sensitive sustained current was 1-87 + 0-2 pA (n = 3) at -10 mV and 4-73 + 1-02 pA (n = 4) at 10 mV. Similar results were obtained in the high-Ba21 solution (Fig. 10C), which indicates that this sustained current does not depend on the kind of divalent cation passing through L-type Ca2+ channels.

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY 469 The measured current values in 1-5 mm Ca2" (0) and 10 mM Ba2+ (@) were converted to current densities and are plotted in Fig. 9B. It is clear that there is good agreement between the theoretical and measured values of the sustained current in both situations, especially given the small sizes of the currents involved. Analysis of A

B

1-5 mM Ca2"

C 20 mM Ca2+

10 mM Ba2+

0

10

0

-20

-10 _-60

-20

-60

-60Q_

20 pA

10 pA 10 pA

Fig. 10. Sustained Ca2+ channel currents measured during 30 s depolarizations in the presence (upper traces in each record) and absence of Cd2+. Each current trace represents the digitally averaged record of three subsequent runs. Voltage protocols and divalent cation concentrations are indicated above. The Cd2+ concentration was 0 5 mm in A and C, and 1 mm in B. Cm was 90 9 (A), 59-6 (B) and 68-2 (C) pF.

the sustained current amplitudes in 20 mm Ca2+ also indicated a good agreement with theoretical values. Assuming that the activation of ICa is shifted to the same extent (23 mV) towards positive potentials as is the steady-state inactivation, and also that the amplitude of the sustained current is increased by 3 1 times in the presence of 20 mm Ca2` in comparison with that measured at 1-5 mm Ca2+, the corrected amplitudes of the sustained ICa measured at -10 and + 20 (0 01 1 and 0-029 ,tA cm-2 respectively) were quite similar to the values predicted on the basis of the constant field theory for ICa at -33 and -13 mV (0-021 and 0-027 ,tA cm-2 respectively) in the presence of 1-5 mm Ca2+. The correlation between the experimentally measured sustained current and the theoretically predicted window current in the normal- and elevated-Ca2+ and high-Ba2+ solutions confirms the suitability of the theoretical approach described in Fig. 9.

Dependence of inactivation of L-type Ca2+ channels in human mesenteric arterial cells on the Ca2+ entry At present it is well known that the inactivation process of L-type Ca2+ channels is controlled by both the membrane potential and Ca2+ ions on the inside of the cell membrane. The availability of L-type Ca2+ channels in the normal-Ca2+ and highBa2+ solutions measured by a two-pulse protocol suggested that both processes also

S. V. SMIRNOV AND P. I. AARONSON occur in human mesenteric arterial cells. In normal physiological saline solution cells were first stepped for 240 ms to a conditioning potential, which was varied, in order to elicit ICa and thereby raise the intracellular Ca2+ concentration to a level dependent upon the degree of Ca2+ channel activation and the driving force. After a 470

Aa

60

10 -60

-60

10L

1 00 ms

b 150 PAR

1 s

50ms

is

150ms

B 1-0 0-

08 C

a)

m 0) N

=

0-6

0*4

0

Z 020o 5 -50

50 0 100 Pre-pulse potential (mV) Fig. 11. Ca2+-dependent inactivation in human mesenteric arterial cells. A, I recorded in the same cell in 1-5 mm Ca2+ with prepulse durations of 240 ms (a) and 30 s (b). The interpulse interval was 8 ms and 20 ms for a and b correspondingly. Note the different digitizing rates in b. Cm = 78-7 pF. B, the mean availability for ICa (six cells, 0) and IBa (five cells, *) studied with a two-pulse protocol; prepulse duration was 240 ms. Peak current amplitudes measured during each of the test pulses were normalized to the peak amplitude measured during the test pulse following the prepulse to -50 mV.

8 ms step back to -60 mV, cells were stepped to +1O mV to examine the effect of the prior Ca2+ influx on channel availability (Fig. 1 lA). Identical experiments were performed in the presence of 10 mm Ba2+ (not shown). The normalized amplitude of the current during the second step is plotted against the potential of the first pulse (Fig. LB). As shown in the figure, inactivation for ICa was observed in a more

Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY 471 negative potential range, and the current inactivated to a greater extent, compared to IBa. Also, at increasingly positive prepulse potentials the fraction of ICa available was increased while that for IBa was quite similar between + 10 and + 100 mV (Fig. 1 1A a and B). Conversely, if 30 s prepulses to + 10 and + 60 mV were used, ICa elicited by the test pulse was inactivated completely (Fig. 1 1A b). DISCUSSION

Our experiments provide evidence for the presence of two types of Ca2+ currents, T- and L-type, in human mesenteric arterial cells. These currents were characterized by different kinetics and voltage ranges of activation and inactivation; similar properties of L- and T-type Ca2+ channels have previously been seen in a variety of SMCs (Benham et al. 1987; Aaronson et al. 1988; Akaike et al. 1989; Loirand et al. 1989; Ganitkevich & Isenberg, 1990), as well as in other tissues (see for review Pelzer et al. 1990). The T-type current was very small under our experimental conditions, making a detailed investigation of its properties difficult even at an elevated divalent cation concentration. At present, an important physiological role for this current in human mesenteric arterial cells seems unlikely, due to its small size, its rapid decay and negative voltage range of inactivation, and because these cells appear to be nonexcitable. On the other hand, the input resistance of these cells seems to be very high (usually more than 10 Gi) and only a small current is necessary to significantly change the membrane potential in these cells. Therefore, we believe that the role of the T-current in these cells needs to be further investigated. In contrast, the L-type current is well developed in human mesenteric arterial cells. We found a good correlation between current amplitude and cell membrane capacitance which suggests a similar density of L-type Ca2+ channels in both large and small human mesenteric arterial cells. At present the in vivo resting potential of these cells is unknown. Our results (Smirnov & Aaronson, 1992b) show that these cells have a well-developed system of potassium currents, and, like other arterial cells, are non-excitable under normal physiological conditions. Also, based on our measurements of the membrane potential in single human mesenteric arterial cells (-44-7 mV, Smirnov & Aaronson, 1992b) with potassium-filled pipettes, and on results obtained on other blood vessels (reviewed by Nelson et al. 1990) we consider it likely that the resting potential is close to -60 mV in an intact tissue. To elucidate the possible physiological role of the L-type Ca2+ channel in human mesenteric arterial cells, we first established that the potential dependency of its activation and inactivation measured in 10 mm Ba2+ were very similar to those observed in 1-5 mM Ca2+. We were then able to take advantage of the greatly increased current amplitude in the high-Ba2+ solution to more easily measure Ca2+ channel current at negative potentials. In addition, the results obtained were independent of the type of current carrier. However, in spite of a more than 3-fold increase of current amplitude in 10 mi Ba2+, the whole-cell inward current was very small in the voltage range between -50 and -40 mV and could be detected only by its abolition upon addition of Cd2+ ions. A theoretical calculation of the current, which correlated closely with experimentally measured current amplitudes in a more positive potential range (Fig. 8D), also predicted a small amplitude of the whole-cell ICa and 16

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S. V. SMIRNOV AND P. I. AARONSON

'Ba in this voltage range. These results might reflect both a small number of activated

Ca2+ channels and their low open probability at the negative potentials, as previously suggested by single-channel recordings in the presence of 10 mm Ba2+ in the pipette in guinea-pig coronary artery (Ganitkevich & Isenberg, 1990) and in 10 mm Ca21 and high-Ba2+ pipette solutions in rabbit basilar artery (Nelson et al. 1990). These results differ, however, from those obtained from rabbit ear artery, where a sustained Ca2+ current was observed in normal [Ca2+]. between -60 and -50 mV, even though the maximal current was somewhat smaller than that present in these cells (Aaronson et al. 1988). It is unlikely that this 20-30 mV difference in the range over which the inward current activated in these two cells types was due to a more prominent T-type current in the ear artery, since current became apparent at -48 mV even at a holding potential of -60 mV, where the T-type current is mostly inactivated; also current near the 'threshold' showed little decay. It may therefore be that the activation range for L-type Ca21 channels varies between different vascular SMCs. A prolonged depolarization in normal Ca2+ and elevated divalent cation concentrations produced a measurable inward current in human mesenteric arterial cells over a 30 s period of depolarization. The amplitude of this current was similar to that calculated from the overlapping of the activation and inactivation curves for L-type Ca2+ channel currents. A window current of this sort was predicted from the overlapping steady-state inactivation and activation curves in urinary bladder (Kl6ckner & Isenberg, 1985). A small sustained inward current was measured in several visceral and vascular SMCs, which was assumed also to be generated by a window current (Imaizumi, Muraki, Takeda & Watanabe, 1989). However, in these studies the simulation of Ca2+ channel current was based on the assumption of a linear relation between current and voltage near the apparent reversal potential for ICa' in spite of the presence of non-linearity in this voltage range (but see Kl6ckner & Isenberg, 1985). In our experiments the Ca2+ channel currents also revealed a curvature near the apparent reversal potential, which was positive to +60 mV indicating a relatively high permeability to divalent cations in comparison with monovalent ones (see also Lee & Tsien, 1984). The Goldman-Hodgkin-Katz nonlinear function we used provided a good fit for the Ca2+ channel currents over the potential range negative to + 60 mV; the breakdown of the fit near the reversal potential is to be expected since a term for the outward current through these channels was not included in the fitting function. As previously determined by direct measurement of the instantaneous I-V relationship for Ca2+ channels in bovine chromaffin cells (Fenwick, Marty & Neher, 1982), snail neurones (Byerly, Chase & Stimer, 1985) and canine atrial cells (Bean, 1985), the activation of Ca2+ channels constructed on the basis of this approach became complete only at membrane potentials positive to + 30 mV (Fig. 8 C). These results, as well as the less steep slope of the activation function, differ from those obtained using a linear relation between the current and the reversal potentials (Klockner & Isenberg, 1985; Imaizumi et al. 1989; Matsuda, Volk & Shibata, 1990). A small window current was predicted even at a membrane potential of -60 mV, which is clearly seen in the presence of 10 mm Ba2+ (Fig. 9B). In normal Ca2+ concentration (1-5 mM) the estimated sustained ICa is 1-69 nA cm2. Given the mean cell capacitance of 46 pF (Smirnov & Aaronson, 1992 b), the charge entering the cell

473 Ca2+ CHANNELS IN HUMAN MESENTERIC ARTERY per second was calculated to be 7-7 x 10-14 C. This amount would be enough to raise intracellular Ca2+ concentration by 04 /tM per second (at room temperature) assuming a cell volume of about 1 pl (Aaronson et al. 1988) and neglecting the binding, sequestration and extrusion of Ca2+. This estimate is similar to that based on single Ca2+ channel analysis (0 3 UM, which increased to about 1 AM at 37 'C, Nelson et al. 1990). It is perhaps surprising that these estimates are also similar to those which can be made from basal 45Ca2+ influx data in the rabbit aorta at 37 'C (Meisheri & van Breemen, 1982). Assuming a cell space of approximately 40%, an influx of 15 JM Ca2+ (kg tissue wet wt-1 min- gives a value of about 0-6 #M Ca2+ So. The significance of the window current for excitation-contraction coupling is suggested by the fact that it is predicted to change by an approximately e-fold factor with a 10 mV shift of the membrane potential from -60 mV in either direction. Dependence of inactivation of L-type Ca2+ channels on Ca2+ entry into cells was described in a variety of tissues (see Eckert & Chad, 1984), including visceral and vascular SMCs (Jmari, Mironneau & Mironneau, 1986, 1987; Ganitkevich, Shuba & Smirnov, 1986, 1987, 1991; Ohya, Terada, Kitamura & Kuriyama, 1986; Ohya, Kitamura & Kuriyama, 1988; Matsuda et al. 1990) and is considered to be a fundamental property of L-type Ca2+ channels. Indications of a Ca2+-dependent inactivation of Ca2+ channels are a U-shaped potential dependence of availability of

ICa measured in a two-pulse protocol and a sensitivity of this process to the type of divalent cation entering through Ca2+ channels. Both these types of evidence for Ca2+-dependent inactivation were found in these cells. Therefore, our results indicate that both potential- and Ca2+-dependent mechanisms for Ca2+ channel inactivation are present. In human mesenteric arterial cells the inactivation of Ca2+ channels was quite dependent on Ca2+ entry during short depolarizations (it should be noted that this effect was probably underestimated due to the presence of a high EGTA concentration in the pipette solution), while with long conditioning depolarizations a potential-dependent process dominated (Fig. lIA and B), suggesting that the potential-dependent process of inactivation is slower than the Ca2 -dependent one. In summary, our results demonstrate the presence of both T-type and L-type Ca2+ currents in human mesenteric arterial cells. The small size of the former, as well as the relatively negative potential range over which it inactivates, suggests that it makes very little contribution to excitation-contraction coupling. Although the apparent 'threshold' of the L-type current is near -30 or -40 mV, calculations based upon its measured activation and inactivation properties suggest that this current contributes a tonic Ca2+ influx which would be strongly influenced by small variations of the membrane potential around its likely resting level. We gratefully acknowledge the assistance of Mr R. J. Nicholls and Mr B. T. Jackson and the other members of Surgical Firm II at St Thomas's Hospital who have provided us with the tissue specimens used in this study. We are similarly indebted to the Wellcome Trust for their financial support of S. V. S. and of this work. REFERENCES

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