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Comparison of the effects of divalent cations on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells

A. S. Aromolaran and W. A. Large

MS 9538 Received 22 April 1999; accepted after revision 18 August 1999.

	ABSTRACT

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1. The facilitatory effects of external Ca²⁺, Sr²⁺ and Ba²⁺ (Ca²⁺_o, Sr²⁺_o and Ba²⁺_o) on the noradrenaline-evoked non-selective cation current (I_cat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques.

2. All divalent cations tested potentiated the amplitude of I_cat and the potency sequence was Ca²⁺_o > Sr²⁺_o > Ba²⁺_o. Ca²⁺_o and Sr²⁺_o increased the amplitude of I_cat by about eight times whereas Ba²⁺_o produced only a threefold facilitation.

3. The current-voltage relationship of I_cat was not changed by Ca²⁺_o, Sr²⁺_o or Ba²⁺_o.

4. From noise analysis the single channel conductance () was approximately 10 pS in divalent cation-free solution but was about 20 pS with Ca²⁺_o, Sr²⁺_o and Ba²⁺_o.

5. From noise and voltage-jump experiments it was apparent that at least three kinetically resolvable channel states are associated with I_cat in divalent cation-free solution. Ca²⁺_o and Sr²⁺_o produced marked changes in the characteristics of the power spectrum and relaxations of I_cat in response to voltage steps, consistent with a shift in the equilibrium between the channel states, whereas Ba²⁺_o produced minimal effects.

6. The data show that Ca²⁺_o, Sr²⁺_o and Ba²⁺_o increase the amplitude of I_cat, which results in part from an increase in the single channel conductance. In addition the results suggest that Ca²⁺_o and Sr²⁺_o alter the kinetic behaviour of the single channels whereas Ba²⁺_o has little effect on the equilibrium between the channel states.

	INTRODUCTION

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In rabbit portal vein smooth muscle cells ₁-adrenoceptor activation evokes a non-selective cation current (I_cat; Byrne & Large, 1988; Inoue & Kuriyama, 1993) which has been suggested to contribute to the depolarization and vasoconstrictor response produced by noradrenaline (Amédée & Large, 1989). Previously it has been demonstrated that the amplitude of I_cat is markedly modulated by external Ca²⁺ ions (Ca²⁺_o; Helliwell & Large, 1996). I_cat can be stimulated in calcium-free external solution, illustrating that Ca²⁺_o is not obligatory for activation, but micromolar concentrations of Ca²⁺_o increased the amplitude by about eightfold (facilitatory action of Ca²⁺_o). Increasing [Ca²⁺]_o above 100 µM produced subsequent depression of the amplitude of I_cat, which is the inhibitory effect of Ca²⁺_o. The difference in the concentration dependence on [Ca²⁺]_o suggested that the facilitatory and inhibitory effects of Ca²⁺_o were mediated at different sites (Helliwell & Large, 1996).

Recently we have used 'noise' and voltage-jump relaxation analysis to determine the basis of the facilitatory action of Ca²⁺_o (Helliwell & Large, 1998). These studies suggested that the facilitatory action of Ca²⁺_o may be explained by two main effects on I_cat. Firstly, micromolar concentrations of Ca²⁺_o increased the single channel conductance of I_cat. Secondly, the spectral density function of I_cat could be described by the sum of two Lorentzian components which indicates that the underlying channels exist in at least three states. It was suggested that micromolar [Ca²⁺]_o shifted the equilibrium between the proposed states which was associated with changes in time constant values estimated from corner frequencies of the Lorentzian components. Thus it appears that Ca²⁺_o also alters the kinetics of the underlying cation channels.

In the present work we have compared the ability of two other divalent cations, Sr²⁺ and Ba²⁺, to potentiate the amplitude of I_cat. It is well known that in some physiological systems Ba²⁺ and Sr²⁺ mimic the actions of Ca²⁺ whereas in other systems Ba²⁺ and Sr²⁺ do not possess the biological activity of Ca²⁺ ions. For example, Ba²⁺ and Sr²⁺ carry the inward current through voltage-gated calcium channels in smooth muscle (e.g. Ganitkevich et al. 1988), but are weak substitutes for Ca²⁺ in triggering contraction in smooth muscle (e.g. Hotta & Tsukui, 1968). The present study was carried out firstly to characterize further the ability of divalent cations to potentiate I_cat and secondly to test the hypothesis posed in the previous paper that divalent cations increased I_cat by altering the amplitude and kinetics of the elementary conductance underlying I_cat. These experiments would provide further insight into the mechanism by which divalent cations facilitate I_cat.

When studying the mechanism of action of the divalent cations it would be desirable to study the characteristics of the unitary conductance at the single channel level. However, noradrenaline-activated cation channels run down rapidly in isolated patches, presumably because of the complex activation and modulation of these channels (see Helliwell & Large, 1998 for a fuller discussion). Consequently we have used noise and voltage-jump analysis of the macroscopic whole-cell current to compare the effects of divalent cations on the kinetic behaviour and single channel conductance of the cation channels underlying I_cat.

	METHODS

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Cell isolation

New Zealand White female rabbits (2-2·5 kg) were killed by intravenous overdose of sodium pentobarbitone. The portal vein was removed and dissected free of connective tissue and fat in normal physiological salt solution (PSS) which contained (mM): NaCl, 126; KCl, 6; CaCl₂, 1·5; MgCl₂, 1·2; glucose, 10; and Hepes, 11; pH adjusted to 7·2 with 10 M NaOH. The tissue was then cut into strips and incubated in low Ca²⁺ PSS (PSS with no added CaCl₂) for 5 min at 37°C. After preincubation the solution was replaced with low Ca²⁺ PSS containing protease Type 1 (Sigma) or protease Type VIII (Sigma) (0·2-0·4 mg ml^-1) for 5 min and then washed with low Ca²⁺ PSS. The low Ca²⁺ PSS solution was replaced with 50 µM Ca²⁺ PSS (PSS containing 50 µM rather than 1·5 mM CaCl₂) containing 0·5-1 mg ml^-1 collagenase Type 1A and the tissue was then incubated for a further 10 min before a final wash in 50 µM Ca²⁺ PSS. Cells were released by trituration of the tissue through a wide-bore pipette in 50 µM Ca²⁺ PSS. The solution containing dissociated cells was then centrifuged at 100 g for 2 min to form a loose pellet which was resuspended in 0·75 mM Ca²⁺ PSS. The cells were stored on coverslips at 4 °C and were used within 8 h.

Electrophysiology

Whole-cell membrane currents were measured at room temperature (20-25°C) with a List EPC-7 patch clamp amplifier (List Electronic, Darmstadt, Germany). Patch pipettes were made from borosilicate glass and had resistances of approximately 6 M. The voltage protocol used to study the current-voltage (I-V) characteristics of the noradrenaline-activated cation current (I_cat) in the various divalent cation solutions involved clamping the membrane at a holding potential (V_h) of -50 mV and stepping to -120 mV for 50 ms before imposing a continuous voltage ramp to +50 mV at a rate of 0·3 V s^-1. When different divalent cation concentrations were used leak currents were subtracted by applying a number of voltage ramps in the appropriate cation solution before activation of I_cat and subtracting the mean current from ramps applied during activation of I_cat. Voltage ramps were generated and the data were recorded on-line with an IBM Pentium PC (Viglen, Middlesex, UK) using CED 1401 interface software (Cambridge Electronic Design Ltd, Cambridge, UK). Data were filtered at 1 kHz and sampled at 5 kHz. Long-term records were played back from a DTR-1205 digital tape-recorder (Intracel Ltd, Royston, Herts, UK), filtered and sampled at 10 and 25 Hz, respectively, using the 1401 and CED Sigavg software. For voltage-jump relaxation analysis voltage steps were applied and currents were also recorded on-line using an IBM Pentium personal computer. Currents were filtered at 1 kHz (-3 dB, Bessel) and sampled at 3 kHz.

Noise analysis

Details of these methods have been given previously (Helliwell & Large, 1998) but, briefly, in order to calculate the current variance _I² membrane currents were first high-pass filtered (0·3 Hz, -3 dB) to remove the DC component before being low-pass filtered (800 Hz, -3 dB), using an eight-pole Butterworth filter (Barr & Stroud Ltd, London, UK). The signal was then amplified (× 10) prior to sampling at 2 kHz using the 1401 plus and spike 2 software (CED). Another current signal was also simultaneously sampled, which was low-pass filtered (0·3 Hz, 3 dB) and used to calculate the mean current (I). Current variance was plotted against mean current to obtain an estimate of the single channel current amplitude (i). Since the probability of channel opening was low

I2 = i I,

(1)

and hence the single channel conductance () was calculated from:

= i/(V - Vrev),

(2)

where V and V_rev are the membrane potential and reversal potential, respectively.

For calculation of the spectral density function of I_cat in the various divalent cations a fast Fourier transform (FFT) of the band-pass signal was performed on the selected section of data where the mean current during I_cat remained constant to give a power spectrum. The spectrum was described by the sum of two Lorentzians according to the equation:

(3)

where G(f) is the spectral density at a given frequency (f), and f_c1 and f_c2 are the corner frequencies of the first and the second components, respectively. G₁(0) and G₂(0) are the zero frequency asymptotes respectively for the first and second components in the spectrum. Time constants were obtained from the corner frequencies ( in Table 2) using the equation:

= 1/(2fc).

(4)

It is possible to obtain another estimate of the single channel conductance using the equation:

(5)

(see Sigworth, 1980 and Helliwell & Large, 1998, for a fuller description).

Voltage-jump relaxation analysis

The kinetic properties of I_cat in various divalent cations were also studied using voltage-jump relaxation analysis (Helliwell & Large, 1998). Relaxations of I_cat were evoked by stepping the potential from -50 to +50 mV for 500 ms and then stepping back to -30 mV for 500 ms before returning to the holding potential (V_h) of -50 mV. The voltage was stepped to +50 mV to switch off the current (because most of the channels are non-conducting at positive potentials, for example see Fig. 4) and then returned to -30 mV to study voltage-dependent channel opening. The value of -30 mV was chosen because the V_rev of I_cat is approximately +10 mV and hence there is a similar, but opposite, driving force at the two test potentials of +50 and -30 mV. Leak-subtracted cation currents were obtained by subtracting the mean of two to three control currents recorded in the absence of noradrenaline from the mean of three to four currents at the peak of the noradrenaline-activated cation current. Exponential fits of the relaxations to obtain time constants and figure preparation were performed using Origin software (Microcal Software Inc., Northampton, MA, USA).

Solutions and drugs

The standard extracellular K⁺-free PSS used in all experiments contained (mM): NaCl, 126; CaCl₂, X; glucose, 10; Hepes, 11; pH was adjusted to 7·2 with NaOH. The value of X was in the range < 10 nM to 5 mM. Cells were superfused using gravity feed at the rate of 5 ml min^-1 and the bath volume was 200 µl. In experiments where the membrane potential was altered 5 µM nicardipine and 100 µM 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) were added to the external solution 2 min before the addition of noradrenaline to block voltage-sensitive Ca²⁺ channels and volume-activated Cl^- channels, respectively. In these conditions these compounds did not alter the characteristics of I_cat. Junction potentials were measured using the technique of Neher (1992) and were less than 3 mV. In experiments where low concentrations of Ca²⁺_o or when Sr²⁺_o and Ba²⁺_o were studied it was necessary to remove contaminating amounts of Ca²⁺. In these cases 1 mM BAPTA was added to nominally Ca²⁺-free PSS (which has the same composition as the standard extracellular K⁺-free PSS but without CaCl₂) to reduce the contaminating [Ca²⁺]_o to < 10 nM, estimated using EQCAL software (Biosoft, Ferguson, MO, USA). At these concentrations Ca²⁺_o has no facilitatory or inhibitory effects on I_cat and therefore this solution is effectively divalent cation-free and is termed 0 Ca²⁺_o. In experiments where low concentrations of Ca²⁺_o (2-5 µM) and all concentrations of Sr²⁺_o and Ba²⁺_o (50 µM to 5 mM) were studied the appropriate amount of XCl₂ estimated by EQCAL was added to 0 Ca²⁺_o to achieve the desired concentration. With concentrations of Ca²⁺_o > 5 µM the appropriate amount of CaCl₂ was added to the Ca²⁺-free PSS. The standard pipette solution contained (mM): CsCl, 18; caesium aspartate, 108; MgCl₂, 1·2; glucose, 10; Hepes, 11; BAPTA, 10; CaCl₂, 1 (free [Ca²⁺]_i predicted by EQCAL was approximately 14 nM); pH adjusted to 7·2 with Trizma base.

All drugs and enzymes were obtained from Sigma.

The values in the text are means ± S.E.M. and the test for significance was Student's t test.

	RESULTS

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Comparison of the facilitatory effect of Ca²⁺, Sr²⁺ and Ba²⁺ on the amplitude of I_cat

In the first series of experiments we compared the ability of different divalent cations to potentiate the amplitude of I_cat. Since there is marked variation in responsiveness to noradrenaline between cells we used an internal control where noradrenaline was applied in 0 Ca²⁺_o PSS (divalent cation-free solution, see Methods). After activation of I_cat in 0 Ca²⁺_o the bathing solution was then exchanged for PSS containing a different concentration of divalent cations with noradrenaline still present. Figure 1 illustrates typical effects of different concentrations of Ca²⁺_o on noradrenaline-evoked I_cat. In Fig. 1A I_cat was first evoked in 0 Ca²⁺_o and just after the peak current had been achieved the PSS was exchanged for PSS containing 5 µM Ca²⁺_o and it can be seen that the amplitude of I_cat increased two- to threefold. When 50 µM Ca²⁺_o was applied to the cell during stimulation of I_cat the current was increased by about eight times (Fig. 1B). When 1-100 µM Ca²⁺_o was added during activation of I_cat the current was first potentiated and then declined spontaneously over a few minutes in the continued presence of noradrenaline (Fig. 1A and B); at present we do not have any experimental evidence on the processes that control this inactivation/desensitization process. However, with [Ca²⁺]_o > 100 µM the initial potentiation of I_cat was followed by rapid inhibition of the response (e.g. with 1·5 mM Ca²⁺_o in Fig. 1C). This rapid decline of I_cat following the potentiation with 1·5 mM Ca²⁺_o represents the inhibitory action of Ca²⁺_o (Helliwell & Large, 1996), which is not the subject of the present work and so was not investigated further. However, since we wanted to compare the facilitatory effects of various divalent cations on the characteristics of I_cat we used 50 µM Ca²⁺_o in subsequent experiments since this concentration produced marked potentiation with little evidence of an inhibitory effect on I_cat. The estimated equilibrium constant for the inhibitory action of Ca²⁺_o is about 400 µM (Helliwell & Large, 1996) and thus on theoretical grounds it is expected that 50 µM Ca²⁺_o would have little inhibitory effect. This was manifest experimentally as the current decayed slowly due to inactivation/desensitization and was similar to that seen with lower concentrations of Ca²⁺_o (e.g. Fig. 1A and B) and there was no rapid inhibition as seen with 1·5 mM Ca²⁺_o (Fig. 1C).

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Figure 1. The facilitatory and inhibitory actions of Ca2+o on noradrenaline-activated Icat Icat was activated by bath-applied noradrenaline (100 µM, horizontal box) in 0 Ca2+o and [Ca2+]o was then changed to 5 µM (A), 50 µM (B) or 1·5 mM (C) as indicated at the top of each record; the change-over is highlighted on the current traces by arrows. Altering [Ca2+]o affected the leak resistance which was assessed at the beginning of each record in the absence of noradrenaline. Standard K+-free conditions were used and Vh was -50 mV.

Figure 2 shows the potentiating effects of Sr²⁺_o and Ba²⁺_o on I_cat elicited in the same manner as described in Fig. 1 for Ca²⁺_o. Addition of 200-1500 µM Sr²⁺_o produced marked potentiation of I_cat (Fig. 2A-C) and the maximum facilitation was about eightfold. Ba²⁺_o also increased the amplitude of I_cat but the degree of facilitation was noticeably less and the maximal potentiation which occurred with 1·5 mM Ba²⁺_o was only two- to threefold (Fig. 2D -F). At concentrations of 0·1-1·5 mM neither Sr²⁺ nor Ba²⁺ produced the rapid inhibitory effect seen with 1·5 mM Ca²⁺_o (for example see Fig. 1C) and therefore in this concentration range Sr²⁺_o and Ba²⁺_o have negligible inhibitory effects but at higher concentrations both Sr²⁺_o and Ba²⁺_o inhibited I_cat. It should be noted that the spontaneous inactivation of I_cat that occurs in the continued presence of noradrenaline also occurred with both Sr²⁺_o and Ba²⁺_o (Fig. 2).

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Figure 2. The facilitatory action of Sr2+o and Ba2+o on Icat Icat was first evoked in 0 Ca2+o and the bathing solution was exchanged for PSS containing 200 µM Sr2+o (A), 500 µM Sr2+o (B), 1·5 mM Sr2+o (C), 50 µM Ba2+o (D), 500 µM Ba2+o (E) or 1·5 mM Ba2+o (F). The divalent cation concentrations are shown above the current records and the arrow in each trace indicates the potentiation of Icat on changing from 0 Ca2+o to 'test' concentrations of Sr2+o and Ba2+o. Changes in leak resistance due to the addition of test concentrations of Sr2+o and Ba2+o in the absence of noradrenaline are shown at the beginning of each record and noradrenaline (100 µM) was applied for the period denoted by the horizontal box.

Quantitative analysis of the facilitatory effects of the divalent cations on I_cat is shown in Fig. 3. The degree of potentiation was estimated as the ratio of currents (b/a) according to the diagram shown in Fig. 3A and takes into account changes in leak current which sometimes occurred with different divalent cation concentrations and was assessed before the application of noradrenaline. From Fig. 3B -D it can be seen that Ca²⁺_o was the most potent divalent cation in potentiating I_cat and the concentration required to increase the amplitude of I_cat to 50 % of the maximum response (EC₅₀) was 29 µM. The corresponding EC₅₀ values for Sr²⁺_o and Ba²⁺_o were 267 and 357 µM, respectively. Another major difference was the maximum degree of potentiation, where both Ca²⁺_o and Sr²⁺_o increased I_cat by about eight times (Fig. 3B and C) whereas Ba²⁺_o increased I_cat by a maximum of about threefold (Fig. 3D). For all divalent cations studied the data points were well fitted by logistic curves and the apparent Hill coefficients were 0·98, 1·65 and 4·26 for Ca²⁺_o, Sr²⁺_o and Ba²⁺_o, respectively. For analysis of their effects in later experiments we used 1·5 mM Sr²⁺_o and Ba²⁺_o since this concentration produced marked potentiation but no inhibition of I_cat.

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Figure 3. Quantification of the facilitatory action of divalent cations on Icat A, the facilitatory action of divalent cations was estimated by dividing the maximum current amplitude (b) in the 'test' divalent cation concentration ([X2+]o) by the amplitude of the current (a) evoked in 0 Ca2+o. Note that the measured amplitude of b allowed for leak currents that occur in bathing solutions of different divalent cation concentrations in the absence of noradrenaline. B-D, the ratio of currents (b/a, from A) was plotted against divalent cation concentration on a logarithmic scale for Ca2+o (B), Sr2+o (C) and Ba2+o (D). The curves were drawn according to the Hill equation in the following form: where y is the current amplitude and EC50, x and nH are the effective concentration at which the potentiation is 50 % of its maximum value, the test divalent cation concentration and the Hill coefficient, respectively. The estimated EC50 values were 29, 267 and 357 µM for Ca2+o, Sr2+o and Ba2+o, respectively. The Hill coefficient values for Ca2+o Sr2+o and Ba2+o were 0·98, 1·65 and 4·26, respectively. Each point represents the mean of 6 cells.

Current-voltage relationship of I_cat in various divalent cation solutions.

I_cat displays marked rectification (Helliwell & Large, 1996) and it is possible that the potentiating effect of divalent cations may be due to the alteration of these rectifying properties. Consequently we plotted current-voltage relationships using voltage ramps during activation of I_cat to test this possibility. In 0 Ca²⁺_o I_cat had an S-shaped current-voltage relationship (Fig. 4A) and little outward current flowed through the conductance at potentials positive to the reversal potential (V_rev, between 0 and +10 mV). Moreover at negative potentials it can be seen that the I-V curve also deviated from linearity. However, similar I-V relationships were obtained for all divalent cations tested with no change in V_rev (Fig. 4B -E). Also with all solutions the data could be described by a Boltzmann distribution and the calculated potential for half-maximal activation and slope factor were similar in all solutions tested (Table 1). It should be noted that these current- voltage curves are similar to those obtained with 1·5 mM Ca²⁺_o (Helliwell & Large, 1996).

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Figure 4. Current-voltage relationships of Icat in various divalent cations The plots are the current-voltage relationships of Icat obtained in 0 Ca2+o (A), 50 µM Ca2+o (B), 1·5 mM Sr2+o (C) and 1·5 mM Ba2+o (D). The membrane potential was stepped from the holding potential of -50 mV to -120 mV for 50 ms before imposing a continuous voltage ramp to +50 mV before and during the application of noradrenaline. Leak subtracted currents were normalized to the value of the current recorded at -40 mV (which is -1) and each point represents the mean of 4-6 cells. All currents were recorded in standard K+-free conditions.

Table 1. Voltage-dependent characteristics of I_cat

where G is the normalized chord conductance and V_m, V_½ and k are the membrane potential, the potential for half-maximal activation and the slope factor, respectively. n, number of cells.

Noise analysis of I_cat in various divalent cation solutions

In these experiments the single channel conductance () was first estimated by plotting the variance of the current against mean current. The plots were linear, confirming low probability of channel opening, and the single channel current amplitude was obtained from the slope from which was calculated (see Methods). Typical plots are shown in Fig. 5 and in 0 Ca²⁺_o was 11 ± 0·7 pS (n = 5). However, when concentrations of divalent cations that potentiated the amplitude of I_cat were included in the PSS the value of was approximately doubled. Thus the value of in 50 µM Ca²⁺_o, 1·5 mM Sr²⁺_o and 1·5 mM Ba²⁺_o was 22 ± 0·9 pS (n = 6), 21 ± 0·6 pS (n = 6) and 21 ± 0·9 pS (n = 6), respectively.

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Figure 5. The effect of divalent cations on the single channel conductance () of Icat The relationship between current variance (I2) and the mean current (I) in < 10 nM Ca2+o (A), 50 µM Ca2+o (B), 1·5 mM Sr2+o (C) and 1·5 mM Ba2+o (D) was linear and the single channel conductance () was calculated from the gradient (i) of the linear relationship (see Methods for details). Note that the value of of Icat in all the divalent cation solutions is approximately twice the estimated value in 0 Ca2+o.

It is possible to obtain information on the kinetics of the underlying channels by analysing the spectral density function of I_cat which also yields an alternative estimate of . Table 2 shows that the values of obtained from analysis of the spectral density function were similar to those obtained from plots of current variance vs. mean. In 0 Ca²⁺_o was 10 pS but in solutions containing divalent cations was 20-22 pS (Table 2).

Table 2. Characteristics of I_cat obtained from the spectral density function of I_cat in the presence of various divalent cations

Divalent cation	1 (ms)	2 (ms)	G2(0)/G1(0)	(pS)	n
0 Ca2+o	11 ± 0·1	1·4 ± 0·06	0·5 ± 0·05	10 ± 0·4	6
50 µM Ca2+o	50 ± 3	1·9 ± 0·03	0·01 ± 0·00	22 ± 0·6	5
1·5 mM Sr2+o	24 ± 2·3	1·5 ± 0·1	0·04 ± 0·01	22 ± 1	6
1·5 mM Ba2+o	12 ± 0·4	1·8 ± 0·3	0·3 ± 0·03	20 ± 1	6

As described previously (Helliwell & Large, 1998), the spectral density function for I_cat in 0 Ca²⁺_o could be described by the sum of two Lorentzian components (Fig. 6A), which indicates that the underlying channels exist in at least three kinetically resolvable states in divalent cation-free external solution. Qualitatively similar spectra were obtained with 50 µM Ca²⁺_o, 1·5 mM Sr²⁺_o and 1·5 mM Ba²⁺_o (Fig. 6B -D) in the bathing solution but there were important differences in the quantitative estimates of the characteristic corner frequencies (f_c values) and the relative contributions of the two Lorentzian components in those different conditions. The latter parameter was estimated as the ratio G₂(0)/G₁(0) where G(0) is the zero frequency asymptote for each Lorentzian component. It can be seen that the corner frequency of the higher frequency Lorentzian component (f_c2), and hence the corresponding time constant (₂), was the same (about 1-2 ms) in all solutions tested (Fig. 6 and Table 2). In contrast, whereas the corner frequency of the lower frequency Lorentzian component (f_c1), and hence the corresponding time constant (₁), was similar in 0 Ca²⁺_o and 1·5 mM Ba²⁺_o, significantly different values were obtained in 50 µM Ca²⁺_o and 1·5 mM Sr²⁺_o (Fig. 6 and Table 2). Thus, ₁ was 11 ms in 0 Ca²⁺_o and 12 ms in 1·5 mM Ba²⁺_o but higher values were obtained in 50 µM Ca²⁺_o (50 ms) and 1·5 mM Sr²⁺_o (24 ms, Table 2). Moreover the G₂(0)/G₁(0) ratio was similar in 0 Ca²⁺_o and 1·5 mM Ba²⁺_o (0·5 and 0·3, respectively, Table 2) but there was a marked decrease in the G(0) ratio in 50 µM Ca²⁺_o and 1·5 mM Sr²⁺_o (0·01 and 0·04, respectively, Table 2). These data show that 50 µM Ca²⁺_o and 1·5 mM Sr²⁺_o produced a marked change in the contribution of the components in favour of the lower frequency Lorentzian component but 1·5 mM Ba²⁺_o had minimal effects.

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Figure 6. The effect of divalent cations on the spectral density function of Icat The spectral density function of Icat obtained in the presence of 0 Ca2+o (A), 50 µM Ca2+o (B), 1·5 mM Sr2+o (C) and 1·5 mM Ba2+o (D). Each spectrum can be described by the sum of two Lorentzian components (continuous lines), where fc1 and fc2 are the corner frequencies of the first and second components, respectively (dotted lines). G1(0) and G2(0) are the zero frequency asymptotes for the first and second components, respectively (dotted lines).

Voltage-jump relaxation analysis of I_cat

Previously we have demonstrated that there are Ca²⁺_o-dependent relaxations of I_cat (Helliwell & Large, 1998) and therefore we carried out similar experiments to see if relaxations of I_cat occurred with Sr²⁺_o and Ba²⁺_o. Figure 7A shows that no relaxations of I_cat were observed in 0 Ca²⁺_o, either when the voltage was stepped from the holding potential of -50 mV to +50 mV or on returning to -30 mV during activation of I_cat, which agrees with previous work (Helliwell & Large, 1998). However, in 50 µM Ca²⁺_o on stepping to +50 mV there was an inward relaxation which was described by a single exponential with a time constant (_relax) of 54 ms (Fig. 7B), reflecting a shift in the equilibrium of channels from the open to the closed state. Moreover on returning to -30 mV the instantaneous current was followed by a large inward relaxation which was described by a single exponential with a time constant of 64 ms. In 50 µM Ca²⁺_o the mean values of the relaxations at +50 and -30 mV were 36 ± 5 and 66 ± 6 ms (n = 6), respectively. This inward relaxation at -30 mV represents an increase in I_cat due to a shift in the equilibrium of channels from the closed to the open state as a consequence of channel opening produced by the hyperpolarizing step. Similar relaxations of I_cat were observed in 1·5 mM Sr²⁺_o (Fig. 7C) although the time constants with Sr²⁺_o were somewhat faster than those seen with 50 µM Ca²⁺_o. In 1·5 mM Sr²⁺_o the mean values of the relaxations at +50 and -30 mV were 15 ± 1 and 29 ± 2 ms (n = 7), respectively. Previously we have shown that the time constants of the relaxations at both positive and negative potentials was not dependent on [Ca²⁺]_o (Helliwell & Large, 1998). Moreover of the relaxation at positive potentials was not dependent on the test voltage (Helliwell & Large, 1998). This also proved to be the case with Sr²⁺_o. Thus in 1·5 mM Sr²⁺_o values of the relaxation at +30 and +70 mV were 14 ± 3 ms (n = 6) and 18 ± 2 ms (n = 5), respectively. At -20 and -80 mV, values of the relaxation in 1·5 mM Sr²⁺_o were 27 ± 2 ms (n = 5) and 26 ± 2 ms (n = 6), respectively.

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Figure 7. The effect of various divalent cations on relaxations of Icat evoked by steps to various voltages Icat evoked in 0 Ca2+o (A), 50 µM Ca2+o (B), 1·5 mM Sr2+o (C) and 1·5 mM Ba2+o (D), whilst applying the voltage-jump protocol illustrated below. Note that no relaxations occurred in 0 Ca2+o (A) and 1·5 mM Ba2+o (D) but marked relaxations were observed in 50 µM Ca2+o (B) and 1·5 mM Sr2+o (C) at both +50 mV and -30 mV. The steps were applied at the peak of the response and the records shown are leak subtracted. Iinst, instantaneous current.

In contrast, usually no relaxations of I_cat were observed with 1·5 mM Ba²⁺_o and the records were similar to those obtained in divalent cation-free solutions (compare Fig. 7A and D). On occasion there appeared to be small relaxations in 1·5 mM Ba²⁺_o but these were too small to quantify. In conclusion voltage-jump relaxation analysis shows that in divalent cation-free solution and 1·5 mM Ba²⁺_o there are no relaxations of I_cat, but Ca²⁺_o and Sr²⁺_o produced relaxations which indicate that Ca²⁺_o and Sr²⁺_o alter the kinetic behaviour of the channels but Ba²⁺_o is without effect.

	DISCUSSION

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The purpose of the present study was to compare the potentiating effects of external divalent cations on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells. It was found that all cations tested (Ca²⁺_o, Sr²⁺_o and Ba²⁺_o) potentiated I_cat but differed in three main respects with regard to their concentration-effect curves. Firstly, there was a marked difference in the potencies of Ca²⁺_o, Sr²⁺_o and Ba²⁺_o. Secondly, there was a difference in the maximum degree of potentiation and thirdly, the Hill coefficients also differed. Whereas the Hill coefficient of Ca²⁺_o was about unity, values of 1·65 and 4·26 were estimated for Sr²⁺_o and Ba²⁺_o, respectively. There is no obvious explanation for the difference in the Hill coefficients and it is possible that the inhibitory action of Ca²⁺_o might lead to an underestimate in the value for Ca²⁺_o. However, this result indicates that Sr²⁺_o and Ba²⁺_o may show positive co-operativity of binding to the facilitatory binding site whereas Ca²⁺_o does not.

The potency order of the ability of the divalent cations to potentiate the amplitude of I_cat warrants some comment. In the present work the EC₅₀ values of Ca²⁺_o, Sr²⁺_o and Ba²⁺_o were 29, 267 and 357 µM, respectively. In a previous publication we found that Mg²⁺_o did not facilitate I_cat (Helliwell & Large, 1996) and therefore the potency sequence of the divalent cations for potentiation of I_cat is Ca²⁺_o > Sr²⁺_o > Ba²⁺_o > Mg²⁺_o. This order is almost the reverse of the aqueous mobility sequence (Ba²⁺ > Sr²⁺ > Ca²⁺ > Mg²⁺) and therefore, presumably, this physicochemical property does not determine the ability to potentiate I_cat. It is worth noting also that the EC₅₀ value of Ca²⁺_o found in this study (29 µM) was slightly higher than the value estimated in a previous study (6 µM, Helliwell & Large 1996). This discrepancy is likely to be due to a difference in the experimental protocols used. In the present study in order to compare different divalent cations it was necessary to apply noradrenaline in 0 Ca²⁺_o and add divalent cations after activation of the current. In the former study I_cat was first activated in 1·5 mM Ca²⁺_o before changing [Ca²⁺]_o.

It is evident that the facilitatory effect of the divalent cations is not brought about by changes in the I-V relationship of I_cat. In 0 Ca²⁺_o and in the presence of divalent cations there were minimal differences in the reversal potential and the parameters describing the voltage dependence of I_cat from the Boltzmann equation were similar in all solutions used (Table 1). These results also reinforce a previous conclusion where it was suggested that the voltage dependence of I_cat is not caused by external cations (Helliwell & Large, 1996).

In contrast, the divalent cations produced significant changes in the conductance and kinetic behaviour of the single channels as revealed by noise analysis of macroscopic I_cat. In 0 Ca²⁺_o was estimated to be about 10 pS but with divalent cations (Ca²⁺_o, Sr²⁺_oand Ba²⁺_o) in the bathing solution was 20-22 pS. Therefore it would seem reasonable to suggest that doubling the single channel conductance would contribute to the potentiation of I_cat by the divalent cations. These results indicate that there are at least two distinct open states of the channel (10 and 20 pS) and that the divalent cations alter the equilibrium in favour of the higher conductance state. A simple explanation for the ability of Ca²⁺_o and Sr²⁺_o to produce a greater potentiation of the amplitude of I_cat than Ba²⁺_o is that Ca²⁺_o and Sr²⁺_o produce a much larger shift in the equilibrium to the higher conductance state than Ba²⁺_o. However, there is also good evidence that Ca²⁺_o and Sr²⁺_o produced a change in the kinetic behaviour of the channels whereas Ba²⁺_o ions did not. In all solutions the spectral density function of I_cat could be fitted by the sum of two Lorentzian components, which suggests that the non-selective cation channels exist in at least three kinetically resolvable states, for example, one closed and two open states (Helliwell & Large, 1998). This is likely to be an oversimplified model (see Helliwell & Large, 1998) but nevertheless gives some insight into a possible mechanism. Ca²⁺_o and Sr²⁺_o produced marked changes in the G(0) ratio such that the spectrum was dominated by the lower frequency component and G₂(0)/G₁(0)) was 0·01 and 0·04 in Ca²⁺_o and Sr²⁺_o, respectively, compared with 0·5 in 0 Ca²⁺_o There were also changes in the time constants associated with the lower frequency Lorentzian components and ₁ in Ca²⁺_o and Sr²⁺_o was 50 and 24 ms, respectively, compared with 11 ms in 0 Ca²⁺_o. These data suggest that the channel transitions associated with this Lorentzian are sensitive to both Ca²⁺_o and Sr²⁺_o. In contrast in Ba²⁺_o the G(0) ratio (0·3) was closer to the value in divalent cation-free solution and moreover ₁ in Ba²⁺_o (12 ms) was similar to the value obtained in 0 Ca²⁺_o. Therefore these data indicate that the transitions associated with the lower frequency Lorentzian are sensitive to Ca²⁺_o and Sr²⁺_o but are not (or little) affected by Ba²⁺_o. The time constant associated with the higher frequency Lorentzian component was similar in 0 Ca²⁺_o and in the presence of divalent cations, which suggests that the channel transitions associated with this Lorentzian are not altered by divalent cations.

The voltage-jump experiments also revealed that Ca²⁺_o and Sr²⁺_o produced changes in the kinetic behaviour of the channels compared with 0 Ca²⁺_o and Ba²⁺_o. In divalent cation-free and 1·5 mM Ba²⁺_o solutions no significant relaxations of I_cat were observed in response to either depolarizing or hyperpolarizing pulses. In contrast, relaxations of I_cat, representing the transition between open and closed channel states, were observed in both 50 µM Ca²⁺_o and 1·5 mM Sr²⁺_o. Moreover the relaxation time constants (_relax at -30 mV) are similar to the time constants of the lower frequency Lorentzian component (₁ in Table 2) which makes by far the greatest contribution to the overall spectrum in both Ca²⁺_o and Sr²⁺_o. Therefore it appears that the relaxations of I_cat to voltage steps correspond to the lower frequency Lorentzian of the power spectrum in that both are sensitive to Ca²⁺_o and Sr²⁺_o, but not to Ba²⁺_o, and that the time constants from noise experiments and relaxation analysis have similar values although the values with Ca²⁺_o are higher than those in Sr²⁺_o. Therefore it appears that Ca²⁺_o and Sr²⁺_o produced a marked shift in the equilibrium of the channel states whereas Ba²⁺_o caused little change in the kinetics of the channels. It is evident that the relaxations observed on returning to -30 mV in Ca²⁺_o and Sr²⁺_o produce an increase in the amplitude of I_cat compared to divalent cation-free solution and Ba²⁺_o where only instantaneous currents were observed. A simple explanation is that two of the channel states may be open conformations with different channel lifetimes and that Ca²⁺_o and Sr²⁺_o shift the equilibrium in favour of the conformation with the longer lifetime which produces a larger current amplitude whereas Ba²⁺_o is not able to produce this effect. An important result from comparing the divalent cations is that the increase in single channel conductance is distinct from the effect of channel kinetics because whereas all divalents increased only Ca²⁺_o and Sr²⁺_o altered the kinetic behaviour of the channels.

In conclusion the divalent cations Ca²⁺_o, Sr²⁺_o and Ba²⁺_o increase the single channel conductance of I_cat to the same extent but only Ca²⁺_o and Sr²⁺_o appear to affect significantly the kinetic behaviour of the underlying channels. It is this latter effect which makes Ca²⁺_o and Sr²⁺_o more efficacious than Ba²⁺_o in increasing the amplitude of I_cat. More precise information on the effects of divalent cations on the properties of the unitary conductance will be available when we establish conditions to prevent channel 'run-down' in isolated patches.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Amédée, T. & Large, W. A. (1989). Microelectrode study on the ionic mechanisms which contribute to the noradrenaline-induced depolarization in isolated cells of rabbit portal vein. British Journal of Pharmacology 97, 1331-1337	[Medline]
Byrne, N. G. & Large, W. A. (1988). Membrane ionic mechanisms activated by noradrenaline in cells isolated from the rabbit portal vein. The Journal of Physiology 404, 557-573	[Abstract]
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Helliwell, R. M. & Large, W. A. (1996). Dual effect of external Ca2+ on noradrenaline-activated cation current in rabbit portal vein smooth muscle cells. The Journal of Physiology 492, 75-88	[Abstract]
Helliwell, R. M. & Large, W. A. (1998). Facilitatory effect of Ca2+ on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells. The Journal of Physiology 512, 731-741	[Abstract/Full Text]
Hotta, Y. & Tsukui, R. (1968). Effect on the guinea-pig taenia coli of the substitution of strontium or barium ions for calcium ions. Nature 217, 867-869	[Medline]
Inoue, R. & Kuriyama, H. (1993). Dual regulation of cation-selective channels by muscarinic and 1-adrenergic receptors in the rabbit portal vein. The Journal of Physiology 465, 427-448	[Abstract]
Neher, E. (1992). Correction for liquid junction potentials in patch clamp experiments. In Methods in Enzymology , vol. 207, ed. Rudy, B. & Iverson, L. E., pp. 123-130. Academic Press, London.
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Acknowledgements

This work was supported by the British Pharmacological Society and The Wellcome Trust.

Corresponding author

W. A. Large: Department of Pharmacology and Clinical Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK.

Email: w.large{at}sghms.ac.uk

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