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

Abstract

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 (Icat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques. 2. All divalent cations tested potentiated the amplitude of Icat and the potency sequence was Ca2+o > Sr2+o > Ba2+o. Ca2+o and Sr2+o increased the amplitude of Icat by about eight times whereas Ba2+o produced only a threefold facilitation. 3. The current-voltage relationship of Icat was not changed by Ca2+o, Sr2+o or Ba2+o. 4. From noise analysis the single channel conductance (gamma) 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 Icat in divalent cation-free solution. Ca2+o and Sr2+o produced marked changes in the characteristics of the power spectrum and relaxations of Icat 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 Icat, 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.

Figure 4. Current-voltage relationships of I_cat in various divalent cations

The plots are the current-voltage relationships of I_cat obtained in 0 Ca_o²⁺ (A), 50 μM Ca_o²⁺ (B), 1.5 mM Sr_o²⁺ (C) and 1.5 mM Ba_o²⁺ (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.

Figure 1. The facilitatory and inhibitory actions of Ca_o²⁺ on noradrenaline-activated I_cat

I_cat was activated by bath-applied noradrenaline (100 μM, horizontal box) in 0 Ca_o²⁺ 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 V_h was −50 mV.

Figure 2. The facilitatory action of Sr_o²⁺ and Ba_o²⁺ on I_cat

I_cat was first evoked in 0 Ca_o²⁺ and the bathing solution was exchanged for PSS containing 200 μM Sr_o²⁺ (A), 500 μM Sr_o²⁺ (B), 1.5 mM Sr_o²⁺ (C), 50 μM Ba_o²⁺ (D), 500 μM Ba_o²⁺ (E) or 1.5 mM Ba_o²⁺ (F). The divalent cation concentrations are shown above the current records and the arrow in each trace indicates the potentiation of I_cat on changing from 0 Ca_o²⁺ to ‘test’ concentrations of Sr_o²⁺ and Ba_o²⁺. Changes in leak resistance due to the addition of test concentrations of Sr_o²⁺ and Ba_o²⁺ 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.

Figure 3. Quantification of the facilitatory action of divalent cations on I_cat

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 Ca_o²⁺. 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 Ca_o²⁺ (B), Sr_o²⁺ (C) and Ba_o²⁺ (D). The curves were drawn according to the Hill equation in the following form:where y is the current amplitude and EC₅₀, x and n_H 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 Ca_o²⁺, Sr_o²⁺ and Ba_o²⁺, respectively. The Hill coefficient values for Ca_o²⁺ Sr_o²⁺ and Ba_o²⁺ were 0.98, 1.65 and 4.26, respectively. Each point represents the mean of 6 cells.

Figure 5. The effect of divalent cations on the single channel conductance (γ) of I_cat

The relationship between current variance (σ_I²) and the mean current (I) in < 10 nM Ca_o²⁺ (A), 50 μM Ca_o²⁺ (B), 1.5 mM Sr_o²⁺ (C) and 1.5 mM Ba_o²⁺ (D) was linear and the single channel conductance (γ) was calculated from the gradient (i) of the linear relationship (see Methods for details). Note that the value of γ of I_cat in all the divalent cation solutions is approximately twice the estimated value in 0 Ca_o²⁺.

Figure 6. The effect of divalent cations on the spectral density function of I_cat

The spectral density function of I_cat obtained in the presence of 0 Ca_o²⁺ (A), 50 μM Ca_o²⁺ (B), 1.5 mM Sr_o²⁺ (C) and 1.5 mM Ba_o²⁺ (D). Each spectrum can be described by the sum of two Lorentzian components (continuous lines), where f_c1 and f_c2 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).

Figure 7. The effect of various divalent cations on relaxations of I_cat evoked by steps to various voltages

I_cat evoked in 0 Ca_o²⁺ (A), 50 μM Ca_o²⁺ (B), 1.5 mM Sr_o²⁺ (C) and 1.5 mM Ba_o²⁺ (D), whilst applying the voltage-jump protocol illustrated below. Note that no relaxations occurred in 0 Ca_o²⁺ (A) and 1.5 mM Ba_o²⁺ (D) but marked relaxations were observed in 50 μM Ca_o²⁺ (B) and 1.5 mM Sr_o²⁺ (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. I_inst, instantaneous current.