Agents that increase tyrosine phosphorylation activate a non-selective cation current in single rabbit portal vein smooth muscle cells

Abstract

The effects of agents that increase tyrosine phosphorylation were studied with whole-cell recording of membrane currents in single smooth muscle cells from the rabbit portal vein. In K+-free conditions with the chloride equilibrium potential at about -50 mV, intracellular application via the patch pipette of 200 microM sodium orthovanadate (Na3VO4), which inhibits tyrosine phosphatases, activated a "noisy" inward current at a holding potential of -50 mV. Intracellular dialysis with 100 microM (pY)EEI, a peptide activator of the cytosolic tyrosine kinase pp60c-src, and bath application of 5 microM insulin, which activates receptor-coupled tyrosine kinases, also evoked a "noisy" inward current. The current-voltage relationships and the reversal potential (about +10 mV) of the Na3VO4-, pp60c-src- and insulin-induced currents were similar to those of the noradrenaline-evoked non-selective cation current (Icat). The inward currents evoked by noradrenaline, Na3VO4, (pY)EEI and insulin were all greatly potentiated when the bathing calcium concentration was reduced from 1.5 mM to 50 microM. The single channel conductance estimated from spectral density analysis of the whole-cell current was about 20 pS for noradrenaline, Na3VO4, (pY)EEI and insulin. Moreover for all agents the spectra were described by the sum of two Lorentzians with similar corner frequencies. Noradrenaline-evoked Icat was inhibited to a similar degree by the tyrosine kinase inhibitors genistein and tyrphostin 23 and their inactive analogues daidzein and tyrphostin A1, respectively. In the presence of Na3VO4, application of noradrenaline evoked a cation current of similar peak amplitude to control Icat although the rate of decay of Icat was enhanced in the presence of Na3VO4. This study shows that stimulation of both cytosolic and receptor-coupled tyrosine kinases evokes a non-selective cation current and the conductance is similar to that activated by noradrenaline.

Figure 1. Comparison of noradrenaline-evoked I_cat and the current activated by intracellular Na₃VO₄

A, I_cat evoked by bath application of 100 μm noradrenaline, denoted by the filled bar. B, inward current evoked by inclusion of 200 μm Na₃VO₄ in the patch pipette. The asterisk indicates the time of achieving the whole-cell configuration. In A and B the holding potential was -50 mV. C, the mean I-V relationship of the noradrenaline-activated I_cat in 5 cells. D, the mean I-V relationship of the Na₃VO₄-activated inward current in 4 cells.

Figure 2. Currents produced by (pY)EEI and insulin

A, inclusion of 100 μm (pY)EEI peptide in the patch pipette activated a ‘noisy’ inward current 20 s after achieving the whole-cell configuration (denoted by the asterisk). B, bath application of 5 μm insulin (denoted by filled bar) activated an inward current with a peak amplitude of 10 pA. In A and B the holding potential was -50 mV. C, the mean I-V relationship of the (pY)EEI-evoked inward current from 3 cells. D, the mean I-V relationship of the insulin-evoked inward current from 4 cells.

Figure 3. Effect of [Ca²⁺]_o on the noradrenaline-, Na₃VO₄-, (pY)EEI- and insulin-activated I_cat

Inward currents were activated by extracellular 100 μm noradrenaline (A), intracellular 200 μm Na₃VO₄(B), intracellular 100 μm (pY)EEI (C) and extracellular 5 μm insulin (D). Currents were initially evoked in 1.5 mm[Ca²⁺]_o which was then lowered to 50 μm[Ca²⁺]_o during activation of I_cat as denoted above the records. In A and D noradrenaline and insulin, respectively, were bath applied as indicated by the horizontal bar. In B and C the asterisk indicates the time of achieving the whole-cell configuration. All the experiments were carried out at a holding potential of -50 mV.

Figure 4. Spectral density functions of the noradrenaline-, Na₃VO₄-, (pY)EEI- and insulin-activated I_cat

A, a typical example of the spectral density function of noradrenaline-activated I_cat in 1.5 mm[Ca²⁺]_o. The spectra could be described by two Lorentzians with corner frequencies of 11 and 89 Hz. The f_c(1) and f_c(2) corner frequencies are represented as the time constants τ₁ and τ₂, respectively. The estimated single channel conductance (γ) of the noradrenaline-activated I_cat was 23 pS. B-D, examples of the spectral density function of the Na₃VO₄-, (pY)EEI- and insulin-evoked inward currents, respectively, showing similar noise characteristics to those of the noradrenaline-activated I_cat. The estimated single channel conductance of the Na₃VO₄-, (pY)EEI- and insulin-activated inward currents was 24, 23 and 19 pS, respectively.

Figure 5. Effects of genistein and daidzein on I_cat

A, control I_cat recorded by bath application of 100 μm noradrenaline. B and C illustrate the inhibition of I_cat by 75 μm genistein and 75 μm daidzein, respectively, applied during the sustained phase of the noradrenaline-evoked I_cat. In A-C the holding potential was -50 mV. D illustrates the concentration-dependent inhibitory effect of genistein where the current amplitude in the presence of genistein was normalised to the current amplitude immediately prior to application of the antagonist. The relative current was plotted against various concentrations of genistein on a logarithmic scale. The data could be described by a logistic equation of the following form: where x denotes the concentration of genistein and n the Hill coefficent. The calculated IC₅₀ value was 30 μm and the Hill coefficient was 2. Each data point represents the mean of 4-5 cells.

Figure 6. Effect of Na₃VO₄ on the noradrenaline-evoked I_cat

A, typical noradrenaline-evoked I_cat in the presence and absence of 200 μm Na₃VO₄ recorded from the same population of cells. Noradrenaline-evoked I_cat was activated in 50 μm[Ca²⁺]_o to increase the peak current amplitude. The current traces were normalised to the peak amplitude to illustrate the changes in the time course, since the two currents had similar peak amplitudes in the presence (210 pA) and absence (180 pA) of Na₃VO₄ in the patch pipette. The holding potential was -50 mV. Note that the peak amplitude and ‘noisy’ appearance of the Na₃VO₄-evoked I_cat are significantly increased on reducing [Ca²⁺]_o from 1.5 mm to 50 μm. B illustrates the time course of the decay of noradrenaline-evoked I_cat in the absence (▪) and presence (□) of 200 μm Na₃VO₄. The current was normalised to the peak amplitude (1.0). Asterisks represent statistically significant differences from control values: *P < 0.05, **P < 0.01 and ***P < 0.001.