Role of Na+/Ca2+ exchange in endothelin-1-induced increases in Ca2+ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943

Abstract

1. The effects of endothelin-1 (ET-1) on intracellular Ca2+ ion level and cell contraction were simultaneously investigated in rabbit ventricular cardiac myocytes loaded with indo-1/A1. The role of Na+/Ca2+ exchange in ET-1-induced positive inotropic effect (PIE) was examined by using KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate), a selective inhibitor of reverse mode Na+/Ca2+ exchange. 2. ET-1 at 0.3 pM - 1 nM increased cell contraction and Ca2+ transient (CaT) with EC50 values of 2.9 pM and 1.2 pM, respectively, and the increase in amplitude of CaT was much smaller relative to the PIE: ET-1 at 1 nM increased peak cell shortening by 237%, while it increased peak CaT by 167%. For a given level of PIE, ET-1-induced increase in CaT was much smaller than that induced by elevation of [Ca2+]o and by isoprenaline. Therefore, ET-1 shifted the relationship between peak CaT and cell shortening to the left relative to the relationship for increase in [Ca2+]o, an indication that ET-1 increased myofibrillar Ca2+ sensitivity. 3. KB-R7943 at 0.1 microM and higher inhibited contraction and CaT induced by 0.1 nM ET-1 and at 0.3 microM it abolished the increase in CaT while inhibiting the PIE by 48.1%. Over concentration range of 0.1-0.3 microM, KB-R7943 neither inhibited baseline contraction and CaT nor the isoprenaline-induced response, although at 1 microM and higher it had a significant inhibitory action on these responses. 4. These results indicate that in rabbit ventricular myocytes both increases in CaT and myofibrillar Ca2+ sensitivity contribute to the ET-induced PIE, and the activation of reverse mode Na+/Ca2+ exchange may play a crucial role in increase in CaT induced by ET-1 in rabbit ventricular cardiac myocytes.

Figure 1

(a) Effects of elevation of [Ca²⁺]_o on the simultaneous recordings of indo-1 fluorescence ratio (upper traces) and cell shortening (lower traces) in a single adult rabbit ventricular cardiac myocyte. [Ca²⁺]_o was increased stepwise from 1.8 mM to 14.4 mM. (b) To facilitate the comparison of changes in time-course induced by elevation of [Ca²⁺]_o tracings at 1.8 mM and 14.4 mM [Ca²⁺]_o were normalized and superimposed. W.O.: washout. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 2

(a) Effects of ET-1 on the simultaneous recordings of indo-1 fluorescence ratio (upper traces) and cell shortening (lower traces) in a single adult rabbit ventricular myocyte. ET-1 was increased from 0.3 pM to 1 nM. (b) To facilitate the comparison of changes in time-course induced by ET-1, tracings at 0.1 nM ET-1 were compared to those at baseline level. W.O.: washout. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 3

Concentration-response curves for the effects of ET-1 administered in a cumulative manner on the cell shortening and indo-1 fluorescence ratio in isolated rabbit ventricular myocytes. B: basal level before the addition of ET-1 was assigned to 100%. Numbers of myocytes are nine (n=9) except for the highest concentration of ET-1 at which two myocytes had deteriorated and excluded from the calculation; therefore numbers of myocytes at 1.0 nM ET-1 are seven (n=7). ^**P<0.01 vs the percentage increase in cell shortening. Statistical analysis was carried out by means of a repeated measures analysis of variance followed by Student's t-test.

Figure 4

The indo-1 ratio-cell shortening trajectory obtained at different concentrations of [Ca²⁺]_o (a) and ET-1 (b) in adult rabbit ventricular cardiac myocytes. Each trajectory was obtained by means of signal averaging of five successive signals.

Figure 5

The relationship between the peak Ca²⁺ transient and peak shortening induced by elevation of [Ca²⁺]_o and ET-1 at various concentrations in rabbit ventricular cardiac myocytes.

Figure 6

(A) Effects of ET-1 at 0.1 nM on the simultaneous recordings of indo-1 fluorescence ratio (middle traces) and cell shortening (upper and lower traces) in a single adult rabbit ventricular myocyte. Upper tracings represent the continuous recordings of cell length. Middle and lower tracings show individual signals recorded at a–f in upper tracings. (B) Effects of isoprenaline at 10 nM on the simultaneous recordings of indo-1 fluorescence ratio (middle traces) and cell shortening (upper and lower traces) in a single adult rabbit ventricular myocyte. Upper tracings represent the continuous recordings of cell length. Middle and lower tracings show individual signals recorded at a–e in upper tracings. W.O.: washout. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 6

(A) Effects of ET-1 at 0.1 nM on the simultaneous recordings of indo-1 fluorescence ratio (middle traces) and cell shortening (upper and lower traces) in a single adult rabbit ventricular myocyte. Upper tracings represent the continuous recordings of cell length. Middle and lower tracings show individual signals recorded at a–f in upper tracings. (B) Effects of isoprenaline at 10 nM on the simultaneous recordings of indo-1 fluorescence ratio (middle traces) and cell shortening (upper and lower traces) in a single adult rabbit ventricular myocyte. Upper tracings represent the continuous recordings of cell length. Middle and lower tracings show individual signals recorded at a–e in upper tracings. W.O.: washout. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 7

Influence of KB-R7943 on the simultaneously recorded indo-1 fluorescence ratio and cell shortening in the presence of 1.8 mM [Ca²⁺]_o in an isolated rabbit ventricular myocyte. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 8

Influence of KB-R7943 on 0.1 nM ET-1-induced increase in indo-1 fluorescence ratio (upper tracings) and cell shortening (lower tracings) in an isolated rabbit ventricular myocyte. Individual tracings were obtained by means of signal averaging of five successive signals.

Figure 9

Influence of KB-R7943 on the increases in indo-1 fluorescence ratio and cell shortening induced by ET-1 at 0.1 nM (a) and by isoprenaline at 10 nM (b). Numbers in parentheses represent numbers of myocytes. KB: KB-R7943. ^*P<0.05; ^**P<0.01 and ^***P<0.001 vs the values with ET-1 or isoprenaline alone, respectively. Statistical analysis was carried out by means of one-way analysis of variance.