Involvement of tyrosine phosphorylation in the positive inotropic effect produced by H(1)-receptors with histamine in guinea-pig left atrium

Abstract

We investigated the effect of stimulation of H(1)-receptors with histamine on protein tyrosine phosphorylation levels in guinea-pig left atrium and evaluated the influences of tyrosine kinase inhibitors on the positive inotropic effect mediated by H(1)-receptors in this tissue. Histamine induced an increase in tyrosine phosphorylation in four main clusters of proteins with apparent molecular weights of 25, 35, 65 and 150 kDa. Tyrosine phosphorylation of these proteins attained a peak around 2 - 3 min following histamine stimulation and then declined to or below basal levels. Histamine-induced protein tyrosine phosphorylation was antagonized by the H(1)-receptor antagonists mepyramine (1 microM) and chlorpheniramine (1 microM), but not by the H(2)-receptor antagonist cimetidine (10 microM). The positive inotropic effect of histamine was depressed in a concentration-dependent manner by the tyrosine kinase inhibitors tyrphostin A25 (50 to 100 microM) and genistein (10 to 50 microM) but not by the inactive genistein analogue daidzein (50 microM). The positive inotropic effect of isoprenaline was unchanged by tyrphostin A25 and genistein. At a concentration of 1 microM histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Treatment with tyrphostin A25 (100 microM) and genistein (50 microM), but not daidzein (50 microM), significantly attenuated the two components of the inotropic response, although genistein suppressed the initial component more markedly than the late component. We conclude that increased protein tyrosine phosphorylation may play an important role in initiating at least some part of the positive inotropic effect of H(1)-receptor stimulation in guinea-pig left atrium.

Figure 1

Histamine-induced protein tyrosine phosphorylation in guinea-pig left atrium. Following unexposure (control) or 3 min exposure to 10 μM histamine, the atrium was homogenized, run on SDS-polyacrylamide gel, electrophoretically transferred to PVDF and probed with antiphosphotyrosine antibody as described under ‘Methods'. Lane 1, control (C): lane 2, 10 μM histamine for 3 min (H). The immunoblots shown are representative of four experiments.

Figure 2

Time course of histamine stimulation of protein tyrosine phosphorylation in guinea-pig left atrium. The tissues were stimulated with 10 μM histamine for various times as indicated. (a) A representative immunoblot showing tyrosine phosphorylation of the 25-, 35-, 65- and 150-kDa protein bands. (b) Quantitation of relative tyrosine phosphorylation levels of the 25-kDa (open bars), 35-kDa (closed bars), 65-kDa (hatched bars) and 150-kDa (dotted bars) protein bands. The values are shown as relative intensity where each band derived from unstimulated tissue is assigned a value of 100%, and are mean±s.e.mean of six experiments. ^*P<0.05 compared to the corresponding control.

Figure 3

Concentration relationship of histamine stimulation of protein tyrosine phosphorylation in guinea-pig left atrium. The tissues were stimulated for 3 min with different concentrations of histamine as indicated. (a) A representative immunoblot showing tyrosine phosphorylation of the 25-, 35-, 65- and 150-kDa protein bands. (b) Quantitation of relative tyrosine phosphorylation levels of the 25-kDa (open bars), 35-kDa (closed bars), 65-kDa (hatched bars) and 150-kDa (dotted bars) protein bands. The values are shown as relative intensity where each band derived from unstimulated tissue is assigned a value of 100%, and are mean±s.e.mean of six experiments. ^*P<0.05 compared to the corresponding control.

Figure 4

Concentration-dependent effects of histamine on tyrosine phosphorylation of the 25-, 35-, 65- and 150-kDa protein bands in the absence (open bars) and presence of 10 μM cimetidine (dotted bars), 1 μM mepyramine (hatched bars) and 1 μM chlorpheniramine (closed bars) in guinea-pig left atrium. The tissues were stimulated for 3 min with different concentrations of histamine. The antagonists were given at least 30 min before the addition of histamine. The values are shown as relative intensity where each band derived from unstimulated tissue in the absence of any antagonist is assigned a value of 100% and are mean±s.e.mean of at least three experiments. ^*P<0.05, ^**P<0.01 and ^***P<0.001 compared to the corresponding value without any antagonist.

Figure 5

Influences of 100 μM tyrphostin A25 on the concentration-response curves for the positive inotropic effects of histamine (a) and isoprenaline (b) in guinea-pig left atrium electrically driven at 0.5 Hz. Tyrphostin A25 and its vehicle were applied to the tissues at least 30 min before the addition of the agonists. Points represent the net increases in force of contraction expressed as a percentage of the basal value recorded before any treatment and are shown as mean±s.e.mean of 4–6 experiments. Basal force of contraction was 527±36 mg (n=22). ^***P value indicates that the curves obtained in the presence of tyrphostin A25 and its vehicle are significantly different from each other (<0.001); n.s.=not significant.

Figure 6

Influences of 50 μM genistein (a) and 50 μM daidzein (b) on the concentration-response curves for the positive inotropic effect of histamine in guinea-pig left atrium electrically driven at 0.5 Hz. Genistein, daidzein and their vehicles were applied to the tissues at least 30 min before the addition of histamine. Points represent the net increases in force of contraction expressed as a percentage of the basal value recorded before any treatment and are shown as mean±s.e.mean of six experiments. Basal force of contraction was 603±69 mg (n=18). ^***P value indicates that the curves obtained in the presence of the drugs and their vehicles are significantly different from each other (<0.001); n.s.=not significant.

Figure 7

Influences of 100 μM tyrphostin A25 (a), 50 μM genistein (b) and 50 μM daidzein (c) on the time course of changes in force of contraction after application of 1 μM histamine in guinea-pig left atrium electrically driven at 0.5 Hz. These drugs and their vehicles were applied to the tissues at least 30 min before the addition of the agonists. Ordinates: increases in force of contraction expressed as a percentage of the basal value recorded before any treatment. Abscissae: time after the addition of histamine. Basal force of contraction was 518±32 mg (n=23). Points are mean±s.e.mean of 5–10 experiments. ^***P value indicates that the curves obtained in the presence of the drugs and their vehicles are significantly different from each other (<0.001); n.s.=not significant.