Activated platelets contribute to stimulation of cardiac afferents during ischaemia in cats: role of 5-HT(3) receptors

Abstract

Myocardial ischaemia activates blood platelets and cardiac sympathetic afferents, which mediate chest pain and cardiovascular reflex responses. We have demonstrated that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents. Platelets absorb and release 5-hydroxytryptamine (5-HT) when they are activated. In the present study we hypothesized that, by releasing 5-HT, activated platelets stimulate cardiac afferents during ischaemia through a 5-HT(3) receptor mechanism. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were obtained from cats. Activation of platelets in PRP was induced by thrombin (5 units ml(-1)) or collagen (2 mg kg(-1)). Using high-performance liquid chromatography, we observed that the concentration of 5-HT was increased significantly in suspensions of platelets activated with thrombin (PRP+thrombin, 28 +/- 1.7 microM) or collagen (PRP+collagen, 27 +/- 2.5 microM) compared with suspensions of unactivated platelets (PRP+saline, 2.3 +/- 0.8 microM) and PPP. During myocardial ischaemia and reperfusion, tirofiban, a specific inhibitor of platelet glycoprotein (GP) IIb-IIIa receptors (100 microg kg(-1), I.V., followed by 5 microg kg(-1) min(-1)), significantly reduced the increase in the concentration of 5-HT in cardiac venous plasma from ischaemic region. Nerve activity of single-unit cardiac afferents was recorded from the left sympathetic chain (T2-T5) in anaesthetized cats. Eighty ischaemically sensitive and seven ischaemically insensitive cardiac afferents were identified. Tirofiban reduced the ischaemia-related increase in activity of seven cardiac sympathetic afferents by 50 %. Injection of 1.5 ml of PRP+collagen or PRP+thrombin into the left atrium (LA) increased activity of 16 cardiac afferents. Tropisetron (300 microg kg(-1), I.V.), a selective 5-HT(3) receptor antagonist, eliminated the afferent's responses to platelets activated with collagen or thrombin. Moreover, LA injection of 5-HT (20-40 microg kg(-1)) and PBG (100 microg kg(-1)), a 5-HT(3) receptor agonist, stimulated nine ischaemically sensitive cardiac sympathetic afferents, significantly increasing the activity of these afferents. However, injection of alpha-M-5-HT (100 microg kg(-1), LA), a 5-HT(2) receptor agonist, stimulated only two of the nine ischaemically sensitive cardiac afferents, and thus did not significantly alter impulse activity of this group of afferents. Both the 5-HT(1) (5-CT, 100 microg kg(-1), LA) and 5-HT(4) receptor agonists (SC53116, 100 microg kg(-1), LA) did not stimulate any of the nine afferents tested. Tropisetron (300 microg kg(-1), I.V.) also eliminated the response of seven ischaemically sensitive cardiac afferents to exogenous 5-HT and attenuated the ischaemia-related increase in activity of nine cardiac sympathetic afferents by 41 %. Conversely, LA injection of 5-HT (40 microg kg(-1)) did not stimulate any of seven ischaemically insensitive cardiac afferents, although this group of afferents consistently responded to bradykinin (3 microg, LA). These data indicate that during myocardial ischaemia the activated platelets stimulate cardiac sympathetic afferents, at least in part, through a 5-HT(3) receptor mechanism.

Figure 1. Concentration of 5-HT in sample of platelets activated with thrombin

A, representative chromatograph of 5-HT, 5-HIAA and tryptophan from standards (5 ng each). B, chromatograph of 5-HT and tryptophan from 0.1 ml of plasma containing platelets activated by thrombin (5 units ml⁻¹). Detection was accomplished coulometrically. Recovery of 5-HT, 93 ± 1.5 %. Limit of detection of 5-HT, 5 pg.

Figure 2. Responses of ischaemically sensitive cardiac afferents to PRP+collagen before and after the blockade of 5-HT₃ receptors

A, discharge frequencies of nine cardiac sympathetic afferents before (□) and after (▪) injection of PRP+collagen (1.5 ml, LA). B, effect of PRP+collagen on mean activity of eight separate cardiac afferents before (- tropisetron) and after (+tropisetron) tropisetron (300 μg kg⁻¹, i.v.). C, neurograms displaying responses of an ischaemically sensitive cardiac C-fibre (CV = 0.45 m s⁻¹) to PRP+collagen (1.5 ml, LA) before (Ca) and after (Cb) administration of tropisetron (300 μg kg⁻¹, i.v.). This cardiac afferent innervated the posterior wall of the left ventricle. Data are presented as means ± s.e.m. * P < 0.05 compared with control; † P < 0.05, post-tropisetron vs. pre-tropisetron.

Figure 3. Effect of tropisetron on responses of cardiac afferents to PRP+thrombin

A, reproducibility of responses of nine cardiac sympathetic afferents to PRP+thrombin (1.5 ml, LA). B, responses of eight other cardiac sympathetic afferents to PRP+thrombin before (-tropisetron) and after (+ tropisetron) treatment with tropisetron (300 μg kg⁻¹, i.v.). Columns and error bars represent means ± s.e.m. * P < 0.05 compared with control; † P < 0.05 post- tropisetron vs. pre- tropisetron

Figure 4. Influence of tirofiban on the response of cardiac afferents to myocardial ischaemia

Neurohistograms showing summed 5 s discharge activity of seven cardiac sympathetic afferents during myocardial ischaemia before (A) and after (B) administration of tirofiban (100 μg kg⁻¹, i.v. followed by infusion of 5 μg kg⁻¹ min⁻¹ for 25-30 min). Neurograms 1-4 are representative tracings of activity of a cardiac afferent innervating the anterior wall of the left ventricle (CV = 1.55 m s⁻¹) during pre-ischaemia (1 and 3) and ischaemia (2 and 4) in the absence (1 and 2) and presence (3 and 4) of tirofiban.

Figure 9. Effect of tropisetron on group responses of cardiac afferents during myocardial ischaemia

A, bar graph summarizing changes in activity of seven ischaemically sensitive cardiac afferents before (□) and during (▪) 5 min of repeated myocardial ischaemia. B, bar graph displaying discharge frequency of nine cardiac afferents before and during 5 min of ischaemia before (- tropisetron) and after (+ tropisetron) administration of tropisetron (300 μg kg⁻¹, i.v.). C, neurohistograms provide summed 2.5 s discharge activity of all nine cardiac afferents during ischaemia before (Ca) and after (Cb) treatment with tropisetron. Columns and error bars represent means ± s.e.m. * P < 0.05vs. pre-ischaemia; † P < 0.05, post-tropisetron vs. pre-tropisetron.

Figure 5. Representative discharge activity of a cardiac sympathetic afferent during myocardial ischaemia and stimulation with 5-HT and specific 5-HT receptor agonists

A, myocardial ischaemia increased average discharge activity of this afferent from 0.67 to 2.73 impulses s⁻¹. B-F, responses of this afferent during injection of 5-HT (B), 5-CT (C), α-M-5-HT (D), PBG (E) or SC53116 (F) into the left atrium (LA). This C-fibre afferent (CV = 0.53 m s⁻¹) innervated the posterior wall of the left ventricle.

Figure 6. Responses of ischaemically sensitive cardiac afferents to 5-HT and specific 5-HT receptor agonists

Bar graph showing peak activity of nine ischaemically sensitive cardiac afferents before (□) and after (▪) left atrial injection of 5-HT (20-40 μg kg⁻¹), 5-CT (100 μg kg⁻¹), α-M-5-HT (100 μg kg⁻¹), PBG (100 μg kg⁻¹) or SC53116 (100 μg kg⁻¹). Data are presented as means ± s.e.m. * P < 0.05vs. control.

Figure 7. Responses of ischaemically sensitive and insensitive cardiac afferents to 5-HT before and after tropisetron

A, bar graph showing discharge activity of seven cardiac afferents during 5 min of myocardial ischaemia, stimulation with 5-HT (40 μg kg⁻¹, LA) and bradykinin (3 μg, LA). B, bar graph showing reproducibility of responses of nine ischaemically sensitive cardiac afferents to repeated LA administration of 5-HT (40 μg kg⁻¹). C, bar graph summarizing responses of six other ischaemically sensitive cardiac afferents to 5-HT (40 μg kg⁻¹, LA) before (-tropisetron) and after (+ tropisetron) treatment with tropisetron (300 μg kg⁻¹, i.v.). Data are presented as means ± s.e.m. * P < 0.05vs. respective control; † P < 0.05, post-tropisetron vs. pre-tropisetron.

Figure 8. Response of a cardiac afferent to myocardial ischaemia before and after the 5-HT₃ receptor blockade

A, histograms showing increase in activity of a cardiac C-fibre afferent (CV = 1.93 m s⁻¹) from 0.09 to 2.90 impulses s⁻¹ during 5 min of myocardial ischaemia. B, tropisetron (300 μg kg⁻¹, i.v.) attenuated the increase in discharge activity of this afferent (from 0.12 to 1.40 impulses s⁻¹) during repeated ischaemia. This cardiac afferent innervated the anterior wall of the left ventricle. Neurograms 1-6 are representative tracings of the afferent at times indicated by arrows above histograms.