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Keywords:

  • aggregation;
  • Beraprost;
  • cAMP;
  • carp thrombocyte;
  • IBMX;
  • Iloprost;
  • rat platelet

Fish thrombocytes, the equivalent of mammalian platelets, are known to take part in inhibition of bleeding physiologically,1 but molecular mechanism for thrombocyte aggregation in fish remains unclear. Cyclic AMP (cAMP), whose level in cells rises by activation of adenylyl cyclase or inhibition of phosphodiesterase, is an important signal transduction molecule in cells and is related to various cellular functions.2 In mammals, cAMP plays a role in inhibitory regulation of platelet aggregation by promoting protein phosphorylation and influencing intracellular calcium mobilization.2 5-[Hexahydro-5-hydroxy-4-(3-hydroxy-4-methyl-1-octen-6-ynyl)-2(1H)-penta-lenylidene] pentanoic acid ([Iloprost] Funakoshi Pharmaceuticals, Tokyo, Japan)3 and 2,3,3a,8b-Tetrahydro-2-hydroxy-1-(3-hydoroxy-4-methyl-1-octen-6-ynyl)-1H-cyclopenta(b)-benzofuran-5-butanoic acid ([Beraprost] Toray Industries, Tokyo, Japan)4 are stable prostacyclin (PGI2) analogs that inhibit platelet aggregation in mammals by binding to the PGI2 receptor on the platelet membrane, resulting in an activation of adenylyl cyclase in the cell. Similarly 3-isobutyl-1-methylxanthine ([IBMX] Wako Pure Chemical, Osaka, Japan) inhibits platelet aggregation in mammals by inhibition of platelet phosphodiesterase,5 resulting in elevation of cAMP level.

Similar to the report of Kayama et al.,7 Matsushita et al.6 showed some differences in the aggregation behavior of thrombocytes or platelets in response to various aggregating agents between carp and rats. In fact, collagen caused both carp thrombocyte and rat platelet aggregation, but ADP and arachidonic acid almost failed to induce aggregation in carp thrombocytes, dissimilar to rat platelets. To clarify a part of the molecular mechanisms of fish thrombocytes aggregation, the participation of cAMP in signal transduction of carp thrombocytes triggered by collagen was studied. In a preliminary experiment,8 N6, O2-dibutyryl cyclicAMP (db-cAMP),9 a cell membrane permeable cAMP analog and can mimic cAMP action, inhibited the carp thrombocyte aggregation, suggesting a possible participation of cAMP in the mechanism. In the present study to confirm the possible involvement of cAMP in the carp thrombocyte signal transduction, the effects of Iloprost, Beraprost and IBMX, which are intracellular cAMP elevating agents, on the carp thrombocyte aggregation, were examined as compared with the rat platelet aggregation.

Carp were obtained from the culture ponds in Ono Limnological Facility, National Fisheries University (Ube, Yamaguchi Prefecture) and were used for the experiments. Male rats (Rattus norvegics), Wistar strain, 8–12-weeks old, were supplied from Seakku Yoshitomi (Chikujo-gun, Fukuoka Prefecture) and maintained for 3–8 weeks with chow and water ad libitum in a light : dark cycle of 12 h (light period 07:00–19:00 hours). The experiments with rats were conducted in accordance with protocols approved by the University's Animal Care and Use Committee. Carp (500–1000 g in mass) were anesthetized with quinaldine (20 p.p.m.), arterial blood (9 mL) was drawn from the caudal aorta, and 1 mL of 3.8% sodium citrate was added as anticoagulant. Rats (300–500 g in mass) were anesthetized by diethyl ether and arterial blood (10–15 mL) was collected through a polyethylene tube inserted into the carotid artery. One-ninth volume of 3.8% sodium citrate was added.

Measurements of aggregation were carried out with a whole blood aggregometer10,11 of Chrono-log (type 560-VS; Haverton, PA, USA). The temperatures of aggregation reaction in carp and rat were 25 and 37°C, respectively. The cuvette holding 1000 μL of blood was set into the aggregometer and the stability of baseline in the recorder was confirmed. The blood (1000 µL) was preincubated with 3 mM CaCl2 for 2 min. Then, 1 µL of Iloprost solution (in ethanol), 10 µL of Beraprost solution (in water) or 1 µL of IBMX solution (in DMSO) was added to the blood and further incubated for 6–8 min. Final concentrations of Iloprost, Beraprost and IBMX were 0–3 µM, 0–1 µM and 0–0.3 mM for carp thrombocytes, and 0–1 µM, 0–0.03 µM and 0–0.3 mM for rat platelets, respectively. One-hundredth volume (i.e. 10 µL) of 0.1 mg/mL collagen solution (type I, from horse tendon; Moriya-sangyo, Tokyo, Japan) was added to induce the aggregation (final concentration 1 µg/mL). The extent of aggregation was expressed as the maximum change of impedance (ohm) until approximately 12 min after the addition of collagen. Statistical analyses were carried out by Dunnett's test.

Thrombocyte aggregation, induced in the carp by collagen, was inhibited by Iloprost and Beraprost in a concentration-dependent manner (Fig. 1a,b) and was inhibited significantly at 1 and 3 µM for Iloprost (Fig. 1a) and at 0.1 and 1 µM for Beraprost (Fig. 1b). A similar result was obtained for rat platelet aggregation (Fig. 2a,b). The inhibitory effect to rat platelet aggregation, however, was significant at 0.1 and 1 µM for Iloprost (Fig. 2a), and at 0.03 µM for Beraprost (Fig. 2b). The effective concentrations of these drugs inhibiting aggregation were slightly high in carp thrombocytes compared with the case of rat platelets, probably showing low sensitivity of carp thrombocytes to the prostacyclin (PGI2) analogs. Thrombocyte aggregation was also inhibited in a concentration-dependent manner by IBMX with a significant inhibitory effect at 0.1 and 0.3 mM (Fig. 1c). Similarly, platelet aggregation, induced in the rat by collagen, was inhibited in a concentration-dependent manner by IBMX with a significant inhibitory effect at 0.3 mM (Fig. 2c). The small difference in the effective concentrations of IBMX inhibiting aggregation between carp thrombocytes and rat platelets might be due to the divergence in sensitivity of phosphodiesterase to IBMX in carp and rat.

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Figure 1. Effects of (a) Iloprost, (b) Beraprost and (c) IBMX treatment on carp thrombocyte aggregation induced by collagen. Carp thrombocytes were preincubated with 3 mM CaCl2 at 25°C for 4–8 min in the presence of Iloprost (0, 0.1, 1, 3 µM), Beraprost (0, 0.1, 1 µM) or IBMX (0, 0.03, 0.1, 0.3 mM). Then collagen (1 µg/mL) was added to induce the aggregation. The extent of aggregation was expressed as the maximum change of impedance (ohm) until about 12 min after the addition of collagen. Each column and horizontal bar represents the mean and standard deviation of 3–5 determinations, respectively. **P < 0.01, *P < 0.05 versus control. NS, not significant versus control.

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image

Figure 2. Effects of (a) Iloprost, (b) Beraprost and (c) IBMX treatment on rat platelet aggregation induced by collagen. The experimental procedure was similar to that described in the caption of Figure 1.

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In the present study, either Iloprost, Beraprost or IBMX inhibited thrombocyte aggregation in carp blood as similar to rat platelet. Therefore, these results, in addition to our preliminary results using db-cAMP,8 clearly show the possible involvement of cAMP to be an important inhibitory mediator of fish thrombocyte functions, such as aggregation, as in mammalian platelets. The present conclusion should be further supported by some experiments demonstrating actual cAMP levels in thrombocyte and reversal by cAMP antagonist such as Rp-cAMP of the inhibitory effect of cAMP elevating agents.

REFERENCES

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