Asian Cardiovasc Thorac Ann 2000;8:357-360
© 2000 Asia Publishing EXchange Pte Ltd
Amlodipine, Nitric Oxide, and Platelet Aggregation
Ravi P Shankar, MBBS,
Vinod K Bhargava, PhD,
Anil Grover, DM,1,
Siddharta Mazumdar, PhD,2,
Santosh K Garg, PhD
Department of Pharmacology
1 Department of Cardiology
2 Department of Experimental Medicine & Biotechnology
Postgraduate Institute of Medical Education and Research
Chandigarh, India
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For reprint information contact: Vinod K Bhargava, PhD Tel: 91 172 74 7585 Fax: 91 172 74 4401 email: medinst{at}pgi.chd.nic.in Department of Pharmacology, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India.
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Abstract
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This study examined the effect of a single dose of 0.4 mg•kg–1 amlodipine on platelet aggregation with and without the nitric oxide synthase inhibitor, Nw-nitro-L-arginine. Blood samples were collected from rhesus monkeys at 0, 1, 2, and 6 hours after administration of amlodipine. Aggregation in platelet-rich plasma was stimulated by 10 µM adenosine diphosphate, 20 µM epinephrine, or 2 µg•mL–1 collagen. Administration of Nw-nitro-L-arginine alone significantly increased platelet aggregation for up to 2 hours. This effect was antagonized by amlodipine administered 30 minutes after Nw-nitro-L-arginine. The findings suggest that in platelet-rich plasma, the inhibition of platelet aggregation by amlodipine might be mediated by nitric oxide, a potent endogenous inhibitor of aggregation.
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Introduction
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The intracellular calcium ion concentration plays a crucial role in platelet activation and aggregation.1 During platelet activation, increased cytosolic Ca2+ results from both release of calcium from internal stores and extracellular calcium influx; the relative contribution of each process may differ, depending on the agonists used.2 Calcium channel blockers inhibit platelet aggregation in vivo and in vitro but the mechanism of this action is not clear.3–5 Although calcium channel blockers lower intraplatelet Ca2+ concentrations, no L-type calcium channels have been detected in the platelet membrane.6–8 Therefore, inhibition of platelet aggregation may be due to other properties of calcium channel antagonists. Nitric oxide reduces platelet responses by increasing platelet cyclic guanosine monophosphate content.9 The enzyme responsible for the formation of nitric oxide in the platelet, nitric oxide synthase, is controlled by the level of intracellular calcium-calmodulin.10 This study was conducted to determine the antiaggregation properties of amlodipine and the possible involvement of nitric oxide in its mechanism, using the nitric oxide synthase inhibitor, Nw-nitro-L-arginine (L-NNA).
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Materials and Methods
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Twenty-four adult male rhesus monkeys (Macaca mulatta) weighing 4 to 7 kg were acclimatized for 6 weeks under standard laboratory conditions with a 12-hour day-night cycle and food and water ad libitum. The study conformed with the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (NIH publication no. 85–23, revised 1996). The animals were divided into 4 groups of 6 monkeys each. Group 1 received 1 mL•kg–1 normal saline via nasogastric tube. Group 2 received 0.4 mg•kg–1 amlodipine (Torrent Pharmaceuticals, Ahmedabad, Gurajat, India) in normal saline via nasogastric tube. Group 3 received 40 mg•kg–1 Nw-nitro-L-arginine (Sigma, St. Louis, MO, USA) in 10 mL normal saline as a slow intravenous bolus dose. Group 4 received 40 mg•kg–1 L-NNA intravenously 30 minutes prior to administration of 0.4 mg•kg–1 amlodipine. In all experiments, drugs were administered at 8 am after overnight fasting. Blood samples were collected at 0, 1, 2, and 6 hours after drug administration. A 4.5-mL blood sample was obtained from the small saphenous vein with a plastic syringe and placed in a plastic vial containing 0.5 mL 3.8% w/v sodium citrate. Platelet-rich plasma was prepared by centrifugation at 800 rpm for 10 minutes. Platelet-poor plasma was prepared after separation of the platelet-rich plasma, by centrifugation at 3000 rpm for 10 minutes. The platelet count of the platelet-rich plasma was determined by a Neubauer counting chamber (Fein-Optik, Blankenburg, Germany) and adjusted with autologous platelet-poor plasma to 3.5 to 5 x 105 platelets per microliter.
Platelet aggregation was carried out using a double-channel aggregometer by the turbidimetric method of Born.11 Aggregation was induced with adenosine diphosphate (ADP) in a final concentration of 10 µM, or epinephrine (20 µM), or collagen (2 µg•mL–1). Platelet aggregation was recorded for 3 minutes and the extent was determined by measuring the maximum height of the aggregation curve. Blood pressure was measured using a sphygmomanometer with a pediatric-sized cuff and auscultation of the Korotkoff sounds over the brachial artery. Phase V, or disappearance of the sound, indicated the diastolic pressure. Heart rate was measured by palpation of the radial artery at the wrist. The monkeys were accustomed to the procedure for 2 to 3 days before the study. The hemodynamic measurements were carried out at 0, 1, and 6 hours after drug administration.
Data were expressed as mean ± standard error and analyzed by two-way repeated measure analysis of variance. A p value of < 0.05 was regarded as significant.
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Results
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The effects of amlodipine, L-NNA, and L-NNA plus amlodipine on ADP-stimulated platelet aggregation are illustrated in Figure 1
. Amlodipine caused a statistically significant decrease in platelet aggregation at 2 and 6 hours. Administration of L-NNA significantly increased aggregation at 1 and 2 hours. In animals treated with amlodipine 30 minutes after the administration of L-NNA, aggregation was higher at 1, 2, and 6 hours, thus the effect of L-NNA on ADP-stimulated platelet-aggregation was antagonized by amlodipine. Figure 2 shows the effects of amlodipine, L-NNA, and L-NNA plus amlodipine on epinephrine-stimulated platelet aggregation. Amlodipine caused a significant decrease in platelet aggregation at 1, 2, and 6 hours. Administration of L-NNA significantly increased aggregation compared to the control at 1 and 2 hours. Amlodipine antagonized the effect of L-NNA on epinephrine-stimulated platelet-aggregation. Similar findings were observed with collagen-stimulated platelet aggregation (Figure 3), where amlodipine significantly decreased aggregation at all sample points, L-NNA significantly increased the aggregation at 1 and 2 hours, and treatment with amlodipine after L-NNA, decreased aggregation. Table 1 lists the hemodynamic parameters in the control animals and those given amlodipine alone or amlodipine plus L-NNA. The differences were not statistically significant.
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Figure 1. Effects of amlodipine, L-NNA, and L-NNA plus amlodipine on ADP-stimulated platelet aggregation in rhesus monkeys. *p < 0.05 compared to control values. **p < 0.01 compared to control values.
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Figure 2. Effects of amlodipine, L-NNA, and L-NNA plus amlodipine on epinephrine-stimulated platelet aggregation in rhesus monkeys. **p < 0.01 compared to control values.
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Figure 3. Effects of amlodipine, L-NNA, and L-NNA plus amlodipine on collagen-stimulated platelet aggregation in rhesus monkeys. *p < 0.05 compared to control values. **p < 0.01 compared to control values.
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Discussion
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Amlodipine, a dihydropyridine calcium blocker, signifi-cantly decreased platelet aggregation stimulated by ADP, epinephrine, and collagen. These 3 aggregation agents were used because each has a different mechanism of action. Administration of L-NNA increased platelet aggregation more effectively in the 1-hour sample compared to subsequent samples, reflecting the falling concentration of L-NNA following the bolus dose. L-NNA inhibits nitric oxide (NO) synthesis in a concentration-dependent and stereospecific manner both in vitro and in vivo. L-NNA was chosen for its high potency in inhibiting NO synthase and for its lack of muscarinic antagonist activity when administered intravenously.12,13 NO reduces platelet responses by increasing their cyclic guanosine monophosphate content. Guanylate cyclase is a heterodimer formed by 2 polypeptides containing a heme moiety as a prosthetic group.14 The interaction between its heme group and NO appears to be an important modulator of its activity.15 Cyclic guanosine mono-phosphate acts by lowering the intraplatelet calcium concentration. As an indirect test of the hypothesis that NO is involved in amlodipine-induced antiaggregation, NO synthesis was blocked by the specific competitive inhibitor (L-NNA) to see whether this reduced the action of amlodipine. Alternative approaches would be to test for abolition of the antiaggregation effect of amlodipine by the NO scavenger oxyhemoglobin or to measure NO in the platelet directly. Recently, it was found that dihydropyridines enhance NO release from endothelial cells, which results in an increased vasorelaxing effect of dihydropyridines in vessels with preserved endothelium.16,17 It appears that amlodipine increases the synthesis and release of NO in platelets also, and thereby increases cyclic guanosine monophosphate levels that in turn reduce aggregation by decreasing intracellular calcium.
The limitations of this study were that the levels of intraplatelet NO were not measured, higher doses of amlodipine were not assessed, and the intracellular Ca2+ was not measured due to technical limitations. In addition, the oxyhemoglobin method was not used to confirm the involvement of NO. However, the findings suggest that the antiaggregation effect of amlodipine may be mediated via nitric oxide, although further studies are required to confirm this.
Presented at the Achari Prize Session, 31st Annual Conference of the Indian Pharmacological Society, Lucknow, India, December 17, 1998.
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Acknowledgments
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Amlodipine was a generous gift from Torrent Pharma-ceuticals, Ahmedabad, Gurajat, India.
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References
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Abstract
Introduction
