Nicotine stereoisomers and cotinine stimulate prostaglandin E2 but inhibit thromboxane B2 and leukotriene E4 synthesis in whole blood

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Abstract

The effects of (−)-nicotine (0.0005–500 μM), (+)-nicotine (0.0005–50 μM) and (−)-cotinine (0.0005–500 μM) on arachidonic acid metabolism were investigated in Ca2+ ionophore A23187 (calcimycin)-stimulated human whole blood in vitro. (−)-Nicotine and (−)-cotinine stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis, as has been observed previously in A23187-stimulated polymorphonuclear leukocytes and platelet-rich plasma [Saareks, V., Riutta, A., Mucha, I., Alanko, J., Vapaatalo, H., 1993. Nicotine and cotinine modulate eicosanoid production in human leukocytes and platelet rich plasma. Eur. J. Pharmacol., 248, 345–349.]. (+)-Nicotine also stimulated prostaglandin E2 but inhibited thromboxane B2 synthesis. High concentrations of (−)-nicotine and (−)-cotinine and even nanomolar concentrations of (+)-nicotine inhibited leukotriene E4 synthesis. These results indicate that (−)-nicotine and (−)-cotinine stimulate cyclooxygenase but inhibit thromboxane synthase and 5-lipoxygenase in whole blood in vitro. (+)-Nicotine is capable of affecting in the same direction as well.

Introduction

Smoking is a major risk factor for many cardiovascular and pulmonary diseases, but the precise mechanisms involved are not clearly understood. As well as activating platelets (Rival et al., 1987; Blache et al., 1992) and leukocytes (Bridges et al., 1993), smoking and the different components in tobacco smoke may contribute to the pathogenesis of these diseases by altering eicosanoid production.

Among eicosanoids, prostaglandin E2 and thromboxane A2 are cyclooxygenase and leukotriene B4 and cysteinyl leukotrienes (leukotrienes C4, D4 and E4) 5-lipoxygenase products of arachidonic acid metabolism. Pathophysiologically, prostaglandin E2 and leukotriene B4 have an important role in inflammatory diseases. Cysteinyl leukotrienes are assumed to be of importance in reperfusion injury (Shappel et al., 1990) and in asthma (for a review, see the work of Arm and Lee (1993)). Increased generation of thromboxane A2 is associated with various cardiovascular diseases.

Daytime plasma (Höfer et al., 1992) and blood (Benowitz et al., 1987) levels of nicotine and cotinine in habitual smokers smoking ad libitum are routinely maintained at constant levels of approximately 0.1 μM and 1 μM, respectively, although the sustained peak level of nicotine after smoking a cigarette may last only for 30 min (Srivastava et al., 1990). Cotinine is a major mammalian metabolite of nicotine, with a more constant plasma concentration and a considerably longer elimination time than that of the parent compound. Another source of nicotine is nicotine substitution therapy, which is widely used as an aid for smoking cessation. In volunteers using nicotine patches giving 22 mg/day, serum nicotine and cotinine levels are 0.06 μM and 0.85 μM, respectively (Hurt et al., 1993). Average blood nicotine and cotinine concentrations in persons chewing nicotine gum (48 mg nicotine per day) are 0.09 μM and 1.14 μM, respectively (Benowitz et al., 1987). Transdermal nicotine has been an effective therapy in ulcerative colitis in some (Sandborn et al., 1997; Pullan et al., 1994) but not in all (Thomas et al., 1995, Thomas et al., 1996) studies. In addition, nicotine may have therapeutic importance in Alzheimer's disease (Van Duijn and Hofman, 1991). However, the safety of chronic nicotine exposure has not been established.

In our previous study (Saareks et al., 1993), nicotine and cotinine increased prostaglandin E2 synthesis but reduced leukotriene B4 synthesis in A23187-stimulated human leukocytes in vitro. In kidney microsomes, nicotine has been reported to inhibit the formation of cyclooxygenase products in arachidonic acid metabolism (Alster et al., 1983). In platelet rich plasma (Toivanen et al., 1986; Saareks et al., 1993) and in macrophage-like cells (Goerig et al., 1992) there is evidence that nicotine reduces thromboxane B2 formation. In A23187-stimulated whole blood ex vivo, smoking cessation without nicotine substitution normalized the enhanced prostaglandin E2, leukotriene B4 and E4 as well as thromboxane B2 formation in smokers (Riutta et al., 1995). However, in volunteers chewing nicotine gum no significant decreases were seen, suggesting that nicotine and cotinine may explain the differences found in eicosanoid synthesis between smokers and non-smokers (Riutta et al., 1995).

Little is known about the actions of cotinine on arachidonic acid metabolism. The same applies to the actions of (+)-nicotine, present in tobacco smoke in a proportion ranging from 3 to 12% of total nicotine content (Klus and Kuhn, 1977; Nwosu et al., 1988), although it is known that the effects of the optical isomers of a drug on eicosanoid production may differ; this has been shown with indobufen, for instance (Patrignani et al., 1990). The effects of nicotine stereoisomers and cotinine on leukotriene E4 synthesis have not been studied before. We carried out the present study to establish the effects of nicotine stereoisomers and cotinine on cyclooxygenase and 5-lipoxygenase by measuring prostaglandin E2, thromboxane B2 and leukotriene E4 synthesis capacity in vitro, using A23187-stimulated human whole blood as a model. This model is regarded as one of the best for studying the effects of drugs on cyclooxygenase and 5-lipoxygenase both in vitro (Patrignani et al., 1990; Spaethe et al., 1992) and ex vivo (Sirois et al., 1991; Surette et al., 1994) because it takes into account the complex interaction between the different cell types capable of modulating arachidonic acid metabolism, although the important contribution of endothelial cells to the eicosanoid spectrum synthesized is excluded in this model.

Section snippets

Chemicals

Calcium ionophore A23187 (calcimycin), (−)-cotinine and (+)-nicotine were purchased from Sigma (St. Louis, MO, USA). (−)-Nicotine was obtained from BDH Chemicals (Poole, UK). Heparinized glass vacutainer tubes (143 U.S.P. heparin) were from Becton Dickinson (Meylan, France). All other reagents whose origins are not mentioned here were commercial and of the highest purity available.

Study protocol and whole blood incubation

Six healthy non-smoking volunteers (age 25–40 years) who had abstained from drugs for at least 2 weeks before

Results

Under basal, unstimulated conditions, whole blood did not release detectable amounts of prostaglandin E2, thromboxane B2 and leukotriene E4. Before drug administration, A23187-stimulated prostaglandin E2, thromboxane B2 and leukotriene E4 production in whole blood was 2.8±0.4, 97.2±11.6 and 58.9±11.8 ng/ml, respectively.

(−)-Nicotine (Fig. 1) dose-dependently increased A23187-stimulated prostaglandin E2 formation. At the concentration found in the plasma of smokers (0.1 μM), (−)-nicotine

Discussion

In the present study, we investigated the effects of nicotine stereoisomers and cotinine on arachidonic acid metabolism in calcium ionophore A23187-stimulated human whole blood in vitro. The whole blood stimulated by A23187 is one of the widely used models for studying the effects of drugs on eicosanoid synthesis in vitro (Patrignani et al., 1990; Spaethe et al., 1992) and ex vivo (Sirois et al., 1991; Surette et al., 1994), because it makes possible to explore simultaneously, both the

Acknowledgements

This work was supported by the Academy of Finland, the Emil Aaltonen Foundation, the Finnish Cultural Fund (Pirkanmaa), the Finnish Ministry of Social and Health Affairs, the Ida Montin Foundation and the Yrjö Jahnsson Foundation.

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