Morphine and Anandamide Stimulate Intracellular Calcium Transients in Human Arterial Endothelial Cells: Coupling to Nitric Oxide Release1
Introduction
Like morphine, cannabinoids have been used by humans for thousands of years. They have common biomedical properties, including analgesia, anti-inflammation and immunosuppression [1]. It is becoming evident that morphine and endocannabinoids also exhibit many biochemical similarities as well in regard to their signalling systems [2], including the stimulation of constitutive nitric oxide synthase (cNOS) in diverse tissues and animals 2, 3, 4, 5, 6.
The link between these two signalling systems is further strengthened by the fact that receptors for anandamide (CB1), an endocannabinoid, and morphine (μ3) have been demonstrated on the same cells, i.e., neurons and immunocytes in evolutionary diverse organisms 2, 7, 8, 9. A cannabinoid receptor cloned from macrophages [1]also was found to exhibit the morphine-selective μ3 receptor [8]. The link between opioids and cannabinoids is further strengthened by the observation that, in cerebellar granule cells, cannabinoid and opioid receptors appear to co-exist on the same cells and the activation of either receptor produces similar biological responses [10]. This is also the case for the macrophage cell line RAW 264.7; these cells have both cannabinoid and μ3 opiate receptors 11, 12.
Given these similarities, we investigated the role of intracellular calcium in endothelial cells because they also have these receptors that are coupled to NO release by cNOS 2, 3, 4, 5, 6. This is important because cNOS is a calcium-dependent enzyme 6, 13. In the present report, we demonstrate, in cultured human arterial endothelial cells loaded with fura 2, that both morphine and anandamide stimulate intracellular calcium transients in a concentration- and receptor-mediated phenomenon. Furthermore, the calcium transient is followed by NO release in these cultured endothelial cells. Thus, the study demonstrates that rapid signal transduction through intracellular calcium takes place in endothelia in response to both endogenous signalling molecules.
Section snippets
Intracellular Calcium Levels Monitored by Calcium Imaging
Human arterial endothelial cells purchased from Cell Systems (Eugene, OR, USA) were grown in chamber slides (Nunc Int.) with the use of CS-C medium (Cell Systems) supplemented with 10% foetal calf serum and endothelial growth factor, at 37°C, in a 5% CO2 atmosphere. Intracellular calcium levels were measured by dual-emission microfluorimetry with the use of the fluorescent dye fura-2/AM. Cells were loaded with the fluorescent ion indicator as follows. They were washed twice in the incubation
Results
Both morphine and anandamide significantly (P<0.001) stimulated endothelial intracellular calcium level increases in a concentration-dependent manner (Fig. 1). Morphine was found to be more potent than anandamide (P<0.001 at 10−6 to 10−8 M). The [Ca]i for anandamide stimulation is approximately half that found for morphine (Fig. 1, Fig. 2Fig. 3). The EC50 (50% increase of ligand-stimulated [Ca]i) for morphine and anandamide is 86±12 and 83±9.2 nM (S.E.M.), respectively. These values are within
Discussion
Opiate receptors are known to inhibit neuronal P/Q and N type calcium channels, by a G protein-dependent mechanism [18]. However, morphine has been reported to modulate intracellular calcium (transients) levels that affect many neuronal cellular functions, including cell growth and development, as well as analgesia [19]. Morphine stimulates morphologic differentiation in type-1 astrocytes and cellular hypertrophy in astroglial cells by μ-opioid-receptor-induced Ca2+ mobilization 20, 21. It also
Acknowledgements
This work was supported in part by the following grants: NIMH COR 17138 (GBS, SP and TVB), NIDA 09010 and the Research Foundation and Central Administration of the State University of New York (GBS, SP) and NIH Fogarty INT 00045 (MS, SP and GBS).
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Abbreviations: NO–Nitric oxide, cNOS–constitutive nitric oxide synthase.