1 Experiments were designed to determine whether anandamide a ects cytosolic Ca 2+ concentrations in endothelial cells and, if so, whether CB 1 cannabinoid receptors are involved. To this e ect, human umbilical vein-derived EA.hy926 endothelial cells were loaded with fura-2 to monitor changes in cytosolic Ca 2+ using conventional¯uorescence spectrometry methods. 2 Anandamide induced an increase in Ca 2+ in endothelial cells which, in contrast to histamine, developed slowly and was transient. Anandamide caused a concentration-dependent release of Ca 2+ from intracellular stores without triggering capacitative Ca 2+ entry, contrary to histamine or the endoplasmic reticulum Ca 2+ -ATPase inhibitor thapsigargin. 3 Anandamide pretreatment slightly reduced the mobilization of Ca 2+ from intracellular stores that was evoked by histamine. The mobilization of Ca 2+ from intracellular stores evoked by anandamide was impaired by 10 mM ca eine. 4 Anandamide and histamine each signi®cantly increased NO synthase activity in EA.hy926 cells, as determined by the enhanced conversion of L-The CB 1 cannabinoid receptor antagonist SR141716A (1 mM) only produced a marginal reduction of the mobilization of Ca 2+ produced by 5 mM anandamide. However, at 5 mM SR141716A elicited the release of Ca 2+ from intracellular stores. This concentration strongly impaired the mobilization of cytosolic Ca 2+ evoked by either anandamide, histamine or thapsigargin. 6 Pretreatment of the cells with either 200 mM phenylmethylsulphonyl¯uoride (to inhibit the conversion of anandamide into arachidonic acid) or 400 ng ml 71 pertussis toxin (to uncouple CB 1 cannabinoid receptors from G i/o proteins) had no signi®cant e ect on the mobilization of cytosolic Ca 2+ evoked by either anandamide, or histamine. 7 Taken together the results demonstrate that anandamide mobilizes Ca 2+ from a ca eine-sensitive intracellular Ca 2+ store that functionally overlaps in part with the internal stores mobilized by histamine. However, a classical CB 1 cannabinoid receptor-mediated and pertussis toxin-sensitive mechanism does not mediate this novel e ect of anandamide in endothelial cells. 8 The mobilization of cytosolic Ca 2+ in endothelial cells may account for the endotheliumdependent and NO-mediated vasodilator actions of anandamide. Due to its non-speci®c inhibition of Ca 2+ signalling in endothelial cells, SR141716A may not be used to assess the physiological involvement of endogenous cannabinoids to endothelium-dependent control of vascular smooth muscle tone.

Increased aggregation of platelets might contribute to the development of vascular complication in diabetes mellitus. In this study release of superoxide anions, intracellular Ca2+ signalling and nitric oxide formation stimulated by the receptor-dependent agonist adenosine 5 '-diphosphate (ADP) and the receptor-independent stimulus thapsigargin, were compared in platelets isolated from patients with Type II (non-insulin-dependent) diabetes mellitus and healthy control subjects. Diabetes augmented intracellular Ca2+ release and Ca2+ entry to ADP by 40 and 44% (control subjects: n = 11; diabetic: n = 6), while the median effective concentration (EC50) of ADP to initiate Ca2+ signalling was similar in both groups. The effect of thapsigargin on Ca2+ concentration was increased by 69% in diabetic patients (control subjects: n = 22; diabetic patients: n = 9). In addition, release of superoxide anions was 70% greater in diabetic patients (control subjects: n = 9; diabetic patients: n = 6). Treatment of platelets from control subjects with the superoxide anion-generating mixture xanthine oxidase and hypoxanthine or buthioninesulphoximine (BSO) mimicked the effect of diabetes on platelet Ca2+ signalling. The antioxidant glutathione normalized enhanced Ca2+ response in the diabetic group (control subjects: n = 5: diabetic patients: n = 6). Basal and thapsigargin-evoked nitric oxide synthase activity was reduced in the diabetic group by 85 and 64%, respectively (control subjects: n = 13; diabetic subjects: n = 13). The nitric oxide-donor 2-(N,N-diethylamino)-diazenolate-2-oxide sodium (DEA/NO) normalized enhanced Ca2+ signalling in platelets preincubated with xanthine oxidase and hypoxanthine (n = 12) and in those from diabetics (control subjects: n = 6; diabetic patients: n = 6). Inhibition of nitric oxide synthase by N-nitro-L-arginine (L-NA) augmented thapsigargin-induced Ca2+ signalling by 51% (n = 8). These data indicate that in diabetes platelet Ca2+ signalling might be enhanced by excessive superoxide production and an attenuated negative direct or indirect feedback control by nitric oxide, due to its reduced production.

Hyperlipidemia represents a major risk factor for development of vascular dysfunction and atherosclerosis. Although the unfortunate role of low-density lipoprotein has been clearly demonstrated, the mechanistic pathways through which triglyceride-derived free fatty acids (FFAs) contribute to vascular disorders are not completely understood. Thus, the present study was designed to elucidate the effects of FFAs on cultured endothelial cells. The Ca(2+) signaling, endothelial nitric oxide synthase (eNOS) activity, and production of superoxide anions (.O(2)(-)) were monitored in cells treated with bovine serum albumin-conjugated FFA. FFA-loaded cells showed enhanced intracellular Ca(2+) release in response to ATP, histamine, or the SERCA inhibitor thapsigargin. This effect corresponded to an overall increase in intracellularly stored Ca(2+). In contrast, autacoid-triggered elevation of cytosolic free Ca(2+) concentration was blunted in FFA-loaded cells due to inhibition of capacitative Ca(2+) entry. In agreement with the reduced Ca(2+) signaling, the Ca(2+)-dependent activity of eNOS was reduced under basal conditions and if cells were stimulated with ATP, histamine, or thapsigargin. The attenuated eNOS activity was associated with.O(2)(-) release in FFA-loaded cells. These data indicate that FFAs significantly affect endothelial Ca(2+) signaling, eNOS activity, and.O(2)(-) release and, thus, might contribute to vascular dysfunction in atherogenesis.

Aims/hypothesis. Diabetes mellitus is associated with endothelial dysfunction in human arteries due to the release of superoxide anions ( · O 2 -) that was found to occur predominantly in smooth muscle cells (SMC). This study was designed to elucidate the impact of high glucose concentration mediated radical production in SMC on EC. Pre-treatment of vascular SMC with increased D-glucose enhanced release of · O 2 -. Methods. Microscope-based analyses of intracellular free Ca 2+ concentration (fura-2), immunohistochemistry (f-actin) and tyrosine kinase activity were performed. Furthermore, RT-PCR and Western blots were carried out. Results. Interaction of EC with SMC pre-exposed to high glucose concentration yielded changes in endothelial Ca 2+ signalling and polymerization of f-actin in a concentration-dependent and superoxide dismutase (SOD) sensitive manner. This interaction activated endothelial tyrosine kinase(s) but not NFκB and AP-1, while SOD prevented tyrosine kinase stimulation but facilitated NFκB and AP-1 activation. Erbstatin, herbimycin A and the src family specific kinase inhibitor PP-1 but not the protein kinase C inhibitor GF109203X prevented changes in endothelial Ca 2+ signalling and cytoskeleton organization induced by pre-exposure of SMC to high glucose concentration. Adenovirus-mediated expression of kinase-inactive c-src blunted the effect of pre-exposure of SMC to high glucose concentration on EC. Conclusions/interpretation. These data suggest that SMC-derived · O 2 -alter endothelial cytoskeleton organization and Ca 2+ signalling via activation of c-src. The activation of c-src by SMC-derived radicals is a new concept of the mechanisms underlying vascular dysfunction in diabetes. [Diabetologia (2003) 46:773-783]