1. Endothelial cell activation is correlated with increased cytosolic Ca¥ concentration, often monitored with cytoplasmic Ca¥ dyes, such as fura_2 and Calcium Green-1. We tested the hypothesis that during weak stimulation of porcine coronary artery endothelial cells, focal, subplasmalemmal Ca¥ elevations occur which are controlled by cell membrane Na¤-Ca¥ exchange near mitochondrial membrane and superficial endoplasmic reticulum (SER). 2. Bulk Ca¥ concentration ([Ca¥]b) was monitored using fura_2 or Calcium Green-1 and subplasmalemmal Ca¥ concentration ([Ca¥]sp) was determined with FFP-18. The distribution of the SER network was estimated using laser scanning and deconvolution microscopy. 1 nmol l¢ Bk or 10 mmol l¢ NaF yielded focal [Ca¥] elevation in the subplasmalemmal region with no increase in the perinuclear area. 6. Treatment with 10 ìmol l¢ nocodazole caused the SER to collapse and unmasked Ca¥ release in response to 1 nmol l¢ Bk and 10 mmol l¢ NaF, similar to low-Na¤ conditions, while the effect of thapsigargin was not changed. 7. These data show that in endothelial cells, focal, subplasmalemmal Ca¥ elevations in response to small or slow IP× formation occur due to vectorial Ca¥ release from the SER towards the plasmalemma followed by Ca¥ extrusion by Na¤-Ca¥ exchange. While these local Ca¥ elevations are not detectable with Ca¥ dyes for the determination of [Ca¥]b, prevention of Ca¥ extrusion or SER disruption yields increases in [Ca¥]b partially due to CICR. 8. All of the data support our hypothesis that in weakly stimulated endothelial cells, intracellular Ca¥ release and [Ca¥] elevation are limited to the subplasmalemmal region. We propose that the SER co-operates with associated parts of the plasma membrane to control Ca¥ homeostasis, Ca¥ distribution and Ca¥ entry. The existence of such a subplasmalemmal Ca¥ control unit (SCCU) needs to be considered in discussions of Ca¥ signalling, especially when cytoplasmic Ca¥ dyes, such as fura_2 or Calcium Green-1, are used.

Alterations of vascular smooth muscle function have been implicated in the development of vascular complications and circulatory dysfunction in diabetes. However, little is known about changes in smooth muscle contractility and the intracellular mechanisms contributing to altered responsiveness of blood vessels of diabetic patients. Therefore, smooth muscle and endothelial cell function were assessed in 20 patients with diabetes and compared with 41 age-matched control subjects. In rings from uterine arteries, smooth muscle sensitivity to K+, norepinephrine (NE), and phenylephrine (PE) was enhanced by 1.4-, 2.3-, and 9.7-fold, respectively, and endothelium-dependent relaxation was reduced by 64% in diabetic patients, as compared with control subjects. In addition, in freshly isolated smooth muscle cells from diabetic patients, an increased perinuclear Ca2+ signaling to K+ (30 mmol/l >73%; 60 mmol/l >68%) and NE (300 nmol/l >86%; 10 micromol/l >67%) was found. In contrast, subplasmalemmal Ca2+ response, which favors smooth muscle relaxation caused by activation of Ca2+-activated K+ channels, was reduced by 38% in diabetic patients as compared with control subjects, indicating a significant change in the subcellular Ca2+ distribution in vascular smooth muscle cells in diabetic patients. In contrast to the altered Ca2+ signaling found in freshly isolated cells from diabetic patients, in cultured smooth muscle cells isolated from control subjects and diabetic patients, no difference in the intracellular Ca2+ signaling to stimulation with either K+ or NE was found. Furthermore, production of superoxide anion (*O2-) in intact and endothelium-denuded arteries from diabetic patients was increased by 150 and 136%, respectively. Incubation of freshly isolated smooth muscle cells from control subjects with the *O2- -generating system xanthine oxidase/hypoxanthine mimicked the effect of diabetic patients on subcellular Ca2+ distribution in a superoxide dismutase-sensitive manner. We conclude that in diabetic subjects, smooth muscle reactivity is increased because of changes in subcellular Ca2+ distribution on cell activation. Increased *O2- production may play a crucial role in the alteration of smooth muscle function.

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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]

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