Both ACh and levcromakalim evoke smooth muscle cell hyperpolarization and associated relaxation in rat mesenteric resistance arteries. We investigated if they could evoke conducted vasodilatation along isolated arteries, whether this reflected spreading hyperpolarization and the possible mechanism involved. Focal micropipette application of either ACh, to stimulate endothelial cell muscarinic receptors, or levcromakalim, to activate smooth muscle K ATP channels, each evoked a local dilatation (88 ± 14%, n = 6 and 92 ± 6% reversal of phenylephrine-induced tone, n = 11, respectively) that rapidly spread upstream (at 1.5 mm 46 ± 19%, n = 6 and 57 ± 13%, n = 9) to dilate the entire isolated artery. The local dilatation to ACh was associated with a rise in endothelial cell [Ca 2+ ] i (F/F t=0 = 1.22 ± 0.33, n = 14) which did not spread beyond 0.5 mm (F/F t=0 = 1.01 ± 0.01, n = 14), while the local dilatation to levcromakalim was not associated with any change in endothelial cell [Ca 2+ ] i . In contrast, ACh and levcromakalim both stimulated local (12.7 ± 1.2 mV, n = 10 and 13.5 ± 4.7 mV, n = 10) and spreading (at 2 mm: 3.0 ± 1.1 mV, n = 5 and 4.1 ± 0.7 mV, n = 5) smooth muscle hyperpolarization. The spread of hyperpolarization could be prevented by cutting the artery, so was not due to a diffusible agent. Both the spreading dilatation and hyperpolarization were endothelium dependent. The injection of propidium iodide into either endothelial or smooth muscle cells revealed extensive dye coupling between the endothelial cells, but limited coupling between the smooth muscle cells. Some evidence for heterocellular spread of dye was also evident. Together, these data show that vasodilatation can spread over significant distances in mesenteric resistance arteries, and suggest this reflects an effective coupling between the endothelial cells to facilitate [Ca 2+ ] i -independent spread of hyperpolarization.

There is overwhelming evidence for an involvement of reactive oxygen species (ROS) in the pathogenesis of diabetes-associated vascular complications. However, neither the exact source of the ROS initiating cascades leading to cell dysfunction in diabetes nor their chemical nature is fully understood. Furthermore, despite our knowledge of the crucial role of ROS in diabetes, little is known about the actual targets and the molecular consequences of the interaction of ROS with cellular signalling pathways.Therefore, we aim to provide an overview of ROS (i. e. O 2 · , NO · , ONOO  and H 2 O 2 ) and their vascular sources in diabetes and to summarise recent knowledge on the mechanisms underlying increased ROS production within the vascular wall. In addition, possible targets of diabetes and ROS within the vasculature are discussed. These include, the effects of ROS on small guanine nucleotide binding proteins, the cytoskeleton, protein kinases (e. g. tyrosine kinases), metalloproteinases, ion homeostasis and transcriptional regulation.Such analysis makes it clear that the generation of ROS could affect a large number of various signalling pathways and proteins. Thus, a better knowledge of the functional diversity and pathological consequences of each individual pathway activated by ROS is essential to understand the mechanisms of diabetesassociated vascular complications. [Diabetologia (2002) 45:476±494]

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1. Using single cell microfluorometry to monitor changes in bulk Ca¥ concentration ([Ca¥]bulk) and the whole-cell configuration of the patch clamp technique to measure K¤ currents (voltage clamp) and membrane potential (current clamp), the mechanisms of histamineinduced Ca¥ oscillations in the umbilical vein endothelial cell-derived cell line EA.hy926 were studied. 2. In single cells, histamine (10 ìÒ) evoked sinusoidal Ca¥ oscillations in low extracellular Ca¥ concentrations ([Ca¥]ï = 10-30 ìÒ). In contrast, histamine did not initiate Ca¥ oscillations either in the absence of extracellular Ca¥ (10 ìÒ EGTA) or in the presence of 2·5 mÒ extracellular Ca¥. 3. Ca¥ oscillations were accompanied by rhythmic activation of Ca¥-activated K¤ (KCa) channels and membrane hyperpolarization of 18·1 ± 3·9 mV. Hence, cell depolarization with 70 mÒ extracellular K¤ or the inhibition of non-selective cation channels (NSCCs) and KCa channels by 10 ìÒ Loe 908 and 10 mÒ tetrabutylammonium prevented histamine-evoked Ca¥ oscillations. 4. Preventing Na¤-Ca¥ exchange (NCX) by 10 ìÒ 2',4'-dichlorobenzamil, or removal of extracellular Na¤, abolished histamine-induced Ca¥ oscillations. Lowering the extracellular Na¤ concentration and thus promoting the reversed mode of NCX (3Na¤ out and 1Ca¥ in) increased the amplitude and frequency of histamine-induced Ca¥ oscillations by 25 and 13%, respectively. Hence, in the absence of extracellular Ca¥, 10 ìÒ histamine induced an elevation of intracellular Na¤ concentration in certain subplasmalemmal domains. 5. The inhibitor of sarcoÏendoplasmic reticulum Ca¥-ATPase (SERCA) 2,5-di-tert-butyl-1,4-benzo-hydroquinone (15 ìÒ) prevented histamine-induced Ca¥ oscillations. In addition, blockage of ryanodine-sensitive Ca¥ release (RsCR) by 25 ìÒ ryanodine blunted Ca¥ oscillations. 6. In endothelial cells that were treated for 16 h with 10 ìÒ nocodazole to collapse the superficial endoplasmic reticulum (sER), no histamine-induced Ca¥ oscillations were found. 7. We conclude that in low [Ca¥]ï conditions histamine-induced Ca¥ oscillations depend on transmembrane Na¤ loading through NSCCs that leads to Ca¥ entry via NCX. Cation influx is controlled by KCa channel activity that triggers membrane hyperpolarization and, thus, provides the driving force for cation influx. Hence, the Ca¥ entering needs to be sequestrated via SERCA into sER to become released by RsCR to evoke Ca¥ spiking. These data further support our previous work on localized Ca¥ signalling as a key phenomenon in endothelial Ca¥ homeostasis. Keywords:

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.