The present study demonstrates the action of the hypoglycaemic drugs repaglinide and glibenclamide in cultured newborn rat islet cells and mouse beta TC3 cells. In cell-attached membrane patches of newborn rat islet cells repaglinide (10 nmol/l) and glibenclamide (20 nmol/l) decrease the open probability of single ATP-sensitive K(+)-channels to approximately 10% of the activity prior to addition of the drugs in short-term experiments (< 5 min). The influence of repaglinide and glibenclamide on the ATP-sensitive K+ current was studied using the whole-cell patch clamp configuration. A half-maximal steady-state inhibition of the ATP-sensitive K+ currents is observed at 89 pmol/l repaglinide and at 47 pmol/l glibenclamide in whole-cell experiments of longer duration (30 min). Applying digital Ca2+ imaging on single beta TC3 cells we found that repaglinide and glibenclamide induced a concentration-dependent increase in intracellular free Ca2+ concentration ([Ca2+]i) with a half-maximal effect at 0.5 nmol/l for both drugs in long-term experiments (30 min). The rise in [Ca2+]i results from Ca2+ entry through voltage-dependent L-type Ca(2+)-channels since it is inhibited by verapamil (10 mumol/l). The effect of repaglinide and glibenclamide is partly reversible (approximately 80%).

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.

In electron microscopy studies of the endothelial vesicles in frog mesenteric capillaries, an accidental observation was made concerning vesicular organization. When tannic acid was added to already fixed tissue, the mordant reached apparently free vesicles in the cytoplasm under conditions in which vesicular movement was excluded and in which the impermeability of the cell membranes was preserved. This indicates a spatial continuity between the vesicles and the cell exterior. It is proposed that cytoplasmic vesicles in endothelial cells are elements of branching, permanent or semipermanent invaginations of the plasmalemma.Palade discovered numerous vesicles in the capillary endothelium and suggested that they ferry small amounts of material between blood and the pericapillary space (1). This picture implies that the vesicles go through a cycle. They are temporarily attached to the cell surface, where they appear as invaginations of the plasmalemma. In this state the vesicular content equilibrates with the extracellular fluid. Subsequently, the vesicles pinch off from the surface and move through the cytoplasm to the opposite cell front where the vesicular membrane fuses with the plasmalemma and the content is discharged to the cell exterior (2). The process is assumed to be bidirectional. Subsequent observations have been interpreted in support of the above hypothesis of "vesicular transport" (3-1). Although some authors have expressed doubts about the hypothesis (12-16), it nevertheless stands as a generally accepted idea, explaining, in particular, macromolecular transport in capillaries.During an ultrastructural study of endothelial vesicles in frog mesenteric capillaries we made an accidental observation that indicates that almost all vesicles are members of a system of invaginations of the plasmalemma. By using tannic acid after aldehyde and osmium fixation to increase membrane contrast, we observed in many cases a connection between vesicles deep in the cytoplasm and the surface. This observation invites the proposal that the cytoplasmic, plasmalemmal membranes in endothelial cells, rather than being organized as freely moving vesicles, represent elements in an organized system of a more permanent or semipermanent nature.The picture we propose reconciles several conflicting interpretations of the behavior of macromolecular tracers in endothelia.MATERIALS AND METHODS The experiments were carried out on mesenteric capillaries of eight frogs (Rana temporaria), each weighing approximately 40 g. The animals, which had been kept for 6-8 months at 40C, were anesthetized by exposure to 5% urethane for about 5 min until mouth respiration had stopped.Preparation for Electron Microscopy. The frog mesenteries were fixed in situ by injecting 8-10 ml of 1% formaldehyde and 1.25% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) into the abdominal cavity. After 10 min the mesenteries were excised and left for 20 hr in the above fixative. After a buffer rinse, the tissue was postfixed in 2% OS04 in 0.1 M ...

A ~ S 7" a A C T This paper reports a description of methods for determining the diffusional permeability to potassium ions of single capillaries in the frog mesentery. By means of micropipettes, injections or infusions were delivered into a single capillary. The subsequent concentration variations in and about the capillary were followed with K+-sensitive microelectrodes. A theoretical analysis is provided which gives a quantitative frame of reference for evaluating the observed timeconcentration curves in terms of capillary permeability. The advantage of single capillary studies is that the surface area through which diffusion occurs is known as is the concentration difference across the capillary membrane. Three different techniques are: (a) the "single injection" method which represents an application of the indicator diffusion technique where a high-K + bolus is injected into a single capillary; (b) the "sack" method which determines the rate of K + disappearance from within and immediately outside an occluded capillary segment, after a brief increase in intracapillary K + concentration; and (c) the "interstitial diffusion" method which records time and spatial distribution of K + in the interstitial space after a step-change in intracapillary K + concentration. The methods gave an average potassium permeability of the capillary membrane of 67 x 10 -s cm s -I (SD: 23, n = 26) at room temperature.These figures are clearly higher than those previously reported in mammalian capillary studies using whole-organ techniques. In terms of the Pappenheimer pore model, this estimate of capillary permeability is consistent with the behavior of a membrane with a thickness of 1.0 t~m which possesses equivalent pores with a radius of 110 ~, a fractional pore area of 0.3%, and a pore density of 8 ~m -2.