Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells,...

Ca2+-triggered catecholamine exocytosis from chromaffin cells involves SNAP-25, synaptobrevin and syntaxin (known as SNAREs). Synaptotagmin I has been implicated as the Ca2+-sensor because it binds Ca2+, and this enhances its binding to syntaxin, SNAP-25 and phospholipids in vitro. However, most of these interactions are only mediated by [Ca2+]i two orders of magnitude higher than that needed t...

The activity of the Ca2+/calmodulin-dependent protein kinase IV/Gr (CaMKIV/Gr) is shown to be strictly regulated by phosphorylation of three residues both in vitro and in response to antigen receptor-mediated signaling in lymphocytes. One residue, Thr-200, is indispensable for enhancement of Ca2+/calmodulin-dependent basal activity by CaMKIV/Gr kinase. This event requires Ca2+/calmodulin in the...

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radio-active filter technique. Trypsin treatment of vesicles resulted in a 3-fold...

When human polymorphonuclear leukocytes (PMN) are placed on various surfaces, they attach and spread rapidly, increasing their diameter severalfold. The spreading is associated with extensive changes in the cytoskeleton. Since many cytoskeletal events are regulated by Ca2+, we measured the cytosolic free calcium concentration ([Ca2+]i) in individual human PMN as they spread. [Ca2+]i was measure...

The relationship between the depletion of IP3-releasable intracellular Ca2+ stores and the activation of Ca(2+)-selective membrane current was determined during the stimulation of M1 muscarinic receptors in N1E-115 neuroblastoma cells. External Ca2+ is required for refilling Ca2+ stores and the voltage-independent, receptor-regulated Ca2+ current represents a significant Ca2+ source for refilli...

SNAREs such as VAMP, SNAP-25 and syntaxin are essential for intracellular trafficking, but what are their exact molecular roles and how are their interactions with other proteins manifest? Capitalizing on the differential sensitivity of SNAREs to exogenous proteases, we quantified the selective removal of identified SNAREs from native secretory vesicles without loss of fusion competence. Using ...

The mechanisms by which flow-imposed shear stress elevates intracellular Ca2+ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca2+ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca2+ concentrations...

This study examines the early effects of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone on cytosolic free calcium concentration ([Ca2+]i in primary cultures of fetal rat hypothalamic neurons. Microspectrofluorimetry of fluorescent Ca2+(-)sensitive indicator Fura-2 was used to quantify these changes. Allopregnanolone (1 pM to 100 nM) increased [Ca2+]i within 2-3 sec, in a dose dependen...

The effects of aspirin on gastric acid secretion were studied in isolated rabbit parietal cells (PC). Aspirin (10(-5) M) potentiated histamine-, dibutyryl cyclic AMP (dbcAMP)-, forskolin- and 3-isobutyl-1-methylxanthine-stimulated acid secretion without affecting basal acid secretion. Augmentation of secretagogue-stimulated acid secretion by aspirin was dependent on calcium (Ca2+) since potenti...