PubMed1997
Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca2+ signaling pathway, the ATP-dependent Ca2+ store and the store-coupled Ca2+ influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca2+, ATP, and IP3. The entry of Ca2+ into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP3-sensitive Ca2+ store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996) FEBS Lett. 395, 137-142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca2+ signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the "diffusion barrier" concept of Ca2+ signaling: Irrespective of the type of the applied stimulus, activation of the Ca2+ influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca2+]i of 200-250 nM as repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca2+ level to about 100 nM. Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca2+ signaling pathway by vasopressin, as well as by the IP3-independent acting Ca2+ store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca2+]i induced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca2+/Fura-2 compartment which is rapidly depleted from Ca2+ by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca2+ influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca2+ via the microvillar tip region in the cytoplasm.