Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Abstract Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca 2+ signaling pathway, the ATP-dependent Ca 2+ store and the store-coupled Ca 2+ 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 Ca 2+ , ATP, and IP 3 . The entry of Ca 2+ 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 IP 3 -sensitive Ca 2+ 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 Ca 2+ 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 Ca 2+ signaling: Irrespective of the type of the applied stimulus, activation of the Ca 2+ 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 [Ca 2+ ] i of 200–250 n M 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 Ca 2+ level to about 100 n M. Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca 2+ signaling pathway by vasopressin, as well as by the IP 3 -independent acting Ca 2+ store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca 2+ ] 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 Ca 2+ /Fura-2 compartment which is rapidly depleted from Ca 2+ by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca 2+ 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 Ca 2+ via the microvillar tip region in the cytoplasm.