In Vitro And In Vivo Activation of Rat Hepatic Stellate Cells Results In De Novo Expression of L–Type Voltage–Operated Calcium Channels

Abstract

Following chronic liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblast-like cells, which develop contractile properties and contribute to increased resistance to blood flow. We investigated whether this phenotypic activation includes changes in the expression of L-type voltage-operated Ca 2؉ channels (VOCC), which mediate Ca 2؉ influx and regulate cell contraction in vascular cell types. Rat HSCs were studied in the quiescent phenotype and after their activation in vitro (cultured on plastic for 14 days) and in vivo (isolated from rats with CCl 4 -induced cirrhosis). Patch-clamp studies showed Ca 2؉ currents through L-type VOCC in HSCs activated both in vitro and in vivo, whereas no currents were detected in quiescent HSCs. Moreover, binding studies with 3 H-isradipine, a specific L-type VOCC antagonist, showed a large number of binding sites in activated HSCs, while no specific binding was found in quiescent HSCs. In chronic liver diseases, hepatic stellate cells (HSCs) transdifferentiate from a quiescent phenotype into a myofibroblastlike phenotype, a process also named "activation." 1,2 The most relevant consequence of this phenotypic activation is the increased secretion of components of the extracellular matrix, a key feature in liver fibrogenesis. 3 In addition, recent data indicate that another important consequence of this process is the acquisition of contractile properties. 4 Due to their strategic anatomical location around hepatic sinusoids, tonic contraction of activated HSCs in response to agonists may increase sinusoidal resistance to blood flow and thus play an important role in the pathogenesis of portal hypertension in advanced chronic liver diseases. 5,6 In the last decade, much attention has been focused on the molecular mechanisms responsible for the increased collagen synthesis that display HSCs following phenotypic activation. 7 In clear contrast with these advances, the mechanisms by which the phenotypic activation of HSCs results in increased cell contractility remain largely unknown. Studies using in vitro and in vivo models of HSC activation have constantly shown that activated HSCs express high amounts of ␣-smooth muscle actin and myosin, cytosolic proteins essential for cell contractility, which are absent in quiescent HSCs. 8,9 Moreover, recent data indicate that in vitro activation of HSCs results in increased sensitivity to vasoconstrictor and depolarizing substances when assessed in terms of Ca 2ϩ mobilization. 10,11 However, the mechanisms underlying this increased Ca 2ϩ response are unknown. In human activated HSCs, an agonist-induced increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) is largely mediated by the opening of L-type voltage-operated Ca 2ϩ channels (VOCC), 12,13 which are known to regulate Ca 2ϩ entry and thereby cell contraction in other vascular cell types. 14 We hypothesized that the process of activation of HSCs is associated with changes in the expression of L-type VOCC that mediate Ca 2ϩ mobilization and cell contraction. ...

Both Ca2+-dependent and -independent pathways are involved in rat hepatic stellate cell contraction and intrahepatic hyperresponsiveness to methoxamine

“…Because of their apparent key role, considerable effort has been made to elucidate the regulation that governs contractile force generation in these cells. At present, it is generally assumed that activated HSCs have a smooth muscle cell-like Ca 2ϩ -dependent contraction pattern (2,5,(22)(23)(24). This contention is founded on different observations, such as 1) a functional and ultrastructural resemblance to pericytes (22); 2) the expression of smooth muscle proteins (like ␣-SMA and myosin II) (5, 26); 3) the expression of L-type voltage-operated Ca 2ϩ channels (2); and 4) the fact that agonists that are known to cause contraction in HSCs are associated with increases in intracellular Ca 2ϩ (22)(23)(24).…”

Cellular mechanisms of direct-current electric field effects: galvanotaxis and metastatic disease

“…dcEF stimulation also more than doubles [Ca 2+ ]i in a rat osteoblast-like cell line (Wang et al, 1998). Moreover, note that Ca 2+ influx induced by various factors, including membrane depolarization, has a role in cellular contraction (see below) in hepatic stellate cells (Bataller et al, 2001), pulmonary artery smooth muscle (Zhang et al, 1997) and skeletal muscle cells (Mickleson and Louis, 1996). The commonly seen cathodal galvanotaxis of cells can be explained if one assumes that, as a consequence of the dcEF, a rise in [Ca 2+ ]i in a given part of the cell causes contraction of that side whereas the opposite occurs at the other side.…”

Mechanisms of Hepatic Fibrogenesis

“…Even in earlier stages of fibrosis, activated stellate cells already show features of smooth muscle-like cells, characterized by expression of a number of contractile filaments including α smooth muscle actin 147 and myosin, 148 which generate calcium-dependent and calcium-independent contractile forces that contribute to cellular contractility. [149][150][151] Culture models increasingly recapitulate many of these smooth muscle features, in part by restoring a more physiologic substratum, as well as by including other resident cell types in a co-culture system, especially Kupffer cells. 152 As reviewed recently, stellate cells are recognized as liver-specific pericytes that contribute to angiogenesis in liver development, regeneration, and the response to injury.…”