The primary physiological role of the exocrine pancreas is to produce pancreatic juice—an important conduit for the initial digestion of ingested nutrients in the small intestine. Neural and hormonal stimulation of the exocrine pancreas following a meal results in the production of a fluid rich in HCO3−
and containing a complex mixture of proteins (Williams and Yule, 2006
). The proteins are predominately inactive precursors of digestive enzymes that are subsequently activated in the lumen of the duodenum. Two epithelial cell types are primarily responsible for secretion from the gland. Acinar cells synthesize, store, and undergo regulated exocytosis of secretory granules while duct cells are responsible for the aqueous component of the secretion. Together, these cells result in the formation and delivery of pancreatic juice to the duodenum. A third, less studied cell type, pancreatic stellate cells (PSC), are also resident in the exocrine pancreas. PSC are present in a periacinar and periductal localization (Apte et al.
; Bachem et al.
; Omary et al.
). In common with hepatic stellate cells, they are characterized as retinol/lipid-storing cells expressing a variety of intermediate filament proteins including desmin and glial fibrillary acid protein (GFAP) (Wake, 1980
; Kordes et al.
). Under physiological conditions stellate cells appear to be benign, and relatively little is known regarding their contribution to the normal function of the gland.
PSC have, however, attracted considerable interest because of their prominent role in the etiology of pancreatic pathology (Omary et al.
). For example, in disease states of the pancreas including chronic pancreatitis and pancreatic cancer, stellate cells undergo a phenotypic transformation to a so-called activated form. Activated PSC (aPSC) are highly proliferative and adopt a myofibroblastic state characterized by enlargement of the endoplasmic reticulum (ER) and nucleus together with the expression of α-smooth muscle actin (α-SMA). Significantly, aPSC, again similarly to hepatic stellate cells, produce large amounts of extracellular matrix proteins (ECM) resulting in the extensive pancreatic fibrosis observed in disease states (Wake, 1980
; Apte et al.
; Haber et al.
; Casini et al.
; Omary et al.
; Kordes et al.
In vivo, it is likely that the signal transduction pathways that initiate and maintain the activated phenotype are elicited by events triggered by insult and mediated by surrounding cells including infiltrating leukocytes and damaged acinar cells. Much of the current understanding of the stellate cell signal transduction has been gleaned from studies of rodent PSC, isolated and maintained in culture. Initially the cultured cells express markers consistent with a quiescent phenotype, including the presence of prominent cytoplasmic lipid droplets (Apte et al.
). However, following short-term culture, cells transform to an activated, proliferative phenotype expressing abundant α-SMA and ECM proteins (Haber et al.
). Using this paradigm, inflammatory agents, growth factors, reactive oxygen species, and autocrine/paracrine factors have been shown to act on stellate cells (Omary et al.
). For example, aPSC proliferate in response to platelet-derived growth factor (PDGF) and transforming growth factor-β as well as proinflammatory cytokines (Apte et al.
; Luttenberger et al.
; Shek et al.
). These studies have implicated the MAP kinase cascade and JAK/STAT and SMAD pathways as contributing to the processes of transformation and proliferation in PSC (Masamune et al.
; Ohnishi et al.
; Kikuta et al.
). There is, however, a relative lack of data relating to other signaling systems that could potentially result in cell growth and/or transformation. In hepatic stellate cells, intracellular Ca2+
signals have been reported to induce proliferation (Soliman et al.
). Furthermore, agents proposed to act on PSC, including growth factors and angiotensin II, have the potential to couple to signal transduction pathways, which results in intracellular Ca2+
elevations (Luttenberger et al.
; Hama et al.
). Little information is available regarding the agonists linked to intracellular Ca2+
signaling in PSC together with the specific temporal and spatial characteristics of these signals. Moreover, data are lacking relating to the functional consequences of elevations in intracellular Ca2+
Given this gap in knowledge, the goals of the present study were to characterize Ca2+ signaling events and the consequences of these signals in cultured PSC in the quiescent (qPSC) and the transformed activated state. An important consideration is that in culture these events may not be identical to those taking place during disease progression in situ. Therefore, to study these events under conditions where the architecture of the pancreas and structural relationship between cell types are relatively unperturbed, the studies are extended to investigate Ca2+ signaling in lobules of intact pancreas using multiphoton (MP) microscopy in both physiological situations and in a model of chronic pancreatitis. Data are presented that provide evidence that the complement of cell surface receptors coupled to Ca2+ signaling is altered during activation of PSC, effectively providing a diagnostic feature of the activated phenotype. Further, the specific spatial characteristics of the Ca2+ signal are important for enhancing proliferation of aPSC. In total, these data are consistent with Ca2+ signaling events triggered by factors, including those likely released following initial acinar cell damage, resulting in proliferation of PSC and contributing to the pathology of disease.