Activated pancreatic stellate cells (PSCs) are involved in the excessive deposition of extracellular matrix (ECM) proteins resulting in the development of fibrosis in alcoholic pancreatitis or fibrotic stroma in pancreatic tumours [1]. In response to tissue injury, quiescent fibroblast-like PSCs undergo activation, that is assume a myofibroblast-like phenotype characterised by upregulation of α-SMA expression, increased contractile capacity and production of ECM components [2]. This phenotype transition significantly affects the physiology of PSCs, including changes in cell signalling and metabolism, which may have profound implications in diseases of the pancreas.

To investigate this, we used quiescent and TGF-β-activated (for 48 h or 7 days) human PSCs, in which we compared pathophysiological Ca²⁺ signals, mitochondrial potential and cell death in response to ethanol (EtOH) and palmitoleic acid (POA) – the major inducers of alcoholic pancreatitis. We also measured mitochondrial parameters using the Seahorse Cell Mito Stress Test and compared the expression of Ca²⁺ channels and pumps between the quiescent and activated phenotypes.

Our data show that, compared to quiescent PSCs, activated cells differ significantly, in terms of Ca²⁺ signalling and metabolic activity. Activated PSCs are much less prone to EtOH/POA-induced cytosolic Ca²⁺ overload and cell death, predominantly due to downregulation of the TRPA1 channel (a decrease to 17.4% and 23.2% in PSCs 48 h and 7 days post-activation, respectively) [3]. In quiescent PSCs, inhibition or silencing of TRPA1 expression reduces cytosolic Ca²⁺ responses to 50 mM EtOH / 50 µM POA (from 4342.5±486.9 a.u. to 1508.0±205.4 a.u., p=0.0051) and protects these cells from cell death (a decrease of cell death from 70.5% to 19.8%, p=0.0006), mimicking the activated phenotype. In addition, activated PSCs had their basal respiration, ATP production and spare respiratory capacity increased (by approx. 1.6x, 1.4x and 12-16x respectively), compared to quiescent cells. EtOH/POA disrupted the mitochondrial potential in quiescent PSCs (an average decrease of 231.9±15.8 a.u. below baseline levels), but this effect was inhibited in cells 7 days post-activation (30.1±8.0 au, p<0.0001). Activated PSCs were also less sensitive to menadione-induced oxidative stress compared to quiescent cells.

Our results reveal significant changes in Ca²⁺ signalling machinery and the condition of mitochondria between quiescent and activated PSCs. These changes are directly responsible for the increased resilience of activated PSCs to noxious signals, which likely allows them to divide and deposit collagen and other components of the ECM even under harsh pathophysiological conditions such as ongoing inflammation. Better understanding of the activation-induced alterations in the cellular physiology of PSCs provides new insights into the mechanisms of pancreatic disorders, particularly those associated with fibrosis.