Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C022

Oral Communications

pH-dependent regulation of pancreatic stellate cell activation and proliferation

Z. D. Pethö1, J. Froese1, B. Fels1, A. Schwab1

1. Institute of Physiology II, University of Münster, Münster, Germany.

Pancreatic stellate cells (PSCs) are stromal cells of the pancreas that can become activated in various diseases such as chronic pancreatitis and pancreatic cancer. Upon activation, PSCs secrete a dense fibrous, so-called desmoplastic stroma. In the healthy pancreas, PSCs are exposed to intermittent changes in the interstitial pH. When pancreatic ductal cells secrete HCO3- into the pancreatic duct, they extrude an equimolar amount of H+ through their basolateral membrane domain, thereby acidifying the interstitium of the pancreas. Upon the tumorous transformation of the pancreatic ductal cells, the impaired ductal HCO3- secretion eventually leads to a relative alkalization of the interstitial pH of the pancreas. In this study we aim to elucidate in what manner changes in extracellular pH (pHe) affect the intracellular pH (pHi) homeostasis and activation of pancreatic stellate cells. We isolated PSCs from healthy adult wild-type C57/BL6 mice. Following isolation, we cultured PSCs in culture for up to 120 hours in media buffered to pH 6.0, 6.6, 7.0 or 7.4. To investigate the impact of pHe on the expression of potential pH sensors, regulators and pH-regulated transporters of PSCs, we performed RT-qPCR measurements. To further evaluate the functional relevance of these transporters, we performed pHi measurements using BCECF as fluorophore. As readout for cellular activation, we measured alpha-smooth muscle actin (α-SMA) positivity and cellular area using immunocytochemistry. Moreover, we assessed cellular proliferation and performed cell cycle analysis using flow cytometry. Alkalization of the extracellular milieu substantially increases cell size, α-SMA positivity as well as cellular proliferation. Moreover, cell cycle progression is also facilitated in an alkaline pHe environment. Our results also imply that multiple pH sensors and regulators, especially those that are regulated by HIF-1α, are differentially expressed in cells cultured in pHe 7.4 compared to freshly isolated cells and cells cultured in pHe 6.6. Also, intracellular pH acidifies moderately upon cellular activation taking place at alkaline pHe. When cells are cultured in an acidic pHe environment, the pHi acidifies to pHi ≤ 7.0. We conclude that alkaline pHe is a potent activator of PSCs as it induces cell cycle progression and the HIF1a-signaling pathway.

Where applicable, experiments conform with Society ethical requirements