Submitted on July 1, 2002
Accepted on February 15, 2003
Effects of Helicobacter pylori on Intracellular Ca2+ Signaling in Normal Human Gastric Mucous Epithelial Cells
Katie M. Marlink1, Kathy D. Bacon1, Brett C. Sheppard1, Hassan Ashktorab2, Duane T. Smoot2, Timothy L. Cover3, Clifford W. Deveney4, and Michael J. Rutten1*
1 Department of Surgery, Oregon Health and Sciences University, Portland, OR, USA
2 Department of Medicine, Howard University, Washington, DC, USA
3 Departments of Medicine, Microbiology, Immunology and VAMC, Vanderbilt University School of Medicine, Nashville, TN, USA
4 Department of Surgery, Oregon Health and Sciences University, Portland, OR, USA; Department of VAMC, Oregon Health and Sciences University, Portland, OR, USA
* To whom correspondence should be addressed. E-mail: ruttenm{at}ohsu.edu.
In the stomach, Helicobacter pylori (Hp) adheres to gastric mucous epithelial cells and initiates several different signal transduction events. Alteration of intracellular Ca2+ [Ca2+]i is an important signaling mechanism in numerous bacteria-host model systems. However, changes in [Ca2+]i induced by Hp in normal human gastric mucous epithelial cells have not yet been described. In the following studies, we therefore examined the effects of Hp on [Ca2+]i in normal human gastric mucous epithelial cells and in a nontransformed gastric mucous cell epithelial cell line (HFE-145 gastric cells). Cultured cells were grown on glass slides, porous filters, or 96-multiwell plates and loaded with either Fura-2 or Fluo-4. H. pylori wild-type strain 60190 and vacA-, cagA-, and picB-/cagE- isogenic mutants were incubated with the cells, and changes in [Ca2+]i were recorded with a fluorimeter or fluorescence plate reader. We found that wild-type Hp produced dose-dependent biphasic transient [Ca2+]i 'peak' and 'plateau' changes in the primary gastric cultures and in the HFE-145 cell line. The Hp vacA- isogenic mutant produced changes in [Ca2+]i that were similar to those produced by the wild-type strain. In comparison to the wild-type strain, the cagA- and picB-/cagE- isogenic mutants produced lower [Ca2+]i 'peak' changes and did not generate a [Ca2+]i 'plateau' change. Preloading the cultures with the intracellular Ca2+-chelator BAPTA blocked all Hp-induced [Ca2+]i changes. Thapsigargin pretreatment of the cultures to release Ca2+ from internal stores reduced the 'peak' change. Extracellular Ca2+ removal reduced the 'plateau' response. The Hp-induced [Ca2+]i 'peak' response was sensitive to inhibitors of G-proteins and phospholipase-C (PLC), whereas the Hp-induced [Ca2+]i 'plateau' change was sensitive to inhibitors of G-proteins, src-kinases, and phospholipase-A2. These findings are the first to show that H. pylori alters [Ca2+]i in normal gastric mucous cells through a Ca2+ release/influx mechanism which depends on the expression of the cagA and picB/cagE genes.
