Activation of K⁺ channels and increased migration of differentiated intestinal epithelial cells after wounding

Abstract

Early mucosal restitution occurs by epithelial cell migration to reseal superficial wounds after injury. Differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene migrate over the wounded edge much faster than undifferentiated parental cells in an in vitro model. This study determined whether these differentiated intestinal epithelial cells exhibit increased migration by altering voltage-gated K(+) (Kv) channel expression and cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). Stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1 cells nor parental IEC-6 cells expressed voltage-dependent Ca(2+) channels. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells was associated with an increase in whole cell K(+) currents, membrane hyperpolarization, and a rise in [Ca(2+)](cyt). The migration rates in differentiated IEC-Cdx2L1 cells were about four times those of parental IEC-6 cells. Inhibition of Kv channel expression by polyamine depletion decreased [Ca(2+)](cyt), reduced myosin stress fibers, and inhibited cell migration. Elevation of [Ca(2+)](cyt) by ionomycin promoted myosin II stress fiber formation and increased cell migration. These results suggest that increased migration of differentiated intestinal epithelial cells is mediated, at least partially, by increasing Kv channel activity and Ca(2+) influx during restitution.

Depolarization and decreased surface expression of K+ channels contribute to NSAID-inhibition of intestinal restitution

“…This cell line was developed and characterized initially by Suh and Traber [30], and then characterized further by others [23,31,32]. The forced expression of the Cdx2 gene in IEC-6 cells results in development of a differentiated phenotype [23,31]. The LacSwitch expression vector system (Stratgene, La Jolla, CA) is used to direct conditional expression of the Cdx2 gene, with isopropyl-β-D-thiogalactopyranoside (IPTG) serving as the inducer for gene expression.…”

“…In contrast to the dearth of information regarding the effects of NSAIDs on intestinal restitution, extensive knowledge of the molecular basis for polyamine-modulation of this process exists [21][22][23][24][25]. Depletion of intracellular polyamines inhibits intestinal cell migration by decreasing the expression of delayed rectifier potassium channels, thereby depolarizing membrane potential (E m ), decreasing the driving force for calcium-influx, and inhibiting downstream [Ca 2+ ] i -dependent signaling events [21][22][23][24][25].…”

“…Depletion of intracellular polyamines inhibits intestinal cell migration by decreasing the expression of delayed rectifier potassium channels, thereby depolarizing membrane potential (E m ), decreasing the driving force for calcium-influx, and inhibiting downstream [Ca 2+ ] i -dependent signaling events [21][22][23][24][25]. NSAIDs have been shown to deplete the polyamine content of immortalized colon cancer cells by enhancing polyamine catabolism [26].…”

The role of humoral autoimmunity in gastrointestinal neuromuscular diseases

“…Pharmacological and molecular evidence points to a contribution of K V 1 channels to both of these currents Glazebrook et al 2002;Dang et al 2004). Functionally within the gut, K V 1 currents are associated with a number of processes including epithelial cell maturation and migration (Rao et al 2002), nutrient absorption (McDaniel et al 2001), modulation of enteric motor nerve excitability (Suarez-Kurtz et al 1999;Vianna-Jorge et al 2000) and generation of smooth muscle 'slow-wave' contractions ; Carl 1995) As such, in a similar manner channel characterisation in the CNS and PNS, several studies have aimed to elucidate the specific K V subunit expression in the gut. In the mouse, Hatton and co-workers reported K V 1.1 immunoreactivity expression in murine (as well as guinea pig and canine) myenteric ganglia and ICC (Hatton et al 2001).…”

“…In comparison, knowledge regarding K V 1 channel expression in the GI tract was limited. Studies of small mammals had begun to identify K V (including K V 1) currents associated with epithelial cell maturation, migration and nutrient absorption (McDaniel et al 2001;Rao et al 2002). Modulation of enteric motor nerve excitability (Suarez-Kurtz et al 1999;Vianna-Jorge et al 2000) and generation of smooth muscle 'slow-wave' contractions Carl 1995) have been suggested to involve K V 1 channel activity.…”