• PI3K/AKT pathway
• Raf/MEK/ERK pathway
• calcium influx
• colorectal cancer
• direct-current electrical field
• galvanotaxis
• patient-derived low-passage cell lines

• 889027 University Medicine Rostock FORUN Program

Colonic epithelial cells are responsible for maintaining a delicate balance between luminal secretion and the absorption of fluids and ions. This review aims to discuss and update the model of colonic electrolyte secretion and absorption via the cystic fibrosis transmembrane regulator (CFTR), epithelial sodium channel (ENaC), Na-K-Cl cotransporters (NKCC1 and 2), Na-H exchangers (NHE1–4), colonic H,KATPase, and several other key components involved in multi-level transepithelial ion transport. Developments in our understanding of the activity, regulation, localization, and relationships of these ion transporters and their interactions have helped forge a more robust understanding of colonic ion movement that accounts for the colonic epithelium’s role in mucosal pH modulation, the setting of osmotic gradients pivotal for fluid retention and secretion, and cell death regulation. Deviations from homeostatic ion transport cause diarrhea, constipation, and epithelial cell death and contribute to cystic fibrosis, irritable bowel syndrome (IBS), ulcerative colitis, and cancer pathologies. Signal transduction pathways that regulate electrolyte movement and the regulatory relationships between various sensors and transporters (CFTR as a target of CaSR regulation and as a regulator of ENaC and DRA, for example) are imperative aspects of a dynamic and comprehensive model of colonic ion homeostasis.

• Autism
• Brain injury
• Bumetanide
• Epilepsy
• NKCC1
• Neonatal
• Prodrugs

• Bartter syndrome
• Na+-K+-Cl− cotransporter 2
• cation-Cl− cotransporter
• high blood pressure
• loop of Henle
• renal physiology

• Alternative Splicing/genetics
• Amino Acid Sequence
• Animals
• Humans
• Ion Transport
• Loop of Henle/metabolism
• Models, Biological
• Solute Carrier Family 12, Member 3/chemistry
• Solute Carrier Family 12, Member 3/genetics
• Solute Carrier Family 12, Member 3/metabolism

• n/a CIHR
• Kidney Foundation of Canada

• Chloride cotransport
• KCC3
• Membrane traffic
• NKCC2
• Protein phosphorylation
• SYK

Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl^-. The present studies employing Ussing chamber technique identified two opposing short-circuit current (I_sc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl^--secretory I_sc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated I_sc in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na⁺-K⁺-2Cl^- cotransporter (NKCC) in crypt cells and basolateral K⁺-Cl^- cotransporter (KCC) in surface epithelium, we proposed this stimulatory I_sc could represent a KCC-mediated Cl^- absorptive current. In support of this hypothesis, ion substitution experiments established Cl^- dependency of this absorptive I_sc and transport inhibitor studies demonstrated the involvement of an apical Cl^- conductance. Current distribution and RNA sequencing analyses revealed that this Cl^- absorptive I_sc is closely associated with epithelial Na⁺ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 μM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl^- absorptive I_sc senses dietary Cl^- content; thus when dietary Cl^- was low, Cl^- absorptive I_sc was up-regulated. In contrast, when dietary Cl^- was increased, Cl^- absorptive I_sc was down-regulated. We conclude that an active Cl^- extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.

Na⁺K⁺2Cl⁻ co-transporters (NKCCs) effect the electroneutral movement of Na⁺-K⁺ and 2Cl⁻ ions across the plasma membrane of vertebrate cells. There are two known NKCC isoforms, NKCC1 (Slc12a2) and NKCC2 (Slc12a1). NKCC1 is a ubiquitously expressed transporter involved in cell volume regulation, Cl⁻ homeostasis and epithelial salt secretion, whereas NKCC2 is abundantly expressed in kidney epithelial cells of the thick ascending loop of Henle, where it plays key roles in NaCl reabsorption and electrolyte homeostasis. Although NKCC1 and NKCC2 co-transport the same ions with identical stoichiometry, NKCC1 actively co-transports water whereas NKCC2 does not. There is growing evidence showing that NKCC2 is expressed outside the kidney, but its function in extra-renal tissues remains unknown. The present study shows molecular and functional evidence of endogenous NKCC2 expression in COS7 cells, a widely used mammalian cell model. Endogenous NKCC2 is primarily found in recycling endosomes, Golgi cisternae, Golgi-derived vesicles, and to a lesser extent in the endoplasmic reticulum. Unlike NKCC1, NKCC2 is minimally hybrid/complex N-glycosylated under basal conditions and yet it is trafficked to the plasma membrane region of hyper-osmotically challenged cells through mechanisms that require minimal complex N-glycosylation or functional Golgi cisternae. Control COS7 cells exposed to slightly hyperosmotic (~6.7%) solutions for 16 h were not shrunken, suggesting that either one or both NKCC1 and NKCC2 may participate in cell volume recovery. However, NKCC2 targeted to the plasma membrane region or transient over-expression of NKCC2 failed to rescue NKCC1 in COS7 cells where NKCC1 had been silenced. Further, COS7 cells in which NKCC1, but not NKCC2, was silenced exhibited reduced cell size compared to control cells. Altogether, these results suggest that NKCC2 does not participate in cell volume recovery and therefore, NKCC1 and NKCC2 are functionally different Na⁺K⁺2Cl⁻ co-transporters.

• COS7
• Golgi
• N-glycosylation
• NKCC2A
• cell volume

• Brain disorders
• Cation-chloride cotransporters
• Diuresis
• Epilepsy
• Ototoxicity

• Animals
• Anticonvulsants/pharmacology
• Anticonvulsants/therapeutic use
• Bumetanide/analogs & derivatives
• Bumetanide/pharmacology
• Diuretics/pharmacology
• Epilepsy/drug therapy
• Humans
• Oocytes
• Solute Carrier Family 12, Member 2/drug effects
• Solute Carrier Family 12, Member 2/genetics
• Structure-Activity Relationship
• Xenopus

• Ca(2+)
• Na(+)–K(+)–2Cl(−) cotransporter (NKCC)
• Phosphorylation
• Vasopressin
• cAMP

AIM: To investigate whether Na⁺-K⁺-2Cl^- cotransporter (NKCC2) is expressed in the mouse distal colonic epithelia and whether it is regulated by vasopressin in the colon.

METHODS: The mRNA expression of NKCC2 in the mouse colonic mucosa was examined by reverse transcription-polymerase chain reaction. NKCC trafficking in the colon stimulated by 1-D-amino(8-D-arginine)-vasopressin (dDAVP) infusion (10 ng/mouse, intraperitoneal injection ) within 15 min, 30 min and 1h was investigated by laser confocal scanning microscopy. Total and membrane NKCC2 expression in the colonic mucosa from control and dDAVP-treated mice was detected by Western blotting. Short circuit current method was performed to determine regulation of NKCC2 by vasopressin in the colon.

RESULTS: NKCC2 was predominantly located in the apical region of the surface of the distal colonic epithelia; by comparison, a large amount of NKCC1 was distributed in the basolateral membrane of the lower crypt epithelia of the mouse distal colon. Short-term treatment with dDAVP, a V2-type receptor-specific vasopressin analog, induced NKCC2 re-distribution, i.e., NKCC2 traffics to the apical membrane after dDAVP stimulation. In contrast, no obvious NKCC1 membrane translocation was observed. Western blotting results confirmed that membrane NKCC2 had significantly higher abundance in the dDAVP-treated mouse colonic mucosa relative to that in the untreated control, which is consistent with our immunostaining data. Moreover, the short-circuit current method combined with a NKCC2 inhibitor demonstrated that NKCC2 was also activated by serosal vasopressin in isolated distal colonic mucosa.

CONCLUSION: Our results provide direct evidence that vasopressin also plays an important role in the colonic epithelia by stimulating NKCC2 trafficking to the apical membrane and inducing NKCC2-mediated ion transport.

• Na⁺-K⁺-2Cl^- cotransporter
• Apical membrane
• Vasopressin
• Distal colonic epithelia
• Trafficking

• Animals
• Colon/drug effects
• Colon/metabolism
• Deamino Arginine Vasopressin/administration & dosage
• Deamino Arginine Vasopressin/pharmacology
• Infusions, Parenteral
• Intestinal Mucosa/drug effects
• Intestinal Mucosa/metabolism
• Ion Transport
• Male
• Membrane Potentials
• Mice, Inbred C57BL
• Protein Transport
• RNA, Messenger/metabolism
• Solute Carrier Family 12, Member 1/drug effects
• Solute Carrier Family 12, Member 1/genetics
• Solute Carrier Family 12, Member 1/metabolism
• Time Factors

• Animals
• Cells, Cultured
• Female
• Gene Silencing/physiology
• Glucose Intolerance/genetics
• Glucose Intolerance/metabolism
• Glucose Intolerance/pathology
• Homozygote
• Insulin/metabolism
• Insulin Secretion
• Islets of Langerhans/metabolism
• Islets of Langerhans/pathology
• Male
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• Rats
• Sodium-Potassium-Chloride Symporters/genetics
• Sodium-Potassium-Chloride Symporters/physiology
• Solute Carrier Family 12, Member 2
• Up-Regulation