Stimulation of human cheek cell Na+/H+ antiporter activity by saliva and salivary electrolytes: amplification by nigericin.
Proton-dependent, ethylisopropylamiloride (EIPA)-sensitive Na+ uptake (Na+/H+ antiporter) studies were performed to examine if saliva, and ionophores which alter cellular electrolyte balance, could influence the activity of the cheek cell Na+/H+ antiporter. Using the standard conditions of 1 mmol/l Na+, and a 65:1 (inside:outside) proton gradient in the assay, the uniport ionophores valinomycin (K+) and gramicidin (Na+) increased EIPA-sensitive Na+ uptake by 177% (p < 0.01) and 227% (p < 0.01), respectively. The dual antiporter ionophore nigericin (K(+)-H+) increased EIPA-sensitive Na+ uptake by 654% (p < 0.01), with maximal Na+ uptake achieved by 1 min and at an ionophore concentration of 50 mumol/l, with an EC50 value 6.4 mumol/l. Pre-incubation of cheek cells with saliva or the low molecular weight (MW) components of saliva (saliva activating factors, SAF) for 2 h at 37 degrees C, also significantly stimulated EIPA-sensitive Na+ uptake. This stimulation could be mimicked by pre-incubation with 25 mmol/l KCl or K(+)-phosphate buffer. Pre-incubating cheek cells with SAF and the inclusion of 20 mumol/l nigericin in the assay, produced maximum EIPA-sensitive Na+ uptake. After pre-incubation with water, 25 mmol/l K(+)-phosphate or SAF, with nigericin in all assays, the initial rate of proton-gradient dependent, EIPA-sensitive Na+ uptake was saturable with respect to external Na+, with Km values of 0.9, 1.7, and 1.8 mmol/l, and Vmax values of 13.4, 25.8, and 31.1 nmol/mg protein/30 sec, respectively. With 20 mumol/l nigericin in the assay, Na+ uptake was inhibited by either increasing the [K+]o in the assay, with an ID50 of 3 mmol/l. These results indicate that nigericin can facilitate K+i exchange for H+o and the attending re-acidification of the cheek cell amplifies 22Na+ uptake via the Na+/H+ antiporter. The degree of stimulation of proton-dependent, EIPA-sensitive Na+ uptake is therefore dependent, in part, on the intracellular [K+]i.
Patten
G S
GS
Division of Human Nutrition, CSIRO, O'Halloran Hill, South Australia.
Leifert
W R
WR
Burnard
S L
SL
Head
R J
RJ
McMurchie
E J
EJ
D000328:Adult; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D002610:Cheek; D004573:Electrolytes / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D009061:Mouth Mucosa / Q000378:metabolism*; D009550:Nigericin / Q000494:pharmacology*; D011188:Potassium / Q000494:pharmacology; D012463:Saliva / Q000502:physiology*; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*; D014634:Valinomycin / Q000494:pharmacology
D004573:Electrolytes; D007476:Ionophores; D017923:Sodium-Hydrogen Exchangers; D006096:Gramicidin; D014634:Valinomycin; D000584:Amiloride; D012964:Sodium; D009550:Nigericin; D011188:Potassium; C039614:ethylisopropylamiloride