Phorbol ester activation of an NHE-like electroneutral Na+/H+ antiporter in isolated E-cells of lobster (Homarus americanus) hepatopancreas
✍ Scribed by Duerr, Jeffrey M.; Ahearn, Gregory A.
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 217 KB
- Volume
- 281
- Category
- Article
- ISSN
- 0022-104X
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✦ Synopsis
The basolateral membrane of Atlantic lobster (Homarus americanus) epithelium possesses an electroneutral Na + /H + antiporter that functionally resembles members of the vertebrate NHE family. Regulatory mechanisms of this antiporter in purified hepatopancreatic E-cell suspensions, produced with a centrifugal elutriation technique, were investigated. Suspensions routinely consisted of greater than 95% E-cells displaying greater than 90% viability. Intracellular pH (pH i ) was monitored by loading cells with the fluorescent dye 2´,7´-bis(carboxyethyl)-5,6carboxyfluorescein (BCECF), and placing suspensions in a spectrofluorometer. Recovery from induced acid-loading was mediated by a Na + -dependent, dimethylamiloride-sensitive proton efflux. Antiport activation was a sigmoidal function of pHi at values below 7.0. Addition of 20 nM phorbol 12-myristate 13-acetate (PMA) to cells suspended in a lobster physiological saline (pH o = 7.4) increased pH i from 7.2 to 7.5 over a 10-min interval. Phorbol ester-induced activation of the Na + / H + antiporter was due to an increased affinity for internal H + (apparent pK was shifted toward more alkaline values) at the level of an internal H + -binding allosteric modifier site. No effect was observed when cells were exposed to 2 µM 8-Br-cAMP. Phorbol ester activation of the lobster NHE-like Na + /H + antiporter was inhibited by 10 nM bisindoylmaleimide I, a potent protein kinase C inhibitor. These results taken together suggest a remarkable conservation of Na + /H + antiport across phyla.