A study of the ouabain-insensitive sodium efflux in barnacle muscle fibres using phorbol dibutyrate as a probe.

1. The resting ouabain-insensitive Na+ efflux in muscle fibres isolated from the barnacle, Balanus nubilus, is stimulated by external or internal application of phorbol 12,13-dibutyrate (PD). The response occurs fairly promptly and may not decay at all, or more commonly, decay rather slowly. The magnitude of the response to external or internal application of PD is dose-dependent, the minimum effective concentration being about 10(-8) M. 2. The response to PD fails to occur in the nominal absence of external Ca2+. Sudden removal of external Ca subsequent to peak stimulation by PD leads to almost complete reversal of the response. The response to PD of fibres suspended in Li(+)-ASW (artificial sea water) is similar in magnitude to that of fibres suspended in Na(+)-ASW. However, it differs in that it is of a sustained nature. 3. Calcium channel blockers, e.g. verapamil, completely prevent the response to PD from occurring. Both Cd2+ and Co2+ are less effective than verapamil. 4. Pre- but not post-injection of EGTA reduces the response to PD. Pre- or post-injection of Mg2+ reduces the response considerably. 5. Fibres pre-injected with GTP show a reduced response to PD. Fibres pre-injected with PD show a reduced response to GTP. Pre-injection of protein kinase inhibitor is without effect on the response to PD. 6. Furosemide, piretanide and bumetanide are without effect on the response to PD. 7. DIDS (4,4'-diisothiocyanostilbene-2,2-disulphonic acid) is a potent inhibitor of the response to PD but not amiloride. Pyridoxal 5-phosphate and benzolamide are also powerful inhibitors. Pyridoxal 5-phosphate in combination with benzolamide fails to completely abolish or reverse the response to PD. 8. Luminescence from aequorin is promptly increased by PD in a dose-dependent manner, the minimal effective concentration being in the nanomolar range. The signal is monophasic or multiphasic in shape, and is often less than 5 min in duration. Not infrequently, however, the aequorin response fails to completely decay and the new level of resting glow remains above the original baseline level. 9. Collectively, these observations accord with a tentative general hypothesis stating that the stimulatory response of the ouabain-insensitive Na+ efflux to PD is triggered by two mechanisms. One involves a rise in myoplasmic free [Ca2+] resulting from the entry of external Ca2+ via opened Ca2+ channels which is followed by the operation of the Na(+)-Ca2+ exchanger in the reverse mode.(ABSTRACT TRUNCATED AT 400 WORDS)