Lack of effect on the sodium efflux of the microinjection of d-Ins(1,4,5)P3 into ouabain-poisoned barnacle muscle-fibers

A study has been carried out using relatively intact mature fibers from the barnacle Balanus nubilus to see whether d - Ins (1,4,5)P 3 stimulates the ouabain-insensitive Na efflux following its microinjection and whether this is accompanied by a contraction of the fiber. Part of the impetus for a study of this type came from the on-going debate between Vergara, Rojas and co-workers and Lea and co-workers, the former group holding the view that skinned barnacle fibers and skeletal fibers in general are responsive to this isomer. The evidence brought forward indicates that the injection of d - Ins (1,4,5)P 3 in concentrations in the range of 10−2 M to 10−6 M into cannulated unskinned fibers pretreated with ouabain fails to increase the residual efflux, and additionally fails to elicit a contraction. A similar picture emerges with the use of non-hydrolyzable dl - Ins (1,4,5)P 3 [S] 3 analog following its injection in a concentration of 0.5 μM. Higher concentrations of the analog were unavailable for use. This work also involved verification of the idea that an effect might be obtainable in depolarized fibers. However, doubling or tripling K0+ and injection of the isomer or the analog simultaneously failed to support this idea. Since nothing is known as to whether d - Ins (1,4,5)P 3 influences the behavior of the Na+Ca2+ exchanger when operating in the reverse mode, experiments were done to check this possibility. ATPNa2 which is thought to activate Na+Ca2+ exchange was injected prior to the isomer or the analog but no significant results were obtained. A similar line of reasoning was followed, that of activating the L-type Ca2+ channel by injecting GTPNa2 which in addition is known to activate adenylate cyclase. Again, neither the isomer nor the analog were effective. Thus, the only conclusion possible is that in relatively intact, mature barnacle fibers there is no coupling between the phosphoinositide signalling pathway and two other keys systems, viz. the Na+Ca2+ exchanger when operating in the reverse mode and the L-type Ca2+ channel. Equally clear is that for some unknown reason the ouabain-insensitive Na efflux and the contractile apparatus are insensitive to d - Ins (1,4,5)P 3 [S] 3 .