Zinc is an inhibitor of the stimulatory response of the sodium efflux to the microinjection of cyclic AMP and forskolin in single barnacle muscle fibers.

The main aim of the experiments with which this paper deals was to test the hypothesis that Zn is an inhibitor of the stimulatory response of the ouabain-insensitive component of the Na efflux to the injection of cAMP, DcAMP (dibutyryl-cAMP) and FD (forskolin derivative) in barnacle muscle fibers. The results obtained were as follows: (1) External application of Zn caused a fall in the Na efflux in fibers poisoned with ouabain beforehand; (2) external application of Zn prior to the injection of cAMP, DcAMP and FD led to a marked reduction in the response of the Na efflux in fibers pre-treated with ouabain; (3) the response obtained by the injection of DcAMP and FD into ouabain-poisoned fibers pre-exposed to Zn was small but sustained; and (4) external application of Zn following peak stimulation by injecting DcAMP or FD led to reversal of this response. (Parallel experiments involving the injection of cAMP were not done, since the response following the onset of peak stimulation decays quite rapidly.) Taken together, these results support the hypothesis that Zn behaves as an inhibitor of the stimulatory response obtained by the injection of cAMP, DcAMP and FD.

Deregulation by zinc of the sodium efflux in barnacle muscle fibers.

Single muscle fibers from the barnacle Balanus nubilus were employed to study the behavior of the resting Na+ efflux toward external and internal application of zinc (Zn2+). This involved both unpoisoned and ouabain-poisoned fibers. The results obtained are as follows: (i) External application of Zn2+, e.g., 2 mM (a maximal dosage) in 10 mM Hepes-ASW (pH 7.3) causes a fall in the resting Na+ efflux which exceeds that caused by 10(-4) M ouabain in companion controls. (ii) The buffer of choice is found to be Hepes, rather than HCO3- or imidazole. (iii) The observed fall in the resting Na+ efflux caused by external application of Zn2+ is concentration-dependent, the IC50 being 10 microM. (iv) The inhibitory effect of Zn2+ is partially reversible; occasionally, however, reversibility is not seen. (v) The Zn(2+)-insensitive component of the Na+ efflux is reduced by 10(-4) M ouabain. (vi) The ouabain-insensitive component of the Na+ efflux is reduced by external application of Zn2+. This response is concentration-dependent. (vii) Preinjection of EGTA reduces the sensitivity of the Na+ efflux to external application of Zn2+. This is true of both unpoisoned and ouabain-poisoned fibers. (viii) (a) The resting Na+ efflux is reduced by injecting Zn2+. Ouabain application reduces the remaining Na+ efflux. (b) Injection of Zn2+ reduces the ouabain-insensitive component of the Na+ efflux. (c) External application of Zn2+ following the injection of Zn2+ reduces the remaining Na+ efflux. Ouabain is ineffective when applied after both maneuvers. (d) Injection of Zn2+ after its external application is without effect. Subsequent application of ouabain is also without effect. (e) Injection or external application of Zn2+ reduces the ouabain-insensitive Na+ efflux. Whereas in the former case subsequent external application of Zn2+ reduces the remaining Na+ efflux, in the latter case Zn2+ injection after external application of Zn2+ is ineffective. Collectively, these results provide evidence in support of the hypothesis that Zn2+ is a potent inhibitor of the ouabain-sensitive and ouabain-insensitive components of the Na+ efflux, and that the inhibitory effect is partly due to the entry of Zn2+ into the myoplasm. They also raise the possibility that the inhibitory effect caused by Zn2+ injection may be the result of Zn2+ leakage from the fiber interior.

Influence of pentachlorophenol on light emission from single barnacle muscle fibers preloaded with aequorin.

Experiments show that the resting ouabain-insensitive sodium efflux in giant fibers from the barnacle Balanus nubilus is stimulated by external application of pentachlorophenol (PCP). This work has now been extended to include a study of muscle fibers preloaded with the Ca2+ indicator aequorin to determine whether PCP is able to increase light emission; and whether its potency depends on the number of chlorine atoms and external pH. The results obtained are as follows: 1) PCP causes a dose-dependent, multiphasic rise in light emission; the threshold concentration in fibers not poisoned with ouabain was in the low micromolar range. 2) The efficacy of PCP is considerably greater than that of less-chlorinated phenols and phenol. 3) The response to PCP is a sigmoidal function of external pH both in unpoisoned and ouabain-poisoned fibers. Reducing external pH potentiates its efficacy. 4) The response to PCP depends on the external Ca2+ concentration, and the requirement for Ca2+ is usually absolute.

Concerning stimulation by injected fluoroaluminate of the sodium efflux in barnacle muscle fibers.

Single barnacle muscle fibers from Balanus nubilus were used primarily to examine the validity of two ideas: first, that the injection of KF stimulates the ouabain-insensitive Na+ efflux, and that this action is potentiated by adding AlCl3 (Al) in a low concentration to the solution of KF prior to injection. And second, that the injection of a KF-AlCl3 solution into ouabain-poisoned, K(+/-)-depolarized fibers elicits a stimulatory response resembling that obtained by injecting GTP. The results of this study are as follows: injection of 0.5 M KF into unpoisoned fibers causes a sustained rise in the resting Na+ efflux. However, injection of a 0.5 M KF, 10(-3) M AlCl3 solution leads to a reduced rather than an augmented response. Whereas injection of 0.5 M KF into ouabain-poisoned fibers elicits a marked stimulatory response, the injection of 0.5 M KF, 10(-3) M AlCl3 reduces the remaining Na+ efflux. Injection of KF-AlCl3 in equimolar concentrations, e.g., 0.25 M, elicits a response that is significantly larger than that obtained by injecting 0.25 M KF. A dose-response curve indicates that a 0.2 M solution of fluoroaluminate probably represents an optimal concentration. Injection of 0.3 M KF following peak stimulation by injecting 0.3 M AlCl3 completely reverses this response to Al. In sharp contrast, injection of a 0.3 M KF, 0.3 M AlCl3 mixture following peak stimulation by injecting 0.3 M AlCl3 is ineffective. Injection of KF into ouabain-poisoned, K+ depolarized fibers does not always cause sustained stimulation of the remaining Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

The reduction of the inhibitory effect of aluminum on Na+ efflux in barnacle muscles fibers by preinjecting phosphate.

The object of the present study was to test the hypothesis that the pre-enrichment of single muscle fibers from the barnacle Balanus nubilus with inorganic phosphate may protect the basal Na efflux from the inhibitory effect of Al injection. This approach was adopted in the light of evidence that the preinjection of ATP fails to stop the Na efflux in unpoisoned fibers from falling following the injection of Al. The results of the experiments are as follows: (i) Preinjection of K2HPO4 into unpoisoned fibers reduces the magnitude of the inhibitory effect on the basal Na efflux of injected Al in a dose-dependent manner but fails to completely stop it from occurring. (ii) Injection of K2HPO4 following Al into unpoisoned fibers fails to arrest or reverse the inhibitory effect of injected Al. (iii) Injection of K2HPO4 in a concentration as high as 0.5 M is without effect on the course of the basal Na efflux. (iv) Injection of K2HPO4 into ouabain-poisoned fibers fails to stop Al from stimulating the ouabain-insensitive Na efflux. Injection of K2HPO4 following peak stimulation by injecting Al is also without effect. (v) Injection of K2HPO4 in a concentration as high as 0.5 M is without effect on the course of the ouabain-insensitive Na efflux. Collectively, the results obtained with unpoisoned 'hypersensitive' fibers are consistent with the view that a significant fraction of the injected inorganic phosphate binds Al3+, and hence protects the basal Na efflux from the untoward action of Al3+.

Abolition with chloramine-T of inactivation in barnacle muscle fibers results in stimulation of the ouabain-insensitive sodium efflux.

The hypothesis that chloramine-T stimulates the basal Na+ efflux in barnacle fibers as the result of the entry of trigger Ca2+ into the myoplasm from the bathing medium was examined in this study. Two reasons for doing so can be given. One is that the oxidant is known to abolish inactivation in sodium and potassium channels. The other is that L-type Ca2+ channels are present in barnacle fibers, and an increase in internal free Ca2+ in these fibers is known to stimulate the Na+ efflux, particularly in ouabain-poisoned fibers. The results of the experiments are as follows: (i) Chloramine-T exerts a biphasic effect on the Na+ efflux: inhibition is followed by stimulation, the threshold concentration being 10(-5) M. This is also found to be the threshold concentration for shortening of these fibers. (ii) The kinetics of the inhibitory effect resemble those of ouabain. (iii) Ouabain is without effect on the stimulatory phase caused by chloramine-T. (iv) Application of chloramine-T after the full effect of 10(-4) M-ouabain is reached elicits solely a stimulatory response. (v) The dose-response curves for the stimulatory action of chloramine-T in unpoisoned and ouabain-poisoned fibers are alike except that the threshold concentration is less than 10(-5) M in poisoned fibers. (vi) Basal light emission from unpoisoned and ouabain-poisoned fibers loaded with the photoprotein, aequorin, some 60 min beforehand increases as soon as they are exposed to 10(-4) M chloramine-T. The response recorded in unpoisoned fibers is monophasic and usually transitory, whereas it is multiphasic and usually sustained in ouabain-poisoned fibers. (vii) The dose-response curve for chloramine-T shows a shift to the left in poisoned fibers. (viii) The magnitude of the rise in light emission depends on the external Ca2+ concentration. A rise fails to take place in the nominal absence of external Ca2+. Taken together, these results support the above hypothesis that chloramine-T causes the entry of trigger Ca2+ into the myoplasm from the outside and provide evidence that stimulation of the Na+ efflux is associated not only with this event but also with a reduced Na+ gradient resulting from inhibition of the membrane Na+/K(+)-ATPase system by the oxidant. Thus, the suggestion put forward is that this oxidant promotes reverse Na+/Ca2+ exchange and is able to exert multiple effects on membrane transport.

Citrate as an aluminum chelator and positive effector of the sodium efflux in single barnacle muscle fibers.

The injection of citrate produces considerably greater stimulation of the Na efflux in ouabain-poisoned fibers (from the barnacle Balanus nubilus) than in unpoisoned fibers. When injected in excess together with Al into unpoisoned fibers it is without effect. Its injection is also without effect on the decline in the Na efflux elicited by injecting Al beforehand. Citrate injection into ouabain-poisoned fibers following peak stimulation by injecting Al produces a further rise which is a function of the Al concentration. Al injection after peak stimulation of the ouabain-insensitive Na efflux by citrate is usually without significant effect. Injection of aspartate into poisoned fibers causes a small rise in the remaining Na efflux and fails to prevent the response to Al injection from occurring. Taken together, these observations are in keeping with the view that citrate is not only a powerful chelator of Al but also a powerful activator of reverse Na+/Ca2+ exchange in ouabain-poisoned fibers, presumably because of its ability to raise myoplasmic pMg.

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 muscle fibers from the barnacle Balanus nubilus to see whether D-Ins(1,4,5)P3 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)P3 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)P3[S]3, analog following its injection in a concentration of 0.5 microM. 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)P3 influences the behavior of the Na(+)-Ca2+ exchanger when operating in the reverse mode, experiments were done to check this possibility. ATPNa2 which is though 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 key 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)P3[S]3.

The ability and inability of ATP to stop aluminum from reducing the sodium efflux in unpoisoned barnacle muscle fibers.

A study has been made in single barnacle muscle fibers with the object of determining whether ATP is able to protect the resting Na efflux from the effects of injected aluminum (Al) and whether Al is able to reduce or abolish the stimulatory action of ATP on the efflux. The results of the experiments show that neither ATPMg nor ATPNa2 preinjection stops Al from reducing the basal Na efflux in unpoisoned fibers which undergo a large fall (hypersensitive fibers). Preinjection of Al into such fibers reduces or abolishes the stimulatory response of the Na efflux to ATP injection. In less hypersensitive fibers, however, ATPMg is protective. This is also true of ATPNa2 preinjection in both classes of fibers showing stimulation. Injection of a mixture of AlCl3-ATPNa2 into unpoisoned fibers causes less inhibition than AlCl3 injection. The hypothesis that both ATPMg and ATPNa2 are protective is also supported by the results obtained with ouabain-poisoned fibers: (i) Al injection after ATP fails to reverse the stimulatory response to ATP, while ATP injection after Al exerts only a small or no effect. (ii) Mg2+ injection fails to reverse the stimulatory response to Al injection in poisoned fibers. And (iii) Anti-proteolysis agents e.g. leupeptin and pepstatin, upon preinjection, do not alter the kinetic results obtained by injecting Al into unpoisoned and ouabain-poisoned fibers.

ATP as a positive effector of the sodium efflux in single barnacle muscle fibers.

A study has been made of the mechanism by which the injection of ATPNa2 stimulates the ouabain-insensitive Na efflux in fibers from the barnacle, Balanus nubilus. The results of this study are as follows: ATPNa2 is found to be a more potent effector of the Na efflux in unpoisoned fibers than ATPMg on an equimolar basis, but not more potent than ADPNa2. In ouabain-poisoned fibers ATPNa2 and ATPMg are equipotent but the former is more potent than ADPNa2. The magnitude of the response to ATPNa2 injection into ouabain-poisoned fibers depends on: (i) the ouabain concentration used; (ii) the concentration of ATPNa2 injected, and (iii) the external Ca2+ concentration. Ouabain is without effect when it is applied at the time of ATPNa2 injection. Responsiveness to ouabain, however, is found to return if the glycoside is applied after complete decay of the response to ATP. Under these conditions, the effect of ouabain in fibers injected with ATPNa2 is significantly less than in fibers injected with ATPMg. Preinjection of EGTA in high concentrations fails to reduce the size of the response to ATPNa2 injection. Injection of Mg2+ following peak stimulation by ATP almost completely reverses the response. The response to Mg2+ is concentration-dependent. Ryanodine but not neomycin reduces the response to ATP. ATP gamma S is not as effective as ATPNa2. Nor is AMP-PNP consistently as effective as ATPNa2. Collectively, these results support the hypothesis that the response of the Na efflux to ATPNa2 injection involves the operation of the putative Na(+)-Ca2+ exchanger in the reverse mode and that a raised Cai2+ is not an absolute requirement. They also strongly suggest that two other governing factors are the Na+ gradient across the sarcolemma and the myoplasmic pMg. Mg2+ seems to act as an inhibitor.

An investigation of the sensitivity of the ouabain-insensitive sodium efflux in single barnacle muscle fibers to pentachlorophenol.

The aim of the present work was to explore the possibility that pentachlorophenol (PCP) influences the behavior of the resting Na efflux in single muscle fibers from the barnacle, Balanus nubilus. It is shown here that PCP causes a transitory rise in the Na efflux in both unpoisoned and ouabain-poisoned fibers and that the response is dose-dependent, the minimal effective concentration in ouabain treated fibers being less than 10(-6) M. The efficacy of PCP is significantly greater than that of 2,3,4-trichlorophenol. 2,3-Dichlorophenol is ineffective. This is also the case with phenol. The magnitude of the response to PCP is a function of external pH. Lowering pHe increases the response. The response has an absolute requirement for external Ca2+ and is a sigmoidal function of external Ca2+ concentration. Since treatment of these fibers with PCP in high concentration leads to prompt contraction, experiments were designed to determine whether the observed rise in ouabain-insensitive Na efflux is due to a fall in myoplasmic pCa and whether trigger Ca2+ originates from the bathing medium. The results obtained show that prior injection of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA) leads to a drastic reduction in the response to PCP. They also show that prior external application of verapamil or devapamil stops the response to PCP from occurring. Both Cd2+ and Co2+ are also effective but only temporarily. Last, the effects of ryanodine and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were tested, since the former is known to block the sarcoplasmic reticulum Ca2+ release channel, and the latter to impair the action of agents known to release Ca2+ from internal depots. Both ryanodine and TMB-8 are found to reduce the response to PCP. Taken together, these observations support the hypothesis that PCP stimulates the ouabain-insensitive Na efflux by increasing the internal free Ca2+ and that the increase in internal Ca2+ is due to the entry of trigger Ca2+ from the outside via Ca2+ channels, as well as release of Ca2+ by the sarcoplasmic reticulum via its channel. They also indicate that the efficacy of PCP depends on the 5 Cl atoms present in its aromatic ring and pHe.

Protection by GTP from the effects of aluminum on the sodium efflux in barnacle muscle fibers.

The idea that guanine nucleotides act as chelators of Al3+ and that Al interrupts the mechanism by which GTP or Gpp(NH)p stimulates the Na efflux in single muscle fibers from the barnacle Balanus nubilus has been tested. As a rule, injection of GTP or Gpp(NH)p into unpoisoned and ouabain-poisoned fibers produces a rise in the 22Na efflux that is usually transitory in nature. Fibers preinjected with GTP show a fall in the Na efflux following the injection of AlCl3 in an equimolar concentration. If, however, the concentration of Al for injection is halved, then GTP is found to be fully protective. Fibers preinjected with AlCl3 show little or no response to the injection of GTP. This is also the case with ouabain-poisoned fibers. Ouabain-poisoned fibers preinjected with GTP also show little or no response to the injection of AlCl3. The stimulatory response to the injection of AlCl3 into fibers preinjected with 0.5 M GTP is dose-dependent. A graded response is also found when 0.5 M AlCl3 is injected into fibers preinjected with GTP in varying concentrations. Gpp(NH)p is fully protective against the inhibitory effect of Al injection in unpoisoned fibers. Further, Gpp(NH)p abolishes the biphasic effect of Al injection on the ouabain-insensitive Na efflux. To strengthen the argument that GTP acts as a chelator of Al, a solution mixture of 0.5 M GTP/0.5 M AlCl3 (pH 1-2) was injected into unpoisoned fibers. This is found to lead to a smaller fall in the resting Na efflux than that obtained by injecting AlCl3 alone or injecting AlCl3 after GTP. It is thus quite clear that the barnacle muscle fiber is a useful preparation for studies of this type.

The behavior of the ouabain-insensitive sodium efflux in single barnacle muscle fibers toward the microinjection of aluminum.

Recent work with single muscle fibers from the barnacle Balanus nubilus has shown that the injection of Al into these fibers leads to inhibition of the resting Na efflux and that this involves both the ouabain-sensitive and the ouabain-insensitive components of the efflux. This work also showed that the injection of Al into ouabain-poisoned fibers often leads to a rise in the remaining Na efflux. This observation suggested the possibility that Al is able to stimulate the ouabain-insensitive Na by increasing myoplasmic free Ca2+ and that trigger Ca2+ reaches the myoplasm via voltage-dependent Ca2+ channels. The results obtained are as follows: (i) The injection of AlCl3 into fibers poisoned with ouabain produces a biphasic or monophasic effect on the remaining Na efflux. That is, stimulation is followed by inhibition, or there is only stimulation. (ii) This response is dose-dependent and is seen to take place following the injection of Al in a concentration as low as 0.01 M. (iii) Both Ga3+ and Sc3+ are able to mimic the effect of Al. (iv) Injection of EGTA following peak stimulation of the ouabain-insensitive Na efflux by injected AlCl3 leads to reversal of this response. If, however, EGTA is injected long after the onset of peak stimulation and after ouabain reaches its maximum effect, it is found to be ineffective. (v) The magnitude of the stimulatory response of the ouabain-insensitive Na efflux is a sigmoidal function of external Ca2+ and is almost completely abolished by verapamil, devapamil, and Cd2+ but is unaffected by injecting Mg2+ before or after AlCl3. (vi) Whereas preinjection of Al reduces the response to ryanodine, external preapplication of ryanodine fails to alter the response to Al. The preinjection of deferoxamine (a potent chelator of Al) fails to stop the stimulatory response to Al injection from occurring. Taken together, these findings support the hypothesis that the stimulatory response elicited by Al injection is due to a fall in myoplasmic pCa resulting from activation of voltage-dependent Ca2+ channels and that it involves the operation of the Na+-Ca2+ exchanger in the reverse mode.

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)

Sensitivity of the sodium efflux in single barnacle muscle fibers to the microinjection of aluminum.

A study has been made of the behavior of the Na efflux in single muscle fibers from the barnacle, Balanus nubilus, toward the microinjection of AlCl3. The effect of microinjecting AlCl3 is either biphasic with inhibition following transitory stimulation, or monophasic with inhibition occurring promptly and taking about 1 hr to reach a maximum. The magnitude of the inhibition is dose dependent and the minimally effective concentration is 10 mM. This is diluted by the myoplasm by a factor of roughly 100. Fibers injected with 1 M AlCl3 are found to be insensitive to 10(-4) M ouabain, whereas fibers injected with 0.5 M AlCl3 show a further fall in Na efflux, which is smaller than that seen in control fibers. The response of the ouabain-insensitive Na efflux to the injection of 0.5 M AlCl3 is biphasic: stimulation is followed by inhibition. The stimulatory phase is largely dependent on the presence of external Ca2+ and is reversed by the sudden omission of Ca2+ from the bathing medium. Fibers also injected with 0.5 M AlCl3 show a marked reduction in the response of the Na efflux to high external K. Injection of ATPMg before or after the injection of AlCl3 fails in most instances to abolish or reverse the inhibitory effect. By contrast, fibers preinjected with deferoxamine show little or no effect following the injection of AlCl3. However, external application of deferoxamine is ineffective.

Further observations on the behaviour of ouabain-insensitive sodium efflux towards proctolin in barnacle muscle fibres.

1. A further study has been made of the stimulatory action of proctolin on the ouabain-insensitive Na+ efflux in single muscle fibres from the barnacle, Balanus nubilus. 2. (i) Strontium (Sr2+) behaves as a substitute for external Ca2+. In this case, however, the response to proctolin fails to decay. (ii) Injection of Sr2+ stimulates the ouabain-insensitive Na+ efflux. This effect is mimicked by injecting Ca2+. 3. Depolarization of the fibre membrane with 30 and 100 mM-external K+ augments the response to proctolin. 4. Pre-injection of GTP or Gpp(NH)p (sodium 5-guanylylimidodiphosphate) prevents the response to proctolin from completely decaying. 5. Pre-injection of guanine nucleotides in conjunction with membrane depolarization stops the response to proctolin from decaying. 6. Measurements of Em before and during treatment with proctolin indicate a prompt but small and reversible fall in the membrane potential. 7. (i) The aequorin response of fibres pre-treated with ouabain to proctolin is monophasic or multiphasic, and concentration dependent, the minimal effective concentration being in the nanomolar range. (ii) The duration of these signals is usually less than 5 min; this is about half the time it takes for the stimulated Na+ efflux to reach a maximum. (iii) The aequorin response to proctolin occurs quite often in fibres suspended in nominally Ca2(+)-free artificial sea water. (iv) Sudden graded elevations in external K+ following complete decay of the aequorin response to proctolin are rapidly followed by stepwise transitory increments in light emission. (v) The aequorin response to 100 mM-external K+ is frequently a triplet. 8. (i) Together, these results are in line with the view that the action of proctolin on the ouabain-insensitive Na+ efflux is the result of a temporary fall in internal pCa and that its point of action is the Ca2+ channel, where a putative G protein in the presence of GTP or Gpp(NH)p is able to maintain constancy of the hormonal effect. (ii). They strengthen the argument that the barnacle muscle fibre as a preparation is especially suitable for studies of this kind.

Nicardipine as a Ca2+ channel blocker in single barnacle muscle fibers.

A study has been made of the efficacy of nicardipine as a Ca2+ channel blocker by determining the magnitude of its effect on the stimulatory response of the ouabain-insensitive Na+ efflux in single barnacle muscle fibers to 100 mM external K+. The results show that nicardipine (at pH 6.5) is a potent inhibitor, the minimal effective concentration being approx. 10(-7) M and the IC(50) about 5.10(-6) M. Nicardipine, however, is not as potent as verapamil (at pH 6.5) on an equimolar basis. This is explained by assuming that the number of dihydropyridine receptors in the t-tubule membranes of barnacle fibers is not high or that verapamil is able to block the sarcoplasmic reticulum Ca2+ release channel in addition to the voltage-dependent Ca2+ channels.

Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate as an antagonist of the stimulatory action of cyclic AMP on the ouabain-insensitive Na efflux in single barnacle muscle fibers.

Single muscle fibers of the barnacle Balanus nubilus have been used as a preparation to test the possibility that the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate, which is the first available analog of cAMP that acts as an antagonist of cAMP, may reduce the magnitude of cAMP-mediated stimulation of the resting ouabain-insensitive Na efflux. The results obtained show that this antagonist is, in fact, able to reduce stimulation of the Na efflux by injected cAMP in a dose-dependent manner.

Urethane as an inhibitor of the firefly light reaction.

A study has been made to test the hypothesis that general anesthetics such as urethane are able to inhibit light from a firefly reaction mixture. Urethane was found to reduce light emission in a dose-dependent manner, the minimal effective concentration being about 20 mM. Dixon plots gave a Ki value in the range of 175 to 215 mM. Lineweaver-Burk plots showed that urethane increases the apparent Km for ATP and reduces Vmax for the reaction. This is taken to mean that urethane acts as both a competitive and non-competitive inhibitor of the firefly light reaction (mixed-type inhibition).

Microinjection of synthetic protein kinase inhibitor into single barnacle muscle fibers before and after cyclic AMP.

Single muscle fibers from the barnacle Balanus nubilus have been used as a preparation to see if a synthetic 20-residue PKI (5-24)-peptide is able to block or reverse the stimulatory response of the ouabain-insensitive Na efflux to injected cAMP. The results obtained show that this peptide behaves as a powerful inhibitor of the cAMP-mediated response and is also able to partially reverse the sustained stimulation of the Na efflux observed in ouabain-poisoned fibers following the injection of subunit A of cholera toxin.