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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. Images Figure 1. Figure 2. Figure 3. A Figure 3. B Figure 4. Figure 5. Figure 6. Figure 7.

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 K 2 HPO 4 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 occuring. (ii) Injection of K 2 HPO 4 following Al into unpoisoned fibers fails to arrest or reverse the inhibitory effect of injected Al. (iii) Injection of K 2 HPO 4 in a concentration as high as 0.5 M is without effect on the course of the basal Na efflux. (iv) Injection of K 2 HPO 4 into ouabain-poisoned fibers fails to stop Al from stimulating the ouabain-insensitive Na efflux. Injection of K 2 HPO 4 following peak stimulation by injecting Al is also without effect. (v) Injection of K 2 HPO 4 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 Al 3+ , and hence protects the basal Na efflux from the untoward action of Al 3+ .

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 .

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)

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.

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.

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. But injection of KF-AlCl3 in equimolar concentration always seems to cause a delayed sustained stimulatory response. Sustained stimulation is also observed after injecting Na2GTP. Although these results provide evidence in support of the hypothesis that the primary point of action of equimolar KF-AlCl3 solutions following injection into barnacle fibers is the membrane adenylate cyclase system, they raise doubts about the validity of the concept that trace amounts of Al are required for KF to act as a positive effector of this system.

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.

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.

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.

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 has been made of the behavior of the Na efflux in single muscle fibers from the barnacle, Balanus nubilus , toward the microinjection of AlCl 3 . The effect of microinjecting AlCl 3 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 m m . This is diluted by the myoplasm by a factor of roughly 100. Fibers injected with 1 m AlCl 3 are found to be insensitive to 10 −4 m ouabain, whereas fibers injected with 0.5 m AlCl 3 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 AlCl 3 is biphasic: stimulation is followed by inhibition. The stimulatory phase is largely dependent on the presence of external Ca 2+ and is reversed by the sudden omission of Ca 2+ from the bathing medium. Fibers also injected with 0.5 m AlCl 3 show a marked reduction in the response of the Na efflux to high external K. Injection of ATPMg before or after the injection of AlCl 3 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 AlCl 3 . However, external application of deferoxamine is ineffective.

1. The efflux of sodium in ouabain-poisoned barnacle muscle fibers is markedly stimulated by injecting ATPNa2 but not by ATPMg. This response is partly or completely reversed by injecting the protein inhibitor of Walsh. Injection of EGTA following ATPNa2 partially reverses the stimulatory response; EGTA followed by protein inhibitor completely reverse together the response but only temporarily. Preinjection of EGTA enhances the response to an injection of ATPNa2. The enhanced response is almost completely reversed by an injection of protein inhibitor. 2. Stimulation of the ouabain-insensitive Na efflux by acidification is completely reversed by injecting the protein inhibitor. It is also almost completely reversed following injection of 1–2 M-solutions of KHCO3. However, injection of KHCO3 fails to modify the response produced by injecting ATPNa2 or the partial reversal produced by injecting protein inhibitor. 3. Benzolamide (5×10−4 M) almost completely abolishes the stimulation caused by external acidification. Its action is dose-dependent, 38% inhibition being observed with a 10−7 M-solution.

1. A study has been made of the action of 5-hydroxytryptamine (5-HT) on the radio-sodium efflux from single barnacle muscle fibres. 2. (i) Stimulation of the Na efflux by external application of 5-HT is seen in both unpoisoned and ouabain-poisoned fibres, (ii) Concentrations of 5-HT as low as 10−9 M are effective, (iii) Characteristically, the response to 5-HT is prompt in onset, reaches a peak within 20 min and then decays rather rapidly. 3. Fibres from certain barnacle specimens are sometimes unresponsive to 5-HT. Such fibres, however, can be rendered responsive by preinjecting into them the non-hydrolysable GTP analogue, Gpp(NH)p. 4. The response of the ouabain-insensitive Na efflux to 5-HT depends on external Ca2+ and, to a certain extent, on external Na+. 5. (i) The response to 5-HT is unaffected by prior external application of Ca2+ antagonists, viz. verapamil, Cd2+ and WB-4101. (ii) The calmodulin antagonist, trifluoperazine (10−5 M), completely abolishes the response to 5-HT, even in fibres preinjected with Gpp(NH)p. (iii) Diphenylhydantoin is less effective than trifluoperazine (TFP). 6. Whereas the receptor antagonist methysergide is ineffective, cyproheptadine is very effective. 7. (i) Prior application of the phosphodiesterase inhibitor 1-propyl-3-methyl-7-(5-hydroxyhexyl)-xanthine (PMX) or the inhibitor 1-isoamyl-3-isobutyl-xanthine (IAX) augments the size of the response to 5-HT, but fails to stop the response from decaying, (ii) Augmentation of the response to 5-HT by IAX is seen despite the presence of 10−5M-TFP. 8. Prior injection of Mg2+ or protein kinase inhibitor (PKI) leads to abolition or reduction of the response to 5-HT. 9. These results demonstrate that barnacle fibres are a useful preparation for investigating the natriferic action of 5-HT. They also support the view that the response to 5-HT involves a receptor-adenylate cyclase complex and is the result of activation by newly formed cAMP of cAMP-dependent protein kinase.

This mini-review attempts to summarize information about the efflux of 22Na from single barnacle muscle fibres, based on the use of the microinjection technique. The view is put forward that the Na efflux consists of three components: an ouabain-sensitive component, an ouabain-insensitive component (representing secondary active transport), and an Na-Na exchange diffusion component. Evidence is brought forward which supports the view that the ouabain-insensitive Na efflux is divisible operationally into 3 phases: (i) the cyclic nucleotide-sensitive phase, (ii) the Cai-sensitive phase, and (iii) the pHe-sensitive phase. It is shown how the barnacle muscle fibre preparation has yielded information about the validity of the cAMP-protein kinase hypothesis and how it can be used to shed some light on the post-translational mechanism of aldosterone action.

Radiosodium efflux from barnacle muscle fibers is a function of pH e , the threshold pH e for stimulation of Na efflux into HCO 3 − -artificial sea water (ASW) being 6.8 and the ‘fixed’ thresholdpCO2 (in an open CO2 system) being approximately 30 mm Hg. Acidification of ASW containing non-HCO 3 − buffer is without effect on the Na efflux. The Na efflux following stimulation by reducing the pH of 10mM HCO 3 − -ASW from 7.8 to 6.3 is reduced by 17.3% as the result of microinjecting 100mM EGTA, and increased by 32.6% as the result of microinjecting 0.5M ATP. The Na efflux into K-free HCO 3 − -ASW is markedly stimulated by external acidification. Ouabain-poisoned fibers are more responsive to a low pH e than unpoisoned fibers. Applying the 2-14C-DMO technique, it is found that fibers bathed in 10mM HCO 3 − -ASW at pH 7.8 have an internal pH of 7.09±0.106 (mean±SD), whereas fibers bathed in 25mM TRIS-ASW at pH 7.8 have a pH i of 7.28±0.112. The relationship between pH i and pH e as external pH is varied by adding H+ is linear. Measurements of the resting membrane potential indicate that external acidification in the presence of HCO 3 − as buffer is accompanied by a fall inE m , the threshold pH e being 7.3 both at 24 and 0°C. This sensitivity amounts to 8.2 mV per pH unit (at 24°C) over a wide range of pH e . Membrane resistance following external acidification remains unchanged. Microinjection of the protein inhibitor of Walsh before external acidification fails to stop depolarization from occurring. Cooling to 0°C also fails to abolish depolarization following acidification. Whereas external ouabain and ethacrynic acid reduceE m in the absence or presence of acidification, DPH hyperpolarizes the membrane or arrests depolarization both at 24 and 0°C. This effect of DPH at 0°C is seen in the absence or presence of acidification. It is suggested that depolarization following acidification of a HCO 3 − -containing medium is due to activation of a Cl−-and/or HCO 3 − -pump and that ouabain and ethacrynic acid reducesE m by abolishing uncoupled Na transport.

1. 1. The efflux of 22 Na in ouabain-poisoned barnacle muscle fibres and its transitory response to injected cAMP has been studied by using a new xanthine derivative, 1-propyl-3-methyl-7-(5-hydroxy-hexyl)-xanthine (PMX). 2. 2. Injection of PMX prior to cAMP fails to significantly alter the behaviour of the ouabain-insensitive Na efflux towards the nucleotide. By contrast, injection of PMX following peak stimulation by injected cAMP stops the rate constant for 22 Na efflux from falling. This effect of PMX is not mimicked by injected HEPES. 3. 3(a). Injection of Mg 2+ following PMX brings about almost complete reversal of the sustained stimulatory response, (b). Injection of trace metals, e.g. Fe and Zn, following PMX brings about complete reversal of the sustained stimulatory response, (c). Injection of R I or R II subunits following PMX brings about partial reversal of the sustained stimulatory response. 4. 4. Partial reversal is also seen with externally applied imipramine (50 μM). 5. 5. These results support the view that the transitory nature of the response of the ouabain-insensitive Na efflux to injected cAMP is due to high phosphodiesterase activity in these fibres and that the major portion of the response itself is due to activation by cAMP of cAMP-dependent protein kinase.

1. A study has been made of the effect of microinjected cholera toxin (CT) on the Na efflux in single barnacle muscle fibers. Characteristically, injected CT causes sustained stimulation of the ouabain-insensitive Na efflux but only after a lag phase. An effect is seen with as little as a 10−7 M-solution of CT. Sustained stimulation after a lag phase is also seen following injection of subunit A fragment. 2. Enrichment of fibers with NAD+ fails to enhance the response to CT. 3. Prior injection of GTP or its non-hydrolyzeable analogue, Gpp(NH)p, markedly reduces the response to CT, whilst prior injection of CT reduces the response to guanine nucleotides. 4. Evidence is also brought forward that omission of external Ca2+ reversibly reduces the response to CT and that pre- or postinjection of EGTA markedly reduces the response to CT. In addition, fibers preinjected with CT show increased aequorin light emission. 5. Whereas verapamil and Cd2+ are ineffective, both Mg2+ and trace metals, e.g. Fe and Zn, reverse the response to CT following injection. 6. Prior injection of protein kirtase inhibitor reduces the response to CT. 7. As for calmodulin inhibitors, e.g. chlorpromazine, imipramine and mepacrine, they are effective in reducing the response to CT but not calmodulin antibody (IgG). 8. Collectively, the above results are compatible with the view that sustained stimulation of the ouabain-insensitive Na efflux by injected CT is due to persistent activation of adenylate cyclase by the toxin and that a fall in myoplasmic pCa facilitates or augments this activation mechanism.

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposcd to aldosterone. 2. The response to injected Gpp(NH)p is not only greater in size than in unexposed fibres but also sustained. 3. Injection of MgCl2 following peak stimulation causes a partial reversal of the response. 4. Injection of ATPNa2 (and 5′-App(NH(p) leads to a sustained stimulatory response which is not significantly greater than that seen in unexposed fibres. 5. MgCl2 injection causes complete reversal of this response. 6. The response of preexposed fibres to injected CaCl2 in varying concentration and to injected cholera toxin is not significantly different from that seen in unexposed fibres. 7. This is also true of Gpp(NH)p when it is injected after peak stimulation by cholera toxin. 8. Prior application of verapamil (10−4 M) drastically reduces the response to injected Gpp(NH)p. 9. The residual response is sustained but markedly reduced by injected Mg2+. Fe or Zn. 10. Injection of PKI following Gpp(NH)p reduces the response, provided PK.I is also injected before Gpp(NH)p. By contrast, injection of Rll subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine. when applied externally (5 × 10−5 M), cause almost complete reversal of the response. 12. The suggestion is made that the response to injected Gpp(NH)p is mainly due to activation of Ca2+ -channels resulting in activation of the calmodulin/Ca-dependent form of adenylate cyclase and that the primary site of aldosterone action is at the level of the calmodulin form of adenylate cyclase.

1. The effects of graded concentrations of caffeine on the Na efflux were investigated. External application of 10 mM caffeine usually caused a biphasic response, viz. a fall, followed by a rise in the Na efflux. 1 and 5 mM caffeine usually caused stimulation. Only the stimulatory phase of this response depended on the presence of external Ca(2+).2. Internal application of 100 mM caffeine caused a small rise in the Na efflux, the magnitude of which was independent of external Ca(2+) and comparable to that obtained with external application of 1 mM caffeine. This action, however, could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain.3. The rise in Na efflux caused by external application of 10 mM caffeine could be greatly augmented by pre-treating the fibre with 5 x 10(-5)M ouabain. The observed stimulatory response was biphasic, more so in the absence of external Ca(2+). Restoration of external Ca(2+) following the onset of the second stimulatory phase resulted in further rise of the Na efflux. Measurements of the Na efflux during treatment with graded concentrations of ouabain and 10 mM caffeine showed that the rate coefficient for Na efflux varied with the ouabain concentration in the range 10(-8)-10(-4)M. Measurements of the ouabain-insensitive Na efflux before and during treatment with 10 mM caffeine in bathing media containing varying concentrations of Ca, disclosed the existence of two Ca(2+)-thresholds, one in the 0-2.5 mM range and the other in the 12.5-15 mM range.4. Comparisons were made between the effects on the Na efflux of 10 mM caffeine followed by external acidification, and external acidification, followed by 10 mM caffeine. The magnitude of the response of the ouabain-insensitive Na efflux to external acidification before treatment with 10 mM caffeine was greater than that found when external acidification followed external application of the alkaloid. It also was considerably greater than that of the response to external application of 10 mM caffeine before external acidification.5. External application of 10 mM procaine prevented 10 mM caffeine from stimulating the Na efflux, and from inducing contractures. Internal application of 100 mM-EGTA reduced the response of the Na efflux to 10 mM caffeine, and also prevented the fibre from contracting. External application of 10(-4)M diphenylhydantoin reduced the response of the Na efflux to 10 mM caffeine but failed to prevent the development of contractures.6. Internal application of 0.05 M-cGMP, cAMP or its dibutyryl derivative caused a large rise in the Na efflux. The magnitude of the effects observed in ouabain-poisoned fibres was often greater than that in unpoisoned fibres. Internal application of 2.5 units/ml. phosphodiesterase beforehand failed to reduce the magnitude of the stimulatory response to injected cyclic nucleotides. Injected phosphodiesterase also failed to reduce the response of the Na efflux to 10 mM caffeine.7. External application of 10 mM caffeine to unpoisoned and ouabain-poisoned fibres caused a fall of approximately 10 mV in the membrane potential. In unpoisoned fibres this effect was transitory. The response of the membrane potential to internal application of graded concentrations of CaCl(2) was biphasic. When low concentrations of CaCl(2) were used the membrane potential underwent a small rise but when high concentrations were used the opposite was found. These results could not be repeated with graded concentrations of MgCl(2).8. The effects of graded concentrations of caffeine on tension development were also studied. Strong contractures were observed with caffeine concentrations as low as 4 mM, while peak tetanus tension was usually exceeded with 7-8 mM concentrations. The tension-external Ca(2+) curve was sigmoidal in shape.9. Electron microscopic studies showed that 10 mM caffeine in ASW caused little or no distension and disorganization of cisternal fine structure. Such structural changes, however, were far more pronounced in fibres suspended in Ca(2+)-free ASW and then treated with 10 mM caffeine in Ca(2+)-free ASW. Fibres soaked in Ca(2+)-free ASW had ruptured mitochondria and mitoplasts, whereas those additionally treated with 10 mM caffeine had relatively intact mitochondria.10. The main conclusions drawn from this work are: (i) that caffeine stimulates the ouabain-insensitive Na efflux (and inhibits the transport enzyme) by raising the internal free Ca(2+) concentration; (ii) that in the presence of inhibition of the transport enzyme, the magnitude of the stimulatory response to 10 mM caffeine depends not only on the external Ca(2+) concentration but mainly on the residual level of activity of the transport enzyme; (iii) that the Ca(2+)-sensitive and CO(2)-sensitive components of the ouabain-insensitive Na efflux, though not the same, may overlap at the level of the plasma membrane or share a common metabolic step away from the membrane; (iv) that cyclic nucleotides participate in the control of the magnitude of the ouabain-insensitive Na efflux, and that the phosphodiesterase system under the present experimental conditions does not seem to be involved in the mechanism underlying the stimulatory action of caffeine; (v) that the membrane potential changes caused by caffeine fail to explain the stimulatory response of the Na efflux, as well as contracture of these fibres; (vi) that the contractile machinery in these fibres is considerably more sensitive to caffeine than that in vertebrate muscle and the muscles of other arthropods, and (vii) that the Ca(2+) released by the sarcoplasmic reticulum which leads to a biphasic response of the Na efflux is the result of cisternal disorganization caused by caffeine.

1. Flash height recorded following the injection of firefly into the external calibration medium depends on the concentration of K-glutamate used, e.g. 120 mM K glutamate reduces flash height by approximately 20 percent. 2. Suspending the fibre in air instead of artificial sea water (ASW) or replacement of NaCl in the bathing medium with Na-glutamate fails to alter flash height. 3. Firefly preparations from DuPont, Packard and SAI give similar myoplasmic ATPMg values viz. 1.1 mM. 4. Analysis of 36 fibres shows the following: myoplasmic ATP = 1.03 +/- 0.06 mM; total ATP (firefly method) = 5.26 +/- 0.12 mmol/kg water; total ATP (enzymic fluorimetry) = 6.27 +/- 0.13 mmol/kg water and ArP = 20.76 +/- 0.59 mmol/kg water. 5. Measurement of ATPMg in samples of myoplasmic aspirate gives a value that is greater than that obtained in situ. 6. Iodoacetate, whether applied externally or internally, reduces resting luminescence in a dose-dependent manner. It also reduces myoplasmic ATP and total ATP. 7. 2-Deoxy-d-glucose fails to reduce myoplasmic ATP but reduces total ATP. 8. Diethylpyrocarbonate, whether applied externally or internally, reduces myoplasmic ATP. It also causes a slow decline in ArP but little change in total ATP. 9. Injection of L-arginine causes a fall in resting luminescence in some fibres while in others it causes a prompt transitory rise. Injection of L-arginine also causes a fall in total ATP. 10. Collectively, these results suggest that the immediate buffering system in the myoplasm is ArP and that ATP supplied by glycolysis lies in a compartment, presumably the interfibrillar space, which is inaccessible to injected firefly.

A study has been made of the mechanism underlying stimulation of the Na efflux in barnacle muscle fibers by protonation of a HCO3-containing bathing medium and by microinjecting Ca2+. This became possible as a result of the availability of the cAMP-dependent protein kinase inhibitor in the lyophilized form. The results obtained are as follows: Injection of the protein kinase inhibitor of Walsh (free of EDTA and phosphate) causes a biphasic effect on the Na efflux: inhibition is followed by stimulation. Omission of external Ca2+ before injecting the protein inhibitor results in abolition of the biphasic response. The delayed stimulation observed in the presence of external Ca2+ is largely abolished by injecting EGTA. Injection of the protein inhibitor causes complete abolition of the stimulatory response of the Na efflux to external acidification. Ouabainpoisoned fibers injected with graded amounts of CaCl2 show a stimulatory response to as little as a 10−6 M-solution. Injection of 0.03 M-protein inhibitor completely reverses the stimulation of the ouabain-insensitive Na efflux caused by injection of 0.1 M-CaCl2. Fibers allowed to soak in Ca2+-free ASW for a short period of time show a marked rise in the ouabain-insensitive Na efflux on restoring external Ca2+ provided the fibers are injected with protein inhibitor beforehand. Injection of 0.1 M-CaCl2 fails to modify the stimulated efflux. Fibers bathed in Ca2+-free ASW for a short period of time show a marked rise in the ouabain-insensitive Na efflux, not following restoration of external Ca2+, but following injection of 0.1 M-CaCl2. This effect is completely reversed by injecting 0.03 M-protein inhibitor. The above results are compatible with the view that a fraction of the ouabain-insensitive Na efflux is modulated by myoplasmic cAMP and that external acidification causes stimulation as the result of activation of protein kinase by newly formed cAMP. They are also compatible with the view that the protein inhibitor of Walsh may act not only as an inhibitor of the cAMP-dependent protein kinase reaction but also as a feedback regulator of the membrane adenyl cyclase system or that the preparation of Walsh may contain an additional substance that has the ability to act as an adenyl cyclase inhibitor.

A study has been made of the efflux of radiosodium in single barnacle muscle fibers cooled to 0°C. Cooling from 24 to 0°C results in a rapid fall in the Na efflux, the magnitude of which averages 81%. Rewarming leads to almost complete restoration of the Na efflux. The Arrhenius plot shows no “breaks” and gives anE a value of 14.2 kcal/mol. External application of 10−4 m-ouabain following cooling to 0°C causes a fall in the residual efflux (∼8%). Rewarming results in partial restoration of the Na efflux. Lowering external pH (pH e ) results in a rise in Na efflux at 0°C, which peaks and declines rather slowly. The magnitude of the stimulatory response to acidification is a function of pH e over the pH e 7.00–5.8 range, the theoretical threshold being pH e 7.1. The magnitude of the response to acidification of cooled, ouabain-poisoned fibers suspended in Li-ASW is the same as that of fibers suspended in Na-ASW. Injection of pure protein kinase inhibitor into fibers maintained at 0°C fails to reduce the size of the response to acidification. Benzolamide but not acetazolamide, ethoxzolamide or Cl 13,580 abolishes the response to acidification. It also reverses the response to acidification. SITS is also able to abolish the response to acidification. An additional new observation is that the Na efflux at 0°C is stimulated following the injection of CaCl2 in a concentration-dependent manner. A similar response is not seen with MgCl2. Acidification (pH e 5.8) following peak stimulation by injection of CaCl2 is without effect. These results add up to a refutation of the concept that the Na efflux at 0°C is wholly passive and that the response to acidification involves Na:Na or Na:Ca exchange. The results also weaken the argument that stimulation of the efflux by acidification is the result of activation of carbonic anhydrase.

1. 1. The Na efflux in barnacle muscle fibres is stimulated by microinjection of 5′-methylene phosphonate analogue of cAMP, more so when fibres are pretreated with ouabain. 2. 2. Concentrations as low as 10−6 M (i.e. 10−8 M in the myoplasm) of the analogue cause 27·4±4·2% stimulation of the Na efflux and 114·1±29·3% stimulation of the ouabain-insensitive Na efflux. Experiments with 100 mM-EDTA and 100 mM-EGTA fail to show that the response of the Na efflux to the analogue depends on myoplasmic pCa. 3. 3. This contrasts sharply with the finding that in the absence of external Ca2+, the response of the Na efflux to microinjection of the analogue is markedly reduced.

1. A study has been made in single barnacle muscle fibres of the effect of micro-injected pure protein kinase inhibitor (PKI) on the response of the Na efflux to injection of cyclic AMP and external acidification. 2. (i) Injection into fibres of 1.6 x 10(-4) M-pure PKI is without effect on the resting Na efflux. (ii) Injection of 1.6 x 10(4) M-pure PKI before 0.03 M-cyclic AMP causes a marked reduction in the magnitude of the response of the Na efflux to the nucleotide. The same is true when 10(-4) M-cyclic AMP is injected after PKI. (iii) Injection of partially pure catalytic subunits causes a sustained stimulation of the ouabain-insensitive Na efflux, which is almost completely reversed by injecting PKI. (iv) Injection of 100 mM-EGTA before PKI fails to alter the lowered response of the ouabain-insensitive Na efflux to injection of 10(-4) M-cyclic AMP. (v) Ouabain (10(-4) M) when applied following the injection of 10(-4) M-cyclic AMP causes a drastic fall in the stimulated Na efflux. 3. (i) Injection of 1.6 x 10(-4) M-pure PKI before or after external acidification fails to abolish or reduce the stimulatory response to acidification. (ii) Injection of 1.6 x 10(-4) M-pure PKI before acidification practically abolishes the response of the ouabain-insensitive Na efflux to 0.03 M-cyclic AMP in the presence of acidification. (iii) Radioimmunoassay of total cyclic AMP and cyclic GMP content in single fibres before and after acidification shows no appreciable alteration in nucleotide content following acidificiation. (iv) Injection of 100 mM-EGTA before acidification enhances the stimulatory response to acidification. (v) External application of Dantrolene (10(-5) M) fails to alter the size of the stimulatory response to acidification. 4. (i) Prior external application of 5 x 10(-4) M-benzolamide results in a marked reduction in the magnitude of the response of the ouabain-insensitive Na efflux to the injection of 3 x 10(-4) M-cyclic AMP. (ii) Benzolamide totally abolishes the response of the ouabain-insensitive Na efflux to the injection of catalytic subunits. 5. The evidence brought forward is compatible with the view that (a) The mechanism by which cyclic AMP stimulates the Na efflux involves activation by cyclic AMP of the cyclic AMP-dependent protein kinase system, and hence release of the catalytic subunit, and (b) the mechanism by which external acidification leads to stimulation of the Na efflux involves activation of a benzolamide-sensitive system, possibly carbonic anhydrase, rather than the adenyl cyclase system. The actions of cyclic AMP and catalytic subunits on the Na efflux are closely linked to activation of the benzolamide sensitive system.

1. 1. The behaviour of the Na efflux towards Li + was studied using single barnacle muscle fibres as a preparation. 2. 2. It is found that the Na efflux into Li + -ASW (artificial seawater) is reduced and that this effect is not fully reversed by returning back to Na + -ASW. 3. 3. Preinjection of 100 mM-EGTA reduces the magnitude of the fall of the Na efflux into Li + -ASW. 4. 4(a). The remaining Na efflux into Li + -ASW is further reduced by external application of 10 −4 M-ouabain. (b) The remaining Na efflux in ouabain-poisoned fibres is reduced by replacing Na e by Li + . However, some fibres show a rise rather than a fall. 5. 5. Fibres loaded with NaCl (by injection) show a prompt and sustained stimulation of the Na efflux when Na e is replaced by Li + . A similar but less pronounced response is often seen with ouabain-poisoned fibres. 6. 6. Injection of LiCl (e.g. a 2 M-solution), causes a 20% fall in Na efflux. Subsequent replacement of Na e by Li + fails to bring about a fall in the remaining efflux. 7. 7. Itis concluded that the Na efflux in these fibres consists of a Na-Na exchange diffusion component which is not mediated by the Na-K pump and that its operation is interrupted by injecting Li + . The relative size of this component is about one-fifth and not one-half of the Na efflux.

Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is ∼20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca2+ artificial seawater (ASW) or in Ca2+-free ASW decreases approximately linearly with increasing logarithm of [K] o . The slope of the plot is slightly steeper for fibers bathed in Ca2+-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK o depends on the external Na+ and Ca2+ concentrations, as well as on external pH, but is independent of external Mg2+ concentration. External application of 10−4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca2+ by injection of 250mm EGTA before exposure of the fiber to 100mm K o leads to ∼60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K o by ∼50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na2 before elevation of [K] o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K o for time periods ranging from 30 sec to 10 min causes a small (∼60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca2+ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.

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).

1. 1. A study has been made of phosphate-containing metabolites in single barnacle muscle fibers using phosphorus-31 nuclear magnetic resonance. 2. 2. Spectra from single fibers (∼50 mg in wet weight) show major resonances from sugar phosphates, inorganic phosphate, arginine phosphate and the α, β and γ phosphorus atoms of ATP. 3. 3. The approximate “free” concentration of each metabolite was determined by integration of the spectrum, using a sample of 1 M-methylene diphosphonic acid as a reference. A notable feature of the results obtained is that the concentrations of SP&Pi in freshly dissected fibers are low. 4. 4. Time-dependent changes in 31P-NMR spectra indicate that ArP declines fairly slowly, while SP and Pi rises. The half-life of ArP at 26°C turns out to be about 8 hr. ATP remains relatively constant for the first 8 hr but disappears following the disappearance of ArP. As the intensity of the Pi resonance increases with time, it broadens and moves upfield, suggesting internal acidosis. 5. 5. These results demonstrate that 31P-NMR can provide useful information about metabolism and its regulation in single barnacle muscle fibers.