E. Edward Bittar

The effects of caffeine on sodium transport, membrane potential, mechanical tension and ultrastructure in barnacle muscle fibres

1. The effects of graded concentrations of caffeine on the Na efflux were investigated. External application of 10 m M caffeine usually caused a biphasic response, viz. a fall, followed by a rise in the Na efflux. 1 and 5 m M caffeine usually caused stimulation. Only the stimulatory phase of this response depended on the presence of external Ca2+.

An investigation of sodium transport in barnacle muscle fibres by means of the microsyringe technique

1. The cation composition of single barnacle muscle fibres following damage by axial insertion of a microsyringe has been measured. The Na and Ca contents of these fibres were raised.

Mode of stimulation by adenosine 3':5'-cyclic monophosphate of the sodium efflux in barnacle muscle fibres.

1. Giant fibres of the barnacle Balanus nubilus have been used as a preparation for studying the mode of action of cAMP on sodium transport. 2. It is shown that a concentration of cAMP as low as 10(‐6)M, when micro‐injected, causes a sharp rise in the radio‐Na efflux. Ouabain fails to reverse the cAMP effect. 3. The magnitude of the response of the Na efflux to cAMP is markedly reduced by pre‐injecting 100 or 500 mM‐EGTA solutions or by omitting Ca2+ from the bathing medium. Both together fail to bring about a greater reduction in the response. 4. The response to cAMP is greatly reduced by pre‐injecting the protein inhibitor of Walsh and practically abolished by pre‐injecting 500 mM‐EGTA and soaking in Ca‐free artificial sea water, ASW. 5. The Ca2+‐independent component of the Na efflux which is also stimulated by cAMP is shown to involve Na for H exchange. The magnitude of this exchange is governed by external pH. 6. The Na efflux into Ca2+‐free, Li+‐ASW is shown to be markedly stimulated by injecting cAMP, an effect which is enhanced by reducing external pH. 7. The Na efflux at 0 degrees C is stimulated by injecting cAMP. This is shown to be related to activation of the protein kinase by cAMP and to depend on the presence of external Ca2+. 8 (i) Ethacrynic acid when injected reduces the ouabain‐insensitive Na efflux into HEPES‐Ca2+‐free ASW at pH 6‐3. These same fibres show a marked response to cAMP. (II) The ouabain‐insensitive Na efflux into HCO3‐, Ca2+‐free ASW from fibres pre‐treated with ethacrynic acid fails to respond to external acidification. This is interpreted as indicating that ethacrynic acid inactivates the CO2‐sensitive adenyl cyclase system. These same fibres when injected with cAMP show a marked response. (iii) Stimulation of the ouabain‐insensitive Na efflux into HCO‐3, Ca2+‐free ASW by external acidification is reversed by injecting ethacrynic acid. These fibres when injected with cAMP show a reduced response. 9. It is concluded that: (i) stimulation of the Na efflux by injected cAMP is mainly due to activation of cAMP‐dependent protein kinase; (ii) the underlying exchange mechanism consists of Na:Ca and Na:H exchange. Interaction of Ca2+ with a phosphorylated membrane, thereby modifying permeability remains as a real possibility; (iii) the site of action of CO2 and ethacrynic acid is the adenyl cyclase system. 10. The implications of activation of the adenyl cyclase system by CO2 and Na:H exchange are briefly touched upon.

Sensitivity of Na efflux from single barnacle muscle fibers to external H+ ions

Abstract BITTAR, Caldwell and Lowe 1 showed that the efflux of Na from single crab muscle fibers is usually exponential and sensitive to the removal of external K ions. These findings have now been extended to muscle fibers from the barnacle Balanus nubilus , and confirmed. Barnacle fibers are about 3–5 cms long and 1–2 mm wide, hence are easily cannulated and ideal for microinjection experiments. The following communication describes experiments meant to shed some light on the part played by external pH in the Ussing mechanism. The results of this work show quite clearly that Na efflux into K-free artificial sea water is markedly stimulated by a low external pH and that the process of stimulation is reversible.

The influence of low pH, high K and microinjected CaCl2 on the ouabain-insensitive component of sodium efflux in barnacle muscle fibers

Abstract Attempts were made to characterize the ouabain-insensitive Na efflux in barnacle muscle fibers. Lowering the external pH to 5.8, raising the external K to 30 mM or microinjecting 1 M CaCl 2 led to a marked rise in the Na efflux from fibers previously poisoned with 5×10 −5 M ouabain. External application of ouabain following stimulation of the Na efflux by acidifying the bathing medium was without effect. Internal application of 2 M MgCl 2 following inactivation of the ouabain-sensitive component of the Na efflux was also without effect.

Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers

SummarySingle 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 100mmKo depends on the external Na+ and Ca2+ concentrations, as well as on external pH, but is independent of external Mg2+ concentration. External application of 10−4m 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 Ko 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 Ko 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 Ko 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 Ko 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.

The influence of injected cyclic AMP protein kinase catalytic subunit on the sodium efflux in barnacle muscle fibres

1. A study has been made of the response of the Na efflux in single barnacle muscle fibres to the injection of cyclic AMP protein kinase catalytic subunit (CSU).

Mode of stimulation by injection of cyclic AMP and external acidification of the sodium efflux in barnacle muscle fibres.

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.

Studies of the mode of stimulation by external acidification and raising the internal free calcium concentration of the sodium efflux in barnacle muscle fibers

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.

An investigation of the effects of external acidification on sodium transport, internal pH and membrane potential in barnacle muscle fibers

SummaryRadiosodium efflux from barnacle muscle fibers is a function of pHe, the threshold pHe for stimulation of Na efflux into HCO3−-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-HCO3− buffer is without effect on the Na efflux. The Na efflux following stimulation by reducing the pH of 10mM HCO3−-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 HCO3−-ASW is markedly stimulated by external acidification. Ouabain-poisoned fibers are more responsive to a low pHe than unpoisoned fibers. Applying the 2-14C-DMO technique, it is found that fibers bathed in 10mM HCO3−-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 pHi of 7.28±0.112. The relationship between pHi and pHe as external pH is varied by adding H+ is linear. Measurements of the resting membrane potential indicate that external acidification in the presence of HCO3− as buffer is accompanied by a fall inEm, the threshold pHe 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 pHe. 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 reduceEm 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 HCO3−-containing medium is due to activation of a Cl−-and/or HCO3−-pump and that ouabain and ethacrynic acid reducesEm by abolishing uncoupled Na transport.