The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres.

We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)2 the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of -0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca2+)/P(K+)=2.4, P(Ca2+)/ P(Na+)=2.7, P(Ca2+)/P(Tris+)=3.1, P(Ca2+)/P(Mg2+)=1.0 and P(Ca2+)/P(Ba2+)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)2 solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)2 solution. The channel activity in symmetrical 50 mM Ca(gluconate)2 solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 microM). The results are in agreement with the conclusion that the sarcoball Ca2+ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca2+-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel.

Ca2+ effluxes from the sarcoplasmic reticulum vesicles of frog muscle: effects of cyclopiazonic acid and thapsigargin.

Ca2+ efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca2+ in the presence of ATP and inorganic phosphate (Pi). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM Pi, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM Pi. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 microM, which completely inhibit Ca2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6-7 nmole/mg/min was recorded. TG (2.5 microM) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca(2+)-ATPase passive channel.

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake, spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae.

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca2+ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca2+ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca2+ content loaded into the SR, (2) the spontaneous Ca2+ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca2+ sensitivity in Triton X-100-treated preparations. Inhibition of Ca2+ loading by TG and CPA increased with time of exposure to the inhibitor over 18-24 min. TG and CPA produced half inhibition of Ca2+ loading at 34.9 and 35.7 microM respectively, when 18-24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 microM TG and 30 microM CPA both abolished the oscillations in this time. The myofilament Ca2+ sensitivity was not affected by 10 and 300 microM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca2+ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation.

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle.

Thapsigargin has been reported to inhibit ATP-dependent Ca2+ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca2+ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca2+ content of the SR. The SR was first depleted of Ca2+ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 microM was required to inhibit Ca2+ loading by 50%. In contrast, another SR Ca2+ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 microM and total inhibition at 50 microM. When cyclopiazonic acid (100 microM) was applied after, rather than during, Ca2+ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 microM), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca2+ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.

Barnacle muscle: Ca2+, activation and mechanics.

In this review, aspects of the ways in which Ca2+ is transported and regulated within muscle cells have been considered, with particular reference to crustacean muscle fibres. The large size of these fibres permits easy access to the internal environment of the cell, allowing it to be altered by microinjection or microperfusion. At rest, Ca2+ is not in equilibrium across the cell membrane, it enters the cell down a steep electrochemical gradient. The free [Ca2+] at rest is maintained at a value close to 200 nM by a combination of internal buffering systems, mainly the SR, mitochondria, and the fixed and diffusible Ca(2+)-binding proteins, as well as by an energy-dependent extrusion system operating across the external cell membrane. This system relies upon the inward movement of Na+ down its own electrochemical gradient to provide the energy for the extrusion of Ca2+ ions. As a result of electrical excitation, voltage-sensitive channels for Ca2+ are activated and permit Ca2+ to enter the cell more rapidly than at rest. It has been possible to determine both the amount of Ca2+ entering by this step, and what part this externally derived Ca2+ plays in the development of force as well as in the free Ca2+ change. The latter can be determined directly by Ca(2+)-sensitive indicators introduced into the cell sarcoplasm. A combination of techniques, allowing both the total and free Ca2+ changes to be assessed during electrical excitation, has provided valuable information as to how muscle cells buffer their Ca2+ in order to regulate the extent of the change in the free Ca2+ concentration. The data indicate that the entering Ca2+ can only make a small direct contribution to the force developed by the cell. The implication here is that the major source of Ca2+ for contraction must be derived from the internal Ca2+ storage sites within the SR system, a view reinforced by caged Ca2+ methods. The ability to measure the free Ca2+ concentration changes within a single cell during activation has also provided the opportunity to analyse, in detail, the likely relations between free Ca2+ and the process of force development in muscle. The fact that the free Ca2+ change precedes the development of force implies that there are delays in the mechanism, either at the site of Ca2+ attachment on the myofibril, or at some later stage in the process of force development that were not previously anticipated.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation and relaxation mechanisms in single muscle fibres.

The effect of Ca2+ on the time course of force generation in frog skinned muscle fibres has been investigated using laser flash photolysis of the caged-calcium, either nitr-5 or DM-Nitrophen. Gradations in the rate and extent of contraction could be achieved by changing the energy of the laser pulse, which varied the amount of caged Ca2+ photolysed and hence the amount of calcium released. The half-time for force development at 12 degrees C was noticeably calcium-sensitive when small amounts of calcium were released (low energy pulses) but did not change appreciably for calcium releases which produced a final tension of more than 50% of the maximal tension at pCa 4.5. This result is unlikely to be due to calcium binding to the regulatory sites of troponin C when on the thin filament, as this process is considered rapid (kon 10(8) M-1 s-1, koff 100 s-1). Our experimental results show that force develops relatively rapidly at intermediate Ca2+ which produce only partial activation (i.e. 50% Pmax or greater). This would not be the case if the affinity of the regulatory sites changes slowly with crossbridge attachment. The kinetics of calcium exchange with the regulatory sites may be much more rapid than crossbridge cycling, so that if calcium binding to a particular functional unit induces crossbridge attachment and force production, the force producing state may be maintained long after calcium has dissociated from that particular functional unit. The relaxation of skinned muscle fibres has also been successfully studied following the rapid uptake of Ca2+ by a photolabile chelator Diazo-2, a photolabile derivative of BAPTA, which is rapidly (> 2000 s-1) converted from a chelator of low Ca2+ affinity (Kd 2.2 microM) to a high affinity chelator (Kd 0.073 microM). We have used single skinned muscle fibres from both frog (actin regulated) and scallop striated muscle (myosin regulated), to study the time course of muscle relaxation. This procedure has enabled us to examine the effects of the intracellular metabolites, ADP, Pi and H+ upon the rate of relaxation.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional characterization of the two isoforms of troponin C from the arthropod Balanus nubilus.

Two isoforms of troponin C (BTnC1 and BTnC2) from the striated muscle of the arthropod Balanus nubilus Darwin (giant barnacle) have been purified (Potter et al., 1987; Collins et al., 1991). Both isoforms were present in all of the white striated muscle fibres studied but not in the red fibres. The ratio of BTnC2 to BTnC1 in different fibre types varied between 3:1 and 1:1. Both forms of TnC could be readily extracted from myofibrillar bundles of barnacle muscle in low ionic strength EDTA solutions, reducing force activation to less than 10%. Both forms either separately or together reassociated with the TnC-depleted fibres in a relaxing (LR) solution (pCa greater than 8.0, [Mg2+] free = 1 mM, I = 0.15 M), and the reconstituted fibres could be subsequently activated in contraction (LA) solution (pCa = less than 3.8, [Mg2+] free = 1 mM, I = 0.15 M). The dissociation of BTnC 1 + 2 is blocked in low ionic strength solutions containing Mg2+ (greater than or equal to 10 mM). The two isoforms of crayfish TnC (CrTnC1 and CrTnC2) were also found to be equivalent to the barnacle TnCs in their ability to reactivate TnC-depleted barnacle myofibrillar bundles. Similar experiments using rabbit skeletal muscle TnC (STnC) (I = 0.15 M) in BTnC-depleted myofibrillar bundles of barnacle showed considerable variability. STnC could associate, although weakly, with the depleted bundles in either LR or LA, and force could be partially restored. In neither situation was it as effective as either BTnC or CrTnC. Interestingly, bovine cardiac TnC (CTnC), although it did not associate at pCa greater than 7.0, did associate and effectively activate force at pCa less than 3.8, but dissociated on return to pCa greater than 7.0 (LR). Neither barnacle TnC isoform associated with TnC-depleted skinned fibres from rabbit skeletal muscle at pCa greater than 7.0, but did associate and activate these fibres at pCa less than 3.8. Once these fibres were returned to LR and then placed in LA at pCa 3.8 all BTnC-restored force was lost, indicating a dissociation of BTnC once the Ca2+ is lowered, as observed with CTnC in barnacle myofibrillar bundles. Finally, the inhibitory effect of BTnI on force and the absence of an effect of calmodulin, trifluoperazine or ATP-gamma-S on force were all taken as evidence for a thin filament regulated Ca2+ control system.

Ca2+ release from the sarcoplasmic reticulum of barnacle myofibrillar bundles initiated by photolysis of caged Ca2+.

1. Ca2(+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum was measured by isometric tension recording from barnacle myofibrillar bundles. Laser-induced photolysis of the caged calcium molecule, nitr-5, was used to generate a rapid jump in free Ca2+ (within 1 ms) at the site of the sarcoplasmic reticulum, thus overcoming delays due to Ca2+ diffusion from the bathing solution. 2. The method consisted of equilibrating a myofibrillar bundle (100 micrograms diameter) in a solution containing 0.1 mM-nitr-5 (initial pCa 6.8-6.6) and then exposing it to a UV laser pulse. The resulting phasic contraction had an amplitude of up to 100% maximum tension (P0) in some preparations and a mean half-time for the rise of tension of 2.3 s at 12 degrees C. Longer half-times were obtained at low pulse energies. 3. Pre-treatment of the myofibrillar bundles with ryanodine (10(-4) M) or the detergent Triton X-100 abolished a large part of the phasic contraction, confirming its dependence on SR Ca2+ release. The small tonic response which remained had a shorter rise half-time than the Ca2(+)-induced Ca2+ release response and was attributed to direct activation of the myofibrils by Ca2+ released from the nitr-5. 4. The size of the photolytic Ca2+ jump was estimated from the amplitude of the fast tension component. By increasing the laser pulse energy or the initial Ca2+ loading of the nitr-5, the post-photolysis pCa was varied from 6.7 to 6.0; the CICR response increased in size over this pCa range. 5. Direct activation of Triton-treated myofibrils by photolysis of 2.0 mM-nitr-5 (initial pCa 6.4) gave contractions of up to 100% P0 and a mean rise half-time of 164 ms at 12 degrees C (n = 9 for contractions greater than 40% P0). Both the amplitude and the rate of these contractions were dependent on the laser pulse energy. 6. The Ca2(+)-induced Ca2+ release responses obtained with nitr-5 photolysis were significantly slower than the fastest rate of tetanus development which has been recorded from intact fibres of barnacle muscle (mean half-time = 177 ms at 12 degrees C). This could mean that either Ca2(+)-induced Ca2+ release is less efficient in isolated myofibrillar bundles than in intact fibres or that Ca2(+)-induced Ca2+ release is not the primary Ca2+ releasing mechanism in excitation-contraction coupling in barnacle muscle.

Rapid activation by photolysis of nitr-5 in skinned fibres of the striated adductor muscle from the scallop.

Photolysis of nitr-5, a caged calcium molecule, has been used for rapid activation of skinned fibre bundles of a myosin-regulated muscle, the striated adductor of the scallop, Pecten maximus. Chemically skinned fibre bundles (diameter 70-200 microns) were equilibrated in solutions containing 1-3 mM nitr-5 (pCa 6.1) and then activated by ultraviolet laser pulse (25 ns). Pulse energies of 60-95 mJ gave contractions of over 90% maximum tension and a mean half-time for tension rise of 43 ms (n = 4) at 12 degrees C. Electrically stimulated bundles of intact fibres develop a tetanus with a rise half-time of 60.2 ms at 10 degrees C (n = 5) (Rall, J.A. (1981) J. Physiol. 321, 287-295, and personal communication). At lower pulse energies the skinned fibres gave smaller amplitude contractions with slower rates of rise (up to 260 ms half-time). In addition, a slower component of tension development (mean rise half-time 13.3 s) was often observed. In ATP-free solutions containing hexokinase and glucose, rigour tension developed with a delayed onset. Rapid release of ATP (0.47-0.59 mM) from photolysis of caged ATP (2 mM) at pCa 4.5 then caused a rapid contraction with a mean half-time for tension development of 17 ms (n = 4). The fast activation rates obtained by the photorelease of Ca2+ from nitr-5 are similar to those obtained with skinned skeletal fibres of actin-regulated muscle. The results imply that the rate-limiting step in excitation-contraction coupling of the scallop muscle is not the increase in sarcoplasmic Ca2+, but rather the activation of the muscle in response to this increase. The half-times of ATP-induced contractions at pCa 4.5 suggest that in a contraction activated by a rapid Ca2+ jump the process comprising ATP hydrolysis and cross-bridge cycling occurs at a somewhat faster rate than the Ca2(+)-dependent activation process which precedes it.

Calcium release from cardiac sarcoplasmic reticulum induced by photorelease of calcium or Ins(1,4,5)P3.

The ability of Ca2+ or inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to release Ca2+ from cardiac sarcoplasmic reticulum (SR) was investigated using saponin-skinned ventricular trabeculae from rats. To overcome diffusion delays, rapid increases in the concentrations of Ca2+ and Ins(1,4,5)P3 were produced by laser photolysis of "caged Ca2+" (Nitr-5) and "caged Ins(1,4,5)P3". Photolysis of Nitr-5 to produce a small jump in [Ca2+] from pCa 6.8 to 6.4 induced a large and rapid force response (t1/2 = 0.89 s at 12 degrees C); the source of the Ca2+ that activated the myofibrils was judged to be the SR, since it was blocked by 0.1 mM ryanodine or 5 mM caffeine. A smaller, slower, and less consistent release of SR Ca2+ was produced by photorelease of Ins(1,4,5)P3. The results demonstrate that these caged compounds can be used to study excitation-contraction coupling in skinned multicellular preparations of cardiac muscle. The data are consistent with a major role for Ca2(+)-induced Ca2+ release in cardiac activation, whereas the role for Ins(1,4,5)P3 may be to modulate, rather than directly stimulate, SR Ca2+ release.

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres.

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sarcoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1 mM EGTA, resulted in a phasic increase in myofibrillar free Ca2+ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca2+ response was higher than the free Ca2+ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca2+ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca2+ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.

An examination of the ability of inositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea.

We have examined the ability of inositol 1,4,5-trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10-500 microM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impermeant anion by Cl-, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg2+ was lowered to 40 microM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0-0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4 degrees to 22 degrees C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3-induced calcium release from rat liver microsomes, had no effect at 50 microM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.

A comparison of the abilities of CO2/HCO3-., protonophores and changes in solution pH to release Ca2+ from the SR of barnacle myofibrillar bundles.

Increases in solution pH from 6.5 to 7.0 to 7.5 at 0.1 microM free Ca2+ concentration had no effect on the isometric tension of barnacle myofibrillar bundles in relaxing solutions containing 0.1-0.16 mM BAPTA. Decreases in pH in the same range were also without effect. Under the same conditions CO2-induced Ca2+ release from the SR could be readily obtained by replacing the Cl(-)-containing containing relaxing solution with one containing HCO3- and 100% CO2 at the same pH. At a higher free Ca2+ of 2.5 microns, there was a contraction on increasing the pH of the Cl(-)-containing solution from 7.0 to 7.5. This response could be abolished by 1 mM procaine suggesting that it was due to Ca2+ release from the SR. The protonophores monensin, gramicidin, CCCP and FCCP at concentrations of 10-100 microM had no effect on resting tension at either free Ca2+ concentration and did not inhibit the response to 100% CO2. It is concluded that dissipation of a possible pH gradient across the SR membrane by protonophores does not release Ca2+ from the SR of barnacle muscle. Since both CO2 (by possibly lowering SR pH) and an increase in solution pH can release Ca2+ at 2.5 microM free Ca2+, the existence of a Ca2+ release channel which is opened by a change in the trans-SR pH gradient cannot be discounted.(ABSTRACT TRUNCATED AT 250 WORDS)

Harmaline distribution in single muscle fibres and the inhibition of sodium efflux.

Harmaline, a known inhibitor of the (Na+ + K+)-ATPase in cell membranes, inhibited 50% of the 22Na efflux from barnacle muscle fibres at an extracellular concentration of 2.4 mM. Injected harmaline inhibited 50% of the efflux at an estimated intracellular concentration of about 8 mM . kg-1, assuming complete equilibration with no binding. Total fibre harmaline was measured in separate fibres by ultraviolet spectrophotometry. Fibres in 3 mM harmaline saline accumulated harmaline with a half-time of 17 min and a final total fibre concentration of 6-12 mM . kg-1. In harmaline-free saline this accumulated harmaline was lost exponentially with a half-time of 35 min; injected harmaline was lost exponentially from fibres with a half-time of 50 min. It is proposed that harmaline crosses the fibre membrane as the uncharged base and that its apparent accumulation against a concentration gradient is mainly due to intracellular binding with an additional contribution from a transmembrane ph gradient. It is concluded that, in fibres exposed to harmaline saline, the intracellular concentration can reach a sufficiently high value, as judged from the results of the injection experiments, to inhibit Na+ efflux at an interior-facing site on the fibre membrane. In contrast, harmaline appears to inhibit the Na+-dependent uptake of L-glutamate at an extracellular site.

Carbon dioxide or bicarbonate ions release Ca2+ from internal stores in crustacean myofibrillar bundles.

The paper describes an investigation into the increase in intracellular free Ca2+ and resting tension of barnacle muscle fibers when exposed to CO2. Isometric tension was recorded in isolated myofibrillar bundles prepared from barnacles and crabs. On replacement of a low relaxing bathing solution (free Ca2+: 20 nM) at pH 7.1 with a similar one containing 100% CO2 and 13 mM HCO3-, also at pH 7.1, the bundles developed a phasic contraction, which aequorin experiments confirmed was due to a release of Ca2+ from a store within the bundles. The source of this Ca2+ is tentatively identified as the sarcoplasmic reticulum (SR) for the following reasons: (1) prior exposure to 20 mM caffeine depleted this Ca2+ store, (2) procaine (10 mM) inhibited the response, and (3) the extracellular space or "clefts" and the mitochondria could be eliminated as possible sources. An effect of the CO2 + HCO3- on the free Ca2+/Mg2+ ratio in the bathing solution was excluded as a possible mechanism. The diuretic furosemide (1 mM) enhanced the response to CO2 + HCO3-. Both furosemide and SITS (1--10 mM), by themselves, also released Ca2+ in myofibrillar bundles. A scheme is put forward to explain these results: it is suggested that diffusion of dissolved CO2 into the SR produces an acidification of the SR lumen, which modifies either the Ca2+/-ATPase or the Ca2+-induced release process in such a way to release Ca2+.

The effect of ouabain on amino acid and orthophosphate influxes in squid giant axons.

1. Some effects of ouabain on the uptake of 14C-labelled amino acids and 32P-labelled orthophosphate into squid axons have been investigated. 2. Ouabain in artificial sea water reduced the uptake of glycine, L-alanine, L-arginine, L-aspartate and L-glutamate. After a period of 3 hr in ouabain the inhibition of glycine and L-glutamate uptake was appreciable. 3. After 3 hr in ouabain the axoplasmic Na concentration had increased by only about 14% and the K concentration had fallen by only about 9%. It therefore seems unlikely that changes in the Na and K gradients across the membrane due to ouabain are the reason for the inhibition of amino acid uptake. This was confirmed when the axons were made to conduct impulses at 200/sec for 40 min. There was no subsequent inhibition of glycine and L-glutamate uptake, even though the axoplasmic Na increased by about 42% and the K decreased by about 37%. 4. A detailed investigation of the time course of the inhibition of amino acid uptake by ouabain in which intracellular scintillator techniques were used, showed that there was no inhibition for 30--100 min, and then it occurred fairly quickly. This course of events was not affected by the protein synthesis inhibitor cycloheximide. It is unlikely that the inhibition of orthophosphate uptake is subject to a similar delay since this had reached a maximum after 40 min. 5. It is concluded that the inhibitory effects of ouabain on amino acid uptake in squid axons are not due to effects on the Na and K gradients brought about by inhibition of the Na-K pump. The delay in the inhibiton may reflect the operation of an intracellular regulatory mechanis, which is initiated when ouabain inhibits the pump but which takes time to operate. If the formation of an inhibitory second messenger molecule is involved this is unlikely to be a protein.