Concerning the stimulation by injected cyclic AMP of the sodium efflux in barnacle muscle fibres and its transient nature

The efflux of 22Na 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). 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 22Na efflux from falling. This effect of PMX is not mimicked by injected HEPES. (a). Injection of Mg2+ 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 RI or RII subunits following PMX brings about partial reversal of the sustained stimulatory response. Partial reversal is also seen with externally applied imipramine (50 microM). 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.     

Mode of action of theophylline on sodium efflux in barnacle muscle fibers

Summary The response of the Na efflux in unpoisoned barnacle fibers to 10mm theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10mm theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500mm EGTA completely abolishes the biphasic action of 10mm theophylline. External application of 10mm theophylline following removal of external Ca²⁺ fails to bring about a biphasic effect. Ca²⁺ restoration, however, results in a moderate rise in the Na efflux. External application of 10mm theophylline stimulates the Na efflux into Ca²⁺-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10mm theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10mm theophylline. Injection of cAMP into ouabainpoisoned fibers, following internal application of Corbin's inhibitor and external application of 10mm theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10mm theophylline, fails to reduce the Na efflux. Fibers injected with 1mm and 100mm EGTA and exposed to 10mm theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10mm. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10mm theophylline. Theophylline (10mm) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3mm-HEPES ASW or 10mm-Ca²⁺-3mm-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa (pCa-log₁₀[Ca²⁺]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.     

The modulatory action of 5-hydroxytryptamine on sodium efflux: the barnacle muscle fibre as a model system

A study has been made of the action of 5-hydroxytryptamine (5-HT) on the radio-sodium efflux from single barnacle muscle fibres. (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. 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. The response of the ouabain-insensitive Na efflux to 5-HT depends on external Ca2+ and, to a certain extent, on external Na+. (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). Whereas the receptor antagonist methysergide is ineffective, cyproheptadine is very effective. (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(-5) M-TFP. Prior injection of Mg2+ or protein kinase inhibitor (PKI) leads to abolition or reduction of the response to 5-HT. These results demonstrate that barnacle fibres are a useful preparation for investigation 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.     

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

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

Increased sensitivity to injected 5'-guanylylimidodiphosphate of the sodium efflux in barnacle muscle fibres preexposed to aldosterone

1. A study has been made of the response to injected Gpp(NH)p of the ouabain-insensitive Na efflux in barnacle muscle fibres preexposed 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 PKI is also injected before Gpp(NH)p. By contrast, injection of R11 subunits causes a partial reversal if injected only once. 11. Imipramine and trifluoperazine, when applied externally (5 X 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.     

Evidence for the presence of protein kinase C in barnacle muscle fibers

1. The efflux of 22Na from single barnacle muscle fibers poisoned with ouabain (strophanthin G) is found to be very sensitive to the tumor-promoting agent, phorbol dibutyrate (PDBu). 2. Injection or external application of PDBu leads to stimulation of the ouabain-insensitive component of the Na efflux. This response is dose-dependent, the minimal effective concentration being about 10(-8)M. 3. The observed stimulatory response is completely dependent on the presence of external Ca2+. 4. Biochemical studies including immunoblot analysis reveal the presence in barnacle muscle of a protein kinase C with a mol. wt of 80,000, the activity of which is dependent on phosphatidylserine and Ca2+. 5. Taken together, these results support the view that barnacle muscle fibers possess protein kinase C. They also raise the possibility that protein kinase C plays a role in modulating the ouabain-insensitive component of the Na efflux.     

Sensitivity to injected cholera toxin of the sodium efflux in single barnacle muscle fibers

A study has been made of the effect of microinjected cholera toxin (CT) on the 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. Enrichment of fibers with NAD+ fails to enhance the response to CT. 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. 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. Whereas verapamil and Cd2+ are ineffective, both Mg2+ and trace metals, e.g. Fe and Zn, reverse the response to CT following injection. Prior injection of protein kinase inhibitor reduces the response to CT. As for calmodulin inhibitors, e.g. chlorpromazine, imipramine and mepacrine, they are effective in reducing the response to CT but not calmodulin antibody (IgG). 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.     

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

A study has been carried out using relatively intact mature muscle fibers from the barnacle Balanus nubilus to see whether D-Ins(1,4,5)P3 stimulates the ouabain-insensitive Na efflux following its microinjection and whether this is accompanied by a contraction of the fiber. Part of the impetus for a study of this type came from the on-going debate between Vergara, Rojas and co-workers and Lea and co-workers, the former group holding the view that skinned barnacle fibers and skeletal fibers in general are responsive to this isomer. The evidence brought forward indicates that the injection of D-Ins(1,4,5)P3 in concentrations in the range of 10(-2) M to 10(-6) M into cannulated unskinned fibers pretreated with ouabain fails to increase the residual efflux, and additionally fails to elicit a contraction. A similar picture emerges with the use of non-hydrolyzable DL-Ins(1,4,5)P3[S]3, analog following its injection in a concentration of 0.5 microM. Higher concentrations of the analog were unavailable for use. This work also involved verification of the idea that an effect might be obtainable in depolarized fibers. However, doubling or tripling K0+ and injection of the isomer or the analog simultaneously failed to support this idea. Since nothing is known as to whether D-Ins(1,4,5)P3 influences the behavior of the Na(+)-Ca2+ exchanger when operating in the reverse mode, experiments were done to check this possibility. ATPNa2 which is though to activate Na(+)-Ca2+ exchange was injected prior to the isomer or the analog but no significant results were obtained. A similar line of reasoning was followed, that of activating the L-type Ca2+ channel by injecting GTPNa2 which in addition is known to activate adenylate cyclase. Again, neither the isomer nor the analog were effective. Thus, the only conclusion possible is that in relatively intact, mature barnacle fibers there is no coupling between the phosphoinositide signalling pathway and two other key systems, viz. the Na(+)-Ca2+ exchanger when operating in the reverse mode and the L-type Ca2+ channel. Equally clear is that for some unknown reason the ouabain-insensitive Na efflux and the contractile apparatus are insensitive to D-Ins(1,4,5)P3[S]3.     

Sensitivity of the sodium efflux in barnacle muscle fibers to the microinjection of ATP

Sodium efflux in barnacle muscle fibers is promptly stimulated by internal application of ATP. This response is markedly augmented by pretreatment of the barnacle fiber with ouabain. ArP is found to be considerably less effective than ATP. It is suggested that the stimulatory response of the ouabain-insensitive Na efflux to the microinjection of ATP may be due to a significant rise in the sarcoplasmic cAMP concentration caused by the catalytic action of an adenyl cyclase system.     

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

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

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

Summary 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.     

Interaction between aldosterone and vasopressin on vascular smooth muscle permeability to sodium

The s.c. injection of aldosterone (10 micrograms/kg) induces a release of vasopressin. The peak of plasma vasopressin level occurs at the same time as the late in vivo effect of aldosterone on passive 22Na efflux from arterial smooth muscle. These results indicate that vasopressin mediates the delayed in vivo effects of aldosterone on ouabain-insensitive 22Na efflux, since on the other hand, it has been possible to show that the action of the peptide is accelerated by a previous exposure to the mineralocorticoid. Indeed, after a 120-min pretreatment with 10(-8) M aldosterone, vasopressin induces an effect on 22Na efflux in 30 min, as opposed to the 120 min needed in the absence of the steroid.     

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

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

The effects of short-chain fatty acids on the neuronal membrane functions ofHelix pomatia. III.22Na efflux from the cells

The effect of short-chain fatty acids on both ouabain-sensitive and ouabain-insensitive fractions of 22Na efflux from the neurons of Helix pomatia was studied. Fatty acids, having fewer than 10 carbon atoms in the hydrocarbon chain, increased the ouabain-sensitive 22Na efflux from the neurons, while fatty acids, having more than 9 carbon atoms, inhibited the 22Na efflux in comparison with that in normal physiological solution. All the fatty acids used had an inhibiting effect on the ouabain-insensitive 22Na efflux from the cells independent on the number of carbon atoms in the hydrocarbon chain. These studies indicate that these short-chain fatty acids can be effective modulators of both ouabain-sensitive and ouabain-insensitive fractions of Na efflux from the cells.     

Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Ouabain-insensitive Na(+)-ATPase activity is an effector protein for cAMP regulation in basolateral membranes of the proximal tubule

This study describes the modulation of the ouabain-insensitive Na(+)-ATPase activity from proximal tubule basolateral membranes by cAMP. An increase in dibutyryl-cAMP (d-cAMP) concentration from 10(-8) to 5x10(-5) M stimulates the ouabain-insensitive Na(+)-ATPase activity. The ATPase activity increases from 6.0+/-0.4 to 10.1+/-0.7 nmol Pi mg(-1) min(-1), in the absence and presence of 5x10(-6) M d-cAMP, respectively. Similarly, the addition of cholera toxin (CTX), forskolin (FSK) or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) also increases the Na(+)-ATPase activity in a dose-dependent manner, with maximal effect at 10(-8) M, 10(-6) M and 10(-7) M, respectively. The effect of 10(-8) M CTX is not additive to the effect of GTPgammaS, and is completely abolished by 200 microM guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of CTX and FSK on the Na(+)-ATPase activity are accompanied by an increase in cAMP formation by the basolateral membranes of the proximal tubule cells. Furthermore, 10(-8) M protein kinase A peptide inhibitor (PKAi) completely abolishes the stimulatory effect of 5x10(-6) M d-cAMP or 10(-4) M FSK on the Na(+)-ATPase activity. Incubation of the basolateral membranes with [gamma-(32)P]ATP in the presence of d-cAMP or FSK increases the global hydroxylamine-resistant phosphorylation and especially promotes an increase in phosphorylation of protein bands of approximately 100 and 200 kDa. This stimulation is not seen when 10(-8) M PKAi is added simultaneously. Taken together these data suggest that activation of a cAMP/PKA pathway modulates the Na(+)-ATPase activity in isolated basolateral membranes of the proximal tubule.     

Lithium transport pathways in human red blood cells

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.     

Na+-Ca2+ exchange and Ca2+ efflux in transfected Chinese hamster ovary cells

The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.     

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.     

The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro

Human leucocytes incubated in tissue culture fluid of low-sodium concentration (2 mM; iso-osmolarity maintained with choline chloride) reached a new equlibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabainsensitive sodium efflux fell by more than 50% and the ouabain-insensitive rate constant increased nearly threefold in the low-sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain-insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low-sodium medium ouabainsensitive potassium influx exceeded ouabain-sensitive sodium efflux and no ouabain-sensitive potassium efflux could be demonstrated. Ouabain-insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low-sodium cells, suggesting reduced activity of a ouabain-insensitive K:K exchange system.