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.     

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

Sodium efflux in barnacle muscle fibers is stimulated by internal application of monensin. Pretreatment of fibers with ouabain leads to marked enhancement of the response of the Na efflux to monensin. This response though independent of the Ca²⁺ gradient can be augmented by protonation of a bathing medium free of HCO₃^?.     

Influence of insulin on sodium efflux in barnacle muscle fibers

Summary The efflux of radiosodium in single muscle fibers from the barnacleBalanus nubilus was irresponsive to internal or external application of insulin. However, this was not the case with fibers isolated from a barnacle specimen pre-exposed overnight to a large dose of insulin. External application of insulin to pre-exposed fibers caused a decrease in the rate of decline of the radiosodium efflux and stopped the decline in the fractional rate constant for Na efflux. Such kinetics were interpreted as indicating that insulin acts either by releasing sequestered Na or abolishing the process of sequestration. Internal application of saline slowed the rate of decline but failed to completely abolish the mechanism of sequestration. Only in the presence of insulin was the fractional loss of Na each second constant. Internal application of insulin caused a prompt step-up in the rate of Na efflux, followed by a reduced efflux rate constant. This meant that injected insulin caused the release of sequestered Na, leading to partial saturation of the efflux. The response of the Na efflux to injected denatured insulin, though resembling that to native insulin was much smaller in size. Internal application of lysozyme produced a transitory step-up in the rate of Na efflux but failed to produce the kinetics observed with native or denatured insulin. Overnight exposure of the barnacle to a dose of denatured insulin failed to render the fiber sensitive to external and internal application of denatured or native insulinin vitro. Experiments with ouabain-poisoned fibers showed that external or internal application of native insulin caused stimulation of the remaining Na efflux. They also showed that a 10-fold increase in the concentration of ouabain failed to further reduce the ouabaininsensitive Na efflux. Microinjection of GTP into ouabain-poisoned fibers pre-exposed to insulin resulted in a striking rise in the remaining Na efflux. The magnitude of this effect was considerably greater than that in unexposed fibers. The response which was dose-dependent could be blunted by prior injection of CaCl₂. Similarly, the response to CaCl₂ injection could be blunted by prior injection of GTP. The evidence brought forward is compatible with the view that insulin acts by abolishing the mechanism of internal Na sequestration and by increasing the activity of the guanylate cyclase system.     

Some aspects of sodium efflux from single barnacle muscle fibres

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.     

Some further observations on the stimulation by monensin of the sodium efflux in barnacle muscle fibers.

Internal application of 20 μg/ml monensin causes a large rise in the Na efflux into Na-free Li-substituted artificial sea water (ASW). A comparable effect is observed in fibers pretreated with ouabain. External acidification fails to significantly augment the response to monensin of the ouabain-insensitive Na efflux into Li-ASW. Fibers cooled to 0°C fail to respond to monensin, when the pH of Li-ASW is adjusted from 7.8 to 6.0. These results are in keeping with the view that the ion exchange induced by momensin does not necessarily involve Na for Na. The cooling experiments establish that momensin can be stopped from acting when the environmental temperature is 0°C, presumably as the result of membrane conformational changes.     

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres.

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 micrometer. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 micrometer ionized Ca and and no ATP is 0.6 pmol-cm-2-s-1. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na+ and Ca2+ from the external medium causes a marked drop in the Ca2+ efflux in the presence of internal ATP.     

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.     

Phosphorylation of the rat Ins(1,4,5)P3 receptor at T930 within the coupling domain decreases its affinity to Ins(1,4,5)P3

The Ins(1,4,5)P₃ receptor acts as a central hub for Ca²⁺ signaling by integrating multiple signaling modalities into Ca²⁺ release from intracellular stores downstream of G-protein and tyrosine kinase-coupled receptor stimulation. As such, the Ins(1,4,5)P₃ receptor plays fundamental roles in cellular physiology. The regulation of the Ins(1,4,5)P₃ receptor is complex and involves protein-protein interactions, post-translational modifications, allosteric modulation, and regulation of its sub-cellular distribution. Phosphorylation has been implicated in the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release observed during oocyte maturation. Here we investigate the role of phosphorylation at T-930, a residue phosphorylated specifically during meiosis. We show that a phosphomimetic mutation at T-930 of the rat Ins(1,4,5)P₃ receptor results in decreased Ins(1,4,5)P₃-dependent Ca²⁺ release and lowers the Ins(1,4,5)P₃ binding affinity of the receptor. These data, coupled to the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release during meiosis, argue that phosphorylation within the coupling domain of the Ins(1,4,5)P₃ receptor acts in a combinatorial fashion to regulate Ins(1,4,5)P₃ receptor function.     

Phosphorylation of the rat Ins(1,4,5)P3 receptor at T930 within the coupling domain decreases its affinity to Ins(1,4,5)P3

The Ins(1,4,5)P₃ receptor acts as a central hub for Ca²⁺ signaling by integrating multiple signaling modalities into Ca²⁺ release from intracellular stores downstream of G-protein and tyrosine kinase-coupled receptor stimulation. As such, the Ins(1,4,5)P₃ receptor plays fundamental roles in cellular physiology. The regulation of the Ins(1,4,5)P₃ receptor is complex and involves protein-protein interactions, post-translational modifications, allosteric modulation, and regulation of its sub-cellular distribution. Phosphorylation has been implicated in the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release observed during oocyte maturation. Here we investigate the role of phosphorylation at T-930, a residue phosphorylated specifically during meiosis. We show that a phosphomimetic mutation at T-930 of the rat Ins(1,4,5)P₃ receptor results in decreased Ins(1,4,5)P₃-dependent Ca²⁺ release and lowers the Ins(1,4,5)P₃ binding affinity of the receptor. These data, coupled to the sensitization of Ins(1,4,5)P₃-dependent Ca²⁺ release during meiosis, argue that phosphorylation within the coupling domain of the Ins(1,4,5)P₃ receptor acts in a combinatorial fashion to regulate Ins(1,4,5)P₃ receptor function.     

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.     

Ins(1,4,5)P3 metabolism and the family of IP3-3Kinases

The release of Ca2+ from intracellular stores is triggered by the second messenger inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3). The regulation of this process is critically important for cellular homeostasis. Ins(1,4,5)P3 is rapidly metabolised, either to inositol (1,4)-bisphosphate (Ins(1,4)P2) by inositol polyphosphate 5-phosphatases or to inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) by one of a family of inositol (1,4,5)P3 3-kinases (IP3-3Ks). Three isoforms of IP3-3K have now been identified in mammals; they have a conserved C-terminal catalytic domain, but divergent N-termini. This review discusses the metabolism of Ins(1,4,5)P3, compares the IP3-3K isoforms and addresses potential mechanisms by which their activity might be regulated.     

The effect of ATP on calcium efflux in dialyzed barnacle muscle fibres

Calcium efflux has been studied in barnacle muscle fibres under internal dialysis conditions. Prolonged dialysis of these fibres, with a medium free of ATP and containing 2 mM cyanide and 1 mM iodoacetate, causes the ATP in the perfusion effluent to fall to less than 20 μM. The mean calcium efflux from fibres dialyzed with EGTA buffered solution containing 0.3 μM ionized Ca and no ATP is 0.6 pmol · cm⁻² · s⁻¹. A two-fold stimulation of the calcium efflux is observed when ATP is added to fibres previously dialyzed with an ATP-free medium. Withdrawal of Na⁺ and Ca²⁺ from the external medium causes a marked drop in the Ca²⁺ efflux in the presence of internal ATP.     

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.     

Antigen-induced Ca2+ mobilization in RBL-2H3 cells: role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.     

45Ca and (14C)EDTA efflux from dialyzed barnacle muscle fibers.

45Ca and 14C-labeled ethylenediamine-N, N'-tetraacetic acid (EDTA) effluxes were measured in internally dialyzed barnacle muscle fibers. In 45Ca experiments the internal ionized 45Ca was fixed at 0.2 muM with ethyleneglycolbis-(beta-aminoethylether)-N, N'-tetraacetic acid(EGTA). The 45Ca efflux was found to increase with internal CaEGTA from 0.05 pmol/cm2.s(CaEGTA equal to 0.02 mM) to 5.0 pmol/cm2.s(CaEGTAequal to 9.6 mM). To determine whether or not most of this increase in efflux was due to the exit of undissociated CaEGTA, comparable experiments were performed with Ca-[14-C]EDTA. Over the same range of internal calcium as studied in the 45Ca experiments, the Ca-[14-C]EDTA efflux was no more than 12% of the 45Ca efflux. We conclude that the exit of undissociated 45Ca cannot account for most of the 45Ca efflux nor can it account for the dependence of 45Ca efflux on internal CaEGTA. The experiments also demonstrated the existence of an endogenous pool of calcium, of 0.43 mmol/kg (about half the total calcium), which remained unexchanged during dialysis.     

Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes

The binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (KD = 6.82+/-2.3 nM, Bmax = 370+/-38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P3 was able to compete with [3H]Ins(1,4,5)P3 whereas Ins(1,3,4)P3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P3 were calculated to be 0.22+/-0.15 microM basal and 2.53+/-1.8 microM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P3 concentrations in the picomolar range.     

Thyrotropin-releasing hormone activates a [Ca2+]i-dependent K+ current in GH3 pituitary cells via Ins(1,4,5)P3-sensitive and Ins(1,4,5)P3-insensitive mechanisms.

The role of Ins(1,4,5)P3 in receptor-induced Ca2+ mobilization in pituitary cells was studied at the single-cell level. Experimental strategies were developed which allowed a comparative analysis of the effects of Ins(1,4,5)P3 with those of receptor activation under identical conditions. These include microfluorimetry as well as a novel technique which permits the controlled and rapid application of intracellular messenger molecules to individual cells. This latter approach is based on the tight-seal whole-cell recording (WCR) technique, and utilizes two patch-clamp micropipettes, one for electrical recording and the second for the controlled pressure injection. Ins(1,4,5)P3, when applied with this dual-WCR (DWCR) technique, leads rapidly to a marked rise in cytosolic free Ca2+ [( Ca2+]i) and a concomitant stimulation of Ca2(+)-activated K+ current; Ins(1,4,5)P3 can thus mimic the effects of thyrotropin-releasing hormone (TRH) in the same cells under identical conditions. In cells dialysed intracellularly with heparin, a potent antagonist of Ins(1,4,5)P3 action, the rapid response to extracellular stimulation with TRH was abolished, as were the effects of intracellular application of Ins(1,4,5)P3. Heparin, which abolished Ins(1,4,5)P3 action completely, blocked responses to TRH in some cells only partially, revealing that Ca2+ mobilization response to TRH is in part slower in onset than the response to Ins(1,4,5)P3. It is concluded (1) that Ins(1,4,5)P3 is an essential element for the action of TRH, providing a rapid mechanism for Ca2+ mobilization induced by the releasing hormone and (2) that TRH action in mobilizing intracellular Ca2+ is sustained by a slower mechanism which is independent of Ins(1,4,5)P3.     

Effects of local anesthetics and hemicholinium-3 on 45-Ca efflux in barnacle muscle fibers.

Benzocaine, which occurs in the uncharged form in the physiological range of pH, caused inhibition of 45-Ca efflux in branacle muscle fibers. By contrast, in the presence of a low external Ca-2+ concentration it produced stimulation of the efflux. Both the inhibitory and stimulatory actions of benzocaine appeared to be less potent than those of procaine. Hemicholinium-3 (HC-3), on the other hand, which exists only in the charged form, caused a large stimulation of the 45-Ca efflux following microinjection, and the potency of this action was found to be at least 10 times greater than that of procaine. External application of HC-3 produced inhibition occasionally. Effects of tetracaine were similar to those produced by procaine; however, its inhibitory action was greater in more alkaline solution, which is the opposite of that observed with procaine. Lidocaine produced a less consistent effect than procaine; the inhibitory action of the former was less potent but the stimulatory action of the two anesthetics were comparable, p-Aminobenzoic acid was without effect on 45-Ca efflux. These results indicate that both the charged and uncharged forms of local anesthetics are capable of causing stimulatory and inhibitory effects on 45-Ca efflux in barnacle muscle fibers, and that the inhibition produced is the result of action on the CA-Ca exchange system whereas the stimulation is the result of release of Ca from internal storage sites.