The role of calcium and guanosine 3':5'-monophosphate in the action of acetylcholine on thyroid metabolism

The role of calcium and guanosine 3':5'-monophosphate (cyclic GMP) in the regulation of thyroid metabolism has been investigated in dog thyroid slices. Carbamoylcholine enhanced glucose carbon-1 oxidation, protein iodination, cyclic GMP accumulation and decreased thyrotropin-induced adenosine 3':5'-monophosphate (cyclic AMP) accumulation and iodine secretion; it did not affect protein synthesis. The effects of carbamoylcholine were reproduced under various experimental conditions by supplementary calcium in the medium, ouabain, and in media in which Na+ had been replaced by choline chloride. They were inhibited by lanthanum. These results further support the hypothesis that free intracellular Ca2+ is the intracellular signal for carbamoylcholine effects and suggest that a Na+ -gradient-driven Ca2+ extrusion mechanism operates in the thyroid cell. Mn2+ reproduced the effect of Ca2+ on glucose oxidation, protein iodination and cyclic GMP accumulation in Ca2+ -depleted slices and medium, and thus mimicked some intracellular effects of Ca2+. On the other hand Mn2+ inhibited the carbamoylcholine effect on thyrotropin-induced thyroid secretion and cyclic AMP accumulation, and Ca2+ inhibited the Mn2+-induced cyclic GMP accumulation. This suggests that the two ions compete for the same channel. Similarly Mn2+ inhibited calcium effects in the presence of ionophore A23187. Procaine inhibited protein iodination under all conditions suggesting a primary effect; it also inhibited all carbamoylcholine and ouabain actions. However the drug did not inhibit the effects of choline chloride and its action was reversed by raising carbamoylcholine but not Ca2+ concentration; it is therefore doubtful that procaine acts by blocking Ca2+ channels. In media without added Ca2+, Mn2+ increased cyclic GMP accumulation but did not decrease thyrotropin-induced cyclic AMP accumulation or iodine secretion, which suggests that cyclic GMP cannot be the sole mediator of the latter two effects of carbamoylcholine.     

The effect of butylated hydroxytoluene, an inhibitor of lipid peroxidation, on the calcium-induced uncoupling of rat liver mitochondria

The accumulation of Ca2+ by rat liver mitochondria in the presence of Pi results in spontaneous activation of respiration, accompanied by progressive loss of the accumulated cation. The lipid peroxidation inhibitor, butylated hydroxytoluene, completely prevents and reverses the loss of accumulated Ca2+ and restores respiration to the state 4 level, but exerts no effect on the rate of Ca2+ accumulation and respiration in the presence of the uncoupler. The strong inhibition by butylated hydroxytoluene of ruthenium red-insensitive Ca2+ efflux has also been observed. No correlation between the BHT-sensitive Ca2+ loss and the formation of malonic dialdehyde in mitochondria has been found. The data obtained suggest that the Ca2+-induced uncoupling of mitochondria is mainly due to the appearance of electrogenic ion fluxes that are controlled by the initial steps of lipid peroxidation.     

Instrumental role of Na+ in NMDA excitotoxicity in glucose-deprived and depolarized cerebellar granule cells

In glucose-deprived cerebellar granule cells, substitution of extracellular Na+ with Li+ or Cs+ prevented N-methyl-D-aspartate (NMDA)-induced excitotoxicity. NMDA stimulated 45Ca2+ accumulation and ATP depletion in a Na-dependent manner, and caused neuronal death, even if applied while Na,K-ATPase was inhibited by 1 mM ouabain. The cells treated with NMDA in the presence of ouabain accumulated sizable 45Ca2+ load but most of them failed to elevate cytosolic [Ca2+] upon mitochondrial depolarization. Na/Ca exchange inhibitor, KB-R7943, inhibited Na-dependent and NMDA-induced 45Ca2+ accumulation but only if Na,K-ATPase activity was compromised by ouabain. In cells energized by glucose and exposed to NMDA without ouabain, KB-R7943 reduced NMDA-elicited ionic currents by 19% but failed to inhibit 45Ca2+ accumulation. It appears that a large part of NMDA-induced Ca2+ influx in depolarized and glucose-deprived cells is mediated by reverse Na/Ca exchange. A high level of reverse Na/Ca exchange operation is maintained by a sustained Na+ influx via NMDA channels and depolarization of the plasma membrane. In cells energized by glucose, however, most Ca2+ enters directly via NMDA channels because Na,K-ATPase regenerating Na+ and K+ concentration gradients prevents Na/Ca exchange reversal. Since under these conditions Na/Ca exchange extrudes Ca2+, its inhibition destabilizes Ca2+ homeostasis.     

关于溴氰菊酯神经毒作用的生物化学研究

 本文利用生物化学的手段,对大鼠进行了急性和亚急性毒性实验,研究溴氰菊酯对动物中枢神经系统离子调节作用的影响。急性实验结果表明:<1>溴氰菊酯能显著抑制脑微粒体上的Ca~(2+)+Mg~(2+)-ATP酶和Na~++K~+-ATP酶活性,但并不降低ecto-Ca~(2+)-ATP酶(细胞表面的Ca~(2+)-ATP酶)的活性;<2>溴氰菊酯对大鼠小脑组织中的环腺苷酸含量无明显影响,但却能显著升高与其作用相反的环鸟苷酸含量。体外实验证明,溴氰菊酯能够减少线粒体对Ca~(2+)的主动摄取。在对大鼠进行的亚急性实验中,发现溴氰菊酯中毒组与对照组大鼠的Ca~(2+)+Mg~(2+)-ATP酶、Na~++K~+-ATP酶和ecto-Ca~(2+)-ATP酶的活性均无显著性差异。根据以上结果推测,在急性中毒的条件下,溴氰萄酯能引起大鼠脑神经细胞内Ca~(2+)和Na~+的浓度增高,致使神经兴奋性发生改变。     

Modulation of salt permeabilities of intestinal brush-border membrane vesicles by micromolar levels of internal calcium

A possible modulation of ion permeabilities of rat intestinal brush-border membrane vesicles by Ca2+, a putative second messenger of salt secretion, was explored by three independent methods: (1) measurements of [3H]glucose accumulation driven by a Na+ gradient; (2) stopped-flow spectrophotometry of salt-induced osmotic swelling; (3) 86Rb+, 22Na+ and 36Cl- flux measurements. Cytoskeleton-deprived membrane vesicles were prepared from isolated brushborders by thiocyanate treatment. Intravescicular Ca2+ levels were varied by preincubating vesicles in Ca-EGTA buffers in the presence of the Ca2+-ionophore A23187. At Ca2+free greater than 10(-5) M, initial Na+-dependent glucose uptake in the presence of a 0.1 M NaSCN gradient (but not in its absence) was inhibited by about 50 per cent as compared to EGTA alone (ED50 approximately equal to 10(-6) M Ca2+). By contrast, initial rates of 22Na+ uptake and reswelling rates of vesicles exposed to a NaSCN gradient were increased at least 2-fold by 10(-5) M Ca2+free. Both observations are compatible with a Ca2+-induced increase of the Na+-permeability of the vesicle membrane. The modulation of ion transport was fully reversible and critically dependent on internal Ca2+, suggesting a localization of Ca2+-sensor sites at the inner surface of the microvillous membrane. As shown by radiotracer and osmotic swelling measurements, micromolar Ca2+ additionally increased the flux rate of K+, Rb+, Cl- and NO-3 but did not change the membrane permeability for small uncharged molecules, including glucose and mannitol. The effect of Ca2+ on ion permeabilities could be blocked by Ba2+ (10(-3) M) or Mg2+ (10(-2) M), but not by amiloride (10(-3) M), apamin (2 X 10(-7) M), trifluoperazine (10(-4) M) or quinine (5 X 10(-4) M). At present it is unclear whether Ca2+ activates a nonselective cation and anion channel or multiple highly selective channels in the vesicle membrane.     

The effects of storage of sarcoplasmic reticulum fragments on the Ca2+, Mg2+-ATPase.

The effects of K+ and Na+ on the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum fragments (SRF) were investigated at 1 mM ATP. There was an alteration of the sensitivity of the ATPase to the monovalent cations during storage of the SRF preparation. The Ca2+, Mg2+-ATPase of freshly prepared SRF was slightly activated by 5-10 mM K+ and Na+. Mg2+-ATPase was inhibited by both the monovalent cations to the same extent, and this response to the ions was independent of the freshness of the preparations. After storage of SRF, however, the Ca2+,Mg2+-ATPase was markedly activated by higher concentrations of K+ and Na+ (0.2-0.3 M). K+ and Na+ reduced the Ca uptake at the steady state in freshly prepared SRF, but did not affect pre-steady state uptake. In the presence of oxalate, the rate of Ca accumulation both in fresh and stored preparations was activated by 0.1-0.2 M K+ and Na+. The Ca2+, mg2+-ATPase with oxalate, so-called "extra ATPase," showed the same response to the ions as did the activity without oxalate during storage.     

Ca2+ transport by intact synaptosomes: the voltage-dependent Ca2+ channel and a re-evaluation of the role of sodium/calcium exchange

The verapamil-sensitive Ca2+ channel in the synaptosomal plasma membrane is investigated. Verapamil is without effect on Ca2+ uptake or steady-state content in synaptosomes with a polarized plasma membrane, but completely inhibits the additional Ca2+ uptake following plasma-membrane depolarization by high [K+], by veratridine plus ouabain or by high concentrations of the permeant cation tetraphenylphosphonium. Verapamil-insensitive Ca2+ influx and steady-state content are identical in polarized and depolarized synaptosomes, even though the Na+ electrochemical potential is greatly decreased in the latter, indicating that Na+/Ca2+ exchange is not a significant mechanism for Ca2+ efflux under these conditions. A transient Na+-dependent Ca2+ efflux can only be observed on addition of Na+ to Na+-depleted depolarized synaptosomes. While 0.2 mM verapamil decreases the ate of 86Rb+ efflux and 22Na+ entry during depolarization induced by veratridine plus ouabain, the final steady-state Na+ accumulation is not inhibited. Ca2+ efflux from synaptosomes following mitochondrial depolarization does not occur by a verapamil-sensitive pathway.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Occurrence of Na(+)-Ca2+ exchange in the ciliate Euplotes crassus and its role in Ca2+ homeostasis.

Ciliates possess diverse Ca2+ homeostasis systems, but little is known about the occurrence of a Na(+)-Ca2+ exchanger. We studied Na(+)-Ca2+ exchange in the ciliate Euplotes crassus by digital imaging. Cells were loaded with fura-2/AM or SBF1/AM for fluorescence measurements of cytosolic Ca2+ and Na+ respectively. Ouabain pre-treatment and Na+o substitution in fura-2/AM-loaded cells elicited a bepridil-sensitive [Ca2+]i rise followed by partial recovery, indicating the occurrence of Na(+)-Ca2+ exchanger working in reverse mode. In experiments on prolonged effects, ouabain, Na+o substitution, and bepridil all caused Ca2+o-dependent [Ca2+]i increase, showing a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis. In addition, by comparing the effect of orthovanadate (affecting not only Ca2+ ATPase, but also Na(+)-K+ ATPase and, hence, Na(+)-Ca2+ exchange) to that of bepridil on [Ca2+]i, it was shown that Na(+)-Ca2+ exchange contributes to Ca2+ homeostasis. In electrophysiological experiments, no membrane potential variation was observed after bepridil treatment suggesting compensatory mechanisms for ion effects on cell membrane voltage, which also agrees with membrane potential stability after ouabain treatment. In conclusion, data indicate the presence of a Na(+)-Ca2+ exchanger in the plasma membrane of E. crassus, which is essential for Ca2+ homeostasis, but could also promote Ca2+ entry under specific conditions.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

An antioxidant prevents and reverses calcium-induced uncoupling of rat liver mitochondria

Accumulation of Ca2+ by rat liver mitochondria in the presence of inorganic phosphate results in spontaneous activation of respiration accompanied by a progressive loss of the accumulated cation. The lipid peroxidation inhibitor, ionol, completely prevents and reverses the Ca2+/phosphate-induced loss of accumulated Ca2+ and restores the respiration to state 4 level without having any effect on the rate of Ca2+ accumulation and respiration in the presence of an uncoupler. No correlation between the ionol-dependent loss of Ca2+ and the formation of malonic dialdehyde in mitochondria was found. The measurements of delta psi across the inner mitochondrial membrane during a progressive loss of Ca2+ suggest that the Ca2+/phosphate-induced "uncoupling" is mainly due to the appearance of electrogenic fluxes (but not Ca2+ cycling) which is under control of some products of initial steps of lipid peroxidation.     

Voltage-Sensitive Calcium Channels in Differentiated Neuroblastoma × Glioma Hybrid (NG108–15) Cells: Characterization by Quin 2 Fluorescence

Depolarization of differentiated neuroblastoma X glioma (NG108-15) cells with KCl (50 mM) or veratridine (50 microM) stimulated Ca2+ accumulation, was detected by quin 2 fluorescence. Intracellular Ca2+ concentrations ([Ca2+]i) were elevated about threefold from 159 +/- 7 to 595 +/- 52 nM (n = 12). Ca2+ entry evoked by high extracellular K+ concentration ([K+]o) was voltage-dependent and enhanced by the dihydropyridine agonists, BAY K 8644 and CGP 28 392, in a dose-dependent manner. CGP 28 392 was less potent and less efficacious than BAY K 8644. The (+) and (-) stereoisomers of 202-791 showed agonist and antagonist properties, respectively. (+)-202-791 was less potent, but as efficacious as BAY K 8644. In the absence of KCl, BAY K 8644 had no effect on Ca2+ entry. Voltage-sensitive calcium channel (VSCC) activity was blocked by organic Ca2+ channel antagonists (nanomolar range) both before and after KCl treatment and also by divalent metal cations (micromolar range). High [K+]o-induced Ca2+ accumulation was dependent on external Ca2+, but not on external Na+ ions ([Na]o), and was insensitive to both tetrodotoxin (3 microM) and tetraethylammonium (10 microM). In contrast, veratridine-induced Ca2+ accumulation required [Na+]o, and was blocked by tetrodotoxin, but not by nimodipine (1 microM). Veratridine-induced Ca2+ accumulation was slower (approximately 45 s), smaller in magnitude (approximately 30% of [K+]o-induced Ca2+ entry), and also enhanced by BAY K 8644 (approximately 50%). VSCC were identified in neuronal hybrid (NG108-15 and NCB-20) cells, but not in glial (C6BU-1), renal epithelial (MDCK), and human astrocytoma (1321N1) cells. NG108-15 cells differentiated with 1.0 mM dibutyryl cyclic AMP showed greater VSCC activity than undifferentiated cultures. These results suggest that cultured neural cells provide a useful system to study Ca2+ regulation via ion channels.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Ca2+- or Mg2+-Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport

Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1-2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM-Sr2+ or Ba2+. Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50-60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal ganglion/spinal nerve preparations, incubated for 20 h in medium containing 0.2 mM-Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM-Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+-dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg+ ATPase in the transport process.     

cGMP-mediated facilitation in nerve terminals by enhancement of the spike afterhyperpolarization

cGMP has long been suspected to play a role in synaptic plasticity, but the inaccessibility of nerve terminals to electrical recording has impeded tests of this hypothesis. In posterior pituitary nerve terminals, nitric oxide enhanced Ca(2+)-activated K+ channel activity by activating guanylate cyclase and PKG. This enhancement occurred only at depolarized potentials, so the spike threshold remained unaltered but the afterhyperpolarization became larger. During spike trains, the enhanced afterhyperpolarization promoted Na+ channel recovery from inactivation, thus reducing action potential failures and allowing more Ca(2+) to enter. Activating guanylate cyclase, either with applied nitric oxide, or with physiological stimulation to activate nitric oxide synthase, increased action potential firing. Thus, the cGMP/nitric oxide cascade generates a short-term, use-dependent enhancement of release.     

Mending Fences: Na,K-ATPase signaling via Ca2+ in the maintenance of epithelium integrity

Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low frequency Ca2+ oscillations. Within a couple of minutes this ouabain mediated signal has resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process and stimulation of embryonic growth processes.The dual function of Na,K-ATPase as an ion pump and a signal transducer is now well established and evaluation of the physiological and pathophysiological consequences of this universal signal emerges as an urgent topic for future studies.     

Ion transport systems as targets of free radicals during ischemia reperfusion injury

Oxidative stress is a recognized pathogenic factor in ischemia/reperfusion injury (IRI). Iron induced generation of reactive oxygen species (ROS) in vitro reduces both the Na+K+-ATPase activity and Na+-Ca2+ exchanger of synaptosomal membranes, concomitantly with alteration of physical state of membranes. Oxidative insult also leads to the loss of ability of endoplasmic reticular membranes (ER) to sequester Ca2+ as well as to the increase of Ca2+ permeability. Furthermore, ROS induces both lipid peroxidation and lipid-independent modifications of membrane proteins. Acute in vivo ischemia alters kinetic parameters of Na+K+-ATPase affecting mainly the dephosphorylation step of ATPase cycle with parallel changes of Na+-Ca2+ exchanger and alterations of physical membrane environment. Subsequent reperfusion after ischemia is associated with decrease of immuno signal for PMCA 1 isoform in hippocampus. In addition, incubation of non-ischemic membranes with cytosol from ischemic hippocampus decreases level of PMCA 1 in non-ischemic tissues. Loss of PMCA 1 protein is partially protected both by calpain- and by non-specific protease inhibitors which suggest possible activation of proteases in the reperfusion period. On the other hand, ischemia does not affect the level of Ca2+ pump (SERCA 2b) and calreticulin of intracellular Ca2+ stores. However, IRI resulted in a decrease of IP3 receptor I and altered active Ca2+ accumulation into the ER. A non-specific alteration of physical properties of total membranes such as the oxidative modifications of proteins as well as the content of lipoperoxidation products can also be detected after IRI. ROS can alter physical and functional properties of neuronal membranes. We discuss our results suggesting that ischemia-induced disturbation of ion transport systems may participate in or follow delayed death of neurons after ischemia.     

Green tea impedes dyslipidemia, lipid peroxidation, protein glycation and ameliorates Ca2+ -ATPase and Na+/K+ -ATPase activity in the heart of streptozotocin-diabetic rats

Diabetes-induced hyperlipidemia, oxidative stress and protein glycation impair cellular calcium and sodium homeostasis associated with abnormal membrane-bound enzyme activities resulting in cardiac dysfunction in diabetes. To explore the cardioprotective mechanism of green tea in diabetes, we measured the changes in the levels of calcium, sodium, potassium and the activities of Na+/K+ -ATPase and Ca2+ -ATPase in green tea treated diabetic rat hearts. The effect of green tea on triglycerides, lipid peroxidation and protein glycation in diabetic heart were also measured to elucidate the underlying mechanisms. Diabetes was induced by streptozotocin (STZ, 60 mg/kg i.p.). Six weeks after the induction of diabetes, some of the diabetic rats were treated orally with green tea extract (GTE) (300 mg/kg/day) for 4 weeks. GTE produced reduction in blood glucose and lowered the levels of lipid peroxides, triglycerides and extent of protein glycation in the heart of diabetic rats. GTE blunted the rise in cardiac [Ca2+] and [Na+] whereas increased the activities of Ca2+ -ATPase and Na+/K+ -ATPase in diabetic rats. In conclusion, the data provide support to the therapeutic effect of GTE and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Ca2+] and [Na+] by ameliorating Ca2+ -ATPase and Na+/K+ -ATPase activities.