Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Assessment of ability of endangered Caspian brown trout, <Emphasis Type="Italic">Salmo trutta caspius</Emphasis> (Salmonidae) in production of semen with desirable quality

To assay the effects of stripping frequency on semen characteristics of Caspian brown trout, Salmo trutta caspius, the chemical composition of seminal fluid, sperm production (semen volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were investigated over four times stripping during the spawning season. According to data, semen volume, sperm density, osmolality and the concentrations of Na⁺, Cl⁻, K⁺, Ca²⁺, Mg²⁺ and total protein gradually decreased by increasing of stripping frequency. The values of glucose and triglyceride had no significant changes over four times stripping. Also, the values of semen pH, the percentage (5 s after activation) and duration of motility were statistically stable until third stripping but a decrease was recorded for these parameters in the fourth stripping. As well as, significant positive correlations were found for sperm density vs. K⁺, Cl⁻, Na⁺, Ca²⁺, Mg²⁺, total protein, spermatocrit; the percentage of motile spermatozoa vs. Ca²⁺, Mg²⁺, K⁺, Cl⁻, Na⁺, total protein, and also the duration of motility vs. K⁺, Cl⁻, total protein and pH. In text, specific objectives of this study have been expressed.     

Spatial distributions and net deposition rates of mineral elements in the elongating wheat (Triticum aestivum L.) leaf under saline soil conditions

Wheat leaf growth is known to be spatially affected by salinity. The altered spatial distribution of leaf growth under saline conditions may be associated with spatial changes in tissue mineral elements. The objective of this study was to evaluate the spatial distributions of mineral elements and their net deposition rates in the elongating and mature zones of leaf 4 of the main stem of spring wheat (Triticum aestivum L. cv. Lona) during its linear growth phase under saline soil conditions. Plants were grown in an illitic-chloritic silty loam with 0 and 120 mM NaCl. Three days after emergence of leaf 4, sampling was begun at 3 and 13 h into the 16-h light period. Spatial distributions of fresh weight (FW), dry weight (DW), and Na⁺, K⁺, Cl⁻, NO⁻ ₃, Ca²⁺, Mg²⁺, total P, and total N in the elongating and mature tissues were determined on a millimeter scale. The patterns of spatial distribution of Na⁺, Cl⁻, K⁺, NO₃ ⁻, and Ca²⁺ in the growing leaves were affected by salinity, while those of Mg²⁺, total P, and total N were not. Sodium, K⁺, Cl⁻, Ca²⁺, Mg²⁺, and total N concentrations (mmol · kg⁻¹ FW) were consistently higher at 120 mM NaCl than at 0 mM NaCl along the leaf axis from the leaf base, whereas NO₃ ⁻ concentration was lower at 120 mM NaCl. Deposition rates of all nutrients were greatest in the elongation zone. The elongation zone was the strongest sink for mineral elements in the leaf tissues. Local net deposition rates of Na⁺, Cl⁻, Ca²⁺, and Mg²⁺ (mmol · kg⁻¹ FW · h⁻¹) in the most actively elongating zone were enhanced by 120 mM NaCl, whereas for NO₃ ⁻ this was depressed. The lower supply of NO⁻ ₃ to growing leaves may be responsible for the inhibition of growth under saline conditions. Higher tissue concentrations of Na⁺ and Cl⁻ may cause ion imbalance but probably did not result in ion toxicity in the growing leaves. Potassium, Ca²⁺, Mg²⁺, total P, and total N are less plausibly responsible for the reduction in leaf growth in this study. Higher tissue K⁺ and Ca²⁺ concentrations at 120 mM NaCl are probably due to the presence of high Ca²⁺ in the soil of this study. Received: 13 March 1997 / Accepted: 9 June 1997     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

The effects of variable water salinity and ionic composition on the plasma status of the Pacific Hagfish (<Emphasis Type="Italic">Eptatretus stoutii</Emphasis>)

Hagfish are the most pleisiomorphic extant craniates, and based on the similarity of ionic concentrations between their internal milieu and seawater (SW), they have long been touted as a model for osmo- and ionoconformation. As a result, the lack of direct symmetry between hagfish plasma and the environment with respect to [Na⁺], [Cl⁻], [Mg²⁺], and [Ca²⁺] have been left largely unexplored. In order to determine the capacity of hagfish to regulate their blood compartment, we exposed Pacific hagfish (Eptatretus stoutii) to 24, 32, 40, and 48 g/l salinity for 48 h, as well as to two treatments where a portion of the water [Na⁺] was replaced with either Mg²⁺ or Ca²⁺ at constant salinity for up to 6 days. Following exposure, we measured plasma ion status, pH, and total carbon dioxide (TCO₂). As expected, our results indicated that hagfish had no capacity to regulate plasma osmolality, [Na⁺], or [Cl⁻], but they did maintain plasma [Mg²⁺] and [Ca²⁺] nearly constant despite fluctuation of environmental salinity or elevated water [Mg²⁺] and [Ca²⁺] (two- and sevenfold, respectively). Furthermore, exposure to elevated water [Mg²⁺] and [Ca²⁺] resulted in a large increase of plasma TCO₂ with little to no increase of plasma pH. We concluded that hagfish may control plasma [Mg²⁺] and [Ca²⁺] at levels below that of their environment via secretion of HCO₃ ⁻, similar to the mechanisms described in the intestine of teleosts. We speculate that secretion of HCO₃ ⁻ likely evolved to maintain plasma [Mg²⁺] and [Ca²⁺] below environmental levels (both of which negatively affect nervous function and muscle contraction if elevated), and was an exaptation for the development of water-absorption mechanisms in the intestine of marine osmoregulators. The ancestors of modern hagfish are thought to have never entered freshwater, thus investigations into their ionoregulatory ability potentially have profound implications regarding the evolution of fishes.     

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca²⁺] levels. Pipettes of tip outer diameter down to 0.4 μm were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204–206) but with very hydrophobic cations replacing Na⁺ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca²⁺] levels. Lowering the Ca²⁺ concentration in the internal electrolyte also increased electrode lifetime.Electrodes showed a Nernstian response to [Ca²⁺] down to 1 μM free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca²⁺. Selectivity for Ca²⁺ over Mg²⁺ and H⁺ was sufficiently high that typical free intracellular levels of Mg²⁺ and H⁺ caused negligible interference. Selectivity for Ca²⁺ over Na⁺ was adequate for cells with 10^?2 M free Na⁺, but higher levels could raise significantly the detection limit for Ca²⁺. Preliminary measurements of [free Ca²⁺] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca²⁺ microelectrodes and optical indicators are discussed.     

Betaine activates a hyperpolarizing chloride conductance in squid olfactory receptor neurons

Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl⁻ and could be reversibly blocked by the Cl⁻ channel blockers 4-acetamido-4′-isothio-cyanatistilbene-2,2′disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl⁻]_i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl⁻ conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K⁺ or Na⁺ ruled out the possibility that the Cl⁻ conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca²⁺ imaging experiments showed that betaine did not affect [Ca²⁺]_i, suggesting that the Cl⁻ current is not Ca²⁺ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons. Accepted: 20 March 1998     

Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na⁺, K⁺, Cl⁻, Mg⁺², and Ca⁺² contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K⁺, Mg⁺² and Ca⁺². The accumulation of Na⁺ and Cl⁻ ions was less in the hydroxyproline resistant calli.     

Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Calcium sensitive non-selective cation current promotes seizure-like discharges and spreading depression in a model neuron

As described by others, an extracellular calcium-sensitive non-selective cation channel ([Ca²⁺]_o-sensitive NSCC) of central neurons opens when extracellular calcium level decreases. An other non-selective current is activated by rising intracellular calcium ([Ca²⁺] _i ). The [Ca²⁺]_o-sensitive NSCC is not dependent on voltage and while it is permeable by monovalent cations, it is blocked by divalent cations. We tested the hypothesis that activation of this channel can promote seizures and spreading depression (SD). We used a computer model of a neuron surrounded by interstitial space and enveloped in a glia-endothelial “buffer” system. Na⁺, K⁺, Ca²⁺ and Cl⁻ concentrations, ion fluxes and osmotically driven volume changes were computed. Conventional ion channels and the NSCC were incorporated in the neuron membrane. Activation of NSCC conductance caused the appearance of paroxysmal afterdischarges (ADs) at parameter settings that did not produce AD in the absence of NSCC. The duration of the AD depended on the amplitude of the NSCC. Similarly, NSCC also enabled the generation of SD. We conclude that NSCC can contribute to the generation of epileptiform events and to spreading depression.     

Roles of Ca2+ and Protein Tyrosine Kinase in Insulin Action on Cell Volume via Na+ and K+ Channels and Na+/K+/2Cl− Cotransporter in Fetal Rat Alveolar Type II Pneumocyte

The aim of the present study was to investigate the roles of Ca²⁺ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca²⁺ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca²⁺ (<1 nm). This insulin action in a Ca²⁺-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na⁺/K⁺/2Cl⁻ cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na⁺ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca²⁺-free solution was completely blocked by quinine (1 mm, a blocker of Ca²⁺-activated K⁺ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm extracellular Ca²⁺, that the stimulatory action of insulin on an amiloride-sensitive Na⁺ channel and a bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter requires Ca²⁺, and that in a Ca²⁺-free solution insulin activates a quinine-sensitive K⁺ channel but not in the presence of 1 mm Ca²⁺. The insulin action on cell volume in a Ca²⁺-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca²⁺-ATPase which depletes the intracellular Ca²⁺ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca²⁺-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K⁺ channel is mediated through PTK activity in a cytosolic Ca²⁺-dependent manner. Lavendustin A, further, completely blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in a Ca²⁺-free solution, but only partially blocked the activity of the Na⁺/K⁺/2Cl⁻ cotransporter in the presence of 1 mm Ca²⁺. This observation suggests that the activity of the Na⁺/K⁺/2Cl⁻ cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca²⁺-independent pathway and the other is a PTK-independent, Ca²⁺-dependent pathway. Further, we observed that removal of extracellular Ca²⁺ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na⁺ channel and the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter, and that removal of extracellular Ca²⁺ abolished the activity of the quinine-sensitive K⁺ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca²⁺ results from diverse effects on the cotransporter and Na⁺ and K⁺ channels. Received: 2 September 1998/Revised: 30 November 1998     

Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.     

The role of ion regulation in the control of the distribution of Gammarus tigrinus (Sexton) in salt-polluted rivers

Fluctuating salinities at different sites on the German salt-polluted rivers Werra and Weser were compared with extracellular ion levels of specimens of Gammarus tigrinus (Sexton; Amphipoda, Crustacea), collected at the same sites. G. tigrinus regulated haemolymph concentrations of inorganic anions (Cl⁻, SO²⁻ ₄, PO³⁻ ₄) and cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) during fluctuations of salt pollution in the upper Weser. This capacity to regulate varying levels of salt pollution in the upper Weser, correlated well with the distribution of the brackish amphipods in this river ecosystem. G. tigrinus tolerated periods of Na⁺ and Cl⁻ stress (>380 mmol l⁻¹) without compensating these maxima by regulating extracellular Na⁺ and Cl⁻. However, during such bursts of Na⁺ and Cl⁻ stress in Werra and Weser, the ability to regulate extracellular [K⁺] at river water K⁺ stress of ≥6.0 mmol l⁻¹ may explain why this brackish species has been more successful in these rivers than its competitors like Gammarus pulex. The present investigation demonstrates that the water salinity affects the [NO⁻ ₃] in the haemolymph of G. tigrinus. With increasing hypo-osmotic stress the animals accumulate increasing amounts of NO⁻ ₃. A simultaneous increase in stream water [NO⁻ ₃] causes an additional accumulation of NO⁻ ₃ in the haemolymph. The high extent of accumulation indicates that active ion transport systems may be involved. The accumulation of NO⁻ ₃ in the haemolymph has low physiological consequences to G. tigrinus, but when hypo-osmotically stressed under anoxic conditions, nitrite formed by the reduction of nitrate may have an adverse affect on the metabolism of G. tigrinus. Accepted: 4 October 1999     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Computer simulations of neuron-glia interactions mediated by ion flux

Extracellular potassium concentration, [K⁺]_o, and intracellular calcium, [Ca²⁺]_i, rise during neuron excitation, seizures and spreading depression. Astrocytes probably restrain the rise of K⁺ in a way that is only partly understood. To examine the effect of glial K⁺ uptake, we used a model neuron equipped with Na⁺, K⁺, Ca²⁺ and Cl⁻ conductances, ion pumps and ion exchangers, surrounded by interstitial space and glia. The glial membrane was either “passive”, incorporating only leak channels and an ion exchange pump, or it had rectifying K⁺ channels. We computed ion fluxes, concentration changes and osmotic volume changes. Increase of [K⁺]_o stimulated the glial uptake by the glial 3Na/2K ion pump. The [K⁺]_o flux through glial leak and rectifier channels was outward as long as the driving potential was outwardly directed, but it turned inward when rising [K⁺]_o/[K⁺]_i ratio reversed the driving potential. Adjustments of glial membrane parameters influenced the neuronal firing patterns, the length of paroxysmal afterdischarge and the ignition point of spreading depression. We conclude that voltage gated K⁺ currents can boost the effectiveness of the glial “potassium buffer” and that this buffer function is important even at moderate or low levels of excitation, but especially so in pathological states.     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Effects of diet and temperature on Morimus funereus larval hemolymph cation concentrations

The effects of diet and different constant temperatures on hemolymph cation concentrations (Na⁺, K⁺, Mg²⁺, Ca²⁺) have been studied in Morimus funereus larvae collected from natural habitat, fed natural (oak or beech bark) or artificial diet, as well as in larvae reared from hatching on an artificial diet. In the hemolymph of larvae maintained under natural conditions Mg²⁺ was dominant, whereas Na⁺ concentration was very low. In their natural diets concentrations of Na⁺ and K⁺ were very low, while those of Ca²⁺ and Mg²⁺ were high. In larvae continuously reared on an artificial diet, hemolymph Mg²⁺ concentration was significantly decreased and Na⁺ concentration increased more than fourfold compared to the results obtained in oak-fed larvae. Na⁺ and K⁺ are the dominant cations in the artificial diet. The concentrations of K⁺ and Ca²⁺ in the hemolymph of larvae fed natural or artificial diet are nearly identical, suggesting the existence of an internal regulatory mechanism in this insect for these cations. The hemolymph cation concentrations of M. funereus larvae are predominantly dependent upon the diet consumed, much less upon the environmental temperatures. The most stable concentrations of cations were observed in larvae continuously fed an artificial diet and exposed to different constant temperatures. There was much less stability in the hemolymph cation concentration in oak larvae fed either natural or artificial food after their transfer to constant temperatures. With respect to the response to the external factors studied, the most sensitive are the Na⁺ concentrations, the most stable seems to be K⁺. © 1992 Wiley-Liss, Inc.     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Accumulation of inorganic and organic osmolytes and their role in osmotic adjustment in NaCl-stressed vetiver grass seedlings

The accumulation of inorganic and organic osmolytes and their role in osmotic adjustment were investigated in roots and leaves of vetiver grass (Vetiveria zizanioides) seedlings stressed with 100, 200, and 300 mM NaCl for 9 days. The results showed that, although the contents of inorganic (K⁺, Na⁺, Ca²⁺, Mg²⁺, Cl⁻, NO₃⁻, SO₄²⁻ and H₂PO₃⁻)) and organic (soluble sugar, organic acids, and free amino acids) osmolytes all increased with NaCl concentration, the contribution of inorganic ions (mainly Na⁺, K⁺, and Cl⁻) to osmotic adjustment was higher (71.50–80.56% of total) than that of organic solutes (19.43–28.50%). The contribution of inorganic ions increased and that of organic solutes decreased in roots with the enhanced NaCl concentration, whereas the case in leaves was opposite. On the other hand, the osmotic adjustment was only effective for vetiver grass seedlings under moderate saline stress (less than 200 mM NaCl).     

Role of the choline exchanger in Na-independent Mg efflux from rat erythrocytes

Two types of Na⁺-independent Mg²⁺ efflux exist in erythrocytes: (1) Mg²⁺ efflux in sucrose medium and (2) Mg²⁺ efflux in high Cl⁻ media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na⁺-independent Mg²⁺ efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K⁺,Cl⁻- and Na⁺,K⁺,Cl⁻-symport, Na⁺/H⁺-, Na⁺/Mg²⁺-, Na⁺/Ca²⁺- and K⁺(Na⁺)/H⁺ antiport, Ca²⁺-activated K⁺ channel and Mg²⁺ leak flux. We suggest that, in choline Cl medium, Na⁺-independent Mg²⁺ efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg²⁺ efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg²⁺ to the same degree. The K_d value for inhibition of [¹⁴C]choline efflux and for inhibition of Mg²⁺ efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg²⁺ efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg²⁺ efflux was reduced to the same degree by these inhibitors as was the [¹⁴C]choline efflux.