Comparison of tetrodotoxin and procaine in internally perfused squid giant axons

Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000–10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 nM blocked the action potential and the early transient current in 2–3 min. Internally perfused procaine at concentrations of 1–10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2–4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.     

Effects of bivalent cations on post-rest adaptation in guinea-pig heart muscle

In isolated papillary muscles of guinea-pig hearts, the inotropic effects of bivalent cations, Ca2+, Ba2+, Sr2+, and Ni2+, were investigated during post-rest adaptation in order to study their individual action on excitation-contraction coupling. Upon exposure to each cation studied, the force of contraction was transiently enhanced, whereas the steady state force was influenced differently: it increased with Ca2+, Ba2+ and Sr2+ and was depressed by Ni2+. The transmembrane action potentials (measured at 90% repolarization) were slightly prolonged by Sr2+ and even more by Ba2+, and were shortened by Ca2+ and Ni2+. After 10 min rest, the post-rest contractions consisted of a late peak (PII) that was enhanced in high Ca2+-solution an by Sr2+. Ni2+ and Ba2+ depressed PII and during adaptation to pre-rest controls an early peak of contraction (PI) prevailed. There was no simple relation between post-rest adaptation of force and the duration of action potential in the presence of the bivalent cations tested. During post-rest adaptation the two components of contraction can be separated. The results are interpreted in terms of a model of excitation-contraction coupling which derives Ca ions for contractile activation from two sources: transmembrane calcium influx and calcium release from cellular stores. From the different effects on post-rest adaptation it is concluded that the individual cations influence excitation-contraction coupling more specifically and not merely by "screening-off" the negative surface charges.     

Membrane currents in a calcium type muscle membrane under voltage clamp.

Four ionic current components were identified in the total membrane current recorded under voltage clamp conditions from the muscle membrane of the crayfish (Astacus fluviatilis). The early inward current component is dependent on the presence of Ca2+ ions, disappears in Ca2+ free solutions and is insensitive to variaton of external Na+ ions and to tetrodotoxin. The outward current consists of at least three components, an early, a late and a slow outward current. The outward currents are sensitive to TEA and their reversal potentials differ. The early potassium current may be separated in a proportion of fibres by a hump from the later potassium current. An insufficient space clamp as a cause of the hump was excluded by comparing the size of the clamped membrane area with the distribution of large membrane clefts in the fibre. The early outward current is critically dependent on the presence of Ca2+ ions and is relatively more sensitive to TEA ions and to conditioning depolarisation than the late outward current.     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

Differences in Na and Ca Spikes As Examined by Application of Tetrodotoxin, Procaine, and Manganese Ions

The effects of tetrodotoxin, procaine, and manganese ions were examined on the Ca spike of the barnacle muscle fiber injected with Ca-binding agent as well as on the action potential of the ventricular muscle fiber of the frog heart. Although tetrodotoxin and procaine very effectively suppress the "Na spike" of other tissues, no suppressing effects are found on "Ca spike" of the barnacle fiber, while the initiation of the Ca spike is competitively inhibited by manganese ions. The initial rate of rise of the ventricular action potential is suppressed by tetrodotoxin and procaine but the plateau phase of the action potential is little affected. In contrast the suppressing effect of manganese ions is mainly on the plateau phase. The results suggest that the plateau phase of the ventricular action potential is related to the conductance increase in the membrane to Ca ions even though Na conductance change may also contribute to the plateau.     

Calcium is necessary for light excitation in barnacle photoreceptors

Summary Illumination of barnacle (Balanus amphitrite) photoreceptors is known to increase the membrane permeability to sodium and Ca²⁺ ions resulting in a depolarizing receptor potential. In this report, we show that lanthanum (La³⁺), a known inhibitor of Ca-binding proteins, reversibly eliminates the receptor potential of barnacle photoreceptors when applied to the extracellular space. Similar reversible elimination of the light response was obtained by removing extracellular Ca²⁺ by application of the calcium chelating agent EGTA. Iontophoretic injection of Ca²⁺, but not K⁺ into the cells protected both the transient and the steady-state phases of the receptor potential from elimination by EGTA while only the transient phase was protected in the presence of La³⁺. The EGTA experiments suggest that internal Ca²⁺ is necessary for light excitation of barnacle photoreceptors while the La³⁺ experiments suggest that La³⁺-sensitive inward current is necessary to maintain excitation during prolonged light.Abbreviations EGTA ethylenglyol-bis-(-aminoethylether) N, N, N¹, N¹-tetraacetate - BAPTA bis-(0-aminophenoxy)-ethane-N, N, N¹, N¹-tetraacetic acid - DMSO dimethyl sulfoxide - trp transient receptor potential - nss no steady state - ASW artificial sea water     

Hemolymph osmolality and cation concentrations in Litopenaeus vannamei during exposure to artificial sea salt or a mixed-ion solution: relationship to potassium flux

Interest in culturing the Pacific white shrimp Litopenaeus vannamei in low-salinity and brackish-well waters has led to questions about the ability of this species to osmo- and ionoregulate in environments containing low concentrations of ions and in environments with ionic ratios that differ from those found in sea water. After seven days, hemolymph osmolality and potassium, sodium and calcium values were all significantly affected by salinity (as artificial sea salt) with values decreasing with decreasing salinity. These decreases were small, however, relative to decreases in salinity, indicating iono- and osmoregulation with adjustment for gradients. The hemolymph osmolality and sodium and calcium concentrations in shrimp exposed to either 2 g/L artificial sea salt or 2 g/L mixed-ion solution (a mixture of sodium, potassium, calcium, and magnesium chlorides that approximate the concentrations and ratios of these cations found in 2 g/L dilute seawater) did not differ significantly. However, hemolymph potassium levels were significantly lower in shrimp held in the mixed-ion environment. Potassium influx rates were similar in shrimp held in either artificial sea salt or mixed ions. The results of this study indicate that salinity affects hemolymph-cation concentrations and osmolality. Further, differential potassium-influx rates do not appear to be the basis for low hemolymph potassium levels observed in shrimp held in mixed-ion environments.     

Osmoregulation in the Extremely Euryhaline Marine Micro-Alga Chlorella autotrophica

Chlorella autotrophica (Clone 580) grows over the external salinity range of 1 to 400% artificial sea water (ASW), can photosynthesize over the range from 1 to 600% ASW, and survives the complete evaporation of seawater. The alga grown at high salinities shows an increase in cell volume and a small decrease in cell water content. Measurements of ion content were made by neutron activation analysis on cells washed in isoosmotic sorbitol solutions which contained a few millimolar of major ions to prevent ion leakage. Cells grown at various ASW concentrations contain large quantities of sodium, potassium, and chloride ions. Measurements of cations associated with cell wall and intracellular macromolecules were made to determine intracellular concentration of free ions. The proline content of cells increases in response to increases in external salinity. Cells in 300% ASW contain 1500 to 1600 millimolar proline.     

Time course of osmotic adaptation and gill energetics of rainbow trout (Salmo gairdneri R.) following abrupt changes in external salinity

Summary The physiological and biochemical responses of rainbow trout (mean weight 250 g) to abrupt increases in salinity have been investigated. An initial crisis period lasting about 30 h was characterized by an increase in plasma and muscle ions, a rapid gill dehydration and a pronounced acidosis following a transient alkalosis. Mortality was low during this period. During the following days, gradual changes resulted in new steady state levels for most parameters examined.Analysis of adenylate pool (ATP, ATP/ADP ratio and energy charge) in the gills demonstrated an increased energy demand exhibiting two phases (4 h and 3 days), and a return to freshwater values. The gill respiration rate was constant during 3 days in sea water and decreased slightly later on. It was not influenced during the reverse transfer of seawater adapted fish into fresh water at the level of either isolated gills or perfused heads.     

Patch-reef morphology as a proxy for Holocene sea-level variability,Northern Florida Keys,USA

A portion of the northern Florida Keys reef tract was mapped with the NASA Experimental Advanced Airborne Research Lidar (EAARL) and the morphology of patch reefs was related to variations in Holocene sea level. Following creation of a lidar digital elevation model (DEM), geospatial analyses delineated morphologic attributes of 1,034 patch reefs (reef depth, basal area, height, volume, and topographic complexity). Morphometric analysis revealed two morphologically different populations of patch reefs associated with two distinct depth intervals above and below a water depth of 7.7 m. Compared to shallow reefs, the deep reefs were smaller in area and volume and showed no trend in topographic complexity relative to water depth. Shallow reefs were more variable in area and volume and became flatter and less topographically complex with decreasing water depth. The knoll-like morphology of deep reefs was interpreted as consistent with steady and relatively rapidly rising early Holocene sea level that restricted the lateral growth of reefs. The morphology of shallow “pancake-shaped” reefs at the highest platform elevations was interpreted as consistent with fluctuating sea level during the late Holocene. Although the ultimate cause for the morphometric depth trends remains open to interpretation, these interpretations are compatible with a recent eustatic sea-level curve that hindcasts fluctuating late Holocene sea level. Thus it is suggested that the morphologic differences represent two stages of reef accretion that occurred during different sea-level conditions.     

A method of microinjection: delivering monoclonal antibody 1223 into sea urchin embryos.

In this paper, a simpler method of microinjecting sea urchin embryos without using the conventional microinjection chamber designed by Kiehart is reported. A trough was made on a surface of 0.6% agarose gel dissolved in artificial sea water. Approximately fifty hatched embryos could be loaded in the trough and, consequently, swimming embryos were trapped in the trough. Monoclonal antibody (mAB) 1223 which blocks spiculogenesis in vitro was delivered into the blastocoels of sea urchin embryos to test whether this antibody inhibits spiculogenesis in vivo and also, whether this new technique is effective for the microinjection of the sea urchin embryos. The embryos were injected with mAB1223 at the hatched blastula, early mesenchyme blastula and early gastrula stages, and 63%, 90% and 97% of the embryos did not form spicules at the late gastrula stage, respectively. Therefore, mAB1223 was shown to also block spiculogenesis in vivo. From the fact that spiculogenesis occurred at a lower rate when mAB1223 was injected at the hatched blastula stage than at later stages, it may be speculated that endogenous proteases degraded the injected antibodies. Using this technique, extracellular events in the blastocoel or the function of certain molecules expressed in blastocoel can be easily investigated in vivo.     

Genome incompatibility between rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) and sea trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) and induction of the interspecies gynogenesis

Rainbow trout (Oncorhynchus mykiss Walbaum) and sea trout (Salmo trutta Linnaeus, 1758) show large karyotypic differences and their hybrid offspring is not viable due to unstable karyotype and chromosome fragmentation. However, gametes from these two species were used to induce gynogenetic development. Rainbow trout eggs activated by UV-irradiated sea trout sperm were subjected to high hydrostatic pressure (HHP) shock to prevent release of the 2nd polar body (early shock) or to inhibit the first cleavage (late shock) in order to produce diploid meiotic gynogenotes and gynogenetic doubled haploids (DHs), respectively. Cytogenetic analysis proved fish that development was induced by the sea trout spermatozoa were rainbow trout. In turn, molecular examination confirmed homozygosity of the gynogenetic DHs. Presumed appearance of the recessive alleles resulted in lower survival of the gynogenetic DH larvae (~25%) when compared to survival of the heterozygous (meiotic) gynogenotes (c. 50%). Our results proved that genomic incompatibilities between studied trout species result in the hybrid unviability. However, artificial gynogenesis including activation of rainbow trout eggs with UV-irradiated sea trout spermatozoa was successfully induced. As both species are unable to cross, application of the UV-irradiated sea trout spermatozoa to activate rainbow trout development assures only maternal inheritance with no contamination by the residues of the paternal chromosomes.     

Nitric oxide-induced eosinophil apoptosis is dependent on mitochondrial permeability transition (mPT), JNK and oxidative stress: apoptosis is preceded but not mediated by early mPT-dependent JNK activation

Background

Eosinophils are critically involved in the pathogenesis of asthma. Nitric oxide (NO) is produced in high amounts in asthmatic lungs and has an important role as a regulator of lung inflammation. NO was previously shown to induce eosinophil apoptosis mediated via c-jun N-terminal kinase (JNK) and caspases. Our aim was to clarify the cascade of events leading to NO-induced apoptosis in granulocyte macrophage-colony stimulating factor (GM-CSF)-treated human eosinophils concentrating on the role of mitochondria, reactive oxygen species (ROS) and JNK.

Methods

Apoptosis was determined by flow cytometric analysis of relative DNA content, by Annexin-V labelling and/or morphological analysis. Immunoblotting was used to study phospho-JNK (pJNK) expression. Mitochondrial membrane potential was assessed by JC-1-staining and mitochondrial permeability transition (mPT) by loading cells with calcein acetoxymethyl ester (AM) and CoCl₂ after which flow cytometric analysis was conducted. Statistical significance was calculated by repeated measures analysis of variance (ANOVA) or paired t-test.

Results

NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) induced late apoptosis in GM-CSF-treated eosinophils. SNAP-induced apoptosis was suppressed by inhibitor of mPT bongkrekic acid (BA), inhibitor of JNK SP600125 and superoxide dismutase-mimetic AEOL 10150. Treatment with SNAP led to late loss of mitochondrial membrane potential. Additionally, we found that SNAP induces early partial mPT (1 h) that was followed by a strong increase in pJNK levels (2 h). Both events were prevented by BA. However, these events were not related to apoptosis because SNAP-induced apoptosis was prevented as efficiently when BA was added 16 h after SNAP. In addition to the early and strong rise, pJNK levels were less prominently increased at 20–30 h.

Conclusions

Here we demonstrated that NO-induced eosinophil apoptosis is mediated via ROS, JNK and late mPT. Additionally, our results suggest that NO induces early transient mPT (flickerings) that leads to JNK activation but is not significant for apoptosis. Thereby, we showed some interesting early events in NO-stimulated eosinophils that may take place even if the threshold for irreversible mPT and apoptosis is not crossed. This study also revealed a previously unknown physiological function for transient mPT by showing that it may function as initiator of non-apoptotic JNK signalling.     

Properties of an endogenous steady current in rat muscle

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.     

Relationship between ion distribution and membrane potential during a steady state

Equations were derived showing the relationship between the membrane potential and the quantities which influence it under steady state conditions. Essentially, the membrane potential is caused by the valence and concentration of the non-permeating ions. The permeating ions can modify the membrane potential by altering the relative concentration of the non-permeating ions with respect to the concentration of the permeating ions. For muscle, the sodium cations act as the non-permeating ions in the extracellular environment by the maintenance of some type of active metabolic process and large anions act as the non-permeating ions in the intracellular environment. Both of these non-permeating ions contribute about equally to the maintenance of the resting membrane potential. When the active metabolic process for sodium extrusion breaks down or when acids are added, the membrane potential should decrease. Water should enter the cell when the sodium metabolic process is diminished; water should leave the cell when acids are added. When acid is added, it is expected that the cations potassium and sodium will leave the cell with little or no shift of the chloride ions.     

Ammonium Ion Currents in the Squid Giant Axon

Voltage-clamp studies on intact and internally perfused squid giant axons demonstrate that ammonium can substitute partially for either sodium or potassium. Ammonium carries the early transient current with 0.3 times the permeability of sodium and it carries the delayed current with 0.3 times the potassium permeability. The conductance changes observed in voltage clamp show approximately the same time course in ammonium solutions as in the normal physiological solutions. These ammonium ion permeabilities account for the known effects of ammonium on nerve excitability. Experiments with the drugs tetrodotoxin (TTX) and tetraethyl ammonium chloride (TEA) demonstrate that these molecules block the early and late components of the current selectively, even when both components are carried by the same ion, ammonium.     

Requirement of divalent cations for fast exchange of actin monomers and actin filament subunits

The exchange of actin monomers and actin filament subunits was investigated in the presence of different cations. At high concentrations of calcium (1.2 mm) or magnesium (0.6 mm) the half-time of exchange was less than two hours. When the calcium ions were chelated by EGTA² and potassium (12 mm) was added the exchange was immediately stopped. In the presence of potassium ions (19 mm) at low concentrations of divalent cations (less than 40 μm) no exchange was observed within 15 hours. Fast exchange was initiated upon addition of calcium ions (1.2 mm).The results were interpreted by the model of head to tail polymerization in which filaments lengthen at one end and shorten simultaneously at the other. At high concentrations of divalent cations association and dissociation reactions occur mainly at different ends, whereas in the presence of potassium and at low concentrations of divalent cations these reactions take place at each end with similar frequencies. The efficiency of the head to tail polymerization depends strongly on the concentration and on the type of cations present.     

Stream chemistry and hydrologic pathways during snowmelt in a small watershed adjacent Lake Superior

In regions with airborne contaminants and large snowpacks, there is concern over the impact that snowmelt chemical pulses — periods of sharp increase in meltwater solute concentration — could have on aquatic resources during spring runoff. A major variable in determining such an effect is the flow path of snowmelt solutes to the stream or lake. From December 1988, to late April 1989, the quality and quantity of precipitation, snowmelt, soil solution and streamwater were measured in a 176-ha gauged watershed on the south shore of Lake Superior. The main objectives were to (1) examine the change in flow path meltwaters take to the stream during distinct winter and spring hydrologic periods, (2) quantify ecosystem-level ion budgets prior to, during, and following snowmelt, and (3) examine if streamwater chemistry might be a sensitive indicator of change in ecosystem flow paths. Prior to peak snowmelt, groundwater made up 80% of stream discharge. During peak snowmelt, the groundwater level rose to the soil surface resulting in lateral water movement through near-surface macropores and as overland flow. Near the end of snowmelt, melt-waters exerted a piston action on deeper soil solution again increasing its relative contribution to streamwater discharge. Net groundwater drawdown during the study resulted in streamwater discharge about equal to precipitation inputs. Unfrozen soils and brief mid-winter thaws resulted in steady snowmelt throughout early and mid-winter. The snowpack lost > 50% of most ions prior to the period of major snowmelt and high stream discharge in late March and early April. Snowmelt and streamwater NO₃ ^– and NH₄ pulses occurred before the period of overland flow and peak streamwater discharge (April 4–24). During overland flow, stream discharge of total N, P, DOC, and AI peaked. Nutrient budgets computed for distinct hydrologic periods were much more helpful in explaining ecosystem pathways and processes than were changes in solute concentration. For the study period, watershed base cation (C_B) discharge was 23 times input and SO₄ ^2– discharge exceeded input by 42%. H⁺ was the most strongly conserved ion with output < 0.2% of input. Also conserved were NH₄ ⁺ with only 1.4% of input leaving the ecosystem and NO₃ ^– with output equal to 9.4% of input.     

Experimental approaches to the study of the formation of axial structures during early development of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The effect of mechanical extension on the differentiation of axial mesoderm in double explants (sandwiches) of Xenopus laevis embryonic tissues isolated during the early gastrula–late neurula developmen-tal period is studied. In explants at the early gastrula stage, artificial extension orients and stimulates isolated differentiation of the notochord and somites as well as their joint formation. Moreover, extension facilitated the formation of the normal anatomical structure of the notochord and affected expression of Chordin gene. At the late gastrula stage, the effect of artificial extension on joint somite–notochord differentiation was weaker. At the stage of late neurula, somites were sometimes formed in explants lacking a notochord anlage. Thus, at earlier stages, the formation of somites was stimulated by contacts with the notochord and joint development of both structures was mechanical dependent, while at the later stages, somites developed inde-pendently of the notochord. Thus, the role of tissue extension is primarily the establishment of normal mor-phology and expression of Chordin was located in the direction of extension.