ON THE ROLE OF CALCIUM IONS IN OOCYTE MATURATION IN THE POLYCHAETE CHAETOPTERUS PERGAMENTACEUS*

The germinal vesicle of mechanically released Chaetopterus oocytes disintegrates in natural sea water (NSW), but not in artificial sea water of normal composition (ASW), calcium-free sea water (CaFSW), magnesium-free sea water (MgFSW) or calcium and magnesium-free sea water (CaMgFSW). Several methods of inducing oocyte maturation using chemically well-defined medium have been established. (1) Germinal vesicle breakdown was induced by the treatment of immature oocytes with KCl (60 mM) in ASW or MgFSW. The presence of Ca²⁺ is necessary for inducing oocyte maturation with high potassium concentration. “Differentiation without cleavage” was observed after this treatment. (2) Trypsin (0.3%) induced oocyte maturation in ASW, but not in CaFSW. Oocytes matured in this manner developed to trochophores upon insemination. (3) Immature oocytes, treated with isotonic CaCl₂ for less than 1 min and then transferred to ASW, underwent germinal vesicle breakdown. The oocytes were arrested at the first meiotic metaphase and upon insemination developed to trochophore larvae. (4) Tetracaine (0.4 mM) induced oocyte maturation in the absence of Ca²⁺ in the medium. In ASW, CaFSW or CaMgFSW containing the drug, oocytes were arrested at the first meiotic metaphase, while in MgFSW with tetracaine they developed parthenogenetically up to the 4- and 8-cell stages. The role of calcium in oocyte maturation was established and its importance was discussed based on the results obtained with the different ways of inducing oocyte maturation.     

DIVALENT CATION SPECIFICITY OF THE CALCIUM REQUIREMENT FOR FAST TRANSPORT OF PROTEINS IN AXONS OF DESHEATHED NERVES

The presence of a requirement for calcium during the fast transport of [³H]protein in axons was assessed in desheathed spinal nerves of bullfrog. The nerves were desheathed locally along 4 mm of their length, and desheathing was judged effective on the basis of an enhanced uptake of [³H]leucine into that region of nerve trunk. Desheathing per se had a slight inhibitory effect on transport. Incubation of desheathed nerve trunks in calcium-free medium reduced transport by 60-80% relative to that in desheathed nerves incubated in normal medium. Addition of Mg²⁺ or Sr²⁺ to the calcium-free medium allowed transport to proceed normally. Addition of Co²⁺ or Mn²⁺ to normal medium did not affect transport in desheathed isolated nerve trunks. When ganglia and nerve trunks were both incubated in medium containing 0.18 mM-CoCl₂, transport was depressed to a similar extent proximal and distal to the desheathed region. This again indicates that Co²⁺ does not inhibit transport in desheathed nerves, whereas it does inhibit transport in the ganglia. Additive inhibitory effects were observed when ganglia were incubated in medium containing 0.018 mM-CoCl₂, and desheathed nerves were incubated in calcium-free medium. Differences in the divalent cation specificities of the axonal and ganglionic calcium requirements suggest that calcium supports transport in nerves in a manner distinct from its role in maintaining transport in spinal ganglia. It is concluded that the ganglionic calcium requirement involves initiation of axonal transport in the soma rather than translocation in the intraganglionic region of axon.     

Some electrical properties of the membrane of the barnacle muscle fibers under internal perfusion

Summary Intracellular perfusion technique has been applied to the muscle fibers of the barnacle species,Balanus nubilus. In these fibers, generation and the form of the calcium spike was governed by the frequency of stimulation and intra- and extracellular calcium concentrations. Voltage-clamp experiments showed that the magnitude of the potassium outward current was controlled by the intracellular calcium concentration whose increase, nearly 10³-fold, raised the resting membrane conductance and the outward potassium current. On the other hand, application of 10mm zinc ions inside the muscle fiber had no effect on either the resting potential or the outward potassium current but suppressed the early inward calcium current. Similarly, the inward calcium current was decreased by low concentration of sodium ions in the extracellular fluid only when its ionic strength was made low by substituting sucrose for the sodium salt. Measurement of outward current with the muscle fiber in calcium-free ASW solution and intracellularly perfused with several cationic solutions established the selectivity sequence TEA相似文献   

Effects of caffeine on the intracellular distribution of calcium in frog sartorius muscle

The influence of caffeine on the intracellular distribution of calcium in the frog sartorius muscle was studied by differential centrifugation in an attempt to identify the locus of action of this alkaloid. The problem was approached in two ways. In the first, the locus of action was sought by relating the kinetic functions of ⁴⁵Ca washout curves of muscles to changes in the distribution of ⁴⁵Ca in the isolated fractions from the same muscles. It was not possible to make any correlation of the ⁴⁵Ca-washout curves to the activity in the fractions; the relative distribution of this nuclide remained essentially unchanged at 1-, 2-, and 3-hour intervals along the curve. The washout curves appear to be the net effect of a complex interaction of the calcium in pools containing both readily exchangeable calcium and calcium which has a slow exchange or turnover rate. The second approach centered upon the examination of the effect of caffeine on the intracellular distribution of ⁴⁵Ca and of calcium among the cellular fractions. Caffeine treatment resulted in a distinct increase in the calcium content of the mitochondrial fraction and a decrease in the calcium of the microsomal fraction. Electron micrographic studies revealed significant morphological changes in the whole muscle and in the isolated mitochondrial fraction after the muscle had been exposed to caffeine in a concentration producing irreversible contracture or rigor (10 mM). The increase in calcium content of the mitochondrial fraction after caffeine treatment may be due to an actual accumulation of calcium by the mitochondria or may be the consequence of the appearance of granular vesicles in the fraction.     

The Time Course of the Loss and Recovery of Contracture Ability in Frog Striated Muscle Following Exposure to Ca-Free Solutions

Using area under the contracture curve to quantitate contractures, the diffusion coefficient of calcium ions within the frog toe muscle during washout in a calcium-free solution and subsequent recovery after reintroduction of calcium to the bathing solution was calculated to be about 2 x 10^-6 cm²/sec. The diffusion coefficient measured during washout was found to be independent of temperature or initial calcium ion concentration. During recovery it was found to decrease if the temperature was lowered. This was likely due to the repolarization occurring after the depolarizing effect of the calcium-free solution. The relation between contracture area and [Ca]_o was found to be useful over a wider range than that between maximum tension and [Ca]_o. The normalized contracture areas were larger at lower calcium concentrations if the contractures were produced with cold potassium solutions or if NO₃ replaced Cl in the bathing solutions. Decreasing the potassium concentration of the contracture solution to 50 mM from 115 mM did not change the relation between [Ca]_o and the normalized area. If the K concentration of the bathing solution was increased, the areas were decreased at lower concentrations of Ca.     

The effect of serum albumin on the efflux of K-42 from frog sartorius muscle

The effect of serum albumin on the washout of K⁴² from isolated frog sartorius muscles, previously labeled in vitro with this isotope, has been investigated. Incorporation of 1% serum albumin in the washout fluid has been found to cause a significant reduction in the rate constant for K⁴² loss from the muscle fibers. A similar reduction in the rat constant for K⁴² efflux was observed when the medium, though not containing protein, was exhaustively dialyzed before use against a solution containing serum albumin. Addition of 10^?6 M HgCl₂ to “dialyzed” Ringer increased the rate of loss of K⁴² from the fibers. Effects similar to those obtained with serum albumin were observed when 10^?4 M cysteine was incorporated in the washout fluid. 3-mercapto-propanol gave rise to transient reductions in the rate of K⁴² efflux, but, following prolonged exposure to this agent, the efflux rate was increased. 2′3-dimercapto-propanol (BAL) increased the rate of K⁴² loss from the fibers. It is suggested that this effect of serum albumin is due to its sequestering action on toxic substances (tentatively identified as heavy metals) normally present in trace amounts in Ringer's solutions.     

Novel function of eubacterial flagella: role in aggregation of a marine bacterium

Pseudomonas marina (ATCC 27 129) rapidly aggregates when suspended in buffered artificial seawater (ASW). Light microscopic observations of stained preparations, showed that flagella-flagella contact was responsible for this phenomenon. Aggregation did not occur if flagella were sheared off, or if motility was inhibited with NaN₃. Aggregates were not observed when Mg²⁺ was omitted from ASW, even though the bacteria remained motile. Other divalent cations, including Ca²⁺, Mn²⁺, and Ba²⁺ could replace Mg²⁺. However, there is no absolute requirement for divalent cations, since aggregation occurred in ASW containing Cs⁺ or Li⁺ instead of Mg²⁺. P. marina aggregates developed from pH 5.8–8.4, but not below pH 5.8 even though motility continued unimpaired to pH 4.5.Abbreviation ASW artificial seawater     

Regeneration and supernumerary limb formation under sparsely innervated conditions

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20⁺ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20⁺ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.     

Demonstration of sensory cutaneous nerve fibers in guinea-pig lip using endogenous oxidase histochemistry

Summary We developed a new histochemical method based on endogenous oxidase activity for the detection of cutaneous nerves in guinea-pig lips. After the application of a reaction mixture containing 0.14 mM 3,3-diaminobenzidine(DAB) and 15 mM nickel ammonium sulfate in Tris-HCl buffer (pH 7.6), many blue-black sensory fibers were observed, i.e., dermal nerve fiber bundles, perifollicular plexuses, Merkel-cell endings, and Meissner-like nerve endings. The autonomic fibers of the skin were not stained. Electron microscopy revealed that the reaction product was mainly localized on mitochondrial cristae. The addition of 0.01 M sodium azide completely inhibited the reaction. This technique is simple and preferentially stains sensory nerves.     

Some properties of the action potentials conducted in the spines of the sea urchin Diadema antillarum

Brief (2-5 msec) electrical pulses applied to the primary spines of the sea urchin Diadema antillarum elicit graded action potentials (ap's). These ap's can be attributed to the electrical activity of a set of 14-21 bundles of neurites, each comprising 1000 processes near the spine base and tapering towards the spine tip. The shape of the ap's varies from a simple diphasic deflection to a complex waveform with 6 or more components. Peak-to-peak amplitude is less than 1mV. The ap's are conducted at a uniform speed of ca. 27 cm/sec. The ap's are not affected by tetrodotoxin (1 microgram/ml) and continue to be produced in Na-free artificial sea water (ASW). The amplitude of the ap's is greatly reduced or totally abolished in Ca-free ASW. However, some electrical activity may continue in the absence of external Ca, due to release of Ca2+ ions from the calcium carbonate crystals of the spine shaft. Replacing the Ca content of ASW by barium ions causes an irreversible blockade of the ap's. Spines equilibrated with ASW containing Sr2+ ions instead of Ca2+ produce ap's of increased amplitude (up to X 2). The ap's are blocked by La3+, Co2+, Cd2+ (2-5 mM) and by the organic Ca channel blocker Bepridil (2 mM). We conclude that the spinal ap's are due to the summation of Ca spikes produced by the activation of Ca channels which are blocked by barium and have a high affinity for, or permeability to Sr vs Ca.     

Sarcoplasmic reticulum. XVI. The permeability of phosphatidyl choline vesicles for calcium

The Ca permeability of phosphatidyl choline vesicles of diverse fatty acid composition was measured. The rate of ⁴⁵Ca release from liposomes equilibrated with 1 mm⁴⁵CaCl₂ was found to be about 8 × 10⁻¹⁸ moles of Ca/cm²/sec for egg lecithin and about 5.3 × 10⁻¹⁷ moles of Ca/cm²/sec for dioleyllecithin at 30 °. Incorporation of cholesterol into dioleyllecithin micelles reduced the rate of Ca release. The Ca permeability of the phosphatidyl choline micelles was insensitive to changes in the pH, calcium or sodium concentration of the medium but increased with increasing temperature. The effect of temperature was most marked with dioleyl lecithin dispersions, but was clearly apparent with dipalmitoyl, plant, bovine, and egg lecithins as well. The activation energy of Ca release fell in the range of 4.2–9.6 kcal/mole. Macrocyclic antibiotics (valinomycin, tyrocidin, and gramicidin) at relatively high concentration increased the rate of Ca release similarly to their effects on fragmented sarcoplasmic reticulum membranes.     

Free calcium concentrations in Krebs-Ringer bicarbonate buffer: Effects on 45Ca- and 32P-transport across the perfused guinea pig placenta

Calcium ion activity was measured in protein-free and protein-containing Krebs-Ringer bicarbonate buffer. Even in protein-free buffer calcium ion activity was 20% less than the total Ca concentration. In solutions containing albumin the calcium ion activity was dramatically reduced, an effect dependent on the albumin concentration and pH of the solution. Acutely changing calcium ion activity from 0.35 mM to 1.25 mM in a Krebs-Ringer bicarbonate buffer (5.4% albumin) perfusing the fetal side of the guinea pig placenta resulted in an immediate increase in transfer (mother to fetus) of ³²P but no change in ⁴⁵Ca transfer. The ratio of ³²P to ⁴⁵Ca clearance was positively related also to maternal plasma calcium ion activity.     

Calcium-Binding Properties of Sarcoplasmic Reticulum As Influenced by ATP,Caffeine, Quinine,and Local Anesthetics

Calcium retained at binding sites of the sarcoplasmic reticulum membranes isolated from rabbit skeletal muscle requires 10^-5 - 10^-4 M ATP to exchange with ⁴⁵Ca added to the medium. The ATP requirement for Ca exchangeability was observed with respect to the "intrinsic" Ca of the reticulum membranes and the fraction of Ca that is "actively" bound in the presence of ATP. Furthermore, a concentration of free Ca in the medium higher than 10^-8 M is required for ATP to promote Ca exchangeability. This exchangeability is not influenced by caffeine, quinine, procaine, and tetracaine, and Ca that is either nonexchangeable (in the absence of ATP) or exchangeable (in the presence of ATP) is released by 1–5 mM quinine or tetracaine, but neither caffeine (6 mM) nor procaine (2–5 mM) has this effect. Quinine or tetracaine also releases Ca and Mg bound passively to the reticulum membranes. A possible role of ATP in maintaining the integrity of cellular membranes is discussed, and the effects of caffeine, quinine, and of local anesthetics on the binding of Ca by the isolated reticulum are related to the effects of these agents on ⁴⁵Ca fluxes and on the twitch output observed in whole muscles.     

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

Summary Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] _o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] _o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is 20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca²⁺ artificial seawater (ASW) or in Ca²⁺-free ASW decreases approximately linearly with increasing logarithm of [K] _o . The slope of the plot is slightly steeper for fibers bathed in Ca²⁺-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK _o depends on the external Na⁺ and Ca²⁺ concentrations, as well as on external pH, but is independent of external Mg²⁺ concentration. External application of 10^–4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca²⁺ by injection of 250mm EGTA before exposure of the fiber to 100mm K _o leads to 60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K _o by 50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na₂ before elevation of [K] _o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K _o for time periods ranging from 30 sec to 10 min causes a small (60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K _o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca²⁺ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.     

Effect of external sodium and calcium on calcium efflux in frog striated muscle

Summary The effect of media with different ionic composition on calcium efflux from the dorsal head of semitendinosus muscles ofRana pipiens was studied. The reduction in the fractional loss of⁴⁵Ca, when going from normal Ringer's solution to an ONa–OCa medium, was 60%. Withdrawal of only Na or Ca from the external medium also caused a significant drop in the fractional loss (33% and 34%, respectively). The effect of different concentrations of Ca (studied in the absence of the external Na) was also studied. It was found that a linear function could describe the relationship between the calcium-dependent calcium efflux and the external calcium concentration. These results indicate that calcium efflux from frog muscle fibers consists of three major components: one that is dependent on the presence of calcium in the external medium, one that is dependent on the presence of sodium in the external medium, and one that persists in the absence of these two cations.     

NEUROCHEMICAL PROPERTIES OF ADRENERGIC NERVES REGENERATED AFTER 6-HYDROXYDOPAMINE

Abstract— The uptake-storage properties and synthesis of noradrenaline, and fluorescence morphology of adrenergic nerves which have been allowed to regenerate for 4 weeks after a chemical sympathectomy produced by 6-hydroxydopamine have been investigated in mouse iris and atrium. The regenerated nerve terminals displayed a lower formaldehyde-induced fluorescence intensity whereas the non-terminal axons exhibited a stronger fluorescence intensity and a more beaded appearance compared with mature nerves. The endogenous noradrenaline concentration after 6-hydroxydopamine was 30% in iris and 45% in atrium compared to control values. Recovery of [³H]noradrenaline uptake was found to be more rapid than that of endogenous noradrenaline concentration after the 6-hydroxydopamine treatment. [³H]Noradrenaline uptake in regenerating and adult mature nerves both obeyed Michaelis-Menten kinetics having identical K_m values. There was a close correlation between [³H]noradrenaline uptake and nerve density of adrenergic nerves regenerated after 6-hydroxydopamine. These results show that [³H]noradrenaline uptake is a better index for the number of regenerated nerve terminals than is the endogenous noradrenaline concentration. The retention of [³H]noradrenaline taken up and accumulated in vitro was about the same in regenerated and mature nerves, although a slight tendency to less effective retention was observed in the regenerated nerves. Subcellular distribution studies showed that relatively less [³H]noradrenaline was recovered in the microsomal fraction after 6-hydroxydopamine treatment. The formation of ¹⁴C-labelled catecholamines from [¹⁴C]DOPA was higher in regenerating nerves than indicated by the endogenous noradrenaline concentration but lower than that indicated by the [³H]noradrenaline. It is concluded that the regenerating nerves contain less endogenous noradrenaline than adult mature nerves and that the uptake mechanism develops promptly, whereas the development of the storage mechanism lags behind.     

Modulations of high‐voltage activated Ca2+ channels in the central neurones of Spodoptera exigua by chlorantraniliprole

The modulation of voltage‐gated calcium channels by chlorantraniliprole in the central neurones isolated from third‐instar larvae of Spodoptera exigua is studied by the whole‐cell patch‐clamp technique. The current of calcium in the third‐instar larvae of S. exigua is identified as a high‐voltage activated Ca²⁺ current. During the 10‐min recording, the current–voltage relationship curves of whole‐cell calcium channels are shifted in a negative direction by 10 mV compared with the control group. The fact that the gravity rundown of calcium current in the treated group is more apparent than in the control group demonstrates that the open channels are constantly inactivated. In addition, chlorantraniliprole inhibits the recorded calcium currents in a concentration‐dependent manner, which is irreversible on washout.     

Calcium and pancreatic β-cell function: 4. Evidence that glucose and phosphate stimulate calcium-45 incorporation into different intracellular pools

β-Cell-rich pancreatic islets were microdissected from ob/ob-mice and used for studies of ⁴⁵Ca uptake and washout. Irrespective of whether the experiments were performed at 21 or 37°C both glucose and phosphate stimulated the net uptake of lanthanum-nondisplaceable ⁴⁵Ca. The stimulatory effect of phosphate was additive to that produced by glucose. ⁴⁵Ca incorporated in response to phosphate differed from that taken up in the presence of 20 mM glucose in being easily washed out although it was not affected by the glucose concentration of the washing medium. The efflux of ⁴⁵Ca was reduced after introducing phosphate into a medium used to perifuse islets which had accumulated ⁴⁵Ca in response to 20 mM glucose. This suggests that the outward calcium transport can be influenced also by intracellular trapping of the cation. The glucose-stimulated insulin release was inhibited by phosphate; an effect reversed by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. It is concluded that a common effect of glucose and phosphate is to trap calcium in the pancreatic β-cells but that there are fundamental differences between their effects on intracellular distribution of calcium and on insulin release.     

Phosphoinositide metabolism in rat superior cervical ganglion, vagus and phrenic nerve: effects of electrical stimulation and various blocking agents

Abstract— Paired vagus nerves, phrenic nerves or superior cervical ganglia from rats were incubated at 37 C for various times in a simple salt solution containing glucose and ³²P_i. One of the pair was usually stimulated electrically for 30 or 60 min. Stimulation of vagus nerve for 30 min increased phosphate incorporation into all the phospholipids studied but the increase was significant only in the case of triphos-phoinositide and diphosphoinositide. This increase was not accompanied by increased labelling of the nucleotide labile phosphate pool. Tetrodotoxin at concentrations sufficient to block transmission had no effect upon phospholipid labelling in vagus or phrenic nerve. Ouabain at blocking concentration did not affect polyphosphoinositide metabolism in vagus nerve but increased [³²P]labelling of the other phospholipids. Hemicholinium-3 increased the labelling of all three phosphoinositides in the sympathetic ganglia but the increase in phosphatidylinositol labelling due to electrical stimulation was not seen in the presence of this inhibitor.     

Increase in cytoplasmic calcium content in internodal cells of Lamprothamnium upon hypotonic treatment

Abstract Internodal cells of Lamprothamnium succinctum, a brackish water Characeae, regulate turgor pressure in response to changes in external osmotic pressure (turgor regulation). When internodal cells were transferred to a hypotonic medium containing 3.9 mol m^?3 Ca²⁺, the cell osmotic pressure decreased and the original turgor pressure was recovered. During turgor regulation Ca content of the cytoplasm increased significantly. Lowering the external Ca²⁺ concentration from 3.9 to 0.01 mol m^?3 inhibited this increase in cytoplasmic calcium content. In a hypotonic medium containing 0.01 mol m^?3 Ca²⁺, turgor regulation was inhibited as previously reported (Okazaki & Tazawa, 1986a). Thus transient increase in cytoplasmic Ca, probably in the ionized form, induced by hypotonic treatment may play an important role in turgor regulation.