The effect on creatine kinase release of altered conditions in the Ca2+ paradox

• 1.1. Release of creatine kinase (CK) in the Ca²⁺ paradox of the Langendorff-perfused rat heart is dependent on the conditions of Ca²⁺ depletion and Ca²⁺ repletion.
• 2.2. CK release is reduced by raising [Ca²⁺]_o during Ca²⁺ depletion and progressively increased by extending the Ca²⁺ free period from 2 to 5 min.
• 3.3. CK release is reduced by decreasing the electrochemical gradient for Ca²⁺ during Ca²⁺ repletion.
• 4.4. The findings are discussed in the light of current hypotheses for the biochemical mechanisms that underlie the Ca²⁺ paradox.

     

Crayfish retinular cells: Influence of extracellular sodium and calcium upon receptor potential

• 1.1. In crayfish, light stimulation of the retinular cells induces a depolarizing receptor potential.
• 2.2. Experiments were designed to determine the role of Na⁺ and Ca²⁺ on receptor potential during dark And light states.
• 3.3. Depolarization depends on Na⁺ and Ca²⁺ availability to the retinular cell.
• 4.4. Repolarization velocity and response duration depend on extracellular Ca²⁺ availability.
• 5.5. Light adaptation increases receptor potential dependence on calcium and sodium ions.
• 6.6. We analyse these results with respect to other invertebrate photoreceptors.

     

Synaptic transmission at high pressure: Effects of [Ca2+]o

• 1.1. The effects of pressure on synaptic currents were examined in crayfish abdominal muscles.
• 2.2. Helium pressure (10.1 MPa) considerably decreased extracellulariy-recorded excitatory junctional potentials associated with increased short-term facilitation.
• 3.3. These effects could be mimicked by a reduction of [Ca²⁺]_o, and partially compensated by an increase in [Ca²⁺]_o.
• 4.4. Pressure also reduced the amplitude of the extracellular nerve terminal potentials (ENTP) by up to 25%, and significantly increased synaptic delay in a [Ca²⁺]_o-dependent manner.
• 5.5. The interaction between compression and various [Ca²⁺]_o were analysed in terms of an existing model of transmitter release. The results were consistent with the hypothesis that high pressure decreases the maximal Ca²⁺ influx into nerve terminals.
• 6.6. The decreased ENTP and increased synaptic delay suggest that additional processes may be involved in pressure effects on synaptic transmission.

     

Tetanus responses under rapid bath solution change: Electrotonic depolarization of transverse tubules may release Ca2+ from sarcoplasmic reticulum of Rana japonica skeletal muscle

• 1.1. Single skeletal muscle fibers were transferred from a normal Ringer solution to Na⁺ ion free solution, and vice versa, and tetanus responses were recorded immediately after the transfer.
• 2.2. Fractional tetanus tension recorded immediately after the displacement from the Na⁺ ion free solution to normal Ringer solution was dependent on fiber diameter.
• 3.3. Diffusion of Na⁺ ions along the transverse tubules was simulated [apparent diffusion constant was 3.11 × 10⁻⁶ (cm²/s)].
• 4.4. Our results suggest that the electrotonic spreading of membrane potential, caused by an action potential in the transverse tubules, could release Ca²⁺ ions from sarcoplasmic reticulum.

     

Ca2+-binding and protein-protein interaction domains of the sea urchin extraembryonic coat protein hyalin

• 1.1. As reported previously (Hopper and Robinson, 1990; Int. J. Biochem. 22, 1165–1170) the sea urchin extraembryonic coat protein hyalin undergoes a Ca²⁺-induced self-association into an insoluble gel (gelation) in the presence of Mg²⁺ and/or NaCl.
• 2.2. A 275 kDa peptide fragment, generated by limited tryptic digestion of hyalin, binds Ca²⁺+ but does not undergo gelation in the presence of Ca²⁺, Mg²⁺ and NaCl.
• 3.3. Comparisons between the capacities of hyalin and the 275 kDa peptide fragment to bind Ca²⁺ indicate that the latter binds 88% less Ca²⁺ than hyalin.
• 4.4. However, the presence of Ca²⁺ alone, at a concentration of 5 mM, protects the 275 kDa peptide fragment from further digestion by trypsin mimicking the effect of this cation in protecting hyalin.
• 5.5. Gel exclusion Chromatographie analyses of the 275 kDa peptide fragment, both in the presence and absence of 5 mM Ca²⁺, indicate that this cation does induce self-association of the fragment.
• 6.6. These results provide information on the organization of the functional domains on hyalin which are required for gel formation.

     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Modulation of the ATP induced [Ca2+]c increase in as-30D hepatoma cells

• 1.1. The regulation of the increase in the cytosolic calcium concentration ([Ca²⁺]c) induced by extracellular ATP in AS-30D hepatoma cells was studied.
• 2.2. Homologous desensitization involving the refilling of intracellular calcium pools and the participation of protein kinase C was found.
• 3.3. Isoproterenol, forskolin and dibutyril-cyclic AMP also induced an increase in [Ca²⁺]c.
• 4.4. Interestingly, synergism was found for isoproterenol or forskolin and ATP.
• 5.5. The results suggest that there are two pathways for mobilizing [Ca²⁺] in AS-30D hepatoma cells; one is activated by ATP receptors and the other by cyclic AMP.

     

The digestive enzymes of the pacific brown shrimp Penaeus californiensis.: I—Properties of amylase activity in the digestive tract

• 1.1. Crude extract of the whole digestive tract from the brown shrimp (P. californiensis) was investigated for digestive amylase activity.
• 2.2. Considerable amylase activity was found at pH 6.5–8.0, with optimum pH at around 7.5.
• 3.3. Optimum temperature was found between 30–40°C, similar to amylases from other crustaceans.
• 4.4. Amylase activity was highly halotolerant, having 50% maximum activity at 3 M NaCl.
• 5.5. Maximum amylase activity was found at 0.01 M NaCl.
• 6.6. Amylase activity was partially inhibited by the divalent ions Hg²⁺, Zn²⁺, Cu²⁺ and Cr²⁺.
• 7.7. Mg²⁺ and Ca²⁺ ions seemed to enhance amylase activity.

     

The sea urchin extraembryonic hyaline layer: Ionic parameters controlling the hyalin gelation reaction

• 1.1. As reported previously (Robinson, 1988) the Ca²⁺-induced self-association reaction of the protein hyalin, purified from the sea urchin extraembryonic hyaline layer, was modulated by both Mg²⁺ and NaCl.
• 2.2. In the presence of 400 mM NaCl the apparent dissociation constant (Ca²⁺) decreased five-fold from 4.8 ± 1.1 mM in the absence to 0.9 ± 0.5 mM in the presence of 20 mM Mg²⁺.
• 3.3. The potentiating effect of Mg²⁺ occurred with an apparent dissociation constant (Mg²⁺) of 4.6 ± 0.5mM.
• 4.4. In the absence of Ca²⁺ or NaCl hyalin dissociated from isolated hyaline layers indicating that the behavior of hyalin within the layer is predictable from results obtained with the purified protein.

     

Electrolyte changes,cell volume regulation and hormonal influences during acclimation of rainbow trout (Salmo gairdnerii) to salt water

• 1.1. Rainbow trout were acclimated to salt water (1.5, 2.0 or 3.0%, which means 40, 60 or 85% concentrated sea-water) and the electrolyte, glucose and cortisol concentrations of the plasma as well as the extra- and intracellular muscle space, the muscle electrolyte concentrations and the ATPase activity were analysed.
• 2.2. Plasma osmolality, Na⁺, Ca²⁺ and Mg²⁺ concentrations of the plasma had a maximum at 24 hr after the start of acclimation when acclimated to 3.0% salt water. Plasma osmolality, Na⁺ and Mg²⁺ concentrations were significantly higher during the whole acclimation time when exposed to 3.0% salt water.
• 3.3. Variations and regulations of ECS and ICS were clearly demonstrated. The intracellular electrolyte concentrations were also maximal at 24 hr.
• 4.4. The plasma glucose level was just slightly elevated, but the cortisol level clearly indicated a stress response at 24 hr.
• 5.5. The activity of gill Na-K-ATPase increased during the acclimation time.
• 6.6. The regulatory processes in trout during acclimation to salt water are compared with those occurring in tilapia and carp.

     

Phosphofructokinase from the anterior byssus retractor muscle of Mytilvs edulis: Modification of the enzyme in anoxia and by endogenous protein kinases

• 1.1. Anoxia exposure resulted in a stable modification of the kinetic properties of 6-phosphofructo-1-kinase (PFK) from the anterior byssus retractor muscle (ABRM) of the sea mussel Mytilus edulis L.
• 2.2. Compared to the aerobic enzyme, the anoxic form of PFK. showed a reduced affinity for both substrates, fructose-6-phosphate (F6P) and ATP, and an increased sensitivity to inhibition by phosphoenolpyruvate.
• 3.3. To analyze the involvement of protein kinases in the modification of PFK, extracts from aerobic or anoxic muscle were incubated with ATP and Mg²⁺ plus protein kinase second messengers cyclic 3',5'-adenosine monophosphate (cAMP), cyclic 3',5'-guanosine monophosphate (cGMP) or Ca²⁺ plus phorbol 12-myristate 13-acetate (PMA).
• 4.4. Both forms of the enzyme responded to the presence of cAMP with a strong increase in affinity for F6P.
• 5.5. In response to cGMP affinity of the aerobic enzyme for F6P decreased whereas that of the anoxic enzyme form was not affected (at 0.5 mM ATP) or increased (at 3 mM ATP).
• 6.6. Incubation with Ca²⁺ + PMA had only a limited effect on PFK kinetics but appeared to enhance the response to cGMP when the three compounds were given together.
• 7.7. Treatment of PFK-aerobic with alkaline phosphatase resulted in a strong decrease in enzyme activity and affinity for F6P; subsequent treatment with cAMP reversed the effect on S_0.5 F6P.
• 8.8. The data indicate that PFK activity is altered during the aerobic-anaerobic transition by a change in the phosphorylation state of the enzyme and that cAMP and cGMP act oppositely to regulate PFK activity, and thereby alter glycolytic rate, during this transition.

     

Phosphatidylethanolamine: Ceramide-ethanolaminephosphotransferase activity in synaptic plasma membrane vesicles. Influence of some cations and phospholipid environment on transferase activity. Further proof of its location

• 1.1. Synaptic plasma membrane vesicles (SPMV) from rat brain synthesized ceramide-phosphoethanolamine (SpE), an analogue of sphingomyelin (SpC) from phosphatidylethanolamine (PE) and ceramide.
• 2.2. This reaction was catalyzed by PE: ceramide-phosphotransferase.
• 3.3. The presence of PC did not modify the SpE synthesis and PI and PS at twice PE concentration seemed to be activators; only PG was an inhibitor at all concentrations.
• 4.4. Some cations (Mg²⁺, Mn²⁺) were without effect, while Ca²⁺ increased transferase activity, so was interesting to study.
• 5.5. Transferase was compared with sialidase (external enzyme).
• 6.6. Kinetics other than those already performed by us were undertaken in order to confirm its location.

     

Myofibril and sarcoplasmic reticulum changes during muscle development: Activity vs inactivity

• 1.1. The purpose of this study was to determine whether biochemical changes of skeletal muscle that occur as a result of exercise in young rats persist into adulthood.
• 2.2. Littermates (10 days old) were assigned to a 3, 6 and 12 week control or training group. In addition, a rest-exercise group (R-E) and exercise-rest (E-R) group were included.
• 3.3. The rest-exercise and exercise-rest rats were maintained for the 12 weeks with the first 6 weeks being either rest or exercise and the condition reversed during the last 6 weeks of the experiment.
• 4.4. Myofibril ATPase activity of rat plantaris increased from the 10d to 12 week animals (P < 0.05). As anticipated, training resulted in a lowered activity at 6 and 12 weeks compared to controls.
• 5.5. The Ca²⁺ uptake and Ca²⁺-ATPase activity of the sarcoplasmic reticulum followed a similar pattern.
• 6.6. With regard to the exercise-rest rats, the myofibril and SR ATPase activities at 12 weeks were comparable to the 12 weeks control rats.
• 7.7. The rest-exercise group approximated the 12 week training group with regard to myofibril and SR ATPase activities (P > 0.05).
• 8.8. The results suggest that the training adaptations that occur during development of skeletal muscle return to normal, when training ceases in the adult rat.
• 9.9. Furthermore, animals that started to train prior to puberty do not have a greater capacity to adapt than animals which initiated training during adulthood.

     

The role of phosphoinositide metabolism in Ca2+ signalling of skeletal muscle cells

• 1.1. The mobilization of Ca²⁺ from intracellular stores by d-myo-inositol 1,4,5-triphosphate[Ins(1,4,5)P₃] is now widely accepted as the primary link between plasma membrane receptors that stimulate phospholipase C and the subsequent increase in intracellular free Ca²⁺ that occurs when such receptors are activated (Berridge, 1993). Since the observations of VoIpe et al. (1985) which showed that Ins(1,4,5)P₃ could induce Ca²⁺ release from isolated terminal cisternae membranes and elicit contracture of chemically skinned muscle fibres, research has focused on the role of Ins(1,4,5)P₃ in the generation of SR Ca²⁺ transients and in the mechanism of excitation-contraction coupling (EC-coupling).
• 2.2. The mechanism of signal transduction at the triadic junction during EC-coupling is unknown. Asymmetric charge movement and mechanical coupling between highly specialized triadic proteins has been proposed as the primary mechanism for voltage-activated generation of SR Ca²⁺ signals and subsequent contraction. Ins(1,4,5)P₃ has also been proposed as the major signal transduction molecule for the generation of the primary Ca²⁺ transient produced during EC-coupling.
• 3.3. Investigations on the generation of Ca²⁺ transients by Ins(1,4,5)P₃ have been conducted on ion channels incorporated into lipid bilayers, skinned and intact fibres and isolated membrane vesicles. Ins(1,4,5)P₃ induces SR Ca²⁺ release and the enzymes responsible for its synthesis and degradation are present in muscle tissue. However, the sensitivity of the Ca²⁺ release mechanism to Ins(l,4,5)P₃ is highly dependent on experimental conditions and on membrane potential.
• 4.4. While Ins(1,4,5)P₃ may not be the major signal transduction molecule for the generation of the primary Ca²⁺ signal produced during voltage-activated contraction, this inositol polyphosphate may play a functional role as a modulator of EC-coupling and/or of the processes of myoplasmic Ca²⁺ regulation occurring on a time scale of seconds, during the events of contraction.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla

• 1.1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca²⁺ stores in chromaffin cells.
• 2.2. The thapsigargin-sensitive compartment of Ca²⁺ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15–30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes.
• 3.3. A Ca²⁺-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca²⁺-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [γ-³²P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca²⁺ ATPases.
• 4.4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca²⁺ uptake.
• 5.5. LMF appears to represent a part of the thapsigargin-sensitive Ca²⁺ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.

     

Some physical and chemical properties of purified nematocysts of Hydra attenuata pall. (Hydrozoa,cnidaria)

• 1.1. A method for purifying undischarged nematocysts from Hydra and other cnidarians is described.
• 2.2. Isolated cysts (relative densities 1.22–1.24) evaginate their tubular content even after previous dehydration.
• 3.3. The cyst wall is permeable to dyes of mol. wts up to 600,000.
• 4.4. Approximately two-thirds of the cyst's dry wt are soluble proteins. Eighty per cent of them are of low mol. wt and highly anionic, presumably serving as binding sites for Ca²⁺ and Mg²⁺.
• 5.5. The other 20% includes 30 different proteins amongst them toxins and enzymes (phospholipase and little proteases but no collagenase, chitinase or hyaluronidase).

     

In situ phosphorylation of contractile proteins of a molluscan Mytilus edulis catch muscle in different functional states

• 1.1. The incorporation of ³²P into the contractile proteins of the anterior byssus retractor muscle of Mytilus edilus L. was analyzed during the different stages of a contraction-catch-relaxatin cycle.
• 2.2. The experiments were performed with saponin-skinned fibers preincubated with γ-³²P-ATP.
• 3.3. The total amount of ³²P incorporated into the fiber proteins was anlyzed by measuring the label of TCA-insoluble protein in a scintillation counter.
• 4.4. The dose incorporated was about twice as high during Ca²⁺ induced contraction and serotonin induced accelerated relaxation as during test and catch.
• 5.5. The molecular mass of the phosphorylated proteins was analyzed by autoradiography of the proteins separated by SDS-PAGE.
• 6.6. Up to 26 protein spots of different molecular masses were labelled, including such well characterized protein spe+cies as myosin heavy and light chains, paramyosin and tropomyosin.

     

Activation of gastric mucosal calcium channels by epidermal growth factor

• 1.1. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin.
• 2.2. The complex following labeling with [³H]PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active ⁴⁵Ca²⁺ uptake into intravesicular space as evidenced by La³⁺ displacement and osmolarity measurements. The ⁴⁵Ca²⁺ uptake was independent of sodium and potassium gradients indicating the electroneutral nature of the process.
• 3.3. The gastric mucosal channels on epidermal growth factor binding in the presence of ATP responded by an increase in protein tyrosine phosphorylation of 55 and 170 kDa subunits of calcium channel.
• 4.4. The phosphorylated channels following reconstitution into vesicles displayed at 48% greater ⁴⁵Ca²⁺ uptake, thus indicating the tyrosine kinase involvement in EGF dependent activation of calcium channel.
• 5.5. The results point towards the importance of epidermal growth factor in the maintenance of gastric mucosal calcium homeostasis.