Ca2+ -free medium enhances the magnitude of slow peak in compound action potential of frog sciatic nerve in vitro

The present investigation was carried out to know the effect of Ca2+ on different peaks of compound action potential (CAP) representing the fibers having different conduction velocity. CAP was recorded from a thin bundle of nerve fibers obtained from desheathed frog sciatic nerve. Suction electrodes were used for stimulating and recording purposes. In Ca2+ -free amphibian Ringer, two distinct peaks (Peak-I and Peak-II) were observed. The threshold, conduction velocity (CV), amplitude and duration of Peak-I were 0.32 +/- 0.02 V, 56 +/- 3.0 m/sec, 2.1 +/- 0.2 mV and 0.75 +/- 0.1 ms, respectively. The Peak-II exhibited ten times greater threshold, eight times slower CV, three times lower amplitude and four times greater duration as compared to Peak-I. Addition of 2 mM Ca2+ in the bathing medium did not alter CAP parameters of Peak-I excepting 25% reduction in CV. But, in Peak-II there was 70-75% reduction in area and amplitude. The concentration-attenuation relation of Peak-II to various concentrations of Ca2+ was nonlinear and 50% depression occurred at 0.35 mM of Ca2+. Washing with Ca2+ -free solution with or without Mg2+ (2 mM)/verapamil (10 microM) could not reverse the Ca2+ -induced changes in Peak-II. Washing with Ca2+ -free solution containing EDTA restored 70% of the response. The results indicate that Ca2+ differentially influence fast and slow conducting fibers as the activity of slow conducting fibers is greatly suppressed by external calcium.     

Dependence of batrachotoxin block of axoplasmic transport on sodium

Batrachotoxin (BTX) in the low concentration range of 19-190 nM blocks axoplasmic transport in the desheathed cat peroneal nerve in vitro. When the level of Na+ in the incubation medium was reduced to 10 mM, the blocking effect of BTX was much diminished, and in an Na+-free medium BTX had no effect on transport at all. The blocking action of BTX with Na+ present was inhibited by increasing the concentration of Ca2+ in the experimental medium. Relatively small increases were effective with a maximum protection seen when the Ca2+ concentrations were 7-10 mM. The results support the view that an increase in axonal Na+ is inhibitory to the transport mechanism. The results are discussed on the basis of the recently developed transport filament model of axoplasmic transport which takes into account an obligatory role for Ca2+ in transport and its axonal regulation. The possible relation of intraaxonal Na+ concentration to the Ca2+ level is also discussed.     

Distribution of desmoplakin in normal cultured human keratinocytes and in basal cell carcinoma cells

In cultured human keratinocytes (NHEK) maintained in medium containing low levels of Ca2+ (0.04 mM) desmoplakin is a component of certain electron-dense bodies in the cytoplasm. These bodies are associated with bundles of intermediate filaments. Upon elevation of the level of Ca2+ in the culture medium to 1.2 mM, desmoplakin first appears at sites of cell-cell contact in association with bundles of intermediate filaments. Subsequently, desmoplakin becomes incorporated into desmosomes in a manner comparable to that seen in mouse keratinocytes (Jones and Goldman: Journal of Cell Biology 101:506-517, 1985). NHEK cells maintained for 24 hr at Ca2+ concentrations between 0.04 mM and 0.18 mM were processed for immunofluorescence, immunoelectron, and conventional electron microscopical analysis. In NHEK cells grown at Ca2+ concentrations of 0.11 mM, desmoplakin appears to be localized in electron-dense bodies associated with intermediate filaments at sites of cell-cell contact in the absence of formed desmosomes. At a Ca2+ concentration of 0.13 mM desmoplakin is arrayed like beads on a "string" of intermediate filaments at areas of cell-cell association. At 0.15 mM, desmosome formation occurs, and desmoplakin is associated with the desmosomal plaque. In basal cell carcinoma cells desmoplakin is not restricted to desmosomes but also occurs in certain electron-dense bodies morphologically similar to those seen in NHEK maintained in low levels of Ca2+ and during early stages of desmosome assembly. We discuss the possibility of "cycling" of desmoplakin through these bodies in proliferative cells.     

Effects of K+ or norepinephrine on oxygen uptake in brown adipose tissue (author's transl)]

This investigation was undertaken to clarify the mechanism of the stimulated--respiration caused by K+ or norepinephrine in brown adipose tissue. 1. The addition of 30 approximately 100 mM K+ stimulated remarkably oxygen uptake in brown adipose tissue, and similarly norepinephrine (0.1 or 1.0 mug/ml) caused a marked stimulation. 2. Even if Na+ in normal Ringer solution was replaced by Choline or Li+, oxygen uptake caused by K+ (30 mM) or norepinephrine (1.0 mug/ml) was unaffected. 3. K+ -induced oxygen uptake was not observed when a Ca2+ -deficient tissue was incubated in Ca2+ -free Ringer, while norepinephrine-induced oxygen uptake clearly observed. And the oxygen uptake of Ca2+ -deficient tissue due to K+ was recovered by the addition of 5 mM Ca2+. 4. Mn2+ (6 mM) or La3+ (10 mM) inhibited significantly oxygen uptake due to K+, but not oxygen uptake due to norepinephrine. 5. K+ -induced oxygen uptake was unaffected by 10(-4) or 10(-3)M ouabain, but norepinephrine-induced oxygen uptake was inhibited considerably by 10(-4)M ouabain. 6. The oxygen uptake due to K+ was unaffected by propranolol (33 muM), whereas that due to norepinephrine was significantly inhibited in the presence of propranolol. 7. In the tissue from reserpine-treated animal, the oxygen uptake caused by K+ was observed. According, from these positive results we are justified to suggest that K+ -induced oxygen uptake is dependent on the presence of Ca2+, and not always caused by catecholamines released secondarily from nerve terminal.     

Differential Calcium Dependence Between the Release of Endogenous Dopamine and Noradrenaline from Rat Brain Synaptosomes

The characteristics of the release of endogenous dopamine and noradrenaline from rat brain synaptosomes were studied using HPLC with an electrochemical detector. The spontaneous release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. Also, the high-K+ (30 mM)-evoked release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. From these results, the ratio of the Ca2(+)-dependent component to the total release of noradrenaline seemed to be similar to that of dopamine. On the other hand, 20 microM La3+ or 1 microM diltiazem inhibited both the spontaneous and 30 mM K(+)-evoked release of dopamine by approximately 50-60% but inhibited neither the spontaneous nor the 30 mM K(+)-evoked release of noradrenaline. The K(+)-evoked rise in intrasynaptosomal Ca2+ concentration was mostly blocked in Ca2(+)-free medium or 100 microM La3(+)-containing medium but was only partially blocked by 20 microM La3+ or 1 microM diltiazem. These data indicate alternative possibilities in that the Ca2(+)-dependent release of noradrenaline might be less sensitive to a change of intracellular Ca2+ concentration than that of dopamine and that the calcium channels directly involved in the noradrenaline release may be more resistant to diltiazem and La3+ than those involved in the dopamine release.     

Protein Kinase C Activation Enhances K+-Stimulated Endogenous Dopamine Release from Rat Striatal Synaptosomes in the Absence of an Increase in Cytosolic Ca2+

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha1-adrenoceptor subtypes mediating noradrenaline-induced contraction of rat epididymal vas deferens in calcium-free medium.

The alpha1-adrenoceptor subtype mediating noradrenaline (NA)-induced contractions of rat epididymal vas deferens in Ca2+-free/EGTA (1 mM) medium was studied using competitive antagonists. The effects of chloroethylclonidine (CEC) was investigated in Ca2+-free and normal Krebs' medium and RT-PCR was used to identify alpha1-adrenoceptor specific mRNA in epididymal vas deferens. In Ca2+-free medium, NA evoked sustained contractions but was less potent (pD2, 5.9) than in normal Krebs' medium (pD2, 7.3). The contractions in Ca2+-free medium were inhibited by prazosin (pA2, 9.3), 5-methylurapidil (pA2, 8.4), spiperone (pA2, 7.6) and BMY 7378 (pK(B), 6.8) consistent with activation of alpha1A-subtype. Repeated pretreatment with CEC (100 microM) reduced the potency of NA and maximum contractions in normal and Ca2+-free media. CEC-sensitivity in normal Krebs' medium was enhanced by prior treatment with phenoxybenzamine. mRNA for alpha1a- and alpha1d- but not alpha1b-adrenoceptors were detected in epididymal vas deferens. These results suggest that NA contracts the tissue in Ca2+-free medium by the stimulation of alpha1A-adrenoceptors. Two factors affecting CEC-sensitivity of NA-induced contractions in this tissue are discussed.     

Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

The Na(+)-Ca2+ exchanger activity in cerebrocortical nerve endings is reduced in old compared to young and mature rats when it operates as a Ca2+ influx or efflux pathway.

The activity of the Na(+)-Ca2+ exchanger, which regulates the entry and the extrusion of Ca2+ ions from nerve endings was investigated in Percoll-purified cerebrocortical synaptosomes of aged rats. 45Ca2+ uptake in a Na(+)-free medium and 45Ca2+ efflux in a 145 mM Na+ medium were significantly reduced in cerebrocortical synaptosomes from aged rats (24 months) as compared to those occurring in young (4 months) and mature (14 months) rats. 45Ca2+ influx induced by 55 mM K+, a concentration of K+ ions which selectively promotes Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), was significantly reduced in mature and aged rats as compared to that occurring in young rats. The impairment of these mechanisms in aged rats is not accompanied by any variation of fura-2 monitored Ca2+ levels under resting and depolarizing conditions.     

Effects of hypoxia on the contractions and lactate release induced by high-K+, ouabain and epinephrine in guinea-pig aorta.

1. The 60 mM K+, 152 mM K+, Na-deficient medium and oubain-induced contractions of aorta were not so affected by severe hypoxia. 2. The 60 mM K+, 152 mM K+, Na(+)-deficient medium-induced responses were greatly reduced by deprivation of external Ca2+ in normoxia. 3. As the concentration of epinephrine increased, the remaining tensions which were expressed as a percentage of the original tensions became progressively greater in hypoxic condition. 4. The percentage of resistant components of the norepinephrine-induced contraction by the lower concentration was further reduced in Ca(2+)-free medium by severe hypoxic condition. 5. The tensions under normoxia and lactate release under severe hypoxia induced by 60 mM K+ or 2.5 x 10(-6) M epinephrine were of the same extent. 6. In conclusion, the inhibition of aortic response to epinephrine with severe hypoxia could not solely be explained by depression of the oxygen supply into the oxidative metabolism. Severe hypoxia did not affect Ca2+ influx through voltage-operated Ca2+ channels, but reduced both receptor-operated Ca2+ influx and intracellular Ca2+ release in the aorta.     

Mg2+- or Ca2+-Activated ATPase in Squid Giant Fiber Axoplasm

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Intracellular calcium and desensitization of acetylcholine receptors

Acetylcholine (ACh) was applied iontophoretically to voltage-clamped endplates in frog muscle. The current induced by prolonged application of ACh decreases progressively as the membrane becomes desensitized. Desensitization was sharply localized, and at a distance of 15 micrometer or less the ACh sensitivity of the membrane remained normal. Desensitization still occurred in muscles exposed to Ca2+-free media for several hours. In these conditions the rate of desensitization was not greatly affected by altering the membrane potential. In normal Ringer (1.8 mM Ca2+) desensitization was more pronounced and ACh application was frequently accompanied by localized contraction of the muscle fibre. Both the desensitization and the contraction were reduced after intracellular injection of EGTA, probably because this opposes the rise in internal Ca2+ normally caused by ACh action.     

Acidosis and Ca2+ distribution in myocardial tissue of flounder and rat

Summary The effect of acidosis on the myocardial Ca²⁺ distribution was examined at 15°C in ventricular strips of the flounder (Platichthys flesus) and at 30°C in atrial strips of the rat (Rattus norvegicus).Lowering the Ringer pH from 7.6 to 6.9 by increasing its CO₂ (flounder 2% to 12%, rat 4% to 14%), resulted in an elevated Ca²⁺ efflux in resting strips as well as in strips stimulated (12/min) to contraction. A decrease in pH of the Ringer used for the flounder myocardium by a lowering of bicarbonate (30 mM to 5 mM) also resulted in an elevation of the Ca²⁺ efflux, but the effect was smaller than that produced by an increased CO₂.With 11 mM Ca²⁺ and 10 mM EGTA added to the Ringer to reduce the amount of⁴⁵Ca²⁺ bound to extracellular sites, an increased CO₂ with a concomitant drop in Ringer pH resulted in an increased Ca²⁺ efflux in both myocardia. The Ca²⁺ efflux was only marginally elevated in the flounder myocardium and unchanged in that of rat when the same drop in Ringer pH was produced with a lowering in bicarbonate.In a nominally Ca²⁺-free Ringer with 0.1 mM EGTA the⁴⁵Ca²⁺ efflux was stimulated for both myocardia by an increase in CO₂.The flounder myocardium was exposed to high CO₂ in a nominally Na⁺, Ca²⁺-free Ringer and again the⁴⁵Ca²⁺ efflux increased. After a return to Na, Ca and low CO₂ in the Ringer, a higher efflux persisted in the strips being subjected to a high CO₂ than in the controls.The Ca²⁺ uptake rate was about the same at high and low CO₂ for both myocardia.Based on these results the measured increase in Ca efflux following an increase in CO₂ or a decrease in bicarbonate probably results from an elevated cytoplasmatic Ca²⁺ activity. It seems unlikely that an increased uptake rate of Ca²⁺ or a direct stimulation of Ca²⁺ transporting mechanisms in the cell membrane are responsible for the change.     

Effects of strontium on calcium-dependent hexose transport in muscle

Hexose transport in isolated perifused rat and guinea pig left atria and in isolated intact rat hemidiaphragms was followed by measuring the tissue/medium distribution of the nonmetabolized glucose analog, 3-O-methyl-D-glucose. Stimulation of 3-methylglucose transport by insulin, hyperosmolar medium, K+-free medium, and ouabain was depressed or absent in Ca2+-free medium. Addition of 2 mM Sr2+ to Ca2+-free media restored the response of transport to the stimulatory factors. Sr2+ also increased basal hexose transport. The Ca2+ dependence and the effect of Sr2 was greatest in guinea pig atria and least in rat hemidiaphragms. It is concluded that Sr2+ plays a Ca2+-like role in the regulation of hexose transport.     

NPC-15199, a novel anti-inflammatory agent, mobilizes intracellular Ca2+ in bladder female transitional carcinoma (BFTC) cells

This report demonstrates that NPC-15199 [(N-(9-fluorenylmethoxycarbonyl)L-leucine)], a novel anti-inflammatory agent, increases intracellular Ca2+ concentration ([Ca2+]i) in human bladder female transitional cancer (BFTC) cells. Using fura-2 as a Ca2+ probe, NPC-15199 (0.1-2 mM) was found to increase [Ca2+]i concentration-dependently. The response saturated at 2-5 mM NPC-15199. The [Ca2+]i increase comprised an initial rise, a slow decay, and a plateau. Ca2+ removal partly inhibited the Ca2+ signals. In Ca2+-free medium, pretreatment with 1 mM NPC-15199 abolished the [Ca2+]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor); and after pretreatment with thapsigargin, NPC-15199-induced Ca2+ release was dramatically inhibited. This indicates that NPC-15199 released internal Ca2+ mostly from the endoplasmic reticulum. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 1 mM NPC-15199 in Ca2+-free medium. Together, the findings suggest that in BFTC bladder cancer cells, NPC-15199 induced Ca2+ release from the endoplasmic reticulum and activating Ca2+ entry.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+-free medium than when preincubated with Ca2+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2+ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+-free medium. Using vesicles preincubated with Ca2+ the apparent Km for ITP found was 2.5 mM at pH 6.0 and 1.0 mM at pH 7.4. The Vmax observed (77 mumol g-1 s-1) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent Km for ATP was 3 microM when preincubated in a Ca2+-free medium. At pH 6.0 the Vmax for ATP varied from 96 to 33 mumol g-1 s-1 depending on whether the vesicles were preincubated in the presence or absence of Ca2+. At pH 7.4 the Vmax for ATP was 90 mumol g-1 s-1 in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2+ concentrations of the reaction medium regardless of the substrate used.     

The effects of pentachlorophenol (PCP) at the toad neuromuscular junction

1. Effects of PCP at the frog neuromuscular junction were studied in vitro in sciatic nerve sartorius muscle of the toad Pleurodema-thaul. 2. Within the concentration 0.003-0.1 mM, PCP caused a dose-time-dependent block of evoked transmitter release acompanied by an increase in the rate of spontaneous quantal release. 3. PCP induced an increase in miniature endplate potential (MEPP) frequency and it was not antagonized in a Ca2(+)-free medium, indicating that it does not depend upon Ca2+ influx from the external medium, but may act by releasing Ca2+ from intraterminal stores. 4. The present data, together with previous results concerning PCP at eighth sympathetic ganglia indicate that 3,4-diaminopyridine (3,4-DAP) counteracts the effects of PCP on synaptic transmission. This result suggests that PCP interfering Ca2+ influx occurs during depolarization of motor nerve terminals.