Hydrolysis of Inorganic Pyrophosphate in Dictyostelium discoideum

The hydrolysis of inorganic pyrophosphate has been studied in cell-free extracts prepared at different stages of development of Dictyostelium discoideum. Two enzyme reactions, pH optima 7.25 and 9.0, appear specific for inorganic pyrophosphate and have an absolute requirement for a divalent cation, preferably Mg²⁺. The enzyme specific activities do not change significantly during differentiation. Neither enzyme reaction is inhibited by orthophosphate and the presence of exogenous potassium phosphate does not affect the levels of pyrophosphalase at any stage. Exogenous glucose raises the pyrophosphatase levels in the sorocarps.     

Isolation of Sclerosporin,a Sporogenic Substance,from Sclerotinia fructicola

The folate-hydrolyzing enzyme was purified 49-fold from the crude extract of Crithidia fasciculata ATCC 12857 by heat treatment, column chromatographies on DEAE-cellulose and Sephadex G-200, and preparative polyacrylamide gel electrophoresis. The final preparation was electrophoretically homogeneous. The enzyme had a molecular weight of 200,000 daltons and consisted of 4 identical subunits of which the molecular weight was about 51,000 daltons. The enzyme hydrolyzed aminopterin, methotrexate, and pABG more effectively than folate. The enzyme hydrolyzed the reduced folates, dihydrofolate and 10-formyltetrahydrofolate, more weakly than folate. The enzyme did not act on pteroly-γ,γ-diglutamylglutamate. The optimum pH for the reaction with each substrate described above was 7.0. Km values for folate, methotrexate, aminopterin, and pABG were 0.13, 0.46, 0.40, and 0.43 mM, respectively. The enzyme activity was inhibited by 2-mercaptoethanol, pCMB, chelating reagents such as α,α′α′′-tripyridyl and bathophenanthroline, divalent cations such as Hg²⁺, Cu^{2 +}, Cd²⁺, Pb²⁺, and Zn²⁺, and by pyrophosphate and orthophosphate.     

Starvation reduces pyruvate dehydrogenase phosphate phosphatase activity in rat kidney

Pyruvate dehydrogenase complex (PDC) from rat kidney or pig heart previously inactivated by phosphorylation (PDHP) was activatedin vitro by PDHP phosphatase from kidneys of starved or fed rats. Starvation for 48 h of the rats from which the PDC was prepared led to a decrease in the rate of activation of PDC at early time periods (<2 min), particularly at submaximal concentrations of Mg²⁺. Using intact permeable kidney mitochondria incubated for 15 sec, it was found that starvation of rats more than doubled the Mg²⁺ concentration at which the half maximal increment of PDC activity (PDC_a) was observed. Reduction of PDHP phosphatase activity due to starvation was also apparent when phosphatase was separated from PDC and recombined with PDC from the same or different animals.

Intraperitoneal injection of insulin and glucose 1 h before sacrifice of starved rats prevented the reduction of PDHP phosphatase activity whether or not protein synthesis was inhibited. The effect of insulin in restoration of PDHP phosphatase activity of starved rats was not mimicked by 5-methylpyrazole 3-carboxylic acid, an inhibitor of lipolysis.

When renal PDHP phosphatase was incubated with pig heart PDC in the presence of 10 mM Mg²⁺ and 0.1 mM Ca²⁺ the increment in PDC_a, in 1 min was 30% of fully activated PDC activity (PDC₁) observed after 15 min. Removal of divalent cations did not affect the increment in 1 min but prevented further increments. Conversely okadaic acid diminished 1 min increment but did not disturb PDC_t. It is suggested that the different behaviour of renal PDC from fed and starved animals may partly be due to different divalent cation independent PDHP phosphatase activity.

     

PHOSPHATE UPTAKE BY SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE): ENHANCEMENT BY CALCIUM ION1

The influence of metallic, cations (added at 10 μM-1 mM) on the uptake of orthophosphate from 0.2–10 μM solution by Synechococcus leopoliensis (Racib.) Komarek was investigated. All cations tested except Mg²⁺ and Zn²⁺ stimulated phosphate uptake. The most pronounced stimulation of phosphate uptake was caused by Ca²⁺·Ca²⁺ markedly decreased the half-saturation concentration for orthophosphate uptake, apparently by acting upon the metabolic processes of phosphate transport into the cell. Phosphate did not influence Ca²⁺ fluxes across the cell-surface.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Metabolism of calcium and effect of divalent cations on respiratory activity of yeast mitochondria

The adsorption of Ca²⁺ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca²⁺ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca²⁺ and Mg²⁺ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca²⁺ in the mitochondria ofTorulopsis utilis and with Co²⁺ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn²⁺ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd²⁺ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca²⁺ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Regulation of phosphoglycerate mutase in developing forespores and dormant spores ofBacillus megaterium by thein vivo levels of phosphoglycerate mutase inhibitor

Bacillus megaterium accumulated 3-phosphoglycerate during sporulation which was utilized during spore germination. During sporulation a protein was synthesized before or at the start of 3-phosphoglycerate accumulation inside the developing spores about 1.5 h before dipicolinic acid accumulation. This protein has an affinity for Mn²⁺ and other divalent metal ions and inhibits phosphoglycerate mutase activity which has been shown to require Mn²⁺ However, the levels of the inhibitor decreased considerably (75–85%) during spore germination. No appreciable amount of the inhibitor was detected in the vegetable cell and mother cell compartment; however, the forespore compartment possesses an activity comparable to that of dormant spores. The partially purified inhibitor has a molecular weight of 11,000 and possesses both high and low affinity binding sites for Mn²⁺ and Ca²⁺ as determined by Scatchard plot analysis.     

Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR

Summary The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using¹H–¹⁵N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by¹⁵N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide¹⁵N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg²⁺, Ca²⁺, or Ba²⁺. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (K_D=2–4 mM for the current divalent ions).     

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     

Role of calcium in the initiation of fast axonal transport of protein: Effects of divalent cations

Fast axonal transport of [³H]protein has been examined in bullfrog primary afferent neurons incubated in media supplemented with divalent cations that can act as agonists or antagonists of calcium ions. Incubation in calcium-free medium (CFM) had no effect on the rate of transport, but reduced the amount of transported [³H]protein by 40–60% relative to transport in the contralateral preparation maintained in normal medium. Preparations incubated in CFM supplemented with 1.8 mM SrCl₂ (equimolar to the CaCl₂ concentration in normal medium) carried out transport at control levels. Incubation conditions in which primary afferent somata were exposed to the Sr²⁺-medium while nerve trunks were maintained in CFM also supported normal transport. By contrast, selective exposure of nerve trunks to Sr²⁺-medium, and somata to CFM resulted in a reduced level of transport similar to that observed when the whole preparation was incubated in CFM. The depression of transport resulting from incubation in CFM was shown to be reversible when preparations were transferred from CFM to either Sr²⁺-supplemented CFM or to normal medium. By contrast to the effects of Sr²⁺, Ba²⁺ (up to 18 mM) did not substitute for Ca²⁺ in the transport process. When normal medium was supplemented with calciumantagonist cations, the amount of transport was depressed (Co²⁺ > Mn²⁺ >> Mg²⁺), with no concomitant effect on the rate of transport. Results of studies with Co²⁺, as well as those with Sr²⁺, suggest that a major locus of action of these cations is within the neuronal soma at a step subsequent to protein synthesis, and prior to the onset of protein translocation via the transport system. Thus, it is inferred that these divalent cations affect a calcium-dependent step that occurs during the initiation phase of fast axonal transport.     

Ins(1,4,5)P3 receptor-mediated Ca2+ signaling and autophagy induction are interrelated

The role of intracellular Ca²⁺ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca²⁺ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca²⁺ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca²⁺-store content and enhanced agonist-induced Ca²⁺ release. The mechanism involved the upregulation of intralumenal ER Ca²⁺-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca²⁺-buffering capacity and reduced the ER Ca²⁺ leak. Second, starvation led to Ins(1,4,5)P₃R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P₃Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P₃-binding domain of the Ins(1,4,5)P₃R. The starvation-induced Ins(1,4,5)P₃R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P₃Rs in ⁴⁵Ca²⁺-flux assays, indicating a direct regulation of Ins(1,4,5)P₃R activity by Beclin 1. Finally, we found that Ins(1,4,5)P₃R-mediated Ca²⁺ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P₃R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca²⁺ signaling and autophagy stimulation as a proximal event in response to starvation.     

Effect of divalent cations on the potassium contracture of slow muscle fibers ofRana temporaria

Summary K contractures of single slow muscle fibers ofRana temporaria were measured isometrically in the presence of normal, reduced, and increased Ca²⁺ concentrations at 18 to 20°C. At normal Ca²⁺ concentration (1.8mm) contracture tension decreased from its peak value of 35.4±8.2 N/cm² to 59.4±23.9% within one minute, and to 48.3±27% within two minutes (30 fibers). Peak tension was virtually unaffected by changes of the Ca²⁺ concentration, but maintenance of tension was impaired by low (0.2mm), and improved by high (10mm) Ca²⁺ concentrations. When Ca²⁺ was added during the K contracture, there was practically no restoration of lost tension. Effects similar to those of Ca²⁺ were observed upon addition of foreign divalent cations to the medium. Co²⁺, Ni²⁺, and Cd²⁺ were slightly more effective than Ca²⁺ and Mn²⁺; the smallest effects were obtained with Mg²⁺, Sr²⁺, and Ba²⁺. The effects of foreign divalent cations were independent of the presence of Ca²⁺. It is concluded that in slow fibers ofRana temporaria maintenance of contracture tension is not due to an influx of Ca²⁺ ions. Instead, binding of divalent cations to superficial sites seems to be essential.     

Phosphate accumulation of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis>

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg²⁺ and glucose, or Mg²⁺ and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by α-ketoglutaric acid in combination with (NH₄)₂SO₄, or arginine, or glutamine, the catabolism of which results in formation of NH₄ ⁺ and α-ketoglutarate.     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

How is glutamine synthetase I activity from Streptomyces aureofaciens regulated ?

Summary Activity of glutamine synthetase I (GSI) from Streptomyces aureofaciens increased markedly during tetracycline production phase. The purified GSI exhibited a low affinity for glutamate but high affinities for ATP and ammonium. Its Mn²⁺-dependent activity was more sensitive to feedback inhibitors than Mg²⁺-dependent activity. Both the activities were significantly stimulated by Co²⁺ but inhibited by other divalent cations. ADP was a strong inhibitor. These results suggest that GSI activity is regulated by availability of substrates, feedback inhibitors, divalent cations, and cell energy charge.     

Calcium and magnesium effect on divalent cation deficientHydrilla verticillata thylakoid electron transport activity

The role of divalent cations like magnesium (Mg²⁺) and calcium (Ca²⁺) was irrvestigated on energy distribution process ofHydrilla verticillata thylakoids. Effect of these cations was tested on relative quantum yield of photosystem (PS) II catalyzed electron transport activity, room and liquid nitrogen temperature fluorescence emission properties and thylakoid light scattering characteristics. The electron transport activity was found to be stimulated in the presence of these cations in a light intensity independent manner. The concentration of cation required for maximum stimulation was nearly 10–12 mM. Comparatively, Ca²⁺ was more effective than Mg²⁺. Cation induced stimulation in electron transport activity was not accompanied by increase in chlorophylla fluorescence intensity either at room (25°C) or liquid nitrogen (77°K) temperatures. Furthermore, 540 nm absorption and 90° light scattering properties of thylakoids remained insensitive towards divalent cations. These facts together suggest that divalent cations inHydrilla thylakoids are not effective in supporting the excitation distribution between the interacting photosystem complexes.     

Treating Urban Wastewater: Nutrient Removal by Using Immobilized Green Algae in Batch Cultures

Essential roles of microalgae during the tertiary treatment of municipal wastewater have been proven. In order to avoid wash out of the biomass and also modify the quality of the treated wastewater; some techniques such as cell immobilization have been developed. To do so, in this study four samples from two species of microalgae (Chlorella vulgaris and Chlamydomonas sp.) were determined and confirmed by taxonomic identification. The samples were immobilized in calcium alginate beads. Within 10 days the amounts of nitrate (No₃^?-N) and orthophosphate (Po₄^3?-P) were measured to calculate the removal efficacy for each individual sample. Based on the standard methods, the amount of nitrate and orthophosphate were determined every 3 days within 10 days. The results indicate that immobilized microalgae are able to remove the nutrients and reduce the amount of nitrate and orthophosphate, significantly. Furthermore, the C. vulgaris (YG02) was the best species in this experience with 72% and 99% of reduction in the amount of nitrate and orthophosphate, respectively.     

Calcium channels and cation transport ATPases in cardiac hypertrophy induced by aortic constriction in newborn rats

In order to evaluate the contribution of pinocytosis to basal (no agonist) and lanthanide-insensitive store-activated Ca²⁺ inflow in freshly-isolated rat hepatocytes, the uptake of extracellular fluid by pinocytosis was measured at 20°C and used to predict the amount of extracellular Ca²⁺ taken up by pinocytosis. This was compared with the measured rate of Ca²⁺ uptake in the basal state, and with the measured lanthanide-insensitive component of divalent cation uptake stimulated by 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the smooth endoplasmic reticulum (Ca²⁺ + Mg²⁺)ATP-ase. Fluid uptake by pinocytosis was measured using [¹⁴C]sucrose. In hepatocytes incubated at 20°C, DBHQ increased the initial rate of sucrose uptake by about 35%. The data for sucrose uptake were used to calculate the volume of extracellular fluid taken up by pinocytosis which, in turn, was used to predict the amount of extracellular Ca ²⁺ taken up through pinocytosis in the basal and DBHQ-stimulated states. Rates of divalent cation inflow in the basal state were determined at 20°C by measuring the uptake of ⁴⁵Ca²⁺. The degree of stimulation of Ca²⁺ inflow by DBHQ and the lanthanide-insensitive component of DBHQ-stimulated divalent cation inflow were determined by measuring the rate of Mn²⁺-induced quenching of intracellular quin-2 in the absence of an agonist, and in the presence of DBHQ or DBHQ plus Gd³⁺. It was calculated that the process of pinocytosis accounts for at least 15% of Ca²⁺ uptake in the basal (no agonist) state, and for about 10% of DBHQ-stimulated lanthanide-insensitive Ca²⁺ uptake. It is concluded that in isolated hepatocytes (i) the release of Ca²⁺ from intracellular stores stimulates pinocytosis and (ii) the process of pinocytosis can account for a substantial proportion of basal Ca²⁺ inflow and a small proportion of DBHQ-stimulated lanthanide-insensitive Ca²⁺ inflow.Abbreviations RACC receptor-activated Ca²⁺ channel - DBHQ 2,5-di-tert-butylhydroquinone - [Ca²⁺] intracellular free Ca²⁺ concentration