Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle

Recent studies have revealed the structural and functional interactions between mitochondria, myofibrils and sarcoplasmic reticulum in cardiac cells. Direct channeling of adenosine phosphates between organelles identified in the experiments indicates that diffusion of adenosine phosphates is limited in cardiac cells due to very specific intracellular structural organization. However, the mode of diffusion restrictions and nature of the intracellular structures in creating the diffusion barriers is still unclear, and, therefore, a subject of active research. The aim of this work is to analyze the possible role of two principally different modes of restriction distribution for adenosine phosphates (a) the uniform diffusion restriction and (b) the localized diffusion limitation in the vicinity of mitochondria, by fitting the experimental data with the mathematical model. The reaction-diffusion model of compartmentalized energy transfer was used to analyze the data obtained from the experiments with the skinned muscle fibers, which described the following processes: mitochondrial respiration rate dependency on exogenous ADP and ATP concentrations; inhibition of endogenous ADP-stimulated respiration by pyruvate kinase (PK) and phosphoenolpyruvate (PEP) system; kinetics of oxygen consumption stabilization after addition of 2 mM MgATP or MgADP; ATPase activity with inhibited mitochondrial respiration; and buildup of MgADP concentration in the medium after addition of MgATP. The analysis revealed that only the second mechanism considered--localization of diffusion restrictions--is able to account for the experimental data. In the case of uniform diffusion restrictions, the model solution was in agreement only with two measurements: the respiration rate as a function of ADP or ATP concentrations and inhibition of respiration by PK + PEP. It was concluded that intracellular diffusion restrictions for adenosine phosphates are not distributed uniformly, but rather are localized in certain compartments of the cardiac cells.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.     

Reversal of Ethionine-Induced Inhibition of Elongation of Avena Coleoptiles by Purines, Adenosine, and Glutamic Acid

Ethionine-induced inhibition of elongation of Avena coleoptile segments has been measured in water and in indol-3yl-acetic acid. In the presence of 10 mM L-ethionine the inhibition amounts to about 70 %. It has been shown previously that the addition of adenosine triphosphate effectively counteracts this inhibition; optimal ATP concentrations are between 0.25 and 0.5 mM. Adenine, adenine sulfate, adenosine, guanine, S-adenosylmethionine, and glutamic acid have now been shown to act similarly to ATP in reversing the ethionine-induced inhibition of elongation.     

Retardation of bean leaf senescence by benzyladenine and its influence on phosphate metabolism

The levels of glucose, sugar phosphates, and adenosine phosphates were determined in primary leaves of intact bean plants during normal senescence and compared to leaves in which senescence was delayed by application of benzyladenine (BA). In both cases there was a rise with time in the levels of glucose 1-phosphate, glucose 6-phosphate, and fructose 6-phosphate, and a decline in 2-phosphoglyceric acid, inorganic phosphate, and the adenosine phosphates (AMP, ADP, ATP). The levels of fructose 1,6-diphosphate remained fairly constant. Although the levels of hexose phosphates, adenosine phosphates, and inorganic phosphate were lower in the BA-treated leaves, the incorporation of ³²P into these compounds by 3- and 6-week-old plants was higher than in the controls. These results suggest that the retardation of leaf senescence by BA in intact bean plants is associated with increased utilization of metabolites, indicating a more rapid turnover of the adenosine phosphates. It is concluded that this effect is brought about by a regulatory coordination of metabolic processes in relation to energy production and utilization.     

Optimized analysis of intracellular adenosine and guanosine phosphates in Escherichia coli.

To investigate the intracellular concentrations of adenosine phosphates in Escherichia coli, especially during bioreactor cultivations, a method that enables reproducible determination of adenosine phosphates in culture solutions containing at least 0.25 g dry cell weight/L has been developed. The detection limits of AMP, ADP, and ATP were found to be as low as 1 pmol. The method involves fast sampling, instantaneous inactivation of cell metabolism, extraction of nucleotides, and quantitative analysis by ion-pair reversed-phase HPLC.     

Comparison of respiratory effects of intravenous adenosine in neonatal and adult rabbits

We administered adenosine by repeated intravenous bolus doses to 34 neonatal rabbits in a dose of 120 micrograms X kg-1 (which we had previously found to stimulate respiration in adult rabbits). In 13 neonatal animals adenosine produced transient respiratory depression. In 15 neonatal animals the change in respiration in response to adenosine did not reach statistical significance. In two animals a transient increase in respiration occurred in response to adenosine. In the neonatal group as a whole intravenous adenosine significantly depressed ventilation. In eleven of the animals studied as neonates, respiratory responses to adenosine were again studied in adulthood. In 10 animals respiratory stimulation occurred in response to adenosine. In the adult group adenosine significantly increased ventilation, in contrast to its effects in the neonatal group. The respiratory effects of intravenous adenosine have not been previously described in neonatal animals. Respiratory stimulation produced by intravenous adenosine in adult rabbits contrasts with the respiratory depression commonly seen in neonatal rabbits in this study. It is suggested that altered responses to adenosine may be involved in the difference between the ventilatory response to hypoxia in adult and neonatal animals.     

Regulation of GH3-cell function via adenosine A1 receptors. Inhibition of prolactin release, cyclic AMP production and inositol phosphate generation.

We examined the mechanism by which adenosine inhibits prolactin secretion from GH3 cells, a rat pituitary tumour line. Prolactin release is enhanced by vasoactive intestinal peptide (VIP), which increases cyclic AMP, and by thyrotropin-releasing hormone (TRH), which increases inositol phosphates (IPx). Analogues of adenosine decreased prolactin release, VIP-stimulated cyclic AMP accumulation and TRH-stimulated inositol phospholipid hydrolysis and IPx generation. Inhibition of InsP3 production by R-N6-phenylisopropyladenosine (R-PIA) was rapid (15 s) and was not affected by the addition of forskolin or the removal of external Ca2+. Addition of adenosine deaminase or the potent adenosine-receptor antagonist, BW-A1433U, enhanced the accumulation of cyclic AMP by VIP, indicating that endogenously produced adenosine tonically inhibits adenylate cyclase. The potency order of adenosine analogues for inhibition of cyclic AMP and IPx responses (measured in the presence of adenosine deaminase) was N6-cyclopentyladenosine greater than R-PIA greater than 5'-N-ethylcarboxamidoadenosine. This rank order indicates that inhibitions of both cyclic AMP and InsP3 production are mediated by adenosine A1 receptors. Responses to R-PIA were blocked by BW-A1433U (1 microM) or by pretreatment of cells with pertussis toxin. A greater amount of toxin was required to eliminate the effect of R-PIA on inositol phosphate than on cyclic AMP accumulation. These data indicate that adenosine, in addition to inhibiting cyclic AMP accumulation, decreases IPx production in GH3 cells, possibly by directly inhibiting phosphoinositide hydrolysis.     

Effects of sodium and potassium ions on oxidative phosphorylation in relation to respiratory control by a cell-membrane adenosine triphosphatase

1. A study has been made of the oxygen consumption of kidney homogenates in relation to the ADP concentration as regulated by the cell-membrane adenosine triphosphatase. Stimulation of this enzymic activity by Na(+) and K(+) caused parallel increases in oxygen consumption and ADP concentration. Similarly, inhibition with ouabain caused a parallel fall. The membrane adenosine triphosphatase concerned in active transport therefore appears to regulate respiration through its control of ADP concentration. 2. The respiration of homogenates and mitochondria was also stimulated by K(+) in a way independent of adenosine-triphosphatase activity. It was shown that K(+) facilitates oxidative phosphorylation and the respiratory response to ADP. A K(+) concentration of 25-50mm was needed for maximum oxidative phosphorylation in the presence of physiological concentration of Na(+). Na(+) counteracted K(+) in the effects on mitochondria. It is concluded that K(+) regulates cellular respiration at two structures, one directly in mitochondria, and the second indirectly through control of ADP production at the cell membrane.     

The structural basis for substrate specificity and inhibition of human S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.     

Parathyroid hormone and prostaglandin E2 stimulate both inositol phosphates and cyclic AMP accumulation in mouse osteoblast cultures.

Parathyroid hormone (PTH) and prostaglandin E2 (PGE2) are physiological agonists which stimulate bone cells to resorb bone, a process by which the mineralized extracellular bone matrix is dissolved. Bone resorption has a key role in the maintenance of plasma calcium levels. It has been established that both PTH and PGE2 activate adenylate cyclase in osteoblasts, but it is apparent that (1) the two agents have qualitatively different effects on osteoblasts, and (2) the generation of cyclic AMP cannot account for all the effects of PTH on bone cell metabolism. Others have demonstrated that PTH and PGE2 may also elevate intracellular calcium levels, but the mechanism by which this is achieved has not been fully defined. Here we have investigated the effects of PTH on neonatal mouse osteoblasts in culture and shown that physiological concentrations of the hormone (50 nM) caused a small increase (22%) in total inositol phosphates accumulation, with a larger increase (40%) in inositol trisphosphate. We found that this activation occurred at lower concentration than was necessary to activate adenylate cyclase. PGE2 was a more effective activator of inositol phosphates accumulation than PTH, causing up to 300% increase in the total inositol phosphates after 30 min. Both PTH and PGE2 stimulated cyclic AMP accumulation, but the activation of adenylate cyclase by forskolin did not enhance inositol phosphates production. We conclude that both PTH and PGE2 stimulate phosphoinositide turnover in mouse osteoblasts and suggest that this mechanism may contribute to their elevation of intracellular calcium in bone cells.     

Influence of low temperature on respiration and contents of phosphorylated intermediates in darkened barley leaves

The aim of this work was to examine the effect of exposure of leaves to low temperatures (5°C) upon the contents of phosphorylated intermediates and respiration in darkened barley (Hordeum vulgare L.) plants which differed in their carbohydrate status. In leaves that had previously been illuminated for 24 hours, there was a large increase in amounts of phosphorylated metabolites at 5°C during the first 3 hours of darkness, compared with control plants kept at 30°C. Hexose phosphates accounted for about two-thirds of this increase, which reached a peak after about 3 hours. At higher temperatures, there was a peak in the amount of fructose 2,6-bisphosphate and the rate of respiration which accompanied the transient increase in phosphorylated intermediates. At 5°C the increase in phosphorylated intermediates was not accompanied by appreciable changes in fructose 2,6-bisphosphate, and there was a rapid decline in the rate of respiration. Leaves that had previously been darkened for 24 hours and that were low in carbohydrate failed to accumulate phosphorylated intermediates when exposed to low temperatures. The results are discussed with respect to the acclimation of carbohydrate metabolism to low temperatures. The results suggest that respiratory carbohydrate metabolism is strictly controlled even when the carbohydrate supply and glycolytic intermediates are abundant. The possibility that accumulation of hexose phosphates may be involved in acclimation of metabolism to low temperature is discussed.     

Effects of purine riboside on nucleic acid synthesis in ascites cells.

Purine riboside (nebularine, 9-beta-ribofuranosylpurine) is a naturally occurring base analog which closely resembles adenosine. It inhibits carcinogenic growth. Purine riboside strongly inhibits RNA and DNA synthesis in different cancer ascites cells. Gel electrophoretic analysis of RNA synthesis in vivo in the presence of purine riboside shows the ribosomal components to be inhibited the most. A method for assaying purine riboside or its phosphates intracellularly has been devised, and by using this it has been shown that purine riboside is extensively phosphorylated in the cells. The triphosphate derivative of purine riboside has been isolated and tested in the Escherichia coli RNA polymerase assay. It appears not to be incorporated into this type of RNA and to competitively inhibit this reaction with regard to ATP.     

A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of approximately 50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.     

Adenosine Inhibition of Histamine-Stimulated Inositol Phospholipid Hydrolysis in Mouse Cerebral Cortex

The effects of adenosine on inositol phospholipid hydrolysis in mouse cerebrocortical slices were examined. Despite having no effect alone, adenosine and some structural analogues inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The responses to carbachol, noradrenaline, 5-hydroxytryptamine, and elevated KCl levels were unaffected. The effect of adenosine was on the maximal response to histamine rather than on its EC50. Several adenosine antagonists competitively blocked the inhibition due to adenosine. The results are discussed in relation to the previously reported enhancement of histamine-stimulated hydrolysis of inositol phospholipids in guinea pig brain.     

Stimulation by ATP of Inositol Trisphosphate Accumulation and Calcium Mobilization in Cultured Adrenal Chromaffin Cells

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.     

Role of S-adenosylhomocysteine hydrolase in adenosine-induced apoptosis in HepG2 cells

Adenosine has been shown to initiate apoptosis through different mechanisms: (i) activation of adenosine receptors, (ii) intracellular conversion to AMP and stimulation of AMP-activated kinase, (iii) conversion to S-adenosylhomocysteine (AdoHcy), which is an inhibitor of S-adenosylmethionine (AdoMet)-dependent methyltransferases. Since the pathways involved are still not completely understood, we further investigated the role of AdoHcy hydrolase in adenosine-induced apoptosis. In HepG2 cells, adenosine induced caspase-like activity and DNA fragmentation, a marker of apoptosis. These effects were potentiated by co-incubation with homocysteine or adenosine deaminase inhibitor, pentostatin, and were mimicked by inhibition of AdoHcy hydrolase by adenosine-2',3'-dialdehyde (Adox). Adenosine-induced effects were significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, whereas inhibitors of adenosine kinase did not affect adenosine-induced changes. Various adenosine receptor agonists and AICAR, an activator of AMP-activated kinase, did not mimic the effect of adenosine. Thus, adenosine-induced apoptosis is likely due to intracellular action of AdoHcy and independent of AMP-activated kinase and adenosine receptors. Because elevated AdoHcy levels are associated with reduced mRNA methylation, we studied mRNA expression in Adox-treated cells by microarray analysis. Since several p53-target genes and other apoptosis-related genes were up-regulated by Adox, we conclude that AdoHcy is involved in adenosine-induced apoptosis by altering gene expression.     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of adenosine-induced cytotoxicity and their clinical and physiological implications

Extracellular ATP (ATPo) and adenosine are cytotoxic to several cancer cell lines, suggesting their potential use for anticancer therapy. Adenosine causes cytotoxicity, either when added exogenously or when generated from ATPo hydrolysis, via mechanisms which are not mutually exclusive and which involve, adenosine receptor activation, pyrimidine starvation and/or increases in intracellular S-adenosylhomocysteine: S-adenosylmethionine ratio. Given that adenosine also appears to protect against cytotoxicity via mechanisms including immunity against damage by oxygen free radicals, an understanding of the contribution of adenosine to ATPo-induced cytotoxicity is thus crucial, when considering any potential therapeutic use for these compounds. However, such an understanding has been largely hindered by the fact that many studies have not focused enough on the possibility that both ATPo and adenosine may mediate cytotoxicity in the same system. Such studies can benefit from use a range of ATPo concentrations when assessing the contribution of adenosine to ATPo-induced cytotoxicity. Whilst future molecular and pharmacological studies are needed to establish the nature of the cytotoxic adenosine receptor, it is possible that more than just one adenosine receptor type is involved and that the cytotoxic receptor(s) type is more likely to have a low affinity for adenosine. Activation of the adenosine receptor(s) would thus lead to cytotoxicity only at relatively high adenosine concentrations, while lower adenosine concentrations mediate non-cytotoxic physiological effects.     

Energy-dependence of calcium accumulation during sporulation of Bacillus megaterium KM.

Ca2+ accumulation and endogenous respiration of sporulating Bacillus megaterium are inhibited to the same extent by electron-transport of inhibitors and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting that Ca2+ is accumulated by an active transport process. Forespores isolated in stage V of sporulation demonstrated Ca2+-specific carrier-mediated Ca2+ uptake, consistent with downhill transfer [Hogarth & Ellar (1978) Biochem. J. 176, 197-203]. In the present studies forespore Ca2+ uptake was unaffected by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and by concentrations of respiratory inhibitor that inhibited forespore endogenous respiration by 85%. These data suggest that Ca2+ enters the isolated forespore by facilitated diffusion. Ca2+ uptake into sporulating protoplasts was completely inhibited by concentrations of respiratory inhibitors that had no effect on either Ca2+ uptake or respiration of stage-V forespores, but which resulted in inhibition of mother-cell membrane NADH oxidase. These results indicate that the mother-cell membrane is a site for active transport of Ca2+ into the sporulating cell. The effects of the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide on mother-cell membrane adenosine triphosphatase, NADH oxidase and protoplast Ca2+ uptake were examined.