Human erythrocytes and neuroblastoma cells are in vitro affected by sodium orthovanadate.

Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.

Impact of adenosine receptors on immunoglobulin production by human peripheral blood B lymphocytes.

Adenosine is an endogenous compound that regulates function of several immune cells including lymphocytes by activating adenosine receptors (ARs). Several reports indicate that stimulation of ARs on lymphocytes affects lymphocyte activation, proliferation and lymphocyte-mediated cytolysis. Unfortunately, most studies focused on T lymphocytes and little information exists on involvement of ARs in B cells regulation. In this study we elucidated the impact of ARs activation on immunoglobulin M (IgM) production by purified human peripheral blood B lymphocytes stimulated in vitro with Staphyloccocus aureus Covan I (SAC) plus IL-2. Performed experiments showed that endogenous adenosine that is released/produced by human B lymphocytes is able to induce cAMP accumulation in the cell through activation of A2A-AR however, this takes place only when other ARs are inhibited by selective antagonists. We observed that accumulated intracellular cAMP suppressed IgM production by B cells stimulated with SAC plus IL-2. Our experiments showed that human B cells cultured at 25 mM glucose produced significantly less IgM in response to stimulation with SAC comparing to cells maintained in media containing 5 mM glucose. However, the high glucose effect on IgM production by B cells stimulated with SAC depended on other factor/s than ARs.

Acetyl-CoA metabolism in amprolium-evoked thiamine pyrophosphate deficits in cholinergic SN56 neuroblastoma cells.

Inhibition of pyruvate (PDHC) and ketoglutarate (KDHC) dehydrogenase complexes induced by thiamine pyrophosphate deficits is known cause of disturbances of cholinergic transmission in the brain, yielding clinical symptoms of cognitive, vegetative and motor deficits. However, particular alterations in distribution of key acetylcholine precursor, acetyl-CoA, in the cholinergic neuron compartment of thiamine pyrophosphate-deficient brain remain unknown. Therefore, the aim of our work was to find out how amprolium-induced thiamine pyrophosphate deficits (TD) affect distribution of acetyl-CoA in the compartment of pure cholinergic neuroblastoma SN56 cells originating from murine septum. Amprolium caused similar concentration-dependent decreases in thiamine pyrophosphate levels in nondifferentiated (NC) and differentiated (DC) cells cultured in low thiamine medium. In such conditions DC displayed significantly greater loss of viability than the NC ones, despite of lesser suppressions of PDHC activities and tetrazolium salt reduction rates in the former. On the other hand, intramitochondrial acetyl-CoA levels in DC were 73% lower than in NC, which explains their greater susceptibility to TD. Choline acetyltransferase activity and acetylcholine content in DC were two times higher than in NC. TD caused 50% decrease of cytoplasmic acetyl-CoA levels that correlated with losses of acetylcholine pool in DC but not in NC. These data indicate that particular sensitivity of DC to TD may result from relative shortage of acetyl-CoA due to its higher utilization in acetylcholine synthesis.

Adenosine 5'-triphosphate is the predominant source of peripheral adenosine in human B lymphoblasts.

Adenosine 5'-triphosphate (ATP) and adenosine are the crucial endogenous signaling molecules in immunity and inflammation. In this study we identified the source of extracellular adenosine in human B lymphoblasts, and evaluate the ATP release and metabolism. We observed that the B cells continuously released substantial quantities of ATP (35 pmol/10(6) cells) when subjected to slow motion in the incubation medium. The adenosine level in the B cell incubation medium was very low, and increased (5-fold) upon inhibition of adenosine deaminase activity with 10 muM of 2-deoxycoformycin (DCF). Inclusion of an inhibitor of equilibrate nucleoside transport (nitrobenzylthioinosine) in the incubation medium in the presence of DCF resulted in the elevation of adenosine level by 9-fold. Inhibition of ecto-ATPase activity with 100 muM of ARL67156 was associated with a 2-fold increase of the extracellular ATP level and a 3-fold decrease of adenosine concentration in the cell culture media. Inclusion of alpha,alpha-methyleneadenosine 5'-diphosphate, a selective inhibitor of ecto-5'-nucleotidase in the incubation medium resulted in a significant decrease (7-fold) the adenosine concentration. In conclusion, our results indicate that ATP released from the B cell is the primary source of peripheral adenosine, and that the activities of ecto enzymes and the efficiency of Ado uptake through the nucleoside transporters determine the Ado level on the B cell surface.

Protein kinase C mediated high glucose effect on adenosine receptors expression in rat B lymphocytes.

Hyperglycemia-induced alterations of adenosine receptors (ARs) expression are implicated in the pathomechanism leading to impaired function of the lymphocytes in diabetes. However, the signaling pathways utilized by glucose to regulate ARs expression are unknown. This work was undertaken to investigate the impact of high glucose level on the ARs expression in rat B lymphocytes. The results presented in this report demonstrate that rat B lymphocytes express all four types of ARs at the mRNA and protein level. Exposing B cells to high glucose (25 mM) suppressed the expression of A(1)-AR, A(2B)-AR, and A(3)-AR, but had no effect on the expression of A(2)A-AR. A selective inhibitor of Ca(2+)-dependent protein kinase C (PKC) isoforms suppressed the high glucose effect on A(1)-AR expression. Inhibition of PKC-delta with rottlerin blocked the high glucose effect on A(1)-AR mRNA level. An inhibitor of Raf-1 kinase completely blocked the high glucose effect on A(2B)-AR expression. The suppression of A(1)-AR and A(2B)-AR mRNA expression induced by high glucose was blocked by an inhibitor (PD98059) of MAPK kinase (MEK). In conclusion, high glucose utilizes a signaling pathway involving some elements of the MAPK pathway and different PKC isoforms to suppress the expression of A(1)-AR, A(2B)-AR, and A(3)-AR in rat B lymphocytes.

Structural effects of Zn(2+) on cell membranes and molecular models.

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1mM Zn and higher concentrations affect cell membrane and molecular models.

Phenotype-dependent susceptibility of cholinergic neuroblastoma cells to neurotoxic inputs.

A preferential loss of brain cholinergic neurons in the course of Alzheimer's disease and other encephalopathies is accompanied by a proportional impairment of acetyl-CoA synthesizing capacity in affected brains. Particular susceptibility of cholinergic neurons to neurodegeneration might results from insufficient supply of acetyl-CoA for energy production and acetylcholine synthesis in these conditions. Exposure of SN56 cholinergic neuroblastoma cells to dibutyryl cAMP and retinoic acid for 3 days caused their morphologic differentiation along with the increase in choline acetyltransferase activity, acetylcholine content and release, calcium content, and the expression of p75 neurotrophin receptors. Acetyl-CoA content correlated inversely with choline acetyltransferase activity in different lines of SN56 cells. In differentiated cells, aluminum (1 mM), amyloid beta(25-35) (0.001 mM), and sodium nitroprusside (1 mM), caused much greater decrease of pyruvate dehydrogenase and choline acetyltransferase activities and cell viability than in nondifferentiated ones. Aluminum (1 mM) aggravated suppressory effects of amyloid beta on choline acetyltransferase and pyruvate dehydrogenase activities and viability of differentiated cells. Similar additive inhibitory effects were observed upon combined exposure of differentiated cells to sodium nitroprusside and amyloid beta(25-35). None or much smaller suppressory effects of these neurotoxins were observed in nondifferentiated cells. Increase in the fraction of nonviable differentiated cells positively correlated with losses of choline acetyltransferase, pyruvate dehydrogenase activities, and cytoplasmic cytochrome c content in different neurotoxic conditions. These data indicate that highly differentiated cholinergic neurons may be more susceptible to aluminum and other neurotoxins than the nondifferentiated ones due to relative shortage of acetyl-CoA, increased content of Ca(2+), and expression of p75 receptors, yielding increase in cytoplasmic cytochrome c and subsequently grater rate of death of the former ones.

Prevalence of unidirectional Na+-dependent adenosine transport and altered potential for adenosine generation in diabetic cardiac myocytes.

Adenosine is an important physiological regulator of the cardiovascular system. The goal of our study was to assess the expression level of nucleoside transporters (NT) in diabetic rat cardiomyocytes and to examine the activities of adenosine metabolizing enzymes. Isolated rat cardiomyocytes displayed the presence of detectable amounts of mRNA for ENT1, ENT2, CNT1, and CNT2. Overall adenosine (10 microM) transport in cardiomyocytes isolated from normal rat was 36 pmol/mg/min. The expression level of equilibrative transporters (ENT1, ENT2) decreased and of concentrative transporters (CNT1, CNT2) increased in myocytes isolated from diabetic rat. Consequently, overall adenosine transport decreased by 30%, whereas Na(+)-dependent adenosine uptake increased 2-fold, and equilibrative transport decreased by 60%. The activity ratio of AMP deaminase/5'-nucleotidase in cytosol of normal cardiomyocytes was 11 and increased to 15 in diabetic cells. The activity of ecto-5'-nucleotidase increased 2-fold in diabetic cells resulting in a rise of the activity ratio of ecto-5'-nucleotidase/adenosine deaminase from 28 to 56.These results indicate that in rat cardiomyocytes diabetes alters activities of adenosine metabolizing enzymes in such a way that conversion of AMP to IMP is favored in the cytosolic compartment, whereas the capability to produce adenosine extracellularly is increased. This is accompanied by an increased unidirectional Na(+)-dependent uptake of adenosine and significantly reduced bidirectional adenosine transport.

Altered expression of adenosine receptors in heart of diabetic rat.

Diabetes results in functional, biochemical, and morphological abnormalities in the heart. Some of these changes may be attributed to altered adenosine action. This study aimed to examine the expression level of adenosine receptors (AR) in heart of streptozotocin-induced diabetic rat. Performed analyses revealed detectable levels of A1-AR, A2a-AR, A2b-AR, A3-AR mRNA and protein in whole heart and isolated cardiac myocytes. An increase in A1-AR protein content with no changes in mRNA level was observed in isolated cardiac myocytes. Diabetes resulted in an increase of A3-AR mRNA and protein levels in heart and in cardiac myocytes. The level of A2a-AR mRNA was increased in whole diabetic heart, but it decreased in cardiac myocytes with no detectable changes in protein content. We did not observe any changes in expression level of A2b-AR in diabetic heart and isolated cardiac myocytes. Administration of insulin to diabetic rat for four days resulted in returning of the ARs mRNA and protein to the levels observed in heart of normal rat. These changes in ARs genes expression, and receptors protein content correspond to some abnormalities characteristic of the diabetic heart, suggesting involvement in pathogenesis of diabetic cardiomyopathy.

Aortic valve replacement and perioperative management in hemodialyzed patient wth antiphospholipid syndrome.

Antiphospholipid syndrome is characterized by the presence of antiphospholipid antibodies, hypercoagulability, and prolonged phospholipid-dependent coagulation indices such as activated clotting time (ACT). Perioperative thrombotic complications are frequent among patients with antiphospholipid syndrome submitted to cardiac surgery, therefore, in these patients, heparin-protamine titration for anticoagulation monitoring is particularly recommended. We demonstrate a case of 42-year-old hemodialyzed patient with antiphospholipid syndrome, submitted to the replacement of stenotic aortic valve. In our patient celite ACT and heparin concentration during cardiopulmonary bypass did not correspond to each other. Anticoagulation based on heparin concentration assessment resulted in safe perioperative hemostatic management.

Interactions between p75 and TrkA receptors in differentiation and vulnerability of SN56 cholinergic cells to beta-amyloid.

NGF modifies cholinergic neurons through its low-p75 and high affinity-TrkA receptors. Native p75(+)TrkA(-) and trkA-transfected p75(+)TrkA(+) SN56 hybrid cholinergic septal cells were used here to discriminate effects mediated by each receptor. In TrkA(-) cells, NGF (100 ng/ml) affected neither choline acetyltransferase nor morphology but depressed pyruvate dehydrogenase activity by about 30%. Aged 25-35 beta-amyloid (1 microM) caused no changes in choline acetyltransferase and pyruvate dehydrogenase activities in nondifferentiated and differentiated TrkA(-) cells. On the contrary, in nondiferentiated TrkA(+) NGF brought about a 2.5-fold increase of choline acetyltransferase. In differentiated TrkA(+) cells, beta-amyloid resulted in no change in PDH but 65% suppression of choline acetyltransferase activity and reduction of their extensions. Thus, activation of TrkA receptors may overcome p75 receptor-mediated inhibitory effects on pyruvate dehydrogenase expression in cholinergic cells. On the other hand, it would make expression of choline acetyltransferase and cell differentiation more susceptible to suppressory effects of beta-amyloid.

Aluminum, NO, and nerve growth factor neurotoxicity in cholinergic neurons.

Several neurotoxic compounds, including Al, NO, and beta-amyloid may contribute to the impairment or loss of brain cholinergic neurons in the course of various neurodegenerative diseases. Genotype and phenotypic modifications of cholinergic neurons may determine their variable functional competency and susceptibility to reported neurotoxic insults. Hybrid, immortalized SN56 cholinergic cells from mouse septum may serve as a model for in vitro cholinotoxicity studies. Differentiation by various combinations of cAMP, retinoic acid, and nerve growth factor may provide cells of different morphologic maturity as well as activities of acetylcholine and acetyl-CoA metabolism. In general, differentiated cells appear to be more susceptible to neurotoxic signals than the non-differentiated ones, as evidenced by loss of sprouting and connectivity, decreases in choline acetyltransferase and pyruvate dehydrogenase activities, disturbances in acetyl-CoA compartmentation and metabolism, insufficient or excessive acetylcholine release, as well as increased expression of apoptosis markers. Each neurotoxin impaired both acetylcholine and acetyl-CoA metabolism of these cells. Activation of p75 or trkA receptors made either acetyl-CoA or cholinergic metabolism more susceptible to neurotoxic influences, respectively. Neurotoxins aggravated detrimental effects of each other, particularly in differentiated cells. Thus brain cholinergic neurons might display a differential susceptibility to Al and other neurotoxins depending on their genotype or phenotype-dependent variability of the cholinergic and acetyl-CoA metabolism.

Relationships between thrombohemorrhagic complications and platelet function in patients with essential thrombocythaemia.

Three subgroups have been distinguished in essential thrombocythaemia (ET) patients, on the basis of clinical and laboratory findings. ET patients with bleeding incidents had smaller platelet volume, lower concentrations of beta-thromboglobulin and platelet factor 4 in their plasma, 10%, 26%, and 26% lower compared to patients without complications, respectively. ATP secretion from platelets of bleeders, clotters, and "no-complications" ET patients was found to be 75%, 36%, and 45%, respectively, lower than in healthy people. Spontaneous platelet aggregation appeared to be normal in about 90% of ET patients with no complications and in all bleeders but only in 35% patients with clotting incidents. All bleeders had abnormal agonist-evoked aggregation assays. Among remaining ET patients 30%-60% displayed normal values of different evoked aggregation tests. Thus, clinically distinguished group of bleeding ET patients may be differentiated from other subgroups on the basis of laboratory findings.

Acute and chronic effects of aluminum on acetyl-CoA and acetylcholine metabolism in differentiated and nondifferentiated SN56 cholinergic cells.

Mechanisms of preferential loss of cholinergic neurons in the course of neurodegenerative diseases are unknown. Therefore, we investigated whether differentiation-evoked changes in acetyl-CoA and acetylcholine metabolism contribute to the susceptibility of cholinergic neuroblastoma to cytotoxic effects of Al. In SN56 cells differentiated with retinoic acid and dibutyryl cAMP (DC), pyruvate utilization and acetyl-CoA content were lower and acetylcholine level higher than in nondifferentiated cells (NC), respectively. In DC Al and Ca accumulations were 50% and 100%, respectively higher than in NC. Acute Al addition caused inhibition, whereas its chronic application had no effect on pyruvate utilization both in NC and in DC. On the other hand, in both experiments, Al evoked a greater decrease of acetyl-CoA level in DC than in NC. Acute addition of Al depressed acetylcholine release from DC to two times lower values than in NC. On the other hand, chronic addition of Al increased ACh release from DC over twofold, being without effect on its release from NC. These findings indicate that higher accumulation of Ca, along with low levels of acetyl-CoA, could make DC more susceptible to neurotoxic inputs than NC. Excessive acetylcholine release, evoked by Al, is likely to increase acetyl-CoA utilization for resynthesis of the neurotransmitter pool and cause deficit of this metabolite in DC. On the other hand, NC, owing to lower Ca accumulation, slower ACh metabolism, and higher level of acetyl-CoA, would be less prone to these harmful conditions.

Acetyl-CoA metabolism in cholinergic neurons and their susceptibility to neurotoxic inputs.

Cholinergic neurons, unlike other brain cells utilize acetyl-CoA not only for energy production but also for acetylcholine (ACh) synthesis. Therefore, suppression of acetyl-CoA metabolism by different neurotoxic inputs may be particularly harmful for this group of cells. Differentiation of SN56 cholinergic hybrid cells increased their choline acetyltransferase (ChAT) activity and ACh content but depressed pyruvate dehydrogenase activity and acetyl-CoA content. Differentiated cells were more susceptible to acute and chronic influences of aluminum, NO and amyloid-beta. Al decreased acetyl-CoA content, ACh release and increased Ca accumulation in differentiated cells (DC) to much higher degree than in non-differentiated ones (NC). NO strongly depressed acetyl-CoA level and increased ACh release in DC but did not affect NC. Additive effects of Al and NO were seen in DC but not in NC. Also long term suppressory effects of amyloid-beta, Al and NO on cholinergic phenotype and morphologic maturation were more evident in DC than in NC. Thus, relative shortage of acetyl-CoA in highly differentiated cholinergic neurons could make them particularly susceptible to degenerative insults in the course of different cholinergic encephalopathies.

[Disturbances of glucose metabolism in epilepsy and other neurodegenerative diseases]. / Zaburzenia metabolizmu glukozy w padaczce i innych chorobach neurodegeneracyjnych.

Disturbances of glucose metabolism in epilepsy and other neurodegenerative diseases. Impairment of glucose and acetyl-CoA metabolism is a characteristic feature of several degenerative brain diseases. Presented paper provides experimental evidences that NO, aluminum and thiamine deficiency result in concordant disturbances in acetyl-CoA campartmentalisation as well as in nonquantal and quantal acetylcholine release in rat brain nerve terminals. Our findings indicate that simultaneous depression of acetyl-CoA synthesis and its increased utilisation for acetylcholine synthesis in the presence of neurotoxic factors is likely to make brain cholinergic neurones particularly prone to neurodegeneration.

Acetylcholine and acetyl-CoA metabolism in differentiating SN56 septal cell line.

The rate of acetylcholine (ACh) synthesis was found to depend on the activity of choline acetyltransferase (ChAT) and on the concentrations of the two substrates of this enzyme, choline and acetyl-CoA. In SN56 cells treated for 3 days with 1 mM dbcAMP activities of ChAT and acetylcholinesterase (AChE) were elevated. It was accompanied by an increased activity of ATP-citrate lyase (ACL)-an enzyme responsible for provision of part of acetyl-CoA for ACh synthesis in cholinergic neurons. In contrast lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) activities were reduced by dbcAMP. Treatment with 0.001 mM all-trans retinoic acid (RA) elevated ChAT and LDH activities but reduced the activities of AChE and ACL. The combined treatment with db-cAMP and tRA increased ChAT activity in supra-additive fashion. The effects of these two compounds on the other enzymes were not additive. Neither compound altered the activities of carnitine acetyl-transferase, acetyl-CoA synthase, or acetyl-CoA hydrolase. On the other hand, they decreased acetyl-CoA content and rate of ACh release. Overall, the results indicate that tRA upregulates only ChAT expression, whereas dbcAMP upregulates several features of cholinergic neurons including ChAT, AChE, and ACL. Low levels of acetyl-CoA in differentiated cells may result in a low rate of ACh release and resynthesis during their depolarization.

[Mechanisms of selective vulnerability of cholinergic neurons to neurotoxic stimuli]. / Mechanizmy selektywnej wrazliwosci neuronów cholinergicznych na bodzce neurotoksyczne.

Preferential loss of cholinergic neurons in course of several encephalopathies may result from the fact that they utilize acetyl-CoA not only for energy production, but also for acetylcholine synthesis. Changes in activities of acetyl-CoA metabolizing enzymes and shifts in acetyl-CoA compartmentalization, found in different animal models of brain pathologies and in human post mortem brain, are discussed in therms of their impact on cholinergic system integrity.

[Measurement of acetylcholine receptor antibodies in serum of patients with myasthenia gravis]. / Badanie poziomu przeciwcial przeciw receptorom acetylocholiny w surowicy chorych z miastenia.

The aim of this study is the evaluation of the level of antiacetylcholine receptor antibodies in patients with different types of myasthenia (according to Osserman's classification) and establishing of correlation between the level of such antibodies and the clinical state of the patients. 63 patients with diagnosed myasthenia and 30 healthy controls without immunological diseases were evaluated. In all of them standard neurological examination was performed and the levels of antiacetylcholine receptor antibodies were measured. In 45 patients electrophysiological investigations were carried out. In 43 cases elevated levels of antiacetylcholine receptor antibodies were noted. In 20 patients the levels were within normal range. The level of antibodies showed correlation with the clinical type of myasthenia according to Osserman (the more severe was myasthenia, the higher was the level of antibodies). But the clinical state of the patient at the moment of examination did not show any clear correlation with the level of antibodies. Acetylcholine receptor antibodies measurement has significant diagnostic value in myasthenia. Nevertheless it should be interpreted with other diagnostic techniques.

Effects of aluminum and calcium on acetyl-CoA metabolism in rat brain mitochondria.

Al complexes are known to accumulate in extra- and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca2+(total) effects were 10-20 times stronger than those of Al. Al decreased mitochondrial acetyl-CoA content in a concentration-dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of Pi in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl-CoA content in Ca-free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca-evoked suppression of mitochondrial metabolism. On the other hand, in Ca-supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil-evoked rise of Ca2+(total) level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO4)OH- complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca2+]total resulting from the Al-evoked rise of cytoplasmic [Ca2+]free, as well as from inhibitory interference with the verapamil binding site on the Na+/Ca2+ antiporter.