Essentiality and toxicity of vanadium supplements in health and pathology.

The biological properties of vanadium complexes have become an object of interest due to their therapeutic potential in several diseases. However, the mechanisms of action of vanadium salts are still poorly understood. Vanadium complexes are cofactors for several enzymes and also exhibit insulin-mimetic properties. Thus, they are involved in the regulation of glucose metabolism, including in patients with diabetes. In addition, vanadium salts may also normalize blood pressure and play a key role in the metabolism of the thyroid and of iron as well as in the regulation of total cholesterol, cholesterol HDL and triglyceride (TG) levels in blood. Moreover, in cases of hypoxia, vanadium compounds may improve cardiomyocytes function. They may also exhibit both carcinogenic and anti-cancer properties. These include dose- and exposure-time-dependent induction and inhibition of the proliferation and survival of cancer cells. On the other hand, the balance between vanadium's therapeutic properties and its side effects has not yet been determined. Therefore, any studies on the potential use of vanadium compounds as supplements to support the treatment of a number of diseases must be strictly monitored for adverse effects.

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.

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.

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.

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.

[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.

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.

Putative significance of shifts in acetyl-CoA compartmentalization in nerve terminals for disturbances of cholinergic transmission in brain.

Acetylcholine and acetyl-CoA metabolism in nerve terminals isolated from rat brain were found to be affected by several neurotoxic and neuroprotective agents, such as aluminium, nitric oxide, beta-hydroxybutyrate, verapamil and thiamine deficiency. The changes evoked by these factors in Ca2+-dependent acetylcholine release were highly significantly correlated (r = 0.98) with changes in concentration of synaptoplasmic acetyl-CoA. On the other hand, in the same experimental conditions, no correlation was found between rates of pyruvate oxidation, intramitochondrial acetyl-CoA levels and different pools of releasable acetylcholine. These data indicate that disturbances in the availability of acetyl-CoA in the cytoplasm of nerve terminals may be a key factor in the pathogenesis of several cholinergic encephalopathies.

Effect of aluminum on acetyl-CoA and acetylcholine metabolism in nerve terminals.

The potential ability of Al to affect cholinergic transmission was studied on synaptosomal fractions of rat brain incubated with pyruvate in depolarizing medium containing 30 mM K+. Addition of 1 mM Ca caused a 266% increase in the acetylcholine (ACh) release despite decreased pyruvate oxidation. Under these conditions, 0.25 mM Al did not affect pyruvate oxidation but raised mitochondrial and decreased synaptoplasmic acetyl-CoA. Simultaneously, a 61% inhibition of Ca-evoked ACh release was observed. Verapamil (0.1 and 0.5 mM) decreased the acetyl-CoA concentration in synaptoplasm and inhibited ACh release. Al (0.012 mM) partially reversed these inhibitory effects. Omission of Pi from the medium abolished suppressive effects of Al on acetyl-CoA content and Ca-evoked transmitter release. We conclude that the Al(PO4)OH- complex may be the active form of Al, which, by interaction with the verapamil binding sites of Ca channels, is likely to restrict the Ca influx to the synaptoplasm. This may inhibit the provision of acetyl-CoA to the synaptoplasm as well as the Ca-evoked ACh release. One may suppose that excessive accumulation of Al in some encephalopathic brains may, by this mechanism, suppress still-surviving cholinergic neurons and exacerbate cognitive deficits caused by already-existing structural losses in the cholinergic system.

Key role of acetyl-CoA in cytoplasm of nerve terminals in disturbances of acetylcholine metabolism in brain.

This paper review data indicating that the concentration of acetyl-CoA in cytoplasm of cholinergic terminals is an important factor which may determine the rate and size of quantal acetylcholine in different physiologic and pathologic conditions.

Pathways of beta-hydroxybutyrate contribution to metabolism of acetyl-CoA and acetylcholine in rat brain nerve terminals.

beta-hydroxybutyrate increased concentration of acetyl-CoA in mitochondria of resting and in cytoplasm of Ca-activated rat brain synaptosomes. Adequate rise of Ca-evoked acetylcholine release was also observed. The activation was abolished by verapamil. It indicates that beta-hydroxybutyrate-derived acetyl-CoA is transported from mitochondria to synaptoplasm by direct Ca-dependent transport mechanism. Presented data evidence that level of synaptoplasmic acetyl-CoA plays an important role in the regulation of cholinergic activity in the brain.

Cholinergic denervation-like changes in rat hippocampus following developmental lead exposure.

We investigated the effects of developmental lead exposure from embryonic day 16 (E16) through postnatal day 28 (PN28), on cholinergic and catecholaminergic markers in the septohippocampal pathway in rats through fourth month of age. Lead exposure resulted in a persistent 30-40% reduction of [3H]hemicholinium-3 ([3H]HC-3) binding in the hippocampus through PN120, and 20-30% reduction of septal and hippocampal choline acetyltransferase (ChAT) activity which persisted through PN84 but returned to control levels in both septum and hippocampus at PN112. The muscarinic ligand [3H]quinuclidinyl benzylate ([3H]QNB) binding was reduced in the septum at PN28 but did not differ significantly from controls at PN56-PN112. Neither short- nor long-term effects of Pb exposure on [3H]QNB binding were seen in the hippocampus. Similar to the effects of fimbria-fornix transection, Pb exposure resulted in a long-term 50-90% increase of tyrosine hydroxylase(TH) activity in the hippocampus, although neither treatment affected TH activity in the septum. The lead-induced increase in hippocampal TH was significantly attenuated by superior cervical ganglionectomy. It is concluded that the effects of perinatal lead exposure resemble in several respects those seen following surgical disruption of the septohippocampal pathway in adult animals. The denervation-like effects in the hippocampus may be an important factor in long-term learning and cognitive impairments following developmental exposure to low-levels of lead.

Disturbances of acetyl-CoA, energy and acetylcholine metabolism in some encephalopathies.

Acetyl-CoA provision to the synaptoplasmic compartment of cholinergic nerve terminals plays a regulatory role in the synthesis of acetylcholine. The disturbances in glucose utilization and in decarboxylation of the end product of its metabolism pyruvate, are considered to be significant factors causing cholinergic deficits in several diseases of the central nervous system. In this article we review data concerning role of acetyl-CoA in patomechanisms of disturbances of cholinergic metabolism in Alzheimers disease, thiamine deficiency, inherited defects of pyruvate dehydrogenase and diabetes.

Reduced densities of sodium-dependent [3H] hemicholinium-3 binding sites in hippocampus of developmental rats following perinatal low-level lead exposure.

We investigated the effect of perinatal, low-level lead exposure on [3H]hemicholinium-3 (HC-3) binding in the hippocampus of postnatal rat. Rat pups were maternally lead-exposed from gestational day 16 through postnatal day 28 (PN28). In control animals, the [3H]HC-3 binding sites increased from 7 fmol/mg protein at postnatal day 1 (PN1) to 14 and 35 fmol/mg protein at PN7 and PN14, respectively, and reached adult values of 50 fmol/mg protein, at PN21 and PN28. In lead-exposed litters, the [3H]HC-3 binding was reduced by 30-40% throughout the early postnatal development and remained 40% below control values in PN60 animals, one month after termination of lead exposure. The Pb-induced reduction in HC-3 binding was associated with a similar decrease in ChAT activity and was comparable to the effect of localized lesion of medial septum. Septal cell counts in the lead-exposed PN21 rats indicated a 22% reduction in the number of ChAT-immunoreactive cells in the medial septum/vertical diagonal band (MS/vDB) complex although cell numbers in the horizontal limb of the diagonal band (hDB) were not altered. These results suggest that perinatal, low-level lead exposure results in a reduced density of cholinergic nerve terminals in the hippocampus, either due to impaired development or degeneration of the cholinergic projection neurons in the MS/vDB complex.

Effect of dichloroacetate on acetyl-CoA content and acetylcholine synthesis in rat brain synaptosomes.

In potassium-depolarized synaptosomes Ca2+ inhibited oxidation of pyruvate (30%) and decreased the level of acetyl-CoA in intrasynaptosomal mitochondria (32%). On the other hand, Ca2+ facilitated provision of acetyl-CoA to synaptoplasm, since under these condition no change of synaptoplasmic acetyl-CoA and twofold stimulation of acetylcholine synthesis were found. However, in Ca(2+)-activated synaptosomes both synaptoplasmic acetyl-CoA and acetylcholine synthesis were suppressed by 1 mM (-)hydroxycitrate by 27 and 29%, respectively. It was not the case in resting synaptosomes. Dichloroacetate (0.05 mM) partially reversed the inhibitory effect of Ca2+ on pyruvate metabolism in synaptosomes and whole brain mitochondria. In Ca(2+)-stimulated synaptosomes, the dichloroacetate overcame suppressive effects of (-)hydroxycitrate on the level of synaptoplasmic acetyl-CoA and acetylcholine synthesis, but not on citrate cleavage. It is concluded that dichloroacetate may improve the metabolism of acetylcholine in activated cholinergic terminals by increasing the production of acetyl-CoA in mitochondria and increasing its provision through the mitochondrial membrane to synaptoplasm by the transport system, independent of the ATP-citrate lyase pathway.

Perinatal low-level lead exposure and the septo-hippocampal cholinergic system: selective reduction of muscarinic receptors and cholineacetyltransferase in the rat septum.

We investigated the effects of low-level lead exposure on the postnatal development of cholinergic muscarinic receptors (mAChR) and a cholinergic marker enzyme cholineacetyltransferase (ChAT) activity in the rat septum and hippocampus. Rat pups were maternally lead-exposed by giving 0.2% lead acetate in drinking water to dams from one week before parturition (gestational day 16) through weaning at postnatal day 28. The lead-exposed litters had blood Pb in the range 20 micrograms/dl and tissue Pb < 0.2 micrograms/g in both the septum and hippocampus. Associated with this level of lead exposure there was a significant 30-40% reduction in the ChAT activity in the septa and hippocampi of PN7 through PN28 animals. In contrast, the levels of glutamic acid decarboxylase (GAD) activity, a GABAergic neuron marker enzyme, were not altered in either brain region. Associated with the selective reduction of ChAT activity there was a parallel 30-40% reduction of the [3H]quinuclidinyl benzilate, [3H]AF-DX 384, and [3H]pirenzepine binding in the septum, however muscarinic ligand binding in the hippocampus of lead exposed animals was not affected. These results indicate preferential vulnerability of septal cholinergic neurons to adverse effects of low-level Pb exposure and suggest that impaired expression of muscarinic receptors and disruption of muscarinic transmission in the septum may be an important factor in cognitive and learning deficits associated with developmental low-level lead exposure.

Effect of angiotensin II and eledoisin on cholinergic neurons in rat hippocampus.

Angiotensin II and eledoisin modulate drinking behaviour in rats that is mediated by monoaminergic and cholinergic neurons. In the present study we have shown that combined intracerebroventricular injections of either 0.1 or 1.0 microgram doses of angiotensin and eledoisin resulted in a decrease of about 25-35% in activities of choline acetyltransferase, ATP-citrate lyase in the hippocampus. In addition, 1 microgram quantities of these peptides depressed activity of carnitine acetyltransferase but did not alter activity of acetylcholinesterase. On the other hand, the application of 0.1 microgram of angiotensin caused no change in activity of monoamine oxidase A, while 1.0 microgram dose brought about its 67% activation. Eledoisin abolished this effect of angiotensin II. These data provide evidence that angiotensin II and eledoisin evoke non related adaptive changes in cholinergic and monoaminergic neurons of the hippocampus.

Differential effects of angiotensin II and eledoisin on monoamine oxidase A and B activities in rat brain.

Intracerebroventricular injections of angiotensin II caused 108, 62, and 54% increases in monoamine oxidase A activities in rat hippocampus, hypothalamus, and striatum, respectively. These activatory effects were abolished by simultaneous injections of eledoisin. No significant changes of monoamine oxidase B activities were found under the same experimental conditions. Neither angiotensin II nor elodoisin changed substrate/inhibitor affinities of both isoenzymes. These data indicate that angiotensin II and tachykinin transmitter systems may exert opposite, long-term regulatory effects on monoaminergic neurons in rat brain.

Evidence for the regulatory function of synaptoplasmic acetyl-CoA in acetylcholine synthesis in nerve endings.

Isolated synaptosomes maintained a relatively stable level of acetyl-CoA during their incubation in the presence of 30 mM-KCl. Addition of Ca2+ resulted in inhibition of pyruvate oxidation and slight activation of acetylcholine synthesis. The cation decreased acetyl-CoA in intrasynaptosomal mitochondria, but did not alter its content in synaptoplasm. Verapamil did not affect pyruvate oxidation, but decreased acetyl-CoA in synaptoplasm and inhibited acetylcholine synthesis in synaptosomes. It indicates that Ca2+ might regulate acetylcholine synthesis through changes in the direct transfer of acetyl-CoA from mitochondria to synaptoplasm.

Regulation of citrate metabolism and acetycholine synthesis by Ca(2+) in rat brain synaptosomes.

The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca(2+) caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca(2+). The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca(2+). (?) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20-0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca(2+) caused no significant changes in [(14)C]ACh synthesis, but it inhibited (14)CO(2) production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca(2+) did not modify these effects of the drug. On the other hand, (?)hydroxycitrate resulted in 22 and 29% inhibition of [(14)C]ACh synthesis in Ca(2+) free and Ca(2+) supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings.