Glutamine protects both transcellular and paracellular pathways of chick intestinal calcium absorption under oxidant conditions.

Glutamine (GLN) avoids the inhibition of the intestinal Ca2+ absorption caused by menadione (MEN) through oxidative stress. The purpose of this study was to elucidate whether molecules of transcellular and/or paracellular pathways of intestinal Ca2+ absorption are involved in the GLN action and underlying mechanisms. One-month old chicks were divided in four groups: 1) controls, 2) MEN treated, 3) GLN treated and 4) GLN + MEN treated. The morphology of intestinal villi, the intestinal Ca2+ absorption and the molecules involved in the transcellular and paracellular pathways were analyzed. Markers of autophagy and inflammation were also evaluated. The data demonstrated that GLN protected both transcellular and paracellular pathways. GLN avoided morphological changes in the intestine caused by MEN. GLN protected the gene expression of transporters involved in the transcellular pathway and the gene and protein expression of molecules belonging to the paracellular pathways altered by MEN. GLN increased the LC3-II protein expression and the number of acidic vesicular organelles, markers of autophagy, and blocked an increase in the NFkB protein expression in the nuclei and in the IL-6 gene expression caused by MEN. In conclusion, GLN protects both transcellular and paracellular pathways of intestinal Ca2+ absorption by increasing autophagy and blocking inflammation.

Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo.

Melatonin displays antioxidant and free radical scavenger properties. Due to its ability with which it enters cells, these protective effects are manifested in all subcellular compartments. Recent studies suggest a role for melatonin in mitochondrial metabolism. To study the effects of melatonin on this organelle we used ruthenium red to induce mitochondrial damage and oxidative stress. The results show that melatonin (10 mg/kg i.p.) can increase the activity of the mitochondrial respiratory complexes I and IV after its administration in vivo in a time-dependent manner; these changes correlate well with the half-life of the indole in plasma. Melatonin administration also prevented the decrease in the activity of complexes I and IV due to ruthenium red (60 microg/kg i.p.) administration. At this dose, ruthenium red did not induce lipid peroxidation but it significantly reduced the activity of the antioxidative enzyme glutathione peroxidase, an effect also counteracted by melatonin. These results suggest that melatonin modulates mitochondrial respiratory activity, an effect that may account for some of the protective properties of the indoleamine. The mitochondria-modulating role of melatonin may be of physiological significance since it seems that the indoleamine is concentrated into normal mitochondria. The data also support a pharmacological use of melatonin in drug-induced mitochondrial damage in vivo.

Alterations of intracellular calcium homeostasis and mitochondrial function are involved in ruthenium red neurotoxicity in primary cortical cultures.

Ruthenium red (RR) is a polycationic dye that induces neuronal death in vivo and in primary cultures. To characterize this neurotoxic action and to determine the mechanisms involved, we have analyzed the ultrastructural alterations induced by RR in rat cortical neuronal cultures and measured its effect on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) and on mitochondrial function. RR produced a dose-dependent, progressive disruption of neurites and plasma membrane of neuronal somata after 8-24 hr of incubation. RR caused also an elevation of both the basal [Ca(2+)](i) and its maximal levels after K(+) depolarization. Mitochondrial oxidative function, assessed by reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and by changes in dihydrorhodamine-123 fluorescence, was significantly diminished after treatment with RR, both in cultured neurons and in isolated brain mitochondria. La(3+) did not prevent but rather potentiated RR-induced cell death. Glutamate receptor antagonists also failed to prevent RR neurotoxicity. Apoptotic electron microscope images were not observed, and protein synthesis inhibitors did not show any protective effect. It is concluded that RR penetrates neurons and that its neurotoxic damage probably is due to intracellular Ca(2+) dishomeostasis and disruption of mitochondrial oxidative function. These results enhance our understanding of the intracellular mechanisms underlying neuronal death.

Ruthenium red neurotoxicity and interaction with gangliosides in primary cortical cultures.

Ruthenium red (RR) is an inorganic polycationic dye able to exert several effects on the nervous system, including neurodegeneration, both in vivo and in cell cultures. Gangliosides have been shown to protect cultured neurons against several damaging conditions, and it has been postulated that RR can interact with the negative charges of the sialic acid residues of these molecules. In the present work we have tested the effect of the trisialoganglioside GT1b and the monosialoganglioside GM1 on the RR-induced neuronal damage in primary cortical cultures, as well as on the binding of RR to synaptosomes. GT1b at 100-200 microM concentrations partially protected against RR-induced neurodegeneration, as judged by light microscopy and by measurement of the reduction of a tetrazolium salt, while GM1 was ineffective. GT1b, but not GM1, also partly blocked both RR binding and its diminution in the culture medium occurring during incubation. These results suggest that the three negative charges of GT1b enable it to interact with RR and as a consequence the entrance of the dye into the cells is blocked and neurotoxicity is diminished, although other mechanisms of protection cannot be excluded. Endogenous polysialic acid-containing molecules do not seem to be involved in RR effects, since the removal of sialic acid residues by treatment with neuraminidase did not prevent the cell damage.

Ruthenium red as a tool to study calcium channels, neuronal death and the function of neural pathways.

The inorganic polycationic dye ruthenium red (RuR) exerts several effects on the nervous system when added in physiological solutions, both in vivo and in vitro. Part of these effects, including the paralysis observed in mammals after the systemic administration of RuR, can be accounted for by the binding of RuR to nerve ending membranes, which results in inhibition of Ca2+ influx through voltage-sensitive calcium channels and the consequent inhibition of neurotransmitter release. On the other hand, the administration of RuR into the cerebrospinal fluid induces intense convulsive activity, and its microinjection into the substantia nigra reticulata or the hippocampus leads to various motor behavior alterations that can be related to hyperexcitability of the neurons of the injected region. In addition, RuR penetrates the neuronal somata present in the area injected and induces cell destruction, which has been interpreted as an excitotoxic action of the dye. The penetration and the toxicity of RuR were also observed in primary neuronal cultures but did not occur in pure glial cultures, suggesting a selective action on neurons. In the present article the in vitro and in vivo effects of RuR are reviewed and discussed in terms of the usefulness of the dye as an interesting tool to study calcium channels linked to transmitter release, neuronal death mechanisms and the function of neural pathways.

Motor alterations and neuronal damage induced by intracerebral administration of Ruthenium red: effect of NMDA receptor antagonists and other anticonvulsant drugs.

The effects of the intracerebroventricular (icv) and the intrahippocampal (ih) microinjection of the inorganic dye Ruthenium red (RuR) on motor activity, and the protective action of excitatory amino acid receptor antagonists and of GABAergic drugs, were studied in the rat. When administered icv, RuR produced intense tonic-clonic convulsions which were refractory to N-methyl-D-aspartate (NMDA) receptor antagonists and to diphenylhydantoin, whereas aminooxyacetic acid (AOA) and valproate only partially protected against seizure activity. The most notable motor effect of the ih RuR administration was the appearance of intense wet-dog shakes (WDS) behavior, which was remarkably attenuated by the icv or intraperitoneal (ip) administration of the NMDA receptor antagonists (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), CGP-37849, and MK-801, but not by their ih coinjection with RuR. Systemic AOA and valproate were also effective in reducing the number of WDS, whereas the non-NMDA receptor antagonist CNQX was ineffective. Light and electron microscopic observations of the RuR-injected brains revealed that the dye was highly concentrated in neuronal somas located in or near the injected areas. In the case of the CA1 region, remarkable damage of the pyramidal neurons was manifested by vacuolization, and 5-9 d after the injection notable cell loss and disruption of the CA1 cell layer organization was apparent. The results indicate that RuR penetrates selectively neuronal bodies and damage them, and suggest that the resulting motor alterations involve hyperactivity of glutamatergic neurotransmission.

Antithetic effects of ryanodine and ruthenium red on osteoclast-mediated bone resorption and intracellular calcium concentrations.

In the process of bone remodeling, osteoclasts are responsible for resorption of bone. High levels of intracellular calcium decrease the bone resorbing activity of osteoclasts and increase detachment of osteoclasts from the bone surface. The regulatory role of intracellular calcium in bone resorption is not clearly understood. To understand this phenomenon, we studied the effects of the intracellular calcium modulators ryanodine and ruthenium red on bone resorption using the disaggregated osteoclast pit assay. Changes in intracellular calcium concentrations after treatment with these compounds were detected with the fluoroprobe fura2. With ryanodine, a significant, dose-dependent decrease in bone resorption was detected. This inhibition of bone resorption was reversible upon the removal of ryanodine. Ryanodine increased intracellular calcium concentrations, suggesting that the mechanism of inhibition by ryanodine was via alterations in intracellular stores of calcium. After treatment with ruthenium red, osteoclasts resorbed significantly more bone compared to vehicle-treated cells. This increase in bone resorption correlated with a decrease in intracellular calcium concentrations. The addition of parathyroid hormone or ruthenium red to osteoclast cultures containing ryanodine did not attenuate the decrease in bone resorption caused by ryanodine, suggesting that the mechanism of ryanodine inhibition of bone resorption may involve the "locking" of a calcium channel in an open position.

Selective neurotoxicity of ruthenium red in primary cultures.

The inorganic dye ruthenium red (RuR) has been shown to be neurotoxic in vivo when injected intracerebrally. In this work the toxicity of RuR was compared in primary cultures of rat cortical neurons, cerebellar granule neurons and cerebellar astroglia. Microscopic examination of the cultures revealed that RuR penetrates the somata of both types of neurons used and produces vacuolization and loss and fragmentation of neurites. In contrast, no RuR was seen inside cultured astrocytes and no morphological signs of damage were observed in these cells. RuR toxicity was also assessed by immunocytochemistry of alpha-tubulin and by biochemical measurement of the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the cultured cells. The morphological alterations in the neurons were closely correlated with loss of tubulin immunoreactivity and particularly with a notable decrement in the ability to reduce MTT. Using the latter parameter, it was found that neuronal damage was independent of the age of the cultures, augmented progressively with time of incubation with RuR, from 8 to 24 h, and showed a clear dose-response curve from 20 to 100 microM RuR. Astrocytes showed only a slight decrease in MTT reduction after 24 h of incubation with 100 microM RuR. It is concluded that RuR seems to be toxic for neurons but not for astroglia, and that this selectivity is probably related to the ability of the neurons to internalize the dye. The possible mechanisms of RuR penetration and neuronal damage are discussed.

Ruthenium red and capsaicin induce a neurogenic inflammatory response in the rabbit eye: effects of omega-conotoxin GVIA and tetrodotoxin.

The effects of ruthenium red, an inorganic dye with known capsaicin antagonist properties, was investigated in the rabbit eye. At a dose of 0.24 nmol ruthenium red inhibited the inflammatory effects of capsaicin (1 or 8 nmol). Unexpectedly, when the dye was injected in doses ranging from 0.24 to 7.4 nmol, it caused an inflammatory response with constriction of the pupil (miosis) and a breakdown of the blood-aqueous barrier, leading to a rise intraocular pressure. Tetrodotoxin (30 nmol) inhibited the ruthenium red-induced rise in intraocular pressure but had less effect on the miotic response. The tachykinin antagonist spantide inhibited the miosis but had no effect on the rise in intraocular pressure. Ruthenium red induced an increase in substance P-like immunoreactivity and calcitonin gene-related peptide-like immunoreactivity in the aqueous humor. These levels were positively correlated with the rise in aqueous humor protein concentration. The ruthenium red-induced miosis and, to a less extent, the rise in intraocular pressure were inhibited by the Ca2+ channel-blocking agent omega-conotoxin GVIA (CTX), indicating a partial dependence on an influx of extracellular Ca2+. CTX also attenuated the miotic effect of capsaicin but had no effect on the capsaicin-induced rise in intraocular pressure. It is concluded that, in the rabbit eye, ruthenium red induces a neurogenic inflammatory response besides its capsaicin antagonist effects.

Qualitative X-ray spectrometric study to demonstrate ruthenium in central nervous system structures, after intraperitoneal injection of ruthenium red to adult rats.

A qualitative X-ray spectrometric study oriented to demonstrate ruthenium (Ru) in central nervous system was made after intraperitoneal (i.p.) injection of ruthenium red (RuR) to adult rats. Ru signals were depicted in the brain synaptosomal fraction since 60 min after RuR i.p. administration, corresponding to the latency period of the convulsive model injecting RuR systemically to adult rats. Ru signals were initially detected in pineal gland and periventricular regions, whereas X-rays from Ru atoms in cerebral cortex were detected at longer time intervals after RuR i.p. injection. It is concluded that RuR, a non-liposoluble substance, when injected systemically, passes from the blood stream to brain parenchyma, probably through areas without blood-brain barrier, reaching the neural elements related to the mechanisms of production of convulsions.

Effects of ruthenium red intraperitoneally injected to adult rats, on the uptake and release of neurotransmitters from brain synaptosomes.

Effect of intraperitoneal (i.p.) injection of ruthenium red (RuR) to adults rats on catecholamine (CA) uptake and dopamine (DA) release was evaluated in brain synaptosomal fractions. No effect on CA uptake by synaptosomal fractions was seen when RuR was added in vitro. There was no effect of RuR injection i.p. to rats, on CA uptake by their brain synaptosomal fractions. A blocking effect on depolarizing induced released of DA from brain synaptosomal fraction obtained from rats previously injected i.p. with RuR was seen. A similar effect was observed on the basal DA efflux from synaptosomes loaded with (14C)DA obtained from rats i.p. injected with RuR. It is concluded that RuR, a non-liposoluble substance, when injected i.p. reaches the brain parenchyma through areas devoid of blood-brain barrier and interferes with Ca(++)-dependent release of neurotransmitters, inducing cerebral hyperexcitability and convulsions.

Interactions of mitochondrial inhibitors with methylmercury on spontaneous quantal release of acetylcholine.

The interaction of methylmercury (MeHg) with various inhibitors of mitochondrial function (dinitrophenol, 50 microM; dicoumarol, 100 microM; valinomycin, 20 microM; and ruthenium red, 20 microM) on spontaneous quantal release of acetylcholine was tested at the neuromuscular junction of the rat. The objective was to determine whether these mitochondrial inhibitors blocked the MeHg-induced increase of spontaneous release of acetylcholine, an effect measured electrophysiologically as increased miniature endplate potential (MEPP) frequency. MEPPs were recorded from myofibers of the rat hemidiaphragm using conventional, intracellular microelectrode recording techniques. When given alone, all four inhibitors increased MEPP frequency from resting levels of 1-2/sec (Hz) to approximately 10-60 Hz after a latency which ranged from 5 to 30 min. MEPP frequency subsequently returned to control levels. Subsequent concomitant application of MeHg (100 microM) with dinitrophenol, dicoumarol, or valinomycin increased MEPP frequency sharply to peak values of 40-60 Hz after 15-20 min. MEPP frequency subsided to pre-MeHg levels 10 min later. The time course and peak MEPP frequency elicited by MeHg after pretreatment with these uncouplers were similar to results obtained in preparations treated with MeHg alone. Ruthenium red, a putative specific inhibitor of the Ca2+ uptake uniporter in mitochondria, increased MEPP frequency to 12 Hz after 8.5 min when given alone. MEPP frequency returned to control levels approximately 10 min later. Subsequent application of MeHg and ruthenium red for up to 80 min failed to increase MEPP frequency. The inability of MeHg to increase MEPP frequency in ruthenium red-treated preparations was not due to depletion of acetylcholine nor to block of postjunctional receptors by ruthenium red since subsequent treatment with La3+ (2 mM) increased MEPP frequency to 12.5 Hz within 10 min. Thus, ruthenium red blocked the stimulatory effect of MeHg on MEPP frequency while uncouplers of oxidative phosphorylation and a K+ ionophore did not. The results with ruthenium red are consistent with the proposal that MeHg may block mitochondrial uptake of Ca2+ or promote its release, leading to an increased free cytosolic Ca2+ concentration which in turn stimulates spontaneous release of acetylcholine.

Potentiation of oxidative cell injury in hepatocytes which have accumulated Ca2+.

Incubation of freshly isolated rat hepatocytes with exogenous ATP, but not with succinate, resulted in intracellular Ca2+ accumulation which was partly prevented when the inhibitor of mitochondrial Ca2+ sequestration, ruthenium red, was also present in the medium. Although the bulk of the accumulated Ca2+ was sequestered by the mitochondria, formation of surface blebs and stimulation of phosphorylase alpha activity during incubation of the hepatocytes with ATP indicate that this treatment was also associated with an increase in cytosolic free Ca2+ concentration. When hepatocytes loaded with Ca2+ by preincubation with ATP were exposed to either 2-methyl-1,4-naphthoquinone or t-butyl hydroperoxide, the cytotoxicity of both agents was markedly potentiated. Our results suggest that ATP-induced Ca2+ accumulation in hepatocytes is not due to contamination of the cell suspension with damaged cells or free intracellular organelles and that the intracellular Ca2+ concentration can affect the response to toxic agents.