Mitri Palmi

Potentiation of mitochondrial Ca2+ sequestration by taurine

The effects of taurine (2-aminoethanesulphonic acid) and its analogues, 2-aminoethylarsonic acid, 2-hydroxyethanesulphonic (isethionic) acid, 3-aminopropanesulphonic acid, 2-aminoethylphosphonic acid, and N,N-dimethyltaurine, were studied on the transport of Ca2+ by mitochondria isolated from rat liver. Taurine enhanced Ca2+ uptake in an apparently saturable process, with a Km value of about 2.63 mM. Taurine behaved as an uncompetitive activator of Ca2+ uptake, increasing both the apparent Km and Vmax values of the process. This effect was not modified in the presence of cyclosporin A (CsA). N,N-Dimethyltaurine also stimulated Ca2+ uptake at higher concentrations, but there was no evidence that the process was saturable over the concentration range used (1-10 mM). Aminoethylarsonate was a weak inhibitor of basal Ca2+ uptake, but inhibited that stimulated by taurine in an apparently competitive fashion (Ki = 0.05 mM). The other analogues had no significant effects on this process. Taurine either in the presence or the absence of CsA had no effect on Ca2+ release induced by 200 nM ruthenium red. Thus, the mechanism of taurine-enhanced Ca2+ accumulation appears to involve stimulation of Ca2+ uptake via the uniport system rather than inhibition of Ca2+ release via the ion (Na+/Ca2+ and/or H+/Ca2+) exchangers or by taurine modulating the permeability transition of the mitochondrial inner membrane. Overall, these findings indicate an interaction of taurine with an as yet unidentified mitochondrial site which might regulate the activity of the uniporter. The unique role of taurine in modulating mitochondrial Ca2+ homeostasis might be of particular importance under pathological conditions that are characterised by cell Ca2+ overload, such as ischaemia and oxidative stress.

Calcium antagonist and antiperoxidant properties of some hindered phenols

1 The calcium antagonist and antioxidant activities of certain synthetic and natural phenols, related to BHA (2‐t‐butyl‐4‐methoxyphenol), were evaluated in rat ileal longitudinal muscle and in lipid peroxidation models respectively. 2 Compounds with a phenol or a phenol derivative moiety, with the exception of 2,2′‐dihydroxy‐3,‐3′‐di‐t‐butyl‐5,5′‐dimethoxydiphenyl (di‐BHA), inhibited in a concentration‐dependent manner the BaCl2‐induced contraction of muscle incubated in a Ca2+‐free medium. Calculated pIC50 (m) values ranged between 3.32 (probucol) and 4.96 [3,5‐di‐t‐butyl‐4‐hydroxyanisole (di‐t‐BHA)], with intermediate activity shown by khellin < gossypol < quercetin < 3‐t‐butylanisole < BHA < nordihydroguaiaretic acid (NDGA) < 2,6‐di‐t‐butyl‐4‐methylphenol (BHT) and papaverine. 3 The Ca2+ channel activator Bay K 8644 overcame the inhibition sustained by nifedipine, BHA and BHT, while only partially reversing that of papaverine. 4 BHA, BHT, nifedipine and papaverine also inhibited in a concentration‐dependent fashion CaCl2 contractions of muscle depolarized by a K+‐rich medium. This inhibition appeared to be inversely affected by the Ca2+‐concentration used. 5 The inhibitory effects of nifedipine, papaverine, BHA and BHT were no longer present when muscle contraction was elicited in skinned fibres by 5 μm Ca2+ or 500 μm Ba2+, suggesting a plasmalemmal involvement of target sites in spasmolysis. 6 Comparative antioxidant capability was assessed in two peroxyl radical scavenging assay systems. These were based either on the oxidation of linoleic acid initiated by a heat labile azo compound or on lipid peroxidation of rat liver microsomes promoted by Fe2+ ions. Across both model systems, di‐t‐BHA, NDGA, BHT, di‐BHA, BHA and quercetin ranked as the most potent inhibitors of lipid oxidation, with calculated pIC50 (m) values ranging between 7.4 and 5.7. 7 Of the 32 compounds studied only 15 phenolic derivatives exhibited both antispasmogenic and antioxidant activity. Within this subgroup a linear and significant correlation was found between antispasmogenic activity and antioxidation. These bifunctional compounds were characterized by the presence of at least one hydroxyl group on the aromatic ring and a highly lipophilic area in the molecule. 8 Di‐t‐BHA is proposed as a lead reference compound for future synthesis of new antioxidants combining two potentially useful properties in the prevention of tissue damage after ischaemia‐reperfusion injury.

Interactions of taurine and structurally related analogues with the GABAergic system and taurine binding sites of rabbit brain

The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [3H]muscimol. Saturation experiments of the binding of [3H]GABA to GABAB receptors showed that SM possess two binding components; twice Triton X‐100‐treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [3H]taurine in a saturable manner (Kd=249.0±6.3 nM and Bmax=3.4±1.0 pmol mg−1 prot). Among the 19 structural analogues of taurine, 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) displaced [3H]taurine binding (Ki=0.13, 0.13, 13.5 and 4.0 μM, respectively). These analogues did not interact with GABAA and GABAB receptors and did not affect taurine‐ and GABA‐uptake systems and GABA‐transaminase activity. 3‐Aminopropanesulfonic acid (OMO), β‐alanine, pyridine‐3‐sulfonic acid, N,N,N‐trimethyltaurine (TMT), 2‐(guanidino)ethanesulfonic acid (GES), ethanolamine‐O‐sulphate, N,N‐dimethyltaurine (DMT), taurine and (±)piperidine‐3‐sulfonic acid (PSA) inhibited [3H]muscimol binding to GABAA receptors with different affinities (Ki=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 μM, respectively). Taurine, 2‐aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [3H]GABA to GABAB receptors with Kis in the μM range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC50=3.72 μM) and PSA GABA transaminase activity (IC50=103.0 μM). In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Rectal temperature and prostaglandin E2 increase in cerebrospinal fluid of conscious rabbits after intracerebroventricular injection of hemoglobin

Abstract Fever accompanies subarachnoid hemorrhage (SAH) in the majority of patients. In a previous study, hemoglobin (Hb) was shown to catalyze in vitro, under aerobic conditions, the conversion of arachidonic acid to prostaglandin E2 (PGE2) and prostaglandin F2α. The aim of the present work was to assess whether this pathway also operates in vivo and to provide a mechanism to explain post-SAH fever. To this end, PGE2 concentration was determined in cerebrospinal fluid (CSF) of conscious rabbits chronically cannulated in the lateral ventricle and cisterna magna, following intracerebroventricular (i.c.v.) injection of 10 µg or 100 µg of commercial rabbit bicrystallized Hb as a model of SAH. Before i.c.v. injection, Hb solutions were filtered on a polimixin-B column to remove substantially, by over 90%, endotoxin-like substances. Results show that in nine rabbits injection of 10 µg Hb did not significantly modify body temperature or significantly alter CSF PGE2 content. On the contrary, in nine rabbits, injection of 100 µg Hb produced a significant increase in core temperature which was accompanied by a significant increase in CSF PGE2. When data related to these two parameters from the 9 control and 18 Hb-treated rabbits were analyzed as a single group, a linear, positive, and highly significant correlation was found. These findings indicate that, once Hb is released into the subarachnoid space during SAH, it enhances CSF PGE2 content and elicits hyperthermia, thus offering an explanation for the fever that is an aggravating condition in most SAH patients.

The Translationally Controlled Tumor Protein Is a Novel Calcium Binding Protein of the Human Placenta and Regulates Calcium Handling in Trophoblast Cells

Abstract The translationally controlled tumor protein (TPT1, also known as TCTP) is a highly conserved, abundantly expressed protein found in mammals as well as in a wide range of other organisms of both the animal and plant kingdom. Initially considered as a growth-related protein, later studies showed TPT1 is endowed with multiple biological activities, including calcium binding. The present study aimed to evaluate the expression of TPT1 in the human placenta and to examine the functional role of the protein in the calcium binding and homeostasis of trophoblast cells. Samples were analyzed by Western blot, reverse transcription-polymerase chain reaction and immunohistochemistry. The effect of TPT1 knockdown by small interfering RNA (siRNA) on calcium uptake and buffering was assessed in the HTR-8/SVneo cell line. TPT1 protein and mRNA were detected in first-trimester and term placenta. In the tissue, TPT1 was localized in the villous trophoblast. TPT1 expression significantly increased during gestation, with the higher protein and mRNA levels reached at term. Recombinant placental TPT1 bound calcium in vitro, while downregulation of the protein levels by siRNA in HTR-8/SVneo cells was associated with a reduced cellular calcium-uptake activity and buffering capacity. These data demonstrate, for the first time, the expression of TPT1 in the human placenta and support a direct role of the protein in placental calcium transport.

A specific taurine recognition site in the rabbit brain is responsible for taurine effects on thermoregulation

Taurine and GABA are recognized as endogenous cryogens. In a previous study, some structural analogues of taurine, namely 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) have been shown to displace [3H]taurine binding from rabbit brain synaptic membrane preparations, without interacting either with GABA‐ergic systems, nor with taurine uptake mechanism, thus behaving like direct taurinergic agents. To answer the question whether the role of taurine as an endogenous cryogen depends on the activation of GABA receptors or that of specific taurine receptor(s), taurine or the above structural analogues were injected intracerebroventricularly in conscious, restrained rabbits singularly or in combination and their effects on rectal (RT)‐ and ear–skin temperature and gross motor behavior (GMB) were monitored. Taurine (1.2 × 10−6–4.8 × 10−5 mol) induced a dose‐related hypothermia, vasodilation at ear vascular bed and inhibition of GMB. CAHS, at the highest dose tested (4.8 × 10−5 mol) induced a taurine‐like effect either on RT or GMB. On the contrary ISE, injected at the same doses of taurine, induced a dose‐related hyperthermia, vasoconstriction and excitation of GMB. AEA and TAG caused a dose‐related hyperthermia, but at doses higher than 1.2 × 10−7 mol caused death within 24 h after treatment. CAHS (4.8 × 10−5 mol) antagonized the hyperthermic effect induced by TAG (1.2 × 10−6 mol), AEA (1.2 × 10−8 mol) or ISE (4.8 × 10−5 mol). In conclusion, these findings may indicate the existence of a recognition site specific for taurine, responsible for its effects on thermoregulation.

Role of the nitric oxide/cyclic GMP/Ca2+ signaling pathway in the pyrogenic effect of interleukin-1β

Interleukin-1β (IL-1β) has a wide spectrum of inflammatory, metabolic, haemopoietic, and immunological properties. Because it produces fever when injected into animals and humans, it is considered an endogenous pyrogen. There is evidence to suggest that Ca2+ plays a critical role in the central mechanisms of thermoregulation, and in the intracellular signaling pathways controlling fever induced by IL-1β and other pyrogens. Data from different labs indicate that Ca2+ and Na+ determine the temperature set point in the posterior hypothalamus (PH) of various mammals and that changes in Ca2+ and PGE2 concentrations in the cerebrospinal fluid (CSF) of these animals are associated with IL-1β-induced fever. Antipyretic drugs such as acetylsalicylic acid, dexamethasone, and lipocortin 5-(204–212) peptide counteract IL-1β-induced fever and abolish changes in Ca2+ and PGE2 concentrations in CSF. In vitro studies have established that activation of the nitric oxide (NO)/cyclic GMP (cGMP) pathway is part of the signaling cascade transducing Ca2+ mobilization in response to IL-1β and that the ryanodine (RY)- and inositol-(1,4,5)-trisphosphate (IP3)-sensitive pools are the main source of the mobilized Ca2+. It is concluded that the NO/cGMP/Ca2+ pathway is part of the signaling cascade subserving some of the multiple functions of IL-1β.

Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines.

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen‐activated protein kinases [extracellular signal‐regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)‐1‐[2‐aminiethyl]‐N‐[2‐ammonioethyl]amino]diazen‐1‐ium‐1,2diolate (DETA/NO) concentrations (10 µm) determined a gradual, moderate elevation in [Ca2+]i (46.8 ± 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 ± 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 ± 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down‐regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50–300 µm) of DETA/NO negatively regulated cell proliferation via a Ca2+‐independent mechanism.

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic–ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low‐oxygen and Ca2+‐free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia‐induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine–NO and inhibited by N‐ω‐nitro‐L‐arginine methyl ester (L‐NAME) or by the NO scavenger 2‐phenyl‐4,4,5,5 tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO). A membrane‐permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U‐373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 ± 13.1% compared to normoxic controls and this effect was inhibited by L‐NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L‐NAME. Although heparin plus RR or RY antagonized hypoxia‐induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO–Ca2+ signalling might be a potential therapeutic target in hypoxia‐induced neuronal degeneration.