[Effect of Galla Chinesis on the demineralization of dental root tissue in pH cycling model].

OBJECTIVE: To evaluate the anti-demineralization efficacy of Galla Chinesis in pH cycling model for elucidating the anti-root caries mechanism. METHOD: Anti-demineralization efficacy evaluation of the natural medicine in the pH-cycling models was used . Sound human root blocks were pH-cycled through the treatment solution, acidic buffer and neutral buffer. The cycling times for demineralization study were 12 times, 2 times per day. The acidic buffers were retained for calcium analysis by atomic adsorption spectroscopy. The sections of blocks were analysed after pH-cycling by CLSM. Treatments were 4 g x L(-1). Galla Chinesis, 1 g x L(-1) NaF solution and distilled water. RESULT: Galla Chinesis was found to inhibit the demineralization in the pH cycling model. Although the effect was not as good as fluoride, there was no significant difference between the two groups. CONCLUSION: These data suggest that Galla Chinesis could modulate the mineralisation behaviour of root tissue in a defined chemical circumstance. These findings support the proposition that Galla Chinesis may be a promising anticaries natural medicine in the future.

[Peculiarities of ion transport of calcium in tumor cells under conditions of irradiation by ionizing radiation, chemopreparations and homeopathic means].

The goal of given investigation was to reveal an effect of different agents on ion transport of Ca2+ in tumor cells (Erlich's carcinomas). Ionizing radiation, antitumor preparation vinkristin as well as homeopathic means - stimulated phosphoric acid diluted at 10-14 were used. Small doses of radiation (0,05 and 0,1 Gr) always had a stimulating effect on ion transport even in combination with vinkristin, which separately always depressed it. Both separately and in any combination stimulated phosphoric acid always reinforced transmembrane ion transport. In regard to Ca2+ a hypothesis about its participation in the process of reparation of tumor cell has been suggested. At increasing of Ca2+ concentrations a transmembrane transport of this ion in the environment increases what induces strengthening of adhesive properties of the cell. However, it is known that in tumors these properties are decreased. Apparently, in this case two contrary processes - strengthening and decrease of adhesive properties take place pointing to the fact that there appear reparative forces in tumor process.

[Influences of some homeopathic preparations on ionic homeostasis at different dilutions].

Considerable number of the investigations are dedicated to the study of the influences of the various agents on ionic homeostasis of the cell. However, there are actually no works related to the impact on these indices of homeopathic preparations (HP). In the present work influence of HP - stimulated phosphoric acid (PA), at low dilutions (10(-14) and 10(-42)) and non-stimulated PA, at high dilutions (10(-200) and 10(-400)), on transmembrane transport of Na+, K+, Ca2+, and enzymes - Na,K-ATPase and Ca-ATPase - was investigated. Experiments were carried out by means of ion-selective electrodes, in order to measure concentration of ions in the Ringer solution. Results have shown that HP at low dilution always stimulated observed indices, while HP at high dilution - suppressed these indices. Therefore, driving force of HP increases with dilution, because number of hydrate complexes increases and so does velocity of information transmission, which is responsible for different effects.

Glycerol Monolaurate (GML) inhibits human T cell signaling and function by disrupting lipid dynamics.

Glycerol Monolaurate (GML) is a naturally occurring fatty acid widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a range of bacteria, fungi, and enveloped viruses but select findings suggest that GML also has immunomodulatory functions. In this study, we have mechanistically examined if GML affects the signaling and functional output of human primary T cells. We found that GML potently altered order and disorder dynamics in the plasma membrane that resulted in reduced formation of LAT, PLC-γ, and AKT microclusters. Altered membrane events induced selective inhibition of TCR-induced phosphorylation of regulatory P85 subunit of PI3K and AKT as well as abrogated calcium influx. Ultimately, GML treatment potently reduced TCR-induced production of IL-2, IFN-γ, TNF-α, and IL-10. Our data reveal that the widely used anti-microbial agent GML also alters the lipid dynamics of human T cells, leading to their defective signaling and function.

Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes.

1. The increase in intracellular CA2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization was investigated in mouse C2C12 myotubes by use of fura-2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca(2+)-entry process. 2. Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC50 = 0.85 +/- 0.09 microM), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 microM) was completely blocked by the nAChR antagonist, pancuronium (3 microM), but was not affected by the muscarinic antagonist, atropine (3 microM), or under conditions when Ca2+ entry was blocked by La3+ (50 microM) or diltiazem (10 microM). Addition of pancuronium (3 microM) during the sustained phase of the carbachol-evoked response did not affect this phase. 3. Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72 +/- 0.08 microM) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP-induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4. Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 microM), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+. 5. Under Ca(2+)-free conditions, the sustained phase of the carbachol- and ATP-evoked responses were abolished. Pre-emptying of depolarization-sensitive stores by high K+ under Ca(2+)-free conditions did not affect the carbachol- or ATP-evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol-induced Ca2+ response to 11%, while in the reverse procedure the ATP-induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6. Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP-evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7. The carbachol-induced Ca2+ response was completely abolished under low Na+/Ca(2+)-free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP- and K(+)-induced responses were changed compared to Ca(2+)-free conditions. 8. ATP (300 microM) induced the formation of Ins(1,4,5)P3 under Ca(2+)-free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 microM) did not affect Ins(1,4,5)P3 levels under Ca(2+)-free conditions. 9. It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P2U-purinoceptors and by high K+. Only the P2U-purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca(2+)-entry is activated by stimulation of nAChRs and P2U-purinoceptors via separate ion-channels, which are different from the skeletal muscle nAChR-coupled cation-channel.

Neuroprotection from glutamate toxicity with ultra-low dose glutamate.

The protective effects of ultra-low doses (ULD) of glutamate against glutamate toxicity was studied in primary rat spinal, cortical and cerebellar neurons. Neurons were exposed to four subtoxic, ultra-low concentrations of glutamate (10(-18) M, 10(-20)M, 10(-22) M and 10(-30) M) for 72 h and then subsequently challenged with toxic concentrations (25 microM) of glutamate. Neuron viability was consistently 10% higher in spinal and cortical neurons pre-exposed to glutamate concentrations of 10(-18) M and 10(-22) M, and in cerebellar neurons pre-exposed to 10(-20) M and 10(-30) M. Using laser scanning confocal microscopy and the fluorescent calcium probe fluo-3, we found no alterations in intracellular calcium dynamics in the protected cells. This protective effect is consistent with a growing body of evidence for tolerance induced by low-dose toxin exposure but is the first time that such tolerance has been demonstrated with ultra-low glutamate exposure. Our data show that pre-exposure of neuronal cells to ULD glutamate can protect against subsequent exposure to toxic levels of glutamate.

Inhibition by vecuronium of carbachol-induced influx of 22Na+, 45Ca2+ and secretion of catecholamines in cultured bovine adrenal medullary cells.

In cultured bovine adrenal medullary cells, vecuronium, pancuronium and D-tubocurarine reduced carbachol-induced 45Ca2+ influx and catecholamine secretion by inhibiting 22Na+ influx via nicotinic receptor-ion channel complex with IC50 values of 0.43, 7.6 and 3.9 mumol/l, respectively. IC50 values of pancuronium and D-tubocurarine observed in adrenal medulla were one order of magnitude higher than the plasma concentrations of these muscle relaxants reported to produce 50% neuromuscular blockade, while IC50 of vecuronium was quite close between adrenal medulla and skeletal muscle.

[Mechanisms of opioid receptor-induced elevation in intracellular calcium by confocal laser scanning microscopy].

OBJECTIVE: To determine the acute and chronic effects of opioid receptor agonists on the intracellular free calcium concentration ([Ca2+]i) in NG-LNCXiNOS cells, stably expressing iNOS gene, and regulation of G-protein on opioid-induced response in [Ca2+]i. METHODS: A single cell [Ca2+]i is measured by confocal laser scanning microscopy using Ca(2+)-sensitive dye Fluo-3 as an new calcium fluorescent probe. RESULTS: DPDPE(D-Pen2, D-Pen5-enkephalin), a delta-opioid receptor agonist, and morphine acutely induced the increase in [Ca2+]i of NG-LNCXiNOS cells. The elevation in [Ca2+]i by DPDPE could be abolished with naloxone. Pretreatment of the cells with pertussis toxin (PTX) at 100 ng/ml for 24 hours almost completely blocked morphine-evoked response. In contrast to acute effect of opioid agonists on [Ca2+]i, the cells exposed to 1 mumol/L DPDPE or 10 mumol/L morphine for 48 hours also appeared to raise [Ca2+]i. However, the elevation in [Ca2+]i was not greater than that caused by acute effect of DPDPE or morphine. After cell "withdrawal" was precipitated by the addition of 10 mumol/L naloxone, the increase in [Ca2+]i could further be intensified. CONCLUSIONS: The opioid agonist-induced increase in [Ca2+]i is mediated by opioid receptor and regulated though PTX-sensitive G-protein. The attenuation of this response in chronically treated cells with opioid agonist is associated with receptor desensitization.

[Effect of sepia on intracellular Ca2+ concentration, nuclear Ca2+/Mg(2+)-ATPase activities and c-jun expression in H22 cancer cells].

The effect of sepia on the intracellular Ca2+ concentration, nuclear Ca2+/Mg(2+)-ATPase activities and the expression of c-jun were studied in H22 cancer cells by using fluorescent probe and immunohistochemical method. The results showed that intracellular Ca2+ concentration decreased 69% and 79%, nuclear Ca2+/Mg(2+)-ATPase activities diminished 21% and 37%, c-jun expression decreased obviously. The results suggested that sepia probably diminished the intracellular Ca2+ concentration, affected Ca(2+)-dependent nuclear Ca2+/Mg(2+)-ATPase activities, thus reduced the amount of Ca2+ transporting into nuclei, so lessened the promoting effect of Ca2+ on c-jun expression, and therefore inhibited cellular differentiation and proliferation. This might be one of the possible anti-cancer mechanisms of sepia.

[Effects of musk glucoprotein on PAF production and cytosolic Ca2+ level in rat polymorphonuclear leukocytes in vitro].

OBJECTIVE: To investigate the effects of Musk glucoprotein on platelet activating factor (PAF) production and the concentration of cytosolic free Ca2+ in polymorphonuclear leukocytes of rat. METHODS: An in vitro incubation system was used, production of PAF and activity of acetyl transferase were measured by isotope incorporation, the concentration of cytosolic free Ca2+ was quantitated using the fluorescent Ca2+ indicator Fura-2. RESULTS: Musk-1 at concentration of 1-100 micrograms.ml-1 can significantly inhibit production of PAF, activity of acetyl transferase and the increase of cytosolic Ca2+ concentration in polymorphonuclear leukocytes of rat. CONCLUSION: Part of mechanisms underlying antiinflammatory action of Musk-1 is through inhibiting the synthesis of PAF and the increase of cytosolic Ca2+ level.

[Pharmacological studies on Chinese bone extract granules].

The Chinese Bone Extract Granules could enhance the immunity of mice with spleen-deficiency, increase the serum calcium content of rats with bone loose, inhibit the gastrointestinal movement of mice with spleen-deficiency and improve the absorption of D-xylose in small intestine of rats with spleen-deficiency. The maximal tolerance dose for mice to take orally twice a day was greater than 50 g/kg. The subacute toxicologic test for 6 weeks of continuous feeding to rats did not show any obvious toxicity.

[Effects of musk glucoprotein on chemotaxis of polymorphonuclear leukocytes in vivo and in vitro].

OBJECTIVE: To investigate the effects of Musk glucoprotein on chemotaxis of Polymorphonuclear leukocytes(PMN). METHOD: The chemotaxis of PMN in abdominal cavity in rat induced by carboxymethyl cellulose(CMC) was used as an in vivo animal model and in in vitro it was evaluated by Boyden chamber. The concentration of cytosolic free Ca2+ was quantitated with the fluorescent Ca2+ indicator Fura-2. RESULT: The water extract of Musk at dose of 5, 20, 80 mg.kg-1 (s.c.) significantly inhibited the chemotaxis of PMN in rat; Musk-1 at concentration of 1-100 micrograms.mL-1 can significantly inhibit the chemotaxis of rabbit PMN in vitro; Musk-1 at concentration of 1-100 micrograms.mL-1 can significantly inhibit the increase of cytosolic Ca2+ concentration in PMN of rat. CONCLUSION: Part of mechanisms underlying antiinflammatory action of Musk is to inhibit the chemotaxis of PMN.

Cardiac electrophysiologic effects of pancuronium.

A microelectrode examination of guinea pig left ventricular papillary muscle was performed to determine whether there was a direct effect of pancuronium on cardiac cells and, if so, to attempt to ascertain the mechanism of this effect. Electrical events were measured before and during superfusion with pancuronium, epinephrine, propranolol, and verapamil; alone and in various combinations. Pancuronium prolonged the duration of the action potential (AP); increased resting potential (Em), AP magnitude, and rate of rise of the AP (dV/dt); and resulted in spontaneity in 12% of the muscles. Epinephrine and pancuronium combined caused spontaneity in 80% of the muscles and oscillatory behavior. Additionally, this combination decreased AP magnitude, Em, and dV/dt in several preparations--a pattern of response similar to that seen in ouabain-treated myocardial cells under the influence of catecholamines. These changes were always reversed by verapamil or by perfusion with a drug-free medium, and were usually reversed by propranolol. The data suggest a combined pancuronium/epinephrine induced increase in cardiac membrane permeability to Ca2+.

Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells.

BACKGROUND: Cardiac glycosides may induce oncolytic effects in cancers. This study was to evaluate bufalin and cinobufagin effects on the proliferation of prostate cancer cell lines named LNCaP, DU145, and PC3. METHODS: Cell proliferation was measured by MTT assay. The cytotoxic effects were determined by lactate dehydrogenase measurements. The intracellular calcium concentration ([Ca(2+)](i)) was measured by a dual-wavelength spectrometer system. TUNEL assay and flow cytometry were performed to measure percentage of apoptotic cells. A colorimetric assay was to measure caspases activities. RESULTS: Bufalin and cinobufagin inhibited proliferation of cancer cells at doses of 0.1, 1, or 10 microM after 2-4 days of culture. Cytotoxicity of bufalin and cinobufagin on the DU145 and LNCaP cells was dose-dependent. Bufalin or cinobufagin increased [Ca(2+)](i) and apoptosis in cancer cells after a 24-hr culture as well as caspase 3 activities in DU145 and PC3 cells and caspase 9 activities in LNCaP cells. CONCLUSIONS: Bufalin and cinobufagin may inhibit the proliferation of prostate cancer cell lines associated with sustained elevation of the [Ca(2+)](i) and that of apoptosis.

Protective effects of polypeptide from Chlamys farreri on Hela cells damaged by ultraviolet A.

AIM: To study the protective effect of polypeptide isolated from Chlamys farreri (PCF) on Hela cells damaged by ultraviolet A (UVA) in vitro. METHODS: Cell proliferation was determined by MTT method; intra-cellular free calcium [Ca2+]i and rates of apoptosis and death were measured by flow cytometry (FCM). RESULTS: PCF (0.5 %-2 %) enhanced the activities of glutathione peroxidase (GSH-px), superoxide dismutase (SOD), and catalase (CAT), and stimulated cell proliferation. The concentration of [Ca2+]i was increased while the amounts of MDA and the rates of apoptosis and death of the cells were decreased. The differences between the PCF groups and control group were significant (P<0.05, P<0.01). CONCLUSION: PCF protected Hela cells against damage by UVA via its anti-oxidative mechanisms.

Effects of 8 years of treatment with tibolone 2.5 mg daily on postmenopausal bone loss.

The objective of this study was to assess the long-term effects of tibolone 2.5 mg daily (Livial; Organon) on bone mineral density in recently postmenopausal women. An 8-year, open, nonrandomized, prospective study was designed to compare the effects of tibolone 2.5 mg daily (n = 59) with an untreated control group (n = 51). The subjects of this study were 110 recently postmenopausal women (6-36 months since last menstrual period). The main outcome measures were bone mineral density of the spine and femur, measured by dual-energy X-ray absorptiometry, and assessment of biochemical markers of bone metabolism. After 8 years of tibolone use, the mean (+/- SEM) increase in bone mineral density compared with baseline was 4.1% +/- 0.8% (p<0.0001) in the spine and 4.6% +/- 1.8% (p = 0.015) in the femoral neck. Over the same period, bone mineral density in the control group decreased in the spine by -7.5% +/- 1.1%, (p<0.0001) and in the femur by -6.7% +/- 1.2% (p<0.0001). The bone resorption marker, calcium/creatinine ratio, decreased in the tibolone group but not in the control group. Serum bone formation markers decreased (alkaline phosphatase) or stayed approximately the same (osteocalcin) in the tibolone group. Adherence was high, with 58% (34 of 59) of the tibolone group continuing treatment for 8 years. We conclude that tibolone 2.5 mg daily prevents bone loss in the lumbar spine and femoral neck over 8 years and adherence to treatment is high. The greater bone density compared with untreated women would be expected to reduce the risk of bone fractures.

Enhancing effect by nicotinic acetylcholine receptor channel blockers, including beta-eudesmol, on succinylcholine-induced inhibition of twitch tension and intracellular Ca++ in mouse diaphragm muscle.

To elucidate the mechanism of neuromuscular block by succinylcholine, nerve-evoked changes in intracellular Ca(++)-aequorin luminescence and twitch tension were measured simultaneously in the presence of several different types of blockers for the nicotinic acetylcholine receptor channel. Mouse diaphragm muscles were pretreated for 30 to 60 min with 3 to 40 microM bupivacaine, chlorpromazine, phencyclidine and beta-eudesmol. The effects of these noncompetitive blockers on the succinylcholine-induced response were also compared with those for pancuronium. These channel blockers potentiated (2- to 10-fold) both the blocking effects on intracellular Ca++ and twitch tension of succinylcholine (13-100 microM), but not the pancuronium (0.3-1.1 microM)-induced block. These channel blockers also suppressed succinylcholine (1.3-5 microM)-induced enhancement of evoked Ca++ transients. On the other hand, the channel blockers inhibited the succinylcholine (2.5-100 microM)-induced increase in basal Ca++ transients. These results suggest that neuromuscular block induced by succinylcholine is mainly due to desensitization of the nicotinic acetylcholine receptor.

Activation of brain acetylcholine receptors by neuromuscular blocking drugs. A possible mechanism of neurotoxicity.

BACKGROUND: Neuromuscular blocking drugs cause excitement and seizures when introduced into the central nervous system. We examined the possibility that these drugs produce paradoxical activation of acetylcholine or glutamate receptors, the chief types of brain receptors involved in excitatory neurotransmission. METHODS: Because activation of central glutamate or acetylcholine receptors causes calcium influx into postsynaptic neurons, we measured intracellular calcium concentration ([Ca2+]i) as an index of receptor activation. Changes in [Ca2+]i were compared in brain slices exposed to neuromuscular blocking drugs or acetylcholine and glutamate receptor agonists. [Ca2+]i was measured with the fluorescent dye fura-2. RESULTS: Pancuronium and vecuronium caused sustained increases in [Ca2+]i in approximately the same potency ratio as for seizure activity in vivo (concentrations at which the increase in [Ca2+]i was 95% of maximal: 100 and 400 microM, respectively). Atracurium and laudanosine did not increase [Ca2+]i in cortical slices. Increases in [Ca2+]i caused by both pancuronium and vecuronium were prevented by the non-subtype-specific nicotinic acetylcholine receptor antagonist D-tubocurarine and were reduced 44-73% by atropine. Blockade of glutamate receptors or voltage-gated calcium or sodium channels had no effect on calcium influx. CONCLUSIONS: The results suggest that the acute excitement and seizures caused by introduction of pancuronium and vecuronium into the central nervous system is due to accumulation of cytosolic calcium caused by sustained activation of acetylcholine receptor ion channels.