Role of tumor necrosis factor-α in the regulation of T-type calcium channel current in HL-1 cells

AIM: Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation( AF). Although tumor necrosis factor( TNF)-α levels are increased in patients with AF,the role of TNF-α in the pathogenesis of AF remains unclear. Recent research has revealed that T-type Ca~(2+)currents( ICa,T) play an important role in the pathogenesis of AF. METHODS: In this study,we used the whole-cell voltage-clamp technique and biochemical assays to explore the role of TNF-α in the regulation of ICa,Tin atrial myocytes. RESULTS: We found that compared with sinus rhythm( SR) controls,T-type calcium channel( TCC)subunit m RNA levels were decreased,while TNF-α expression levels were increased,in human atrial tissue from patients with AF. In murine atrial myocyte HL-1 cells,after cultured for 24 h,12. 5,25 and 50 μg / L TNF-α significantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner. The peak current was reduced by the application of 12. 5 or 25μg / L TNF-α in a concentration-dependent manner [from(- 15. 08 ± 1. 11) p A / p F in controls to(- 11. 89 ± 0. 83) p A / p F and(- 8. 54 ± 1. 55) p A/p F in 12. 5 and 25 μg/L TNF-α groups,respectively]. TNF-α application also inhibited voltage-dependent inactivation of I~(Ca,T)shifted the inactivation curve to the left. CONCLUSION: These results suggest that TNF-α is involved in the pathogenesis of AF,probably via decreasing ICa,Tfunction in atrium-derived myocytes through impaired channel function and down-regulation of channel protein expression. This pathway thus represents a potential pathogenic mechanism in AF.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.     

Lack of involvement of G proteins in the activation of cardiac CFTR Cl– current by genistein

 The involvement of guanine nucleotide-binding proteins (G proteins) in the activation of cardiac adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– current (I _Cl) by the tyrosine kinase inhibitor genistein (GST) was investigated in guinea-pig ventricular myocytes. Pertussis toxin (PTX) and intracellular application of 1 mM non-hydrolysable guanosine-5′-0-(2-thiodiphosphate) (GDPβS) and guanosine-5′-0-(3-thiotriphosphate) (GTPγS) were used to modify G protein activity, and the efficacy of the treatments determined by examining the activation of I _Cl by isoproterenol (ISO) and forskolin (FSK), and its inhibition by 1 μM acetylcholine (ACh). GDPβS inhibited ISO-activated I _Cl by 80–90%, but had little effect on I _Cl activated by different GST regimens (50 μM; 100 μM; 50 μM plus 0.1 μM FSK). GTPγS had little effect on the amplitude of I _Cl activated by 1 μM ISO, whereas it increased the amplitude of the current activated by 50 and 100 μM GST and rendered it insensitive to 1 μM ACh (inhibition of 2±2% versus (PTX-sensitive) inhibition of 94±3% in control myocytes). Unlike I _Cl activated by ISO in GTPγS-dialysed myocytes, I _Cl activated by GST deactivated on removal of the drug. GST (50 μM) reversibly increased I _Cl by nearly 50% in myocytes with G_s selectively activated by 1 μM ISO, and also reversibly increased the I _Cl that was persistently activated after withdrawal of ISO from GTPγS-dialysed myocytes. These results indicate that G proteins are not involved in the pathway between GST binding and CFTR opening, and suggest that enhanced adenylate cyclase activity in GTPγS-dialysed myocytes mediates the potentiated responses to GST. Received: 11 September 1998 / Received after revision: 6 January 1999 / Accepted: 12 January 1999     

Silibinin (Legalon-70®) enhances the motility of human neutrophils immobilized by formyl-tripeptide,calcium ionophore,lymphokine and by normal human serum

Experiments reported here were designed to investigate the effect of silibinin (extracted from Silybum marianum) on human polymorphonuclear leukocyte (PMN) motility and on leukocyte immobilizing activity of lymphokine (leukocyte inhibitory factor, LIF), formyl-Met-Leu-Phe (fMLP), calcium ionophore A-23187 and human sera inactivated by heat (HI-S).In thein vitro experiments, silibinin (1–10 g/ml) failed to influence the random motility of unstimulated PMNS in agarose droplet assay, but enhanced the motility of the PMNs immobilized by fMLP, calcium inophore, LIF or by autologous human sera.In thein vivo study, silibinin (Legalon-70^®) two hours after the administration was effective in enhancing spontaneous motility of leukocytes obtained from healthy volunteers which action could be regarded as a consequence of the decrease of leukocyte immobilizing activity being present in normal human plasma.     

Is inhibition of cyclooxygenase required for the chemopreventive effect of NSAIDs in colon cancer? A model reconciling the current contradiction

NSAIDs are powerful chemopreventive agents for colon cancer, but their mechanism of action remains unknown. Their best recognized pharmacological property is inhibition of the enzyme cyclooxygenase (COX), which catalyzes the synthesis of prostaglandins; however, additional effects are well documented. Current studies on the mechanism of the chemopreventive effect of NSAIDs lead to two contradictory conclusions: NSAIDs prevent colon cancer either by inhibiting the activity of COX, or through mechanisms that do not require COX inhibition. To resolve this apparent conflict, after examining several alternatives, we propose a model, which assumes that both mechanisms are correct but that they exert their effect either on different steps of the multistep process of colon carcinogenesis or on different control mechanisms. This postulated dual action of NSAIDs may explain their remarkable effectiveness in colon cancer prevention. Unraveling these mechanistic details can be very rewarding for the design of more refined approaches to cancer chemoprevention and for a deeper understanding of colorectal carcinogenesis.     

G-protein-independent modulation of P-type calcium channels by μ-opioids in Purkinje neurons of rat

P-type calcium channels play a key role in the synaptic transmission between mammalian central neurons since a major part of calcium entering pre-synaptic terminals is delivered via these channels. Using conventional whole-cell patch clamp techniques we have studied the effect of μ-opioids on P-type calcium channels in acutely isolated Purkinje neurons from rat cerebellum. The selective μ-opioid agonist DAMGO (10 nM) produced a small, but consistent facilitation of current through P-type calcium channels (10 ± 1%, n = 27, p < 0.001). The effect of DAMGO was rapid (less than 10 s) and fully reversible. This effect was both concentration and voltage-dependent. The EC₅₀ for the effect of DAMGO was 1.3 ± 0.4 nM and the saturating concentration was 100 nM. The endogenous selective agonist of μ-opioid receptors, endomorphin-1 demonstrated similar action. Intracellular perfusion of Purkinje neurons with GTPγS (0.5 mM) or GDPβS (0.5 mM), as well as strong depolarizing pre-pulses (+50 mV), did not eliminate facilitatory action of DAMGO on P-channels indicating that this effect is not mediated by G-proteins. Furthermore, the effect of DAMGO was preserved in the presence of a non-specific inhibitor of PKA and PKC (H7, 10 μM) inside the cell. DAMGO-induced facilitation of P-current was almost completely abolished by the selective μ-opioid antagonist CTOP (100 nM). These observations indicate that μ-type opioid receptors modulate P-type calcium channels in Purkinje neurons via G-protein-independent mechanism.     

Parathyroid hormone induces epithelial-to-mesenchymal transition via the Wnt/β-catenin signaling pathway in human renal proximal tubular cells

Epithelial-to-mesenchymal transition (EMT) has been shown to play an important role in renal fibrogenesis. Recent studies suggested parathyroid hormone (PTH) could accelerate EMT and subsequent organ fibrosis. However, the precise molecular mechanisms underlying PTH-induced EMT remain unknown. The present study was to investigate whether Wnt/β-catenin signaling pathway is involved in PTH-induced EMT in human renal proximal tubular cells (HK-2 cells) and to determine the profile of gene expression associated with PTH-induced EMT. PTH could induce morphological changes and gene expression characteristic of EMT in cultured HK-2 cells. Suppressing β-catenin expression or DKK1 limited gene expression characteristic of PTH-induced EMT. Based on the PCR array analysis, PTH treatment resulted in the up-regulation of 18 genes and down-regulation of 9 genes compared with the control. The results were further supported by a western blot analysis, which showed the increased Wnt4 protein expression. Wnt4 overexpression also promotes PTH-induced EMT in HK-2 cells. The findings demonstrated that PTH-induced EMT in HK-2 cells is mediated by Wnt/β-catenin signal pathway, and Wnt4 might be a key gene during PTH-induced EMT.     

Reassessing the role of progesterone in fertilization--compartmentalized calcium signalling in human spermatozoa?

Progesterone is present at micromolar concentrations in the vicinity of the oocyte. Human spermatozoa generate a biphasic rise in intracellular calcium concentration ([Ca(2+)](i)) and undergo the acrosome reaction upon progesterone stimulation, suggesting that the hormone acts as a secondary inducer or 'primer' of the acrosome reaction in association with the zona pellucida. However, the sensitivity of human spermatozoa to progesterone is such that many cells may undergo the acrosome reaction prematurely, compromising their ability to fertilize. We have shown that exposing human spermatozoa to a progesterone gradient, simulating the stimulus encountered as sperm approach the oocyte, results in a novel response. A slow rise in [Ca(2+)](i) occurs, upon which, in many cells, [Ca(2+)](i) oscillations are superimposed. Cells showing this pattern of response do not undergo the acrosome reaction, but instead show an alternating pattern of flagellar activity associated with peaks and troughs of [Ca(2+)](i). A Ca(2+) store in the rear of the sperm head apparently generates this complex signal, functioning as an '[Ca(2+)](i) oscillator'. We propose that: (i) the acrosome reaction and flagellar beat are regulated by separate Ca(2+) stores; (ii) these stores are mobilized through different mechanisms by different agonists; and (iii) progesterone in vivo acts as a switch for the oscillator which regulates the flagellar beat mode.     

Myocardial ultrastructure in cardiac hypertrophy induced by thyroid hormone—an acute study in rats

The early responses of the myocardium ultrastructure under thyroid dysfunction conditions, hemodynamic parameters, cardiac hypertrophy and ultrastructural evaluations were performed in hypothyroid and hyperthyroid rats submitted to different doses [T4–25 and T4–100; 0.025 mg and 0.1 mg kg^–1 body weight (BW) ·per day, respectively)]. All groups were treated for 7 days. The animals were sacrificed, the hearts were excised and weighed and the left ventricle tissue samples were processed for transmission election microscopy. Systolic blood pressure (SBP) was not altered by administration of T4. An increased heart rate and ratio of heart weight to body weight (HW/BW) were found in the hyperthyroid rats. However, the SBP and HW/BW decreased significantly in hypothyroid rats. No significant ultrastructural alterations were detected when the hypothyroid and T4–25 groups were compared with the control group. Alterations of cardiomyocytes nuclei of these groups were also not detected. Notably, disorganization of intercellular junctions was observed in many cardiomyocytes of T4–100 group. The present results indicate that in the early stages of hyperthyroidism, the cardiac hypertrophy development was mainly due to direct effects of thyroid hormone. Despite cardiac hypertrophy development, there is no ultrastructural evidence of myocardial degeneration.     

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation.     

Involvement of intracellular calcium ions in the release of the fluorescent dye calcein by cholinergic and α-adrenergic agonists from rat parotid acinar cells

Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10^–8 M to 10^–6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca²⁺]_i). Addition of NA at 10^–6 M increased [Ca²⁺]_i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10^–6 M CCh, but it abolished the sustained increase in [Ca²⁺]_i. The transient increase in [Ca²⁺]_i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10^–6 M), but not 10^–3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca²⁺]_i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca²⁺]_i by muscarinic and -adrenergic agonists and may represent the initial process of salivary secretion.     

Evaluation of estrogen receptor-β expression in the epithelium of the oral mucosa in menopausal women under hormone replacement therapy

PurposeWe aimed to evaluate the ERβ expression in the epithelium of the oral mucosa in menopausal women treated with oral, transdermal or local (vaginal) menopausal hormone therapy.Material/methodsIn this study, we included 60 women treated with oral, transdermal or vaginal menopausal hormone therapy. The study material was obtained from swabs taken from the buccal mucosa before administering HRT, and after 6 weeks, 3 months, 6 months and 12 months of therapy. We assessed estrogen receptor-β (ERβ) expression levels in subsequent swabs by immunohistochemical analysis.ResultsThe highest increase in the ERβ expression was observed after 3 months of oral and transdermal hormone therapy.ConclusionsOral and transdermal HRT may be an effective method of treatment of oral discomfort in menopausal women.     

Activation of calcium/calmodulin-dependent protein kinase IIα in the striatum by the heteromeric D1-D2 dopamine receptor complex

Synaptic plasticity in the striatum is a key mechanism that underlies processes such as reward related incentive learning and behavioral habit formation resulting from drugs of abuse. Key aspects of these functions are dependent on dopamine transmission as well as activation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). In this study, we examined the ability of a recently identified heteromeric complex composed of D1 and D2 dopamine receptors coupled to Gq/11 to activate striatal CaMKIIα. Using the dopaminergic agonist SKF83959, which selectively activates the D1-D2 complex, we demonstrated phosphorylation of CaMKIIα at threonine 286, both in heterologous cells and in the murine striatum in vivo. Phosphorylation of CaMKIIα by activation of the receptor complex required concurrent agonism of both D1 and D2 receptors and was independent of receptor pathways that modulated adenylyl cyclase. The identification of this novel mechanism by which dopamine may modulate synaptic plasticity has implications for our understanding of striatal-mediated reward and motor function, as well as neuronal disorders in which striatal dopaminergic neurotransmission is involved.     

Effects of β-amyloid (25–35) on learning in the common snail

The effects of neurotoxic β-amyloid fragment (25–35) on the formation of behavioral sensitization and a conditioned defensive reflex to food were studied. Administration of β-amyloid (25–35) to common snails before the start of training led to a significant reduction in sensitization of the defensive reaction, weakening of the formation of the conditioned defensive reflex to food, and impairment of memory. These impairments to behavioral plasticity may be mediated by changes in synaptic plasticity previously observed in the presence of β-amyloid. __________ Translated from Zhurnal Vysshei Nervnoi Deyatel’nosti imeni I. P. Pavlova, Vol. 57, No. 2, pp. 229–236, March–April, 2007.     

3D CFD Simulation of Pulsatile Blood Flow in the Human Aorta

INTRODUCTION　　The human aorta is the majorblood vessel of complex geometry including curva-tures in multiple planes,branches at the apex of the arch,significant tapering andwith distensible vessel wall ( as shown in Fig.1 ) . The blood flow structures in theaorta are very complex and attribute a lot to the development of atherosclerotic le-sions,which always occur in the vicinity of arterial branches,curvatures and bifur-cations〔1～ 5〕.In order to understand the complex nature of the …     

Bortezomib enhances the radiosensitivity of hypoxic cervical cancer cells by inhibiting HIF-1α expression

Objective: This study aimed to investigate the radiosensitivity of bortezomib to cervical cancer and the possible underlying mechanism. Methods: HeLa and SiHa cell lines with or without hypoxia treatment were divided into control, radiation alone, bortezomib alone, and radiotherapy plus bortezomib groups. CCK8 assay, clone formation assay, flow cytometry, and immunofluorescence test were used to measure cell proliferation, colony formation, apoptosis, and DNA double-strand break (DSB). Western blot analysis was performed to detect the expression of HIF-1α, PARP-1, and caspase-3, -8, and -9. Result: Statistical analysis of data revealed that bortezomib at nanomolar level exerted a radiosensitization effect on both cervical cancer cell lines in normoxia or hypoxia. Western blot analysis showed that the drug could inhibit hypoxia-related HIF-1α expression to increase apoptosis-related caspase-3, -8, and -9 activation and DNA DSB-related PARP-1 cleavage. Conclusions: Radiotherapy sensitization of bortezomib on cervical cancer cell lines had a drug-dose relation, and sensitization in hypoxia was more remarkable than in normoxia. Bortezomib may be a potential radiotherapy sensitization drug for cervical cancer.