Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

BACKGROUND AND PURPOSE

The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes.

EXPERIMENTAL APPROACH

Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes.

KEY RESULTS

In the presence of 1–10 μM AEA, suppression of both Na⁺ and L-type Ca²⁺ channels was observed. Inhibition of Na⁺ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [³H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in B_max values but no change in K_d. Further studies on L-type Ca²⁺ channels indicated that AEA potently inhibited these channels (IC₅₀ 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba²⁺ currents. MetAEA also inhibited Na⁺ and L-type Ca²⁺ currents. Radioligand studies indicated that specific binding of [³H]isradipine, was inhibited significantly by metAEA. (10 μM), changing B_max but not K_d.

CONCLUSION AND IMPLICATIONS

Results indicate that AEA inhibited the function of voltage-dependent Na⁺ and L-type Ca²⁺ channels in rat ventricular myocytes, independent of CB₁ and CB₂ receptor activation.     

Long-term increase of hippocampal excitability by histamine and cyclic AMP

The action of histamine (HA) on rat hippocampal CA1 pyramidal cells in vitro was investigated in slices perfused with solution containing 0.2 mM Ca²⁺/4.0 mM Mg²⁺. Extracellular recordings of the spontaneous discharges occurring under these conditions revealed that HA caused a long-lasting increase in cell firing. The HA-effects were dose-dependent, in that low concentrations of HA (0.1–0.5 μM) exhibited an initial transient depression of cell firing and practically no long-lasting action, whereas higher concentrations of HA (1–10 μM) exerted strong, non-declining increases. The H₁-receptor antagonist mepyramine (1 μM) blocked the initial depression of firing and attenuated the long-lasting HA-mediated excitation. Pure H₁-receptor activation, tested with the H₁-receptor agonist 2-(3-fluorphenyl)histamine (1–10 μM) depressed cell firing, similar to the low dose effects of HA. HA-induced excitations were prevented by the H₂-receptor antagonist cimetidine (10–50 μM), and mimicked by the very potent H₂-receptor agonist impromidine (1 or 3 μM) which was, however, less effective compared to equal concentrations of HA. H₃-receptor activation by R-α-methylhistamine had no significant effect on cell firing. Thus, histamine H₁ and H₂ receptors seem to cooperate in producing this long-lasting augmentation of excitability. 8-Bromo-cyclic AMP monophosphate (8-Br-cAMP, 50–100 μM) mimicked the long-term excitation, whereas the adenylyl-cyclase inhibitor 9-tetrahydro-2-furyladenine (THFA, 100–500 μM) or the PKA-inhibitor Rp-adenosine-3′5′-cyclic monophosphate (Rp-cAMPS, 10 μM) blocked it, indicating that the HA-mediated increase of excitability in the hippocampus is dependent on the adenylate cyclase/PKA-signal transduction cascade. -2-Amino-5-phosphonopentanoic acid (APV, 50 μM) significantly attenuated the magnitude of the HA-induced enhancement, indicating an NMDA receptor-dependent component. Other biogenic amines, acting through receptors positively coupled to adenylyl cyclase, elicited similar responses as HA, indicating common mechanisms by which these substances modulate excitability in CA1 pyramidal cells.     

Development of an in vitro drug screening system for Mycobacterium leprae based on the determination of the intrabacterial sodium to potassium ratio of individual bacterial organisms

In vitro drug effects on Mycobacterium leprae (M. leprae) in a cell-free system have been monitored by mass spectrometric determination of the ratio of the intrabacterial concentrations of the sodium and potassium ions (Na⁺, K⁺ ratio) of a limited number of individual bacteria per sample. From the drug-induced increase of the median values of the distributions of the Na⁺, K⁺ ratio, information on the concentration and time dependence of drug effects as well as on antagonistic or synergistic interactions of drugs has been obtained. Moreover, absolute values for the percentage of killed bacteria (% kill) have been derived from the distribution of the Na⁺, K⁺ ratios within a bacterial population. For this, the limiting value of the Na⁺, K⁺ ratio (up to which bacteria are viable) —which had been determined as 0.45 for cultivable bacteria — has been presumed to be valid also for M. leprae. Highest killing rates have been observed for fusidic acid and clarithromycin, followed by rifabutine, rifampin, and clofazimine. Minocycline and dapsone have shown only moderate killing effects and isoniazid and — probably due to the restricted metabolism of M. leprae in a cell-free medium — ofloxacin have been completely inactive. Strong ofloxacin effects, however, have been observed for cultivable mycobacteria and intracellular M. leprae phagocytized by a murine macrophage cell line.     

Characteristics and possible mechanisms of low - Na+ induced contractions in rat aorta

The influence of reducing external Na⁺ concentration ([Na⁺]^ex) upon vascular smooth muscle contractility was investigated using the rat isolated aorta. NaCl from the physiological saline solution (PSS) was replaced with either choline-Cl, sucrose, or LiCl to give the following [Na⁺]_ex (mM): 115, 85, 55, and 25 (115NaPSS to 25NaPSS). Small reductions in [Na⁺]_ex (115NaPSS) induced a biphasic contraction, comparable in amplitude with the control one induced by phenylephrine 10^–6 M. Elimination of the endogenous catecholamine participation using either phentolamine 10^–5 M or guanethidine 3.10^–6 M similarly reduces these contractions to 25% (sucrose replacement). A similar relaxing effect was obtained with D600 10^–5 M, an antagonist of the voltage operated Ca²⁺ channels (25–30% residual tension for all the substitutes). Large reductions in [Na⁺]_ex (25NaPSS) induced contractions comparable in amplitude and shape, but less sensitive to phentolamine and guanethidine (residual tension 65–75 %, sucrose replacement) and insensitive to D600 (all the substitutes). The Na⁺/K⁺ ATPase inhibitor ouabain (10^–4 M) elicited slowly developing contractions, the amplitude being 115% of the phenylephrine 10^–6 M control.Phenylephrine further contracted the 115NaPSS precontracted preparations, but was significantly less effective in 25NaPSS, although the precontraction levels were similar for the same substitute used. The amplitude of the superimposed phenylephrine contractions exhibited [Na⁺]_ex dependence. Phenylephrine 10^–6 M failed to further contract the ouabain 10^–4 M precontracted rings.We conclude that relatively small reductions in [Na⁺]_ex are able to induce contractions of rat aorta primarily through release of endogenous catecholamines, probably through neural Na⁺/Ca²⁺ exchange. Larger reductions in [Na⁺]_ex appear to cause contraction through muscular Na⁺/Ca²⁺ exchange.     

Veratridine-induced intoxication in the isolated left atrium of the rat: Effects of some anti-ischemic compounds

Summary Veratridine-induced Na⁺ and Ca²⁺ uptake was used as a simulation of ischemia-induced Na⁺ and Ca²⁺ uptake. Therefore, electrically driven (1 Hz) isolated left atria of the rat were intoxicated with veratridine and the ⁴⁵Ca²⁺ uptake was determined. Veratridine (10^–4 mol/l) increased the ⁴⁵Ca²⁺ uptake from 575±13 to 2320±86 dpm/mg ww (n=20). The total tissue content of ⁴⁵Ca²⁺ was elevated from 4328±132 to 5136 ±303 dpm/mg ww (n = 13). The veratridine-induced ⁴⁵Ca²⁺ uptake was completely suppressed by tetrodotoxin (10^–7 and 10^–6 mol/l), whereas amiloride (6·10^–6 mol/1) and phentolamine (10^–6 and 10^–5 mol/l) exhibited no effect on the veratridine-induced ⁴⁵Ca²⁺ uptake. Nifedipine (10^–7 and 10^–6 mol/l) was ineffective on veratridine-induced ⁴⁵Ca²⁺ uptake. Verapamil (10^–5 mol/l) suppressed the veratridine-induced ⁴⁵Ca²⁺ uptake, but the ⁴⁵Ca²⁺ uptake in the absence of veratridine was also suppressed by verapamil (10^–6 and 10^–5 mol/l). The novel anti-ischemic compounds R 56865 (10^–8–10^–5 mol/l) and R 59494 (10^–8 -10-5 mol/l) totally abolished veratridine-induced ⁴⁵Ca²⁺ uptake.It is speculated that Ca²⁺ enters the cell via a Na⁺ channel which changes its selectivity upon veratridine treatment. Consequently, R 56865 and R 59494 could display their protective effect by either inhibiting the modified Na⁺ channel or preventing the transition of the normal Na⁺ channel to its altered state. As ischemia- and veratridine-induced Na⁺ and Ca²⁺ uptake share some similarities, it is proposed that veratridine-induced ⁴⁵Ca²⁺ uptake of the isolated left atrium of the rat could be used to study the mechanism of action of novel antiischemic drugs. Send offprint requests to D. Wermelskirchen at the above address     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

Pharmacological properties of ATP-sensitive purinergic receptors expressed in human G292 osteoblastic cells

We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca²⁺ level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC₅₀ of 0.5 ± 0.07 μM. While α,β-methylene-ATP (αβmeATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC₅₀ of 0.7 ± 0.2 μM. UTP and UDP also evoked responses with EC₅₀ of 2.0 ± 0.4 μM and 0.5 ± 0.1 μM respectively, but their responses were much smaller, resulting in an order of the response magnitude: ATP ~ ADP >> UTP ~ UDP. The responses evoked by ATP and ADP were blocked by pyridoxal-5'-phosphate-6-azophenyl-2,4,-disulfonate (PPADS) with IC₅₀ of 3.0 ± 0.05 μM and 5.0 ± 0.4 μM respectively, but not by suramin up to 30 μM. ATP-evoked responses were insensitive to inhibition by trinitrophenyl-ATP (TNP-ATP) and brilliant blue G. ADP-evoked responses were significantly inhibited by 2'-deoxy-N⁶-methyladenosine-3',5'-biphosphate (MRS2179) and 2-chloro-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279) with IC₅₀ of 48 ± 1.9 μM and 7.7 ± 0.9 μM respectively. Taken together, these results provide strong evidence for functional expression of ATP-sensitive P2Y receptors and particularly P2Y₁-like receptor in G292 cells.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na⁺/K⁺-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with –logEC₅₀=7.16±0.11 and maximal effect of 67±4% of the response to acetylcholine (1 mM). Ouabain (10 M)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H₁-receptors and -adrenoceptors, or neuronal Na⁺ channel blockade. The inhibition of ouabain contraction in tissues bathed in K⁺-free medium, and the inhibition by ouabain of the K⁺-induced relaxation confirm that the contractile action of ouabain is mediated by inhibition of Na⁺/K⁺-ATPase. Furthermore, depolarization (16.4±0.9 mV) was observed in human isolated bronchus by intracellular microelectrode recording. Ouabain (10 M)-induced contractions were abolished by a Ca²⁺-free solution but not by blockers of L-type Ca²⁺ channels. In human cultured bronchial smooth muscle cells, ouabain (10 M) produced a sustained increase in [Ca²⁺]_i (116±26 nM) abolished in Ca²⁺-free medium. Incubation with a Na⁺-free medium or amiloride (0.1 mM) markedly inhibited the spasmogenic effect of ouabain thus suggesting the role of Na⁺/Ca²⁺ exchange in ouabain contraction while selective inhibitors of Na⁺/H⁺-antiport, Na⁺/K⁺/Cl^–-antiport, or protein kinase C had no effect. Ouabain (10 M) failed to increase inositol phosphate accumulation in human bronchus. Ouabain (10 M) did not alter bronchial responsiveness to acetylcholine or histamine but inhibited the relaxant effects of isoprenaline, forskolin, levcromakalim, or sodium nitroprusside. These results indicate that ouabain acts directly to produce contraction of human airway smooth muscle that depends on extracellular Ca²⁺ entry unrelated to L-type channels and involving the Na⁺/Ca²⁺-antiporter.     

Functional and morphological properties of pericytes in suburothelial venules of the mouse bladder

Background and Purpose

In suburothelial venules of rat bladder, pericytes (perivascular cells) develop spontaneous Ca²⁺ transients, which may drive the smooth muscle wall to generate spontaneous venular constrictions. We aimed to further explore the morphological and functional characteristics of pericytes in the mouse bladder.

Experimental Approach

The morphological features of pericytes were investigated by electron microscopy and fluorescence immunohistochemistry. Changes in diameters of suburothelial venules were measured using video microscopy, while intracellular Ca²⁺ dynamics were visualized using Fluo-4 fluorescence Ca²⁺ imaging.

Key Results

A network of α-smooth muscle actin immunoreactive pericytes surrounded venules in the mouse bladder suburothelium. Scanning electron microscopy revealed that this network of stellate-shaped pericytes covered the venules, while transmission electron microscopy demonstrated that the venular wall consisted of endothelium and adjacent pericytes, lacking an intermediate smooth muscle layer. Pericytes exhibited spontaneous Ca²⁺ transients, which were accompanied by phasic venular constrictions. Nicardipine (1 μM) disrupted the synchrony of spontaneous Ca²⁺ transients in pericytes and reduced their associated constrictions. Residual asynchronous Ca²⁺ transients were suppressed by cyclopiazonic acid (10 μM), 2-aminoethoxydiphenyl borate (10 μM), U-73122 (1 μM), oligomycin (1 μM) and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (10 μM), but unaffected by ryanodine (100 μM) or YM-244769 (1 μM), suggesting that pericyte Ca²⁺ transients rely on Ca²⁺ release from the endoplasmic reticulum via the InsP₃ receptor and also require Ca²⁺ influx through store-operated Ca²⁺ channels.

Conclusions and Implications

The pericytes in mouse bladder can generate spontaneous Ca²⁺ transients and contractions, and thus have a fundamental role in promoting spontaneous constrictions of suburothelial venules.     

Purification and pharmacological characterization of BmKK2 (α-KTx 14.2), a novel potassium channel-blocking peptide, from the venom of Asian scorpion Buthus martensi Karsch

BmKK2 (α-KTx 14.2) is one of the novel short-chain peptides found in molecular cloning of a venom gland cDNA library from Asian scorpion Buthus martensi Karsch. Based upon its amino acid sequence, the peptide was proposed to adopt a classical α/β-scaffold for α-KTxs. In the present study, we purified BmKK2 from the venom of B. martensi Karsch, and investigated its action on voltage-dependent K⁺ currents in dissociated hippocampal neurons from neonatal rats. BmKK2 (10–100 μM) selectively inhibited the delayed rectifier K⁺ current, but did not affect the fast transient K⁺ current. The inhibition of BmKK2 on the delayed rectifier K⁺ current was reversible and voltage-independent. The peptide did not affect the steady-state activation of the current, but caused a depolarizing shift (about 9 mV) of its steady-state inactivation curve. The results demonstrate that BmKK2 is a novel K⁺ channel-blocking scorpion peptide.     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

Role of Na+-K+ ATPase enzyme in vascular response of goat ruminal artery

Objective:

To study the role of Na⁺, K⁺- ATPase enzyme in the vascular response of goat ruminal artery.

Materials and Methods:

Ruminal artery was obtained in chilled aerated modified Krebs-Henseleit solution (KHS) from a local slaughterhouse and transported in ice for further processing. The endothelium intact arterial ring was mounted in a thermostatically controlled (37 ± 0.5°C) organ bath containing 20 ml of modified KHS (pH 7.4) bubbled with oxygen (95%) and CO₂ (5%) under 2g tension. An equilibration of 90 min was allowed before addition of drugs into the bath. The responses were recorded isometrically in an automatic organ bath connected to PowerLab data acquisition system. In order to examine intact functional endothelium, ACh (10 μM) was added on the 5-HT (1.0 μM) - induced sustained contractile response. Similarly, functional characterization of Na⁺, K⁺-ATPase activity was done by K⁺-induced relaxation (10 μM-10 mM) in the absence and presence of ouabain (0.1 μM/ 0.1 mM), digoxin (0.1 μM) and barium (30 μM).

Results:

ACh (10⁻⁵ M) did not produce any relaxing effect on 5-HT-induced sustained contractile response suggesting that vascular endothelium has no significant influence on the activation of sodium pump by extracellular K⁺ in ruminal artery. Low concentration of Ba²⁺ (30 μM) (IC₅₀: 0.479 mM) inhibited K⁺-induced relaxation suggesting K_ir (inward rectifier) channel in part had role in K⁺-induced vasodilatation in ruminal artery. Vasorelaxant effect of KCl (10 μM-10 mM) in K⁺-free medium is also blocked by ouabain (0.1 μM and 0.1 mM) (IC₅₀:0.398 mM and IC₃₅: 1.36 mM), but not by digoxin (0.1 μM) (IC₅₀ 0.234 mM) suggesting that ouabain sensitive Na⁺, K⁺-ATPase isoform is present in the ruminal artery.

Conclusion:

In the goat ruminal artery functional regulation of sodium pump is partly mediated by K⁺ channel and ouabain sensitive Na⁺, K⁺ ATPase.     

The saluretic effect of the thiazide diuretic bemetizide in relation to the glomerular filtration rate

The effect of the thiazide diuretic, bemetizide, on the excretion of Na⁺, K⁺, Cl^–, Ca²⁺, and Mg²⁺ in relation to the glomerular filtration rate (GFR) was studied in 17 subjects whose creatinine clearances ranged from 133 to 5 ml·min^–1.After a 2-day fluid and salt balanced control period, 25 mg bemetizide given orally induced natriuresis and kaliuresis which lasted for 24 h and were proportional to the GFR of the patients. The ratio of bemetizide-induced K⁺/Na⁺ excretion was always 0.17 irrespective of individual GFR. In renal failure, bemetizide increased the fractional Na⁺ excretion from 3% to about 10%. Kaliuresis was associated with magnesiuria, whereas bemetizide-induced calciuresis was insignificant. The thiazide reversibly lowered GFR in all subjects.     

Effect of heparin treatment on the expression and activity of different ion-motive P-type ATPase isoforms from mouse extensor digitorum longus muscle during degeneration and regeneration after Bothrops jararacussu venom injection

Ca²⁺ ions are essential to myonecrosis, a serious complication of snake envenomation, and heparin seems to counteract this effect. We investigated the effect of local injection of Bothrops jararacussu venom in mouse fast-twitch extensor digitorum longus (EDL) muscle, without or with heparin, on functional/molecular alterations of two central proteins involved in intracellular Ca²⁺ homeostasis, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺/K⁺-ATPase. EDL-specific SERCA1 isoform expression dropped significantly just after venom administration (up to 60% compared to control EDL values at days 1 and 3; p < 0.05) while SERCA2 and Na⁺/K⁺-ATPase α₁ isoform expression increased at the same time (3-6- and 2-3-fold, respectively; p < 0.05). Although not significant, Na⁺/K⁺-ATPase α₂ isoform followed the same trend. Except for SERCA2, all proteins reached basal levels at the 7th day. Intravenous heparin treatment did not affect these profiles. Ca²⁺-ATPase activity was also decreased during the first days after venom injection, but here heparin was effective to reinstate activity to control levels within 3 days. We also showed that B. jararacussu venom directly inhibited Ca²⁺-ATPase activity in a concentration-dependent manner. Our results indicate that EDL SERCA and Na⁺/K⁺-ATPase are importantly affected by B. jararacussu venom and heparin has protective effect on activity but not on protein expression.     

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Aim:

To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.

Methods:

Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na⁺/H⁺ exchanger 1 (NHE1) and calpain, intracellular free Ca²⁺level ([Ca²⁺]_i), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE^−/−) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.

Results:

LPS treatment increased NHE1 activity and [Ca²⁺]_i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca²⁺-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca²⁺]_i. and calpain activity. In the presence of the Ca²⁺ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE^−/− mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.

Conclusion:

LPS stimulates NHE1 activity, increases [Ca²⁺]_i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.     

Antioxidant activities, metal contents, total phenolics and flavonoids of seven Morchella species

Seven Morchella species were analyzed for their antioxidant activities in different test systems namely β-carotene/linoleic acid, DPPH, reducing power, chelating effect and scavenging effect (%) on the stable ABTS⁺, in addition to their heavy metals, total phenolic and flavonoid contents. In β-carotene/linoleic acid system, the most active mushrooms were M. esculenta var. umbrina and M. angusticeps. In the case of DPPH, methanol extract of M. conica showed high antioxidant activity. The reducing power of the methanol extracts of mushrooms increased with concentration. Chelating capacity of the extracts was also increased with the concentration. On the other hand, in 40 μg ml⁻¹ concentration, methanol extract of M. conica, exhibited the highest radical scavenging activity (78.66 ± 2.07%) when reacted with the ABTS⁺ radical. Amounts of seven elements (Cu, Mn, Co, Zn, Fe, Ca, and Mg) and five heavy metals (Ni, Pb, Cd, Cr, and Al) were also determined in all species. M. conica was found to have the highest phenolic content among the samples. Flavonoid content of M. rotunda was also found superior (0.59 ± 0.01 μg QEs/mg extract).