Low-Mg(2+) treatment increases sensitivity of voltage-gated Na(+) channels to Ca(2+)/calmodulin-mediated modulation in cultured hippocampal neurons.

Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

Nucleotides maintain the activity of Cav1.2 channels in guinea-pig ventricular myocytes.

The activity of Cav1.2 Ca(2+) channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca(2+) channels in the presence of 1 µM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca(2+) channels.

[High intracellular Mg²âº affects the activities of L-type calcium channel in guinea- pig ventricular myocytes].

This study is aimed to investigate the effects of high intracellular Mg²âº on L-type calcium channel in guinea-pig ventricular myocytes. The cardiomyocytes were acutely isolated with enzyme digestion method. By adopting inside-out configuration of patch clamp technique, single channel currents of the L-type calcium channel were recorded under different intracellular Mg²âº concentrations ([Mg²âº]i). In control group, which was treated with 0.9 mmol/L Mg²âº, the relative activity of calcium channel was (176.5 ± 34.1)% (n = 7). When [Mg²âº]i was increased from 0.9 to 8.1 mmol/L (high Mg²âº group), the relative activities of calcium channel decreased to (64.8 ± 18.1)% (n = 6, P < 0.05). Moreover, under 8.1 mmol/L Mg²âº, the mean open time of calcium channel was shortened to about 25% of that under control condition (P < 0.05), but the mean close time of calcium channel was not altered. These results suggest that high intracellular Mg²âº may inhibit the activities of L-type calcium channel, which is mainly due to the shortening of the mean open time of single L-type calcium channel.

Repeated oral treatment with polysaccharide sulfate reduces insulin resistance and dyslipidemia in diabetic dyslipidemic rat model.

Polysaccharide sulfate (PSS) is a new type of antiatherosclerotic medicine for its effects of anticoagulation, anti-thrombosis and modulation of dyslipidemia. However, it is still uncertain whether PSS could modulate the diabetic dyslipidemia or not. Here, the rat model of diabetic dyslipidemia was developed and the effects of PSS on glucose and lipid levels were investigated in this animal model. Wistar rats were iv injected with streptozotocin 20 mg x kg(-1) after feeding with high fat diet for one and a half month. Then, rats received orally PSS (30, 90, and 180 mg x kg(-1)) for 1 month. After oral treatment with PSS (90 and 180 mg x kg(-1)) for 1 month, the levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) were significantly reduced and the level of high density lipoprotein-cholesterol (HDL-C) increased, compared with diabetic control rats. Moreover, PSS (30, 90, and 180 mg x kg(-1)) had a tendency to reduce glucose and insulin levels, and significantly increased insulin sensitivity index. Our results suggest that PSS could improve insulin sensitivity and relieve dyslipidemia in diabetic dyslipidemic rats.

Astragaloside IV Inhibits Membrane Ca[Formula: see text] Current but Enhances Sarcoplasmic Reticulum Ca[Formula: see text] Release.

Astragaloside IV (AS-IV) is one of the active ingredients in Astragalus membrananceus (Huangqi), a traditional Chinese medicine. The present study investigated the effects of AS-IV on Ca[Formula: see text] handling in cardiac myocytes to elucidate its possible mechanism in the treatment of cardiac disease. The results showed that AS-IV at 1 and 10[Formula: see text][Formula: see text]M reduced KCl-induced [Ca[Formula: see text]]i increase ([Formula: see text] from 1.33[Formula: see text][Formula: see text][Formula: see text]0.04 (control, [Formula: see text] 28) to 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text], [Formula: see text] 29) and 1.22[Formula: see text][Formula: see text][Formula: see text]0.02 ([Formula: see text] 0.01, [Formula: see text]), but it enhanced Ca[Formula: see text] release from SR ([Formula: see text] from 1.04[Formula: see text][Formula: see text][Formula: see text]0.01 (control, [Formula: see text]) to 1.44[Formula: see text][Formula: see text][Formula: see text]0.03 ([Formula: see text], [Formula: see text]) and 1.60[Formula: see text][Formula: see text][Formula: see text]0.04 ([Formula: see text] 0.01, [Formula: see text]0), in H9c2 cells. Similar results were obtained in native cardiomyocytes. AS-IV at 1 and 10[Formula: see text][Formula: see text]M inhibited L-type Ca[Formula: see text] current ([Formula: see text] from [Formula: see text]4.42[Formula: see text][Formula: see text][Formula: see text]0.58 pA/pF of control to [Formula: see text]2.25[Formula: see text][Formula: see text][Formula: see text]0.12 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) and [Formula: see text]1.78[Formula: see text][Formula: see text][Formula: see text]0.28 pA/pF ([Formula: see text] 0.01, [Formula: see text] 5) respectively, when the interference of [Ca[Formula: see text]]i was eliminated due to the depletion of SR Ca[Formula: see text] store by thapsigargin, an inhibitor of Ca[Formula: see text] ATPase. Moreover, when BAPTA, a rapid Ca[Formula: see text] chelator, was used, CDI (Ca[Formula: see text]-dependent inactivation) of [Formula: see text] was eliminated, and the inhibitory effects of AS-IV on ICaL were significantly reduced at the same time. These results suggest that AS-IV affects Ca[Formula: see text] homeostasis through two opposite pathways: inhibition of Ca[Formula: see text] influx through L-type Ca[Formula: see text] channel, and promotion of Ca[Formula: see text] release from SR.