Cyclic AMP-mediated increase in L-type calcium current (ICa,L) by nitroglycerin in guinea-pig ventricular myocytes.

The effects of nitroglycerin (NG) on the L-type Ca2+ current (ICa,L) were investigated in guinea-pig ventricular myocytes. NG did not affect the basal ICa,L significantly. The ICa,L was increased by NG when the ICa,L was augmented by the pre-treatment with isoproterenol (Iso), and this increase was abolished by the pretreatment with methylene blue (MB), a guanylate cyclase inhibitor. NG also increased ICa,L in the myocytes undergoing the pretreatment with isobutylmethylxanthine (IBMX). The NG-induced increase in ICa,L was also observed in the myocytes intracellularly dialyzed with cyclic adenosine monophosphate (cAMP). The order of the Iso-induced increase in ICa,L was Iso, IBMX and cAMP. Relatively a lower concentration of the extracellularly applied 8bromo-cyclic guanosine monophosphate (8Br-cGMP) increased ICa,L in the myocytes dialyzed with cAMP, whereas the higher concentration of 8Br-cGMP decreased ICa,L. NG produced a marked increase in cGMP levels, and a slight increase in cAMP levels in the ventricular tissues. These results suggest that the stimulatory effect of NG on the ICa,L is due to an inhibition of cGMP-inhibitable cAMP-phosphodiesterase (PDE) and a possible activation of cGMP-dependent protein kinase via the NG-induced increase in cGMP levels in guinea-pig ventricular myocytes.     

Chronic ramelteon treatment in a mouse model of Alzheimer's disease

Prior research has reported beneficial effects of melatonin in rodent models of Alzheimer's disease (AD). This study evaluated the effect of ramelteon (Rozerem, a melatonin receptor agonist) on spatial learning & memory and neuropathological markers in a transgenic murine model of AD (the B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mouse strain; hereafter 'AD mice'). Three months of daily ramelteon treatment (~3mg/kg/day), starting at 3 months of age, did not produce an improvement in the cognitive performance of AD mice (water maze). In contrast to wild-type control mice, AD mice did not show any evidence of having learned the location of the escape platform. The cortex and hippocampus of AD mice contained significant quantities of beta-amyloid plaques and PARP-positive (poly ADP ribose polymerase) cells, indicating apoptosis. Six months of ramelteon treatment, starting at 3 months of age, did not produce any change in these neuropathological markers. The ability of long term melatonin treatment to improve cognition and attenuate neuropathology in AD mice did not generalize to this dosage of ramelteon.     

Effects of K-channel blockers, calcium, and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential. (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao. This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 microM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.     

Chronic leptin treatment stimulates lipid oxidation in immortalized and primary mouse skeletal muscle cells

Leptin administration enhances lipid oxidation in skeletal muscle. Nevertheless, direct and chronic effect of leptin has not been well characterized. Here, we measured the effect of leptin on skeletal muscles and their signaling pathways using differentiated C₂C₁₂ myotubes and primary myotube cultures. Differentiated myotubes expressed both the short and long forms of leptin receptors. Leptin increased lipid oxidation in myotubes in a concentration- and time-dependent manner, with significant induction of lipid oxidation occurring after 6 h. Actinomycin D completely blocked leptin-induced lipid oxidation. Leptin significantly increased phosphorylation of JAK2 and STAT3 in myotubes, and leptin-induced lipid oxidation was abolished by treatment with a JAK2 inhibitor or STAT3 siRNA. We then used mouse myotubes to measure these effects under physiological conditions. Leptin increased lipid oxidation, which again was blocked by a JAK2 inhibitor and STAT3 siRNA. These results suggest that the JAK2/STAT3 signaling pathway may underlie the chronic effects of leptin on lipid oxidation in skeletal muscles.     

Chronic hypoxia induces nonreversible right ventricle dysfunction and dysplasia in rats

The purpose of this study was to evaluate the reversibility of right ventricular (RV) remodelling after pulmonary artery hypertension (PAHT) secondary to 3 wk of hypobaric hypoxia. A group of 10 adult male Wistar rats were studied and were the following: control normoxic (C), after 3 wk of chronic hypoxia (CH), and after 3 wk of exposure to hypoxia followed by 3 wk of normoxia recovery (N-RE). Mean pulmonary artery pressure was 11 +/- 2 mmHg in the C group, 35 +/- 2 mmHg in the CH group, and 14 +/- 3 mmHg in the N-RE group. RV function was assessed by echocardiography. In the CH group, the pulmonary flow measured in Doppler mode depicted a midsystolic notch and a decrease of the pulmonary acceleration time compared with control [17 +/- 1 vs. 34 +/- 1 ms (n = 10), respectively; P < 0.05]. RV thickening measured in M-mode was apparent in the CH group compared with the control group [2.84 +/- 0.40 vs. 1.73 +/- 0.26 mm (n = 10), P < 0.05]. In the N-RE group, the RV wall was significantly thinner compared with the CH group [1.56 +/- 0.08 vs. 1.73 +/- 0.26 mm (n = 10), P < 0.05]. The calculated RV diameter shortness fraction was not different between the CH group and C group (34 +/- 4.2% vs. 36 +/- 2.8%) but decreased in the N-RE group [20 +/- 2.4% (n = 10), P < 0.01]. The E-to-A wave ratio on the tricuspid Doppler inflow was significantly lower in the CH group and N-RE group compared with the C group [0.70 +/- 0.8 and 0.72 +/- 0.1 vs. 0.88 +/- 0.2 (n = 10), respectively; P < 0.05]. In the isolated perfused heart using the Langendorff method, RV compliance was increased in the CH group and decreased in the N-RE group. In the N-RE group, fibrous bands with metaplasia were observed on histological sections of the RV free wall. We conclude that PAHT induces nonreversible RV dysfunction with dysplasia.     

Functional expression of a GFP-tagged Kv1.5 alpha-subunit in mouse ventricle

The experiments here were undertaken to determine the feasibility of increasing the cell surface expression of voltage-gated ion channels in cardiac cells in vivo and to explore the functional consequences of ectopic channel expression. Transgenic mice expressing a green fluorescent protein (GFP)-tagged, voltage-gated K+ (Kv) channel alpha-subunit, Kv1.5-GFP, driven by the cardiac-specific alpha-MHC promoter, were generated. In recent studies, Kv1.5 has been shown to encode the micromolar 4-aminopyridine (4-AP)-sensitive delayed rectifier K+ current (I(K,slow)) in mouse myocardium. Unexpectedly, Kv1.5-GFP expression is heterogeneous in the ventricles of these animals. Although no electrocardiographic abnormalities were evident, expression of Kv1.5-GFP results in marked decreases in action potential durations in GFP-positive ventricular myocytes. In voltage-clamp recordings from GFP-positive ventricular myocytes, peak outward K+ currents are significantly higher, and their waveforms are distinct from those recorded from wild-type cells. Pharmacological experiments revealed a selective increase in a micromolar 4-AP-sensitive current, similar to the 4-AP-sensitive component of I(K,slow) in wild-type cells. The inactivation rate of the "overexpressed" current, however, is significantly slower than the Kv1.5-encoded component of I(K,slow) in wild-type cells, suggesting differences in association with accessory subunits and/or posttranslational processing.     

Potassium conductance in straight proximal tubule cells of the mouse. Effect of barium, verapamil and quinidine

The present study has been performed to test for the influence of verapamil and quinidine on the potential difference across the basolateral cell membrane (PDbl) and on the basolateral potassium conductance of isolated perfused segments of the mouse proximal tubule. PDbl was recorded continuously with conventional microelectrodes during rapid alterations of bath or luminal perfusate composition. The contribution of the basolateral potassium conductance to the conductance of both cell membranes (tk) was estimated from the effects of altered bath potassium concentration on PDbl. Under control conditions tk approaches 0.8, i.e. the basolateral cell membrane is mainly conductive to potassium. Neither quinidine nor verapamil affect PDbl at concentrations below 10 mumol/l. At higher concentrations both substances depolarize the basolateral cell membrane mimicking the effect of 1 mmol/l barium. In the presence of 0.1 mmol/l verapamil tk is virtually abolished at 5 to 10 mmol/l bath potassium concentration but is almost unaffected at bath potassium concentrations between 20 and 40 mmol/l. 1 mumol/l ionophore A-23187 does not change the depolarizing effect of 0.1 mmol/l verapamil on cell membrane potential. In the presence of 0.1 mmol/l quinidine, tk is reduced to some 50%, irrespective of the bath potassium concentration. It is concluded that the potassium conductance in straight proximal tubules is inhibited not only by barium but as well by high concentrations of verapamil and quinidine. The effect is probably direct and not related to alterations in the intracellular calcium activity.     

Effect of verapamil treatment on compensatory renal growth in mice

Calcium has been shown to control the proliferation of various cells in vitro and in vivo. In this study we have attempted to modify compensatory renal growth by pharmacological interventions in mice who have undergone uninephrectomy. The effect of a calcium channel blocker verapamil was investigated. Unilateral nephrectomy of intact male mice produced the expected increase in weight of the remaining kidney by 67.5+/-8.1%. This rise was accompanied by a proportional increase in RNA. In mice, cell hypertrophy was found to be a major factor in compensatory renal growth. Verapamil given in a i.p. dose of 1.0 or 2.0 mg/day/mouse attenuated the growth of the remaining kidney so that its weight rose by only 48.2+/-6% and 28.2+/-4.4 %, respectively. In vivo administration of verapamil decreased the degree of compensatory renal growth and this growth inhibiting effect was directly proportional to the dose.     

Erythromycin resistance in mouse L cells

The sensitivity of mouse cell lines in culture to the macrolide antibiotic, erythromycin stearate, was investigated. Both resistant and sensitive lines were found. Experiments indicated that in sensitive cells erythromycin stearate inhibits mitochondrial protein synthesis. Mutants resistant to erythromycin stearate were selected from the line LM(TK-), and these are also less sensitive to other macrolide antibiotics such as carbomycin and spiramycin. Attempts to transfer the erythromycin resistance of either the mutants or naturally resistant lines by fusion of cytoplasts with sensitive cells were unsuccessful, and it is concluded that resistance to erythromycin stearate is controlled by nuclear genetic factors.     

Carbomycin resistance in mouse L cells

A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.