Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Gonadotropins at menopause: the influence of obesity, insulin resistance, and estrogens

Obese, postmenopausal women have lower FSH levels. To determine whether this is due to higher estrogen exposure, we compared feedback gonadotropin sensitivity and its relation to insulin resistance in four groups of obese and lean, postmenopausal women. Group one was treated with 400 mg troglitazone (TG) daily for two weeks; 150 clomiphene citrate (CC) was added daily for the second week. Group two received 150 mg CC daily for a week. Group three received 1000 mg metformin (MET) daily for two weeks, with 120 mg raloxifene (RAL) added during the second week. Group four received 120 mg RAL for a week. Before and after each period, a serum pool was obtained from samples taken every minute during a 10 ml interval. The women recruited for this study were categorized as obese or lean based on BMI >/= 29 or BMI < 29, respectively. Obese, menopausal women had lower FSH (45.5 IU/l) and LH (16.2 IU/l) values than those of lean (64.1 IU/l and 23.0 IU/l), but the obese menopausal women had higher leptin, DHEAS, glucose, insulin, and HOMA-IR levels. Log [FSH] was associated with BMI (r = -0.53, P < 0.000001) and number of pregnancies (r = -0.37, P = 0.0009). TG treatment did not change HOMA-IR or gonadotropin levels, but DHEAS and androstenedione levels decreased significantly. CC alone or together with TG, diminished FSH (-7.9 and -9.2) and LH (-2.5 and -3.6) concentrations, with a greater reduction in lean women. MET reduced glucose and the HOMA-IR index without affecting gonadotropin or steroid levels. Conclusions: obese, menopausal women have lower FSH levels due to greater estrogen exposure, by mechanisms unrelated to insulin resistance.     

Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations

Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.     

Involvement of endothelin-1 and its receptors in PGF2alpha-induced luteolysis in the rat

The possible mediatory role of endothelin-1 (ET-1) in prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in the rat was examined. The effect of PGF(2alpha) was tested on day 9 of pregnancy either in vivo, by injecting cloprostenol, an analog of PGF(2alpha) or in vitro, in isolated intact corpora lutea incubated with PGF(2alpha). Luteolysis was confirmed by progesterone determination in the peripheral blood serum or in the culture medium, respectively. Administration of cloprostenol (.0025 mg/rat) induced within 1 hr, a significant fall (from 56.8 to 27.6 ng/ml, P < 0.0001) in serum progesterone concentrations that was associated with an increased expression of the mRNA to ET-1 and its protein product in rat luteal tissue. Elevated level of ET-1 were also determined at the spontaneous regression of the CL, upon parturition. Expression of the ET receptors, ETA and ETB was not affected by cloprostenol. On the other hand, this PGF(2alpha) analog induced expression of luteal VEGF mRNA. In vitro experiments demonstrate that the LH (100 ng/ml)-induced increase in luteal progesterone secretion was reduced by PGF(2alpha) (1 microg/ml). The inhibitory effect of PGF(2alpha) was reversed by BQ123 (10(- 7) M), that is a selective ETA receptor antagonist. We conclude that the PGF(2alpha)-induced elevation in luteal expression of ET-1 combined with the reversal of its luteolytic effect by an ETA receptor antagonist suggest that ET-1 may take part in the PGF(2alpha)-induced luteolysis in the rat.     

Altered proteinogram in short term portal vein stenosed rats

The electrophoretic pattern of serum proteins has been studied in short-term prehepatic portal hypertensive rats since atrophy is produced in the liver, which is the main origin of most of these proteins, during this postoperative period. After 28 days of evolution, rats (n = 9) with triple stenosing ligated portal vein showed hypoalbuminemia, hypo-alpha-globulinemia, hyper-alpha2-globulinemia and hyper-gamma-globulinemia, the albumin/globulin ratio decreased with respect to the control animals (n = 8). These alterations are associated with hepatic atrophy, portosystemic and portohepatic (44.4%) collateral circulation. The proteinogram alterations found in rats with short-term prehepatic portal hypertension suggest that hepatic failure exists in spite of potential portohepatic revascularization which is frequently originated by the development of portohepatic collateral circulation.     

Interrelationship between ectoparasites and wild rodents from northeastern Buenos Aires Province,Argentina

Infestation parameters and indices of mites, ticks and fleas associated with wild rodents from northeastern Buenos Aires Province, Argentina, were studied. Host species similarity was also analyzed in relation to their ectoparasites. Fifty-five rodents were captured from January 2000 to March 2001. In total, 1,022 ectoparasites were collected and three ectoparasite-host associations were new records. However, this is the first study on Craneopsylla minerva wolffhuegeli infesting parameters. Ectoparasite total mean abundance and total prevalence were higher in Holochilus brasiliensis (MA = 47.7; P = 100%) and Scapteromys aquaticus (MA = 25.4; P = 95.4%), meanwhile specific richness and diversity were higher in Oligoryzomys flavescens (S = 6; H = 1.3) and Akodon azarae (S = 4; H = 1.0). On the other hand, the only individual of Calomys laucha was not parasited. S. aquaticus-H. brasiliensis, which preferred similar microhabitats, shared the same ectoparasite species (Css = 100). Whereas, A. azarae, which was mostly associated with grassland, showed the highest difference with the other hosts (Css < 0.4). Considering every ectoparasite species, H. brasiliensis showed the highest mean abundance, prevalence and preference. The results suggest that the particular characteristics of this rodent would give it better possibilities not only of being infested by ectoparasites, but also of transmitting them to its progeny.     

Increased glucose uptake promotes oxidative stress and PKC-delta activation in adipocytes of obese,insulin-resistant mice

Increased oxidative stress is believed to be one of the mechanisms responsible for hyperglycemia-induced tissue damage and diabetic complications. In these studies, we undertook to characterize glucose uptake and oxidative stress in adipocytes of type 2 diabetic animals and to determine whether these promote the activation of PKC-delta. The adipocytes used were isolated either from C57Bl/6J mice that were raised on a high-fat diet (HF) and developed obesity and insulin resistance or from control animals. Basal glucose uptake significantly increased (8-fold) in HF adipocytes, and this was accompanied with upregulation of GLUT1 expression levels. Insulin-induced glucose uptake was inhibited in HF adipocytes and GLUT4 content reduced by 20% in these adipocytes. Reactive oxygen species (ROS) increased twofold in HF adipocytes compared with control adipocytes and were largely reduced with decreased glucose concentrations. At zero glucose, ROS levels were reduced to the normal levels seen in control adipocytes. The activity of PKC-delta increased twofold in HF adipocytes compared with control adipocytes and was further activated by H2O2. Moreover, PKC-delta activity was inhibited in HF adipocytes either by glucose deprivation or by treatment with the antioxidant N-acetyl-l-cysteine. In summary, we propose that increased glucose intake in HF adipocytes increases oxidative stress, which in turn promotes the activation of PKC-delta. These consequential events may be responsible, at least in part, for development of HF diet-induced insulin resistance in the fat tissue.