hKv4.3 channel characterization and regulation by calcium channel antagonists.

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.

Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current.

OBJECTIVE: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.

[Prognostic value of thymidine kinase in cancer of the breast]. / Valeur pronostique de la thymidine kinase dans le cancer du sein.

We have measured by a radioenzymatic assay the thymidine kinase in the cytosol of 182 primary infiltrating breast cancers. Maximal follow-up is 95 months. Thymidine kinase was found to be related to SBR grade, tumour size and absence of oestradiol receptors (RE). Univariate analysis has pointed out a significant linkage between overall or metastase free survival and thymidine kinase, using a cut-off level of 80 mU/mg protein which is the most discriminating value. Thymidine kinase appeared to be particularly useful in lymph-node-positive, RE-negative and grade 3 patients. Multivariate analysis of the overall survival and of the metastase free survival (Cox model) revealed that they were strongly related to thymidine kinase status.

Some characteristics of fetal and adult isoenzymes of thymidine kinase in human breast cancers.

Two isoenzymes of Thymidine Kinase were isolated from 90 human breast cancers by ion exchange chromatography. One was the enzyme present in adult tissues (TK-A), the second corresponded with the enzyme found in fetal tissues (TK-F). The presence of both isoenzymes was observed in the 90 tumors investigated. The activity of TK-A varied in the same range as in normal tissues, while the activity of TK-F varied to a larger extent and was higher than that of TK-A in a majority of tumors. Moreover, TK-F activity was independent of the presence or of the level of estradiol and progesterone receptors. Both isoenzymes were further characterized and compared.

[Prognostic value of fetal thymidine kinase measurements in breast cancer]. / Intérêt pronostique du dosage de l'activité de la thymidine kinase foetale dans les cancers du sein.

Estimation of fetal Thymidine Kinase activity in primary tumors could be a useful parameter in the assessment of early relapses in patients with breast cancer. In a group of 39 women followed up for 42 months, the percentage of early relapses and their frequency was significantly higher in patients whose tumors had a high level of enzyme activity than in others.

[Close relation between fetal thymidine kinase and thymidylate kinase activities in breast cancer]. / Relation étroite entre les activités de la thymidine kinase foetale et de la thymidylate kinase dans les cancers du sein.

In human breast cancers, assays on thymidine kinase activity revealed the synthesis of large amounts of d-TTP. This fact suggested the presence of thymidylate kinase closely associated with thymidine kinase. Results obtained with experimental tumors were quite different. These tumors appeared inadequate for the study on thymidine metabolism in mammary cancers.

[Demonstration of fetal-type thymidine kinase in cancer of the breast]. / Mise en évidence de la thymidine kinase de type foetal dans les cancers du sein.

Seventy five human breast cancers were examined in order to search for the presence of thymidine kinase of the fetal-type (TK-F). The presence of TK-F was evidenced in all tumors. Its activity varied from one to another tumor, but it was evident that the increased TK activity observed in mammary cancers could exclusively be related to high TK-F activity. Some relations between TK-F activity and the presence of estradiol and progesterone receptors (ER, PR) were obvious. The highest activities were observed in cancers with high level of ER and PR. Thymidylate kinase activity (d-TTP synthesis) varied in parallel with TK-F activity. In a general way, it was higher in ER+ PR+ than in ER+ PR- cancers.

[In vitro variations of hepatic glycogenolysis after incubation of rat liver slices in the presence of polyamines]. / Variations in vitro de la glycogènolyse hépatique après incubation de coupes de foie de rat en présence de polyamines.

If rat liver histological sections, cut by cryostat, incubated in presence of different putrescine concentrations (diluted in Hanks medium) and stained with P.A.S. show no real changes in glycogenolysis, others in a medium containing spermidine or better spermine, proportionally to the concentration in polyamine and duration of incubation, show an increase in the staining of the hepatic cytoplasms compared with the relative controls which are sections incubated in Hanks medium during the same time. So, in these conditions, putrescine doesn't seem to have an influence on glycogenolysis, but spermidine and spermine have a protective effect on hepatic glycogen.