No abstract

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3, 7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl(-) current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.

No abstract

Cardiac and skeletal type of excitation-contraction coupling (ECC) are quite different. Those differences could be explained by structural ones in the molecular entities involved in ECC, ie dihydropyridines (DHP) receptors (alpha1 subunit of L-type calcium channels) of the sarcolemma or ryanodine receptors of the sarcoplasmic reticulum membrane. As previously demonstrated by means of electrophysiology, the two types of ECC coexist during the first stages of in vitro development of skeletal muscle, whereas the skeletal type predominates at the later ones. In order to see whether evolution of ECC could be correlated with the one of alpha1 subunit expression, we determined by Northern Blotting which isoforms of alpha1 subunit are expressed during the in vitro myogenesis. mRNA corresponding to the cardiac isoform are present in myoblasts (before fusion), but patch-clamp experiments showed that they are not functional. After fusion, skeletal and cardiac mRNA are coexpressed in myotubes, with different intensities: whereas expression of skeletal mRNA (which are the more intensive) stabilized at the later stages tested, cardiac mRNA decreased. We conclude that evolution in mRNA alpha1 subunit isoforms expression could partly explained evolution of ECC features during in vitro myogenesis.

Two dihydropyridine receptor mRNA isoforms (cardiac and skeletal) are expressed in rat skeletal muscle cells in primary culture. The progressive changes in excitation-contraction coupling mode from dual mode (‘skeletal’ and ‘cardiac’) to predominant ‘skeletal’ one during in vitro myogenesis are thought to be linked to the developmental changes in the relative expression of the two types of molecular entity previously observed in this preparation. In order to test this hypothesis, myotube cultures (5- to 7-day-old) were treated with antisense phosphorothioated oligodeoxynucleotides against cardiac or skeletal alpha1 subunit of L-type calcium channel. The oligodeoxynucleotide uptake by cells was checked by means of imaging of fluorescent oligodeoxynucleotide derivatives within the cells. Optimum concentration used (10 microM in the extracellular medium) and incubation time (70 hours) were empirically determined. Antisense directed against the cardiac type led to a 54% decrease in the averaged L-type calcium current peak density at −10 mV. The same type of experiment was performed with antisense against the skeletal isoform and led to a same order of inhibition (46%). This result clearly shows that the two isoforms can work as a calcium channel. Conversely, analysis of the shape of T-V (relative contractile amplitude versus membrane potential) curves shows that the treatment with ‘skeletal’ antisense depressed the contractile response in the medium membrane potential range whereas treatment with ‘cardiac’ antisense had no effect. This and other results taken together suggest that the skeletal isoform of dihydropyridine receptor is involved in both ‘cardiac’ and ‘skeletal’ types of EC coupling mechanisms at work in early stages of myotubes in vitro development. The type of coupling probably depends on the proximity of the skeletal dihydropyridine receptor and the ryanodine receptor.