Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells.

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.

Development of substituted Benzo[c]quinolizinium compounds as novel activators of the cystic fibrosis chloride channel.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells.

The effects of the primary extract roots of Securidaca longepedunculata were tested on sodium, calcium and potassium currents in rat skeletal muscle cells developed in culture. In addition, they were tested on depolarisation-induced contraction and resting intracellular calcium levels. S. longepedunculata extract (10(-6) g/l) increases sodium current at all potentials. No clear effect was observed on calcium current except for a slight increase at negative potentials (-30, -10 mV) revealing a 5 mV shift towards negative potentials of the I(Ca)/V curve, as with potassium current. In contrast, at the same concentration, S. longepedunculata enhanced the contractile response elicited by durable depolarisation. This was not attributable to the slight increase in resting intracellular free calcium concentration which did not change during and following S. longepedunculata application. These results strongly suggest that S. longepedunculata root extract contains one or more components acting on the voltage-sensor of excitation-contraction coupling (dihydropyridine receptors), regardless of its implication as a calcium channel.

Antisense oligonucleotides against 'cardiac' and 'skeletal' DHP-receptors reveal a dual role for the 'skeletal' isoform in EC coupling of skeletal muscle cells in primary culture.

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.

Reversal of the relative expression of cardiac and skeletal alpha1 subunit isoforms of L-type calcium channel during in vitro myogenesis.

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.