Cyclic mechanical strain decreases the DNA synthesis of vascular smooth muscle cells.

In vivo, smooth muscle cells of the vascular wall are rhythmically stretched by the arterial pulse. Here, we test the hypothesis that rhythmical stretch is important for suppressing the growth of vascular smooth muscle (vsm) cells. DNA-synthesis rate, cell number, metabolic activity, and cell death were compared between rhythmically stretched and non-stretched vsm cells from the rat embryonic aortic A10 cell line. Rhythmical stretch (0.5 Hz, 5% elongation, 48 h) did not induce vsm cell proliferation, that is the vsm cell number was constant. Cell damage or necrosis was excluded because the release of lactate dehydrogenase (LDH) was identical. The low rate of apoptosis (0.2%) was not different between stretched cells and control cells. Stretch significantly reduced the DNA-synthesis rate [measured as incorporation of 5-bromo-2'-deoxyuridine (BrdU)] in a time-dependent manner. BrdU incorporation was decreased by 32% after 24 h of cyclic stretching and was further diminished to 50% after 48 h of strain. Metabolic activity (measured by Wst-1 cleavage) was only modestly influenced. The stretch-induced decrease in DNA synthesis was independent of the extracellular matrix. No differences were detected when laminin- or pronectin-coated membranes were used instead of collagen-coated membranes. The effect of stretch was unlikely to be mediated by secretion of an unknown "factor", because vsm cells incubated with medium conditioned by stretched cells did not show a significant decrease in BrdU uptake. The results support the idea that rhythmical stretch is important to keep the rate of DNA synthesis and thereby the proliferation of vsm cells at a low level.

The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl- conductance of Xenopus oocytes.

Oocytes from Xenopus laevis activate a Ca2+ dependent Cl- conductance when exposed to the Ca2+ ionophore ionomycin. This Ca2+ activated Cl- conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (GI- / GCl-) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- conductance, which produces more linear I/V curves with a GI- / GCl- ratio of about 0.52. Ionomycin enhanced CaCC (DeltaG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (microS) 23 +/- 1.9 (n=9) and 23.6 +/- 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 +/- 0.48 microS (n=18) and 6. 9 +/- 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. DeltaF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl- channel in oocytes inhibited CaCC to a lesser degree (DeltaG=12.1 +/- 1.1 microS; n=7). Changes of CFTR and CaCC-Cl- whole cell conductances were also measured when extracellular Cl- was replaced by I-. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca2+. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca2+ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl- currents is paralleled by an inhibition of Ca2+ activated Cl- currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl- conductance. They might explain previous findings in epithelial tissues of CF-knockout mice.

Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.

The three subunits (alpha, beta, gamma) encoding for the rat epithelial Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na+ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At -90 mV and -40 mV, the IC50 values were 5 microM and 10 microM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 microM at pH 6.5 and only 17 microM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at -90 mV were 0.74 microM for the charged and 100.6 microM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC50 values in the range of 0.1-20 microM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC alpha-subunit-deletion mutant, Delta278-283, which lacks a putative amiloride-binding site, induced a Na+ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.

cAMP stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl- and K+ conductances is necessary in many Cl--secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K+ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl- conductance, also upregulates endogenous K+ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl- conductance. Of the cAMP-induced whole-cell current increase, 15-25% was due to a 293B-, Ba2+and TEA+-inhibitable K+ conductance. The cRNA of the mutated CFTR (DeltaF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K+ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K+ channels, might explain the coordinated cAMP-mediated increase in Cl- and K+ conductances.

Culture-dependent expression of Na+ conductances in airway epithelial cells.

According to previous studies, amiloride-sensitive (Amil+) Na+ channels are present in apical membranes of airway epithelial cells. When isolated from intact tissue and grown in primary culture or as immortalized cell lines, these cells tend to lose these Amil+ Na+ channels. The present study examines this issue in immortalized human bronchial epithelial cells (16HBE14o- cell line). The mRNA of one subunit of the Na+ channel alphahENaC) was semi-quantified by polymerase chain reaction of reverse transcribed RNA. Transcripts were significantly increased when cells were exposed to aldosterone and dexamethasone irrespective of whether grown on permeable supports or plastic. When grown on plastic dishes 16HBE14o-cells showed cAMP-dependent Cl- currents in whole-cell (WC) patch-clamp experiments, corresponding to expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Na+ currents could not be detected although cells expressed significant amounts of alphahENaC as demonstrated by Northern blot analysis. In contrast, when cells were grown on permeable supports or cultured in the presence of butyrate (5 mmol/l, plastic or permeable support) or aldosterone and dexamethasone (both 1 micromol/l, plastic or permeable support), amiloride (10 micromol/l) hyperpolarized the membrane voltage (deltaVm) by 2-9 mV, paralleled by small reductions of WC conductances (deltaGm) of 0.4-4.0 nS. The effects of amiloride on deltaVm were gnerally more pronounced (up to 12 mV) when cells were grown on permeable supports. The amiloride effect (deltaVm) was concentration dependent with an inhibitory constant, Ki, of about 0.1 micromol/l. We further examined whether the induction of an Amil+ Na+ conductance was paralleled by additional changes in membrane conductance. In fact, the cAMP-activated Cl- conductance was significantly attenuated by approximately 80% (n=35) in cells responding to amiloride, whilst the ATP-activated K+ conductance remained unaffected. The present data suggest that cellular mechanisms determining differentiation control the function expression of Na+ and Cl- conductances in human airway epithelial cells.

Wild type but not deltaF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes.

Airway epithelial cells bearing mutations of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) possess an increased Na+ conductance along with their well described defect of cAMP dependent Cl- conductance. Currently it is not clear, how this occurs, and whether it is due to a CFTR control of epithelial Na+ conductances which might be defective in CF patients. In the present study, we have tried to identify possible interactions between both CFTR and the epithelial Na+ conductance by overexpressing respective cRNAs in Xenopus oocytes. The expression of all three (alpha, beta, gamma) subunits of the rat epithelial Na+ channel (rENaC) and wild type (wt) CFTR resulted in the expected amiloride sensitive Na+ and IBMX (1 mmol/l) activated Cl- currents, respectively. The amiloride sensitive Na+ conductance was, however, inhibited when the wt-CFTR Cl- conductance was activated by phosphodiesterase inhibition (IBMX). In contrast, IBMX had no such effect in deltaF508 and Na+ channels coexpressing oocytes. These results suggest that wt-CFTR, but not deltaF508-CFTR, is a cAMP dependent downregulator of epithelial Na+ channels. This may explain the higher Na+ conductance observed in airway epithelial cells of CF patients.

Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.

Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.