Hans Joachim Lang

INHIBITION OF IKS IN GUINEA PIG CARDIAC MYOCYTES AND GUINEA PIG ISK CHANNELS BY THE CHROMANOL 293B

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K+ conductance in rat colon crypts. Subsequent studies on cloned K+ channels from the rat demonstrated that 293B blocks specifically IsK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K+ conductances IKs and IKr, and for the cloned guinea pig IsK channel and the human HERG channel, which underly IKs and IKr, respectively, was analyzed. 293B inhibited both the slowly activating K+ conductance IKs in cardiac myocytes and guinea pig IsK channels expressed in Xenopus oocytes with a similar IC50 (2-6 μmol/1). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating IKr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of IKs and the underlying IsK channels.

The role of the IsK protein in the specific pharmacological properties of the IKs channel complex

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage‐dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17‐β‐oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6–100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady‐state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

Effect of inhibitors of Na+/H+‐exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N‐formyl‐Met‐Leu‐Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs. Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling. Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP‐induced cell swelling. Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP. H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP‐induced increase in CV. Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP‐stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited. The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole. Western analysis with a monoclonal anti gastric H+/K+ATPase β‐subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs. The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+‐dependent H+‐extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters suppresses the increase in CV and precludes PMNs from stimulated migration.

KVLQT channels are inhibited by the K+ channel blocker 293B.

Abstract Previous data have indicated that the chromanol 293B blocks a cAMP activated K+ conductance in the colonic crypt, a K+ conductance in pig cardiac myocytes and the K+ conductance induced by IsK protein expression in Xenopus oocytes. We have also shown that cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) up-regulates, apart from the typical Cl–current, a 293B- inhibitable K+ current. Very recently it has been shown that the IsK protein interacts with KVLQT subunits to produce a K+ channel. These data have prompted us to ask the following questions: Is the 293B-inhibitable current in oocytes expressing CFTR and activated by cAMP caused by an endogenous Xenopus KVLQT (XKVLQT), and is mouse KVLQT (mKVLQT) expressed in oocytes inhibited by 293B? Antisense and sense probes for XKVLQT were coinjected with CFTR cRNA into oocytes. After 3–4 days the oocytes were examined by two electrode voltage clamp. It was found that in control oocytes expressing CFTR and stimulated by isobutylmethylxanthine (IBMX, 1 mmol/l) 293B (10 μmol/l) reduced the conductance (Gm). In oocytes coinjected with the sense probe for XKVLQT and pretreated with IBMX 293B still reduced Gm, whilst the 293B-inhibitable Gm was almost completely absent in oocytes coinjected with XKVLQT antisense. In another series a full length clone for mKVLQT was generated by PCR techniques and the cRNA was injected into oocytes. After several days these oocytes, unlike water injected ones, were found to be strongly hyperpolarized and their Gm was increased significantly. The oocytes were depolarized significantly and their Gm was reduced reversibly by 10 μmol/l 293B. These data indicate that CFTR activation by IBMX indeed co-activates an endogenous oocyte XKVLQT channel and that this channel is inhibited by a new class of channel blockers, of which 293B is the prototype.

Cell shrinkage stimulates bradykinin-induced cell membrane potential oscillations in NIH 3T3 fibroblasts expressing the ras-oncogene

In NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) bradykinin leads to sustained oscillations of cell membrane potential due to oscillations of intracellular Ca2+ with subsequent activation of Ca2+-sensitive K+ channels. In cells not expressing the oncogene (-ras), bradykinin leads only to a single transient hyperpolarization of the cell membrane. The present study has been performed to elucidate the possible interaction of cell volume, intracellular pH and bradykinin-induced oscillations of the cell membrane potential. Bradykinin leads to cell shrinkage and intracellular alkalinization of both +ras cells and −ras cells. Inhibition of Na+/H+ exchanger by HOE 694 abolishes the bradykinin-induced alkalinization but does not significantly interfere with the bradykinin-induced oscillations of cell membrane potential. In contrast, prevention of bradykinin-induced cell shrinkage by simultaneous reduction of extracellular osmolarity blunts the oscillations. Thus, cell shrinkage stimulates bradykinin-induced oscillations of cell membrane potential. On the other hand, cell shrinkage alone does not elicit oscillations unless, in addition, Ca2+ entry is stimulated by ionomycin.

EFFECTS OF BRADYKININ ON NIH 3T3 FIBROBLASTS PRETREATED WITH LITHIUM MIMICKING EVENTS OF HA-RAS ONCOGENE EXPRESSION

As shown previously, expression of Ha-ras oncogene in NIH 3T3 fibroblasts (+ ras cells) increases cellular concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and enhances bradykinin induced Ca2+ entry [1-3]. These cells respond to low concentrations of serum or bradykinin with sustained oscillations of the cell membrane potential due to pulsatile release of calcium from internal stores and subsequent activation of calcium sensitive K+ channels [1]. Furthermore Ha-ras oncogene expression leads to depolymerization of the actin filament network and delayed increase of cell volume [4-6]. Pretreatment of the same cells not expressing the oncogene (-ras cells) with Li+ similarly increases Ins(1,4,5)P3 and Ins(1,3,4,5)P4 [2]. As shown in the present study, -ras cells pretreated with Li+ similar to Ha-ras oncogene expressing cells respond to bradykinin with sustained oscillations of cell membrane potential, depolymerization of the actin filament network and increase of cell volume. The oscillations of the cell membrane potential and the depolymerization of the actin cytoskeleton can be inhibited by the calcium channel blocker lanthanum and the bradykinin induced increase of cell volume is inhibited by HOE 694, pointing to involvement of Na+/H+ exchange. The data indicate a close functional linkage of the calcium oscillations, cytoskeletal rearrangement and activation of the Na+/H+ exchanger. Thus, Li+ pretreatment mimicks crucial cellular events triggered by expression of the Ha-ras oncogene. However, unlike in cells expressing the Ha-ras oncogene, Li+ pretreatment alone does not allow for growth factor-independent proliferation of the cells.