Roland Z. Kozlowski

BIOPHYSICAL PROPERTIES OF CA2- AND MG-ATP-ACTIVATED K+ CHANNELS IN PULMONARY ARTERIAL SMOOTH MUSCLE CELLS ISOLATED FROM THE RAT

A novel class of Ca2+-activated K+ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these “KCa,ATP” channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of KCa,ATP channels to voltage, intracellular Ca2+ ([Ca2+]i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K+ and are approximately 20 times more selective for K+ ions than Na+ ions, with a K+ permeability (PK) of 4.6×10−13cm s−1. Ca2+ ions applied to the intracellular membrane surface of KCa,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca2+ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of KCa,ATP channels by Mg-ATP has an EC50 of approximately 50 μM. Although the EC50 is unaffected by [Ca2+]i, channel activation by Mg-ATP is enhanced by increasing [Ca2+]i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of KCa,ATP channels to Ca2+. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of KCa,ATP channels to Ca2+ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca2+ through voltage-activated Ca2+ channels, ultimately leading to increased vascular tone.

Electrophysiological effects of endothelin-1 and their relationship to contraction in rat renal arterial smooth muscle

The electophysiological effects of endothelin‐1 (ET‐1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch‐clamp measurements using freshly isolated smooth muscle cells from this tissue. Pharmacological experiments revealed that ET‐1 produced constriction of renal arteries dependent on the influx of extracellular Ca2+, mediated solely through ETA receptor stimulation. Current‐clamp experiments revealed that renal arterial myocytes had a resting membrane potential of ∼32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET‐1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly. Under voltage‐clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage‐ramps revealed an outward current activated at ∼−30 mV, sensitive to both 4‐AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K+ (KV) and Ca2+‐activated K+ (BKCa) channels. Under voltage‐clamp, ET‐1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ETA receptor stimulation. Thus, in conclusion, activation of ETA receptors by ET‐1 causes inhibition of KV and BKCa channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L‐type Ca2+ channel activity providing an influx pathway for extracellular Ca2+ essential for contraction.

KV2.1 channels mediate hypoxic inhibition of IKV in native pulmonary arterial smooth muscle cells of the rat

OBJECTIVE To determine whether, in native pulmonary arterial smooth muscle cells (PASMC), K(V)2.1 delayed-rectifying K(+) channels are central to the process of hypoxic pulmonary vasoconstriction. METHODS In this study, we tested for the presence of K(V)2.1 channel transcripts in rat small pulmonary arteries using RT-PCR, and for the protein itself using immunolocalisation. The contribution of K(V)2.1 channels to whole-cell K(V) currents (I(KV)) and their role in hypoxic inhibition of I(KV) in native PASMC was investigated utilising patch-clamp recordings. RESULTS K(V)2.1 mRNA expression and AbK(V)2.1 (anti-K(V)2.1 antibody) protein immunoreactivity were both present in small pulmonary arteries. Dialysis of PASMC with AbK(V)2.1 significantly attenuated I(KV) by 67% at +50 mV. Hypoxia ( approximately 20-30 mmHg) inhibited I(KV) by approximately 70% at +50 mV. Ablation of currents associated with K(V)2.1 using AbK(V)2.1 caused a marked reduction in the amplitude of I(KV). Hypoxia in the presence of the antibody did not affect the magnitude of I(KV). CONCLUSIONS These results indicate that K(V)2.1 channel subunits exist within small pulmonary arteries and conduct a significant part of I(KV) within native PASMC. Furthermore, application of AbK(V)2.1 abolishes hypoxic inhibition of I(KV) in native PASMC suggesting that K(V)2.1 channels play a pivotal role in mediating hypoxic pulmonary vasoconstriction.

Endothelial Cells Freshly Isolated From Small Pulmonary Arteries of the Rat Possess Multiple Distinct K+ Current Profiles

This study demonstrates for the first time that endothelial cells freshly isolated from small pulmonary arteries of the rat, based on their electrophysiological profile, possess two distinct populations of cells. Immunohistochemical staining revealed the presence of both anti-Kv1.5 and anti-Kir2.1 immunoreactivity in the endothelium of small pulmonary arteries. Patch-clamp studies demonstrated that 90% of cells studied exhibited an electrophysiological profile that was characterized by a delayed rectifier K+ conductance. However, the remaining 10% of cells studied showed the complete absence of a delayed rectifier K+ current and were characterized by an inward rectifier K+ conductance. Together these results indicate that endothelial cells isolated from rat small pulmonary arteries possess a heterogeneous population of cells that may be distinguished by their markedly different electrophysiological profiles. These different populations of cells may differ in their control of the resting membrane potential of endothelial cells, and thereby altering Ca2+ homeostasis and release of vasoactive compounds. These findings may therefore have important implications for understanding the regulation of pulmonary vascular tone.

Volume‐Sensitive Organic Osmolyte/Anion Channels in Cancer: Novel Approaches to Studying Channel Modulation Employing Proteomics Technologies

Abstract: Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume‐sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull‐down assays, yeast 2‐hybrid, and functional protein arrays have already proved valuable in studying protein‐protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein‐protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein‐protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

Cardiac chloride channels: physiology, pharmacology and approaches for identifying novel modulators of activity

Drugs that block cardiac cation channels have been marketed as the therapeutic answer to cardiac arrhythmia. However, such molecules have been only moderately successful at improving the survival of cardiac patients, and so new targets have been needed for future antiarrhythmic agents. This article outlines the properties and roles of Cl(-) channels, which are one of these new targets, and describes an approach for identifying novel CI(2) channel modulators.

Inhibition of ERK and JNK Decreases Both Osmosensitive Taurine Release and Cell Proliferation in Glioma Cells

Cell swelling is associated with the activation of an increase in the osmosensitive taurine release (OTR) rate, which serves to decrease cell volume as part of a process known as regulatory volume decrease. OTR, which is sensitive to many pharmacological agents including anion channel blockers and signalling pathway modulators, has also been suggested to play a role in cell cycle progression. At non-cytotoxic concentrations, the anion channel blocker NPPB (25 μM), the extra-cellular signal-regulated kinase inhibitor PD98059 (50 μM), and the c-Jun NH2-terminal kinase inhibitor SP 600125 (5 μM) each decreased the OTR rate by ≥50%, decreased cell proliferation, and increased G0/G1 cell cycle arrest.

DIFFERENTIAL EFFECTS OF PYRETHROIDS ON VOLUME-SENSITIVE ANION AND ORGANIC OSMOLYTE PATHWAYS

1. There are no effective ways of screening for potential modulators of volume‐regulated anion channels in their native cell type. Generally, cell lines are used for this purpose. Using HeLa and C6 glioma cells, we identified the pyrethroids as a novel class of compounds that inhibit taurine efflux through volume‐regulated anion transport pathways in these cells. Subsequently, we examined their effects on volume‐regulated anion channels in guinea‐pig ventricular myocytes to determine whether results obtained using cell lines could be extrapolated to other tissues.

Actions of the anti-oestrogen agent clomiphene on outward K+ currents in rat ventricular myocytes

1. The effects of clomiphene (CLM) on cardiac outward K+ current components from rat isolated ventricular myocytes were investigated using the whole‐cell patch‐clamp technique. Clomiphene (10 µmol/L) significantly inhibited both peak (Ipeak) and end‐pulse (Ilate) outward currents (elicited by a 500 msec voltage step from −40 to +50 mV in the presence of K+‐containing intracellular and extracellular solutions) by approximately 37% (n = 6; P < 0.01) and 49% (n = 6; P < 0.01), respectively. In contrast, CLM had no effect on outward currents when K+‐free solutions were used.

Open day review: bridging the gap between academia and industry.

In conclusion, the meeting was viewed positively by academics, industrialists and administrators. The scientists generated considerable interest with some positive follow-up. Above all, it was clear that Bristol University is working to support the bridge that already spans the gap between academic and industrial pharmacology.