Diverse mechanisms underlying the regulation of ion channels by carbon monoxide.

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca(2+) -sensitive K(+) channels. More recent studies have revealed the ability of CO to inhibit T-type Ca(2+) channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.

AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Modulation of O(2) sensitive K (+) channels by AMP-activated protein kinase.

Hypoxic inhibition of K(+) channels in type I cells is believed to be of central importance in carotid body chemotransduction. We have recently suggested that hypoxic channel inhibition is mediated by AMP-activated protein kinase (AMPK). Here, we have further explored the modulation by AMPK of recombinant K(+) channels (expressed in HEK293 cells) whose native counterparts are considered O(2)-sensitive in the rat carotid body. Inhibition of maxiK channels by AMPK activation with AICAR was found to be independent of [Ca(2+)](i) and occurred regardless of whether the alpha subunit was co-expressed with an auxiliary beta subunit. All effects of AICAR were fully reversed by the AMPK inhibitor compound C. MaxiK channels were also inhibited by the novel AMPK activator A-769662 and by intracellular dialysis with the constitutively active, truncated AMPK mutant, T172D. The molecular identity of the O(2)-sensitive leak K(+) conductance in rat type I cells remains unclear, but shares similarities with TASK-1 and TASK-3. Recombinant TASK-1 was insensitive to AICAR. However, TASK-3 was inhibited by either AICAR or A-769662 in a manner which was reversed by compound C. These data highlight a role for AMPK in the modulation of two proposed O(2) sensitive K(+) channels found in the carotid body.

Inhibition of L-type Ca(2+) channels by carbon monoxide.

Inhibition of K(+) channels in glomus cells underlies excitation of the carotid body by hypoxia. It has recently been proposed that hypoxic inhibition involves either activation of AMP activated protein kinase (AMPK) or inhibition of carbon monoxide (CO) production by heme oxygenase 2 (HO-2). In the vasculature, L-type Ca(2+) channels are also O(2) sensitive. Here, we have investigated the possible involvement of either AMPK or CO in the hypoxic inhibition of L-type Ca(2+) channels. Using whole-cell patch clamp recordings from HEK293 cells stably expressing the human cardiac alpha1C(2+)channel subunit, we found that pre-treatment of cells with AICAR (to activate AMPK) was without effect on Ca(2+) currents. CO, applied via the donor molecule CORM-2 caused reversible, voltage-independent Ca(2+) channel inhibition of up to ca. 50%, whereas its inactive form (iCORM) was without significant effect. Effects of CO were prevented by the antioxidant MnTMPyP, but not by inhibition of NADPH oxidase (with either apocynin or diphenyleneiodonium), or xanthine oxidase (with allopurinol). Instead, inhibitors of complex III of the mitochondrial electron transport chain and a mitochondrial-targeted antioxidant (Mito Q), prevented the effects of CO. Our data suggest that hypoxic inhibition of L-type Ca(2+) channels does not involve AMPK or CO. However, the known cardioprotective effects of HO-1 could arise from an inhibitory action of CO on L-type Ca(2+) channels.