The CamKKß Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells.

It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKß, in the acute response to hypoxia in isolated mouse type I cells.Initial data indicated several previously unreported artefacts associated with using the CamKKß inhibitor STO609 and Ca(2+) imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca(2+) ([Ca(2+)](I)). Furthermore, STO609 (100 µM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK(Ca) inhibitor paxilline.Taken together these data suggest that ST0609 should be used with caution during Ca(2+) imaging studies as it can directly interact with Ca(2+) binding dyes. The rapid inhibitory effect of STO609 on BK(Ca) was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK(Ca), inhibiting AMPK's upstream kinases would, if anything, be predicted to have the opposite effect on BK(Ca). Future work will determine if the inhibition of BK(Ca) is via CamKKß or via an off target action of STO609 on the channel itself.

Acutely Administered Leptin Increases [Ca2+] i and BK Ca Currents But Does Not Alter Chemosensory Behavior in Rat Carotid Body Type I Cells.

Obesity related pathologies are the health care crisis of our generation. The fat cell derived adipokine leptin has been shown to be a central stimulant of respiration. Very high levels of leptin, however, are associated with the depressed ventilatory phenotype observed in obesity hypoventilation syndrome. Leptin receptors have been identified on carotid body type I cells but how their activation might influence the physiology of these cells is not known.The acute application of leptin evoked calcium signaling responses in isolated type I cells. Cells increased their Fura 2 ratio by 0.074 ± 0.010 ratio units (n = 39, P < 0.001). Leptin also increased the peak membrane currents in 6 of 9 cells increasing the peak macroscopic currents at +10 mV by 61 ± 14 % (p < 0.02). Leptin administered in the presence of the selective BK(Ca) channel inhibitor Paxilline (0.5 µM) failed to increase membrane currents (n = 5). Interestingly, leptin did not significantly alter the resting membrane potential of isolated type I cells (n = 9) and anoxic/acidic depolarizations were unaffected by leptin (n = 7, n = 6).These data suggest that leptin receptors are functional in type I cells but that their acute activation does not alter chemosensory properties. Future studies will use chronic models of leptin dysregulation.