Hyperpolarization Induced by Lipopolysaccharides but Not by Chloroform Is Inhibited by Doxapram, an Inhibitor of Two-P-Domain K+ Channel (K2P)

Abstract

Bacterial septicemia is commonly induced by Gram-negative bacteria. The immune response is triggered in part by the secretion of bacterial endotoxin lipopolysaccharide (LPS). LPS induces the subsequent release of inflammatory cytokines which can result in pathological conditions. There is no known blocker to the receptors of LPS. The Drosophila larval muscle is an amendable model to rapidly screen various compounds that affect membrane potential and synaptic transmission such as LPS. LPS induces a rapid hyperpolarization in the body wall muscles and depolarization of motor neurons. These actions are blocked by the compound doxapram (10 mM), which is known to inhibit a subtype of the two-P-domain K+ channel (K2P channels). However, the K2P channel blocker PK-THPP had no effect on the Drosophila larval muscle at 1 and 10 mM. These channels are activated by chloroform, which also induces a rapid hyperpolarization of these muscles, but the channels are not blocked by doxapram. Likewise, chloroform does not block the depolarization induced by doxapram. LPS blocks the postsynaptic glutamate receptors on Drosophila muscle. Pre-exposure to doxapram reduces the LPS block of these ionotropic glutamate receptors. Given that the larval Drosophila body wall muscles are depolarized by doxapram and hyperpolarized by chloroform, they offer a model to begin pharmacological profiling of the K2P subtype channels with the potential of identifying blockers for the receptors to mitigate the actions of the Gram-negative endotoxin LPS.

The Effects of Doxapram Blocking the Response of Gram-Negative Bacterial Toxin (LPS) at Glutamatergic Synapses

“…Prior investigations illustrated that LPS from various strains of bacteria enhances the amplitude of the EJP at the crayfish NMJ [ 11 , 16 , 17 , 28 ]. At the larval Drosophila NMJ, doxapram partially blocked the effect of LPS on increasing the heart rate in larval Drosophila [ 29 ].…”

“…Analysis of responses used the amplitudes of the EJPs from the short-term facilitation train of pulses from the 25 or 40 stimuli at 40 Hz or 60 Hz, respectively, as previously described [ 27 ]. The recording details and general experimental procedures examining the effects of LPS have been described previously [ 11 , 16 , 17 ]. The amplitude of the 25th EJPs are measured from the proceeding trough to the peak response ( Figure 1 B).…”

“…Not all the cellular effects of LPS appear to be mediated via the receptor complex mentioned above. There are also direct actions on ion channels such as reducing potassium currents in mammalian cells [ 10 ] and blocking glutamatergic receptors to reduce synaptic efficacy in Drosophila [ 11 ]. In addition, it is of interest to address the rapid acute responses to long-term LPS exposure which likely recruits secondary immune reactions.…”

“…If LPS, as a compound, can avoid being caught by the innate immunity response in the hemolymph of invertebrates and reach tissues, there appears to also be a rapid action of LPS on tissues. This has been demonstrated by using isolated nerves and muscles as well as cardiac tissues flushed with saline to remove any hemolymph after the tissue has been removed from the animal or held in situ [ 11 , 16 , 17 ].…”

“…The direct action of LPS on the larval Drosophila heart and neuromuscular junctions is speculated to alter the membrane potential by transiently activating a K + channel subtype known as a K2P (two-P-domain K + subunits) subtype, which helps to maintain the resting membrane potential of the cells [ 2 , 18 ]. The compound, doxapram (trade names, Stimulex or Respiram), blocked the acute actions of LPS from Serratia marcescens [ 11 , 19 ]. Recently, it was demonstrated that LPS from Serratia marcescens also has rapid effects on the NMJs of crayfish but with different effects than those reported for the NMJs of the larval Drosophila .…”