The effects of doxapram and its potential interactions with K2P channels in experimental model preparations.

The channels commonly responsible for maintaining cell resting membrane potentials are referred to as K2P (two-P-domain K+ subunit) channels. These K+ ion channels generally remain open but can be modulated by their local environment. These channels are classified based on pharmacology, pH sensitivity, mechanical stretch, and ionic permeability. Little is known about the physiological nature of these K2P channels in invertebrates. Acidic conditions depolarize neurons and muscle fibers, which may be caused by K2P channels given that one subtype can be blocked by acidic conditions. Doxapram is used clinically as a respiratory aid known to block acid-sensitive K2P channels; thus, the effects of doxapram on the muscle fibers and synaptic transmission in larval Drosophila and crawfish were monitored. A dose-dependent response was observed via depolarization of the larval Drosophila muscle and an increase in evoked synaptic transmission, but doxapram blocked the production of action potentials in the crawfish motor neuron and had a minor effect on the resting membrane potential of the crawfish muscle. This indicates that the nerve and muscle tissues in larval Drosophila and crawfish likely express different K2P channel subtypes. Since these organisms serve as physiological models for neurobiology and physiology, it would be of interest to further investigate what types of K2P channel are expressed in these tissues. (212 words).

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

Lipopolysaccharides (LPS) associated with Gram-negative bacteria are one factor responsible for triggering the mammalian immune response. Blocking the action of LPS is key to reducing its downstream effects. However, the direct action of LPS on cells is not yet fully addressed. LPS can have rapid, direct effects on cells in the absence of a systemic immune response. Recent studies have shown that doxapram, a blocker of a subset of K2P channels, also blocks the acute actions of LPS. Doxapram was evaluated to determine if such action also occurs at glutamatergic synapses in which it is known that LPS can increase synaptic transmission. Doxapram at 5 mM first enhanced synaptic transmission, then reduced synaptic response, while 10 mM rapidly blocked transmission. Doxapram at 5 mM blocked the excitatory response induced by LPS. Enhancing synaptic transmission with LPS and then applying LPS combined with doxapram also resulted in retarding the response of LPS. It is possible doxapram and LPS are mediated via a similar receptor or cellular responses. The potential of designing pharmacological compounds with a similar structure to doxapram and determining the binding of such compounds can aid in addressing the acute, direct actions by LPS on cells.

The effects of Gram-positive and Gram-negative bacterial toxins (LTA & LPS) on cardiac function in Drosophila melanogaster larvae.

The effects of Gram negative and positive bacterial sepsis depend on the type of toxins released, such as lipopolysaccharides (LPS) or lipoteichoic acid (LTA). Previous studies show LPS to rapidly hyperpolarize larval Drosophila skeletal muscle, followed by desensitization and return to baseline. In larvae, heart rate increased then decreased with exposure to LPS. However, responses to LTA, as well as the combination of LTA and LPS, on the larval Drosophila heart have not been previously examined. This study examined the effects of LTA and a cocktail of LTA and LPS on heart rate. The combined effects were examined by first treating with either LTA or LPS only, and then with the cocktail. The results showed a rapid increase in heart rate upon LTA application, followed by a gradual decline over time. When applying LTA followed by the cocktail, an increase in the rate occurred. However, if LPS was applied before the cocktail, the rate continued declining. These responses indicate the receptors or cellular cascades responsible for controlling heart rate within seconds and the rapid desensitization are affected by LTA or LPS and a combination of the two. The mechanisms for rapid changes which are not regulated by gene expression by exposure to LTA or LPS or associated bacterial peptidoglycans have yet to be identified in cardiac tissues of any organism.

Measuring Electrical Responses during Acute Exposure of Roots and Rhizoids of Plants to Compounds Using a Flow-Through System.

Monitoring electrical signals in plants allows the examination of their acute and chronic physiological changes and responses to stimuli. Understanding how plant roots/rhizoids respond to chemical cues in their environment will provide insight into how these structures acquire resources. Chronic exposure to L-glutamate alters root growth and is known to alter Ca2+ flux inside roots. The ionic flux can be detected by electrical changes. A rapid and relatively easy approach is presented to screen the electrical sensitivity of roots/rhizoids to compounds such as amino acids and known agonists/antagonists to receptors and ion channels. The approach uses a background-flow system of basal salt or water; then, the administered compounds are added to the roots/rhizoids while monitoring their electrical responses. As a proof of concept, the response to flow-through of glutamate (1 mM) was targeted at the root/rhizoids of three plants (Arabidopsis thaliana, Pisum sativum and Marchantia inflexa). Both Arabidopsis thaliana and Pisum sativum produced rapid depolarization upon exposure to glutamate, while M. inflexa did not show an electrical response. In some experiments, simultaneous recordings with impedance measures for acute changes and glass electrodes for chronic electrical potential changes were used. The effect of potassium chloride (300 mM) as a depolarizing stimulus produced responses in both P. sativum and M. inflexa. The protocol presented can be used to screen various compounds in a relatively rapid manner for responsiveness by the roots/rhizoids of plants.