Investigations into hydrogen sulfide-induced suppression of neuronal activity in vivo and calcium dysregulation in vitro.

Acute exposure to high concentrations of hydrogen sulfide (H2S) leads to sudden death and, if survived, lingering neurological disorders. Clinical signs include seizures, loss of consciousness, and dyspnea. The proximate mechanisms underlying H2S-induced acute toxicity and death have not been clearly elucidated. We investigated electrocerebral, cardiac and respiratory activity during H2S exposure using electroencephalogram (EEG), electrocardiogram (EKG) and plethysmography. H2S suppressed electrocerebral activity and disrupted breathing. Cardiac activity was comparatively less affected. To test whether Ca2+ dysregulation contributes to H2S-induced EEG suppression, we developed an in vitro real-time rapid throughput assay measuring patterns of spontaneous synchronized Ca2+ oscillations in cultured primary cortical neuronal networks loaded with the indicator Fluo-4 using the fluorescent imaging plate reader (FLIPR-Tetra®). Sulfide >5 ppm dysregulated synchronous calcium oscillation (SCO) patterns in a dose-dependent manner. Inhibitors of NMDA and AMPA receptors magnified H2S-induced SCO suppression. Inhibitors of L-type voltage gated Ca2+ channels and transient receptor potential channels prevented H2S-induced SCO suppression. Inhibitors of T-type voltage gated Ca2+ channels, ryanodine receptors, and sodium channels had no measurable influence on H2S-induced SCO suppression. Exposures to > 5 ppm sulfide also suppressed neuronal electrical activity in primary cortical neurons measured by multi-electrode array (MEA), an effect alleviated by pretreatment with the nonselective transient receptor potential channel inhibitor, 2-APB. 2-APB also reduced primary cortical neuronal cell death from sulfide exposure. These results improve our understanding of the role of different Ca2+ channels in acute H2S-induced neurotoxicity and identify transient receptor potential channel modulators as novel structures with potential therapeutic benefits.