Ionic mechanisms of toluene cerebrovascular actions

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Acute intoxication with toluene (Tol) constitutes a worldwide public health problem. Human and animal data demonstrate that acute Tol intoxication is associated with brain hypoperfusion. The decrease in blood flow is a significant determinant of Tol-induced long-term neurological deficits and catastrophic acute scenarios, including death. Remarkably, the biological targets and mechanisms underlying Tol-induced reduction in cerebral perfusion are unknown. Our preliminary data from rat and mouse show that, consistent with hypoperfusion, acute exposure to intoxicating concentrations of Tol leads to cerebral artery constriction both in vitro and in live animals. Thus, we will cover the current knowledge gap in neurovascular toxicology by departing from all previous work, which focused on Tol effects on central neuron ion channels, to address this overarching hypothesis: constriction of cerebral arteries by acute Tol exposure is primarily due to drug inhibition of potassium channels of the BK type present in the arterial smooth muscle (SM) itself. This drug action is determined by distinct sensing of Tol by the two BK subunits that give rise to the SM BK phenotype: channel- forming cbv1, which enables drug action through its cytosolic tail domain, and the SM-abundant, regulatory β1, which downregulates Tol actions on both channel and cerebral artery function. We will address three conceptually related, yet independently testable specific aims (SA): SA1 (phenomenology) will establish that Tol at levels reached in blood and brain during acute intoxication constricts cerebral arteries independently of Tol systemic metabolism, circulating or endothelial factors but by primarily inhibiting BK, which only requires the two SM BK subunits in a bare lipid environment. SA2 (mechanism of drug action) will identify the specific roles of cbv1, β1, and allosteric gating processes that determine Tol action on BK activity and cerebral artery diameter. SA3 (translational aspects) will prove that naturally occurring variations in β1 levels determine the differential vulnerability of brain arterial branches to Tol-induced constriction, whereas this subunit can be used as therapeutic target of selective small agents to counteract Tol action on brain vessels. To test the proposed aims, we will use a multidisciplinary approach that includes Tol vapor exposure paradigms and a cranial window in vivo, in vitro myogenic tone determinations, novel and selective pharmacological tools, engineered mice, recombinant DNA and engineered BK subunits, electroporation of tissues with foreign cDNAs, biotinylation and Western blotting, lipid bilayer and patch-clamp electrophysiology, and allosteric gating analysis. We expect to unveil the cellular targets and molecular mechanisms that mediate Tol- induced cerebrovascular constriction and to deliver new selective pharmacological tools for early intervention in Tol-induced brain ischemia, while having minor side effects in other organs.