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 ß1,
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, ß1, 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 ß1 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.