Endothelial Hyperpolarization in Humans

• Forearm Blood Flow [ Time Frame: Immediate ] [ Designated as safety issue: No ]
  

Drug: Tetraethylammonium (TEA)
Short duration (minutes) intra-arterial infusion.
Drug: L-NMMA
Short duration (minutes) intra-arterial infusion.
Drug: Bradykinin
Short duration (minutes) intra-arterial infusion.
Drug: Nitroprusside
Short duration (minutes) intra-arterial infusion.
Drug: S-methyl-L-thiocitrulline (SMTC)
Short duration (minutes) intra-arterial infusion.
Drug: Acetylcholine
Short term (minutes) intra-arterial infusion.

The vascular endothelium synthesizes at least four potent vasodilator substances: nitric oxide (NO), prostacyclin, carbon monoxide and endothelium-derived hyperpolarizing factor (EDHF) that contribute to vasodilator tone, and to inhibition of platelet activation and inflammation. EDHF release is stimulated by receptor-dependent agonists such as acetylcholine and bradykinin (BK), and leads to hyperpolarization of the underlying smooth muscle cells presumably by opening Ca2+-activated K+ channels. Indirect pharmacological evidence suggests that EDHF is a cytochrome P450-derived arachidonic acid metabolite, presumably an epoxide. Although the pivotal role of NO to conduit vessel dilation in response to acute increases in shear stress is well known, its' contribution to dilation with sustained increases in flow are minimal, and may be due to EDHF release.

We have recently demonstrated that miconazole, an inhibitor of cytochrome P-450, antagonized BK-mediated, endothelium-dependent vasodilation after inhibition of NO synthase and cyclooxygenase in the forearm circulation of healthy subjects. Reduced bioavailability of endothelium-derived NO in subjects with hypertension, hypercholesterolemia, diabetes and smoking contribute to the pathogenesis of this condition, but whether EDHF activity is modified in hypercholesterolemia remains unknown and will be investigated in this protocol.

In Specific Aim 1, we will test the hypotheses that in hypercholesterolemia, CYP450-derived epoxide release in response to bradykinin is increased and compensates for the reduced NO activity. We propose to study two different antagonists of CYP450, a non-specific blocker, miconazole and a specific CYP450 2C9 antagonist, sulphaphenazole.

Activation of calcium-dependent potassium channels (K+CA) leading to membrane hyperpolarization and vasodilation is a cardinal feature of EDHF release. Experimentally, in human vasculature K+CA channels are inhibitable by charybdotoxin and apamin. Clinical studies have used tetraethylammonium chloride (TEA) that antagonizes K+CA channels.

In Specific Aim 2, we will test the hypothesis that bradykinin-mediated forearm microvascular vasodilation results from activation of K+CA channels in vivo in healthy humans, and that this is altered in hypercholesterolemic patients. We propose to study whether TEA alone or in combination with CYP450 blockade will inhibit bradykinin-mediated vasodilation, allowing us to investigate whether sources other than CYP450-derived epoxides hyperpolarize smooth muscle cells.

Conductance vessel dilation in response to short periods of hyperemia have long been recognized to be secondary to release of NO, however, vasodilation fter sustained hyperemia is largely independent of NO release. Similarly, physiologic metabolic vasodilation of the microcirculation during exercise has a minor contribution from endothelium-derived NO release and the contribution of EDHF to metabolic forearm vasodilation in vivo remains unknown.

In Specific Aim 4, we will test the hypothesis that flow-mediated vasodilation during sustained hyperemia or during exercise is partly mediated by EDHF and that this contribution varies between healthy and hypercholesterolemic subjects.

Thus, the aim of these studies is to comprehensively establish the activity and identity of EDHF in the human forearm circulation, investigate its physiologic role in vasodilation, and determine how its activity is modulated by hypercholesterolemia, a condition that is accompanied by dysfunction of several endothelium-dependent functions