Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells.
The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E(rev)) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E(rev) of nicotine-induced current as a function of extracellular Na(+) concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K(+)/Na(+) permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca(2+) concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na(+), which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D018679:Cholinergic Agonists / Q000494:pharmacology; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000502:physiology*; D016257:Fura-2 / Q000494:pharmacology; D005786:Gene Expression Regulation; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008833:Microcirculation / Q000502:physiology*; D009538:Nicotine / Q000494:pharmacology; D021122:Protein Subunits / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology
D018679:Cholinergic Agonists; D017931:DNA Primers; D021122:Protein Subunits; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D009538:Nicotine; D012964:Sodium; D000109:Acetylcholine; D016257:Fura-2