Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells.
Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole-cell patch-clamp. Reverse transcription-polymerase chain reaction analysis indicated the presence of alpha2, alpha3, alpha4, alpha5, alpha7, beta2 and beta4 transcripts (alpha6 and beta3 could not be detected). Immunocytochemistry revealed most cells positive for alpha3, beta2, beta4 and alpha5 proteins. Few cells were immunoreactive for alpha2 and alpha4, while none was for alpha7. At single-cell level, colocalization could be demonstrated for alpha3alpha5 and alpha4beta2. Western blot analysis confirmed antibody specificity for alpha3, alpha4, alpha5, beta2 and beta4 subunits. Inward currents elicited by nicotine pulses were insensitive to alpha-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional alpha7 receptors. Partial block of nicotine currents was observed with either AuIB alpha-conotoxin (selective against alpha3beta4 receptors) or MII alpha-conotoxin (selective against alpha3beta2 receptors). With high concentrations of co-applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro-beta-erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise alpha3beta4, alpha3beta2 with the addition of alpha5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the alpha3beta4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.
Di Angelantonio
Silvia
S
Biophysics Sector and INFM Unit, International School for Advanced Studies (SISSA), 34014 Trieste, Italy.
Matteoni
Cosetta
C
Fabbretti
Elsa
E
Nistri
Andrea
A
D000311:Adrenal Glands / Q000187:drug effects / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D020916:Conotoxins / Q000494:pharmacology; D004079:Dihydro-beta-Erythroidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004594:Electrophysiology / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D008564:Membrane Potentials / Q000187:drug effects; D008967:Molecular Biology / Q000379:methods; D018733:Nicotinic Antagonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011978:Receptors, Nicotinic / Q000145:classification / Q000235:genetics* / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D020916:Conotoxins; D018733:Nicotinic Antagonists; D021122:Protein Subunits; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; C098872:alpha-conotoxin MII; C115558:conotoxin AuIB; D004079:Dihydro-beta-Erythroidine