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The endothelin receptor A (ETA) and endothelin receptor B (ETB) are G protein-coupled receptors that are co-expressed in vascular smooth muscle cells. Endothelin-1 (ET-1) activates endothelin receptors to cause microvascular vasoconstriction. Previous studies have shown that heteromerization between ETA and ETB prolongs Ca2+ transients, leading to prolongation of Gαq-dependent signaling and sustained vasoconstriction. We hypothesized that these effects are in part mediated by the resistance of ETA/ETB heteromers to β-arrestin recruitment and subsequent desensitization. Using bioluminescence resonance energy transfer 2 (BRET2), we found that ETB has a relatively equal affinity to form either homomers or heteromers with ETA when co-expressed in the human embryonic kidney 293 (HEK293) cells. When co-expressed, activation of ETA and ETB by ET-1 caused a heteromer-specific reduction and delay in β-arrestin-2 recruitment with a corresponding reduction and delay in ET-1-induced ETA/ETB co-internalization. Furthermore, the co-expression of ETA and ETB inhibited ET-1-induced β-arrestin-1-dependent extracellular signal-regulated kinase (ERK) phosphorylation while prolonging ET-1-induced Gαq-dependent ERK phosphorylation. ETA/ETB heteromerization mediates the long-lasting vasoconstrictor response to ET-1 by the prolongation of Gαq-dependent signaling and inhibition of β-arrestin function.

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was −50 ± 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+ inward current and both sustained and transient outward K+ currents. Sustained outward K+ current was reduced (55–77%) by 5 mM tetraethylammonium and to a lesser extent (42–46%) by superfusion with nominally Ca2+ free external solution. Transient outward current was blocked by 100 μm 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to −50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+ and K+ currents giving rise to action potentials.Key words: fluorescent tracer, patch clamp, neuron, ciliary ganglion.

In bullfrog sympathetic B cells, axotomy decreases the amplitude and decay time of membrane afterhyperpolarization (AHP) and increases action potential (AP) duration. A second (test) axotomy, 7 days after an initial (conditioning) axotomy, did not amplify these changes. No recovery of AHP amplitude or AP duration occurred by 56 days post-axotomy, but AHP decay time recovered 21 days earlier than after test axotomy alone. Conditioning, previously shown to accelerate regeneration, speeds the return to normal of those membrane properties previously shown to recover after axotomy.