Proceedings of The Physiological Society
Durham University (2010) Proc Physiol Soc 21, C02 and PC02
The molecular and cellular identity of peripheral osmoreceptors
S. G. Lechner1, S. Markworth1, K. Poole1, E. S. Smith1, L. Lapatsina1, F. C. Luft3, J. Jordan2, G. R. Lewin1
1. Max-Delbrueck-Centre for Molecular Medicine, Berlin, Germany. 2. Institute for Clinical Pharmacology, Medical School Hannover, Hannover, Germany. 3. ECRC, Charite, Berlin, Germany.
The osmolality of the extra cellular body fluid (ECF) is subject to permanent changes due to salt/water intake and excretion. As large deviations from the normal osmolality have fatal effects on the physical and functional integrity of cells and tissues, afferent systems have evolved that detect hypo- or hyperosmotic shifts in the ECF and trigger homeostatic control to keep osmolality at a well defined physiological set point. It had previously been suggested, that in addition to central osmoreceptors, peripheral osmoreceptors might exist which could contribute to the regulation of ECF osmolality. Here we show that oral administration of 1ml of water in awake mice causes a drop in blood osmolality (-8%) in the liver portal vein (for blood sampling mice were anaesthetised by i.p. injection of 0.3ml PBS containing 10mg/ml Ketavet and 0.04% ROMPUN ), which results in the activation of pERK (phosphorylated form of ERK) in hepatic afferent endings from thoracic DRG neurons. We quantified pERK immunoreactvity in sensory fibers around blood vessels as this is a reliable marker of neuronal activation. Fura-2 calcium imaging experiments on thoracic neurons showed that osmosensitive neurons are enriched in these ganglia (33%, n=167). We further show that the hypo-osmotically induced calcium transients occur concurrently with cell swelling and depend on extracellular Ca2+, but are not affected by depletion of intracellular calcium stores with thapsigargin or by blockade of voltage-gated calcium channels with Cadmium. Patch-clamp recordings with simultaneous fura-2 imaging revealed that a non-selective outwardly rectifying cation channel, which is half-maximally activated at ∼278mOsm, mediates the increases in intracellular calcium. Electrophysiological experiments on retrogradely labelled sensory neurons that project to the liver revealed that almost all thoracic liver afferents are osmosensitive (20/23), but none of the vagal hepatic afferents in the nodose ganglia, are osmosensitive (0/16). Members of the transient receptor potential (TRP) ion channel family, in particular TRPV1 and TRPV4, have been suggested to play a role in osmosensing and consistently the TRP channel blockers Ruthenium Red and Gd3+ abolished calcium transients and osmosensitive inward currents in thoracic sensory neurons. Using qRT-PCR we could show that TRPV4 mRNA is enriched in thoracic DRGs and immunostaining of liver sections confirmed that TRPV4 is present at the peripheral nerve endings of hepatic afferent fibres. Strikingly, in mice lacking TRPV4, hepatic sensory neurons no longer exhibit osmosensitive inward currents and the in-vivo activation of liver afferents following water intake is abolished. Taken together we have identified a new population of thoracic sensory neurons that serve as peripheral osmoreceptors and show that they require the osmosensitive ion channel TRPV4 to sense small ongoing changes in hepatic blood osmolality.
Where applicable, experiments conform with Society ethical requirements