Distinct Receptors, Second Messengers and Conductances Underlying the Dual Responses to Serotonin in an Identified Leech Neurone

Pressure-sensitive mechanosensory (P) neurones of the leech Hirudo medicinalis produce two responses to serotonin (5-HT): activation of a Cl⁻conductance and of a non-selective monovalent cation conductance. The effects of channel blockers, the receptor pharmacology and the second-messenger dependence of these responses were studied in voltage-clamped P cells in culture. Antagonists were applied by superfusion and agonists by pressure ejection.

Zn²⁺ (100μmoll⁻¹) and H⁺ (pH 6.5 and lower) reversibly reduced the Cl⁻conductance activated by 5-HT. The cation conductance was impermeant to calcium ions and was reduced by micromolar concentrations of the Na⁺ channel inhibitors amiloride and 3,4-dichlorobenzamil.

High concentrations of antagonists or agonists of 5-HT₁ receptors and an antagonist of 5-HT₃ receptors had no effect on either response of P cells to 5-HT. Micromolar concentrations of ketanserin or cyproheptadine, which selectively antagonize 5-HT₂ receptors, reduced the cation but not the Cl⁻ conductance. From these results, the receptor underlying the cation conductance appears to be of the 5-HT₂ subtype, whereas the receptor activating the Cl⁻ conductance does not fit within the mammalian classification scheme.

Brief (<500 ms) application of membrane-permeant agonists of the second messenger cyclic AMP elicited a Cl⁻ conductance, whereas antagonists of cyclic-AMP-dependent protein kinase A reversibly suppressed the Cl⁻ conductance elicited by 5-HT and by cyclic AMP agonists. Compounds affecting other second messenger pathways were without effect on the Cl⁻ conductance. It therefore appears that the Cl⁻ conductance is activated by cyclic-AMP-dependent protein kinase A.

Cyclic nucleotide agonists and antagonists were without effect on the cation conductance. However, brief application of phorbol esters, which activate protein kinase C, elicited an amiloride-sensitive cation current. An inhibitor of protein kinase C reduced the cation conductance activated by 5-HT and by phorbol esters. Therefore, the cation conductance appears to depend on activation of protein kinase C.

We conclude that 5-HT activates two types of receptor coupled to separate ionic channels via different second messenger pathways in P cells. A receptor that is distinct from the mammalian subtypes activates Cl⁻ channels via cyclic-AMP-dependent protein kinase A. 5-HT₂ receptors appear to activate cation channels by means of protein kinase C.