Differential actions of pituitary adenylate cyclase activating polypeptide PAC1 receptors on peripheral terminals and cell bodies of primary sensory neurons

É Szoke1, E Bánki0,2, T Bagoly1, D Reglodi0,2, Zs Helyes1. 1University of Pécs, Pharmacology and Pharmacotherapy, 7624, Hungary, 2University of Pécs, Anatomy, 7624, Hungary

Pituitary adenylate-cyclase activating polypeptide (PACAP) acts at G protein-coupled receptors: the specific PAC1 and VPAC1/VPAC2. PACAP6-38 was descibed as a potent PAC1/VPAC2 antagonist in several models. Maxadilan is a selective PAC1 agonist, its fragment, MAXA65, is a specific antagonist. Ala11,22,28VIP is a selective VPAC1 agonist, while BAY 55-9837 is a selective VPAC2 agonist. We have reported earlier that PACAP1-38 (the full-length peptide) and the PACAP6-38 fragment are both able to decrease the electrical-field stimulation-induced release of the sensory neuropeptide calcitonin gene-related peptide (CGRP) from sensory nerve endings of the isolated rat trachea. Therefore, the fragment did not behave as an antagonist in this system.

In the present study we aimed at analysing the actions of peptide fragments considered as agonists and antagonists on sensory neural responses in vitro.

Ratiometric technique of [Ca2+]i measurement with the fluorescent indicator fura-2-AM on primary cultures of trigeminal ganglia (TRG) neurons. Calcitonin gene-related peptide (CGRP) release from the stimulated (8 min) peripheral sensory nerve terminals of the isolated rat trachea was measured by radioimmunoassay and expressed as fmol/mg tissue weight.The PACAP and fragments were dissolved in saline and diluted to the final concentrations in extracellular solution or Krebs solution in the experiments on TRG neurons or on nerve terminals.

Slowly increasing [Ca2+]i indicating Gq protein-coupled receptor activation was detected both after PACAP1-38 and PACAP6-38 administration (90 sec). The percent of neurons responding to PACAP1-38 was 12.19%, 25%, 53,47%, 40.9% and 42.85% after 10, 30, 100, 500 nM and 1µM concentration, respectively. Similar result was detected after similar concentrations of PACAP6-38, the respective values were 3.5%, 8.3%, 54.08%, 48.14% and 50%. The co-administration of agonist and antagonist (100 nM) increased the fluorescence in 60% of the neurons.

The PAC1 receptor agonist maxadilan and the PAC1 receptor antagonist MAXA65 caused similar response, Ca-influx was measured after 100 nM maxadilan administartion (in 25% of neurons), after 300 nM MAXA65 (in 20% of neurons) as well as after their combination (in 22% of neurons) (number of neurons was 90 and 140 in one group). The VPAC2 receptor agonist BAY 55-9837 evoked Ca-influx in 33.7% of the neurons, in contrast, the VPAC1 receptor agonist Ala11,22,28VIP had no effect on [Ca2+]i .

Maxadilan, MAXA65 and BAY55-9837 significantly increased the electrical field stimulation-induced outflow of CGRP from the sensory nerve endings. This release increased by 280%, 250% and 230% after maxadilan, MAXA65 and BAY55-9837, respectively, compared to the control studies. The VPAC1 receptor agonist Ala11,22,28VIP had no significant effect on the CGRP outflow.

Conclusion: peptides considered as both agonist and antagonists in several tisssues at PAC1 and VPAC2 receptors stimulate the cell bodies and the stimulation-evoked response of the terminals of primary sensory neurons. Therefore, interestingly, they act as agonists on the sensory neurons. Presently unknown receptors or splice variants linked to distinct signal transduction pathways might explain these differences. The VPAC1 receptor does not play a role in these processes.