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Front Cell Neurosci
2019 Jan 01;13:114. doi: 10.3389/fncel.2019.00114.
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PACAP-38 and PACAP(6-38) Degranulate Rat Meningeal Mast Cells via the Orphan MrgB3-Receptor.
Pedersen SH
,
la Cour SH
,
Calloe K
,
Hauser F
,
Olesen J
,
Klaerke DA
,
Jansen-Olesen I
.
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Infusion of pituitary adenylate cyclase activating peptide-38 (PACAP-38) provokes migraine attacks in migraineurs and headache in non-migraineurs. Adverse events like long-lasting flushing and heat sensation can be terminated with oral antihistamine treatment, indicating the involvement of mast cell activation after PACAP-infusion. Degranulation of rat peritoneal mast cells was provoked by several isoforms of PACAP via previously unknown receptor pharmacology. The effect might thus be mediated either via specific splice variants of the PAC1-receptor or via an unknown receptor for PACAP-38. In the present study, we characterize degranulation of rat meningeal mast cells in response to PACAP-receptor ligands. Furthermore, we investigate if PACAP-38-induced mast cell degranulation is mediated via PAC1-receptor splice variants and/or via the orphan Mas-related G-protein coupled member B3 (MrgB3)-receptor. To address this, the pharmacological effect of different PACAP isoforms on meningeal mast cell degranulation was investigated in the hemisected skull model after toluidine blue staining followed by microscopic quantification. Presence of mRNA encoding PAC1-receptor splice variants and the MrgB3-receptor in rat mast cells was investigated by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis. The effect of PACAP isoforms on PAC1- and MrgB3-receptor-expressing Xenopus laevis oocytes were performed by two-electrode voltage-clamp (TEVC) electrophysiology. PACAP-38 is a more potent mast cell degranulating agent than Pituitary Adenylate Cyclase Activating Peptide-27 (PACAP-27) in the meninges. Presence of mRNA encoding the PAC1-receptor and its different splice variants could not be detected in peritoneal mast cells by RT-PCR, whereas the orphan MrgB3-receptor, recently suggested to be a mediator of basic secretagogues-induced mast cell degranulation, was widely present. In PAC1-receptor-expressing Xenopus laevis oocytes both PACAP-38, PACAP-27 and the specific PAC1-receptor agonist maxadilan were equipotent, however, only PACAP-38 showed a significant degranulatory effect on mast cells. We confirmed Pituitary Adenylate Cyclase Activating Peptide(6-38) [PACAP(6-38)] to be a PAC1-receptor antagonist, and we demonstrated that it is a potent mast cell degranulator and have an agonistic effect on MrgB3-receptors expressed in oocytes. The present study provides evidence that PACAP-induced mast cell degranulation in rat is mediated through a putative new PACAP-receptor with the order of potency being: PACAP-38 = PACAP(6-38) > > PACAP-27 = maxadilan. The results suggest that the observed responses are mediated via the orphan MrgB3-receptor.
Figure 1. Agarose gel electrophoresis showing the absence of mRNA-expression of PAC1-receptor splice variants in rat peritoneal mast cells (left) and their presence in rat spinal cord (right) using primers directed towards the N-terminal part of the PAC1-receptor (392 bp) and exon 14 and 15 of the Hop-Hop variant of the PAC1-receptor (upper amplicon 471 bp and lower 387 bp). Primers detecting β-actin (422 bp) was used as a positive control and was present in both tissues. The experiment was performed in peritoneal mast cells from three rats.
Figure 2. Agarose gel electrophoresis showing the RT-PCR-product corresponding the presence of mRNA encoding the MrgB3 receptor (259 bp) in rat peritoneal mast cells. No band is seen in the negative control [(−) control] where mRNA was not reverse transcribed to cDNA prior to amplification. The experiment was performed in peritoneal mast cells from three rats.
Figure 3. (A) Two-electrode voltage clamp performed on Xenopus laevis oocytes expressing the PAC1-receptor (n = 8–19, ncontrol = 6–16) showed that increasing concentrations (0.01–10 μM) of PACAP-38 and PACAP-27 cause a significant increase in membrane current. The inward currents are measured at a holding potential of −70 mV. In (B) the Xenopus laevis oocytes is expressing the MrgB3-receptor (n = 4–24, ncontrol = 3–13). In these oocytes only PACAP-38 induces a significant change in current (Vm = −70 mV). (C) Concentration-response curves of PACAP-38 and PACAP-27 on rat meningeal mast cell degranulation after 30 s stimulation at concentrations ranging from 0.1 to 10 μM. It was found that a significant mast cell degranulation only was observed after stimulation with PACAP-38. Values are given as percentage of degranulated mast cells of total number of counted mast cells (n = 5–6). *Represent p < 0.05, ***p < 0.001, and ****p < 0.0001 [two-way analysis of variance (ANOVA), Sidak’s multiple comparisons test]. All values are given as mean ± standard error of the mean (SEM).
Figure 4. Effect of PACAP-38, PACAP-27 and maxadilan (PAC1-receptor agonist) on (A) rat meningeal mast cell degranulation following 30 s of 10 μM ligand stimulation. PACAP-38-induced a strong degranulation of meningeal mast cells. The mast cells were unresponsive to PACAP-27 and maxadilan. Values are given as percentage of degranulated mast cells from the total number of counted mast cells, n = 5–11. (B) Measurements on PAC1-receptor-expressing Xenopus laevis oocytes using two-electrode voltage clamp showed similar changes in current during 60 s perfusion of PACAP-38, PACAP-27 or maxadilan (all ligands 0.1 μM, n = 14–21). (C) In MrgB3-receptor-expressing oocytes PACAP-38 and PACAP-27 were perfused in a concentration of 3 μM and maxadilan at 10 μM (n = 8–12). Only PACAP-38 caused a change in current. All measurements were done at a holding potential of −70 mV. In (A) ***represent p < 0.001 as compared to control (A). In (C)ns, p = 0.2879 (Mann-Whitney non-parametric t-test). **Represent p < 0.01 as compared to PACAP-38 (one-way ANOVA, Tukey’s multiple comparisons test). Values are given as mean ± SEM.
Figure 5. Representative traces showing a change in current after perfusion with PACAP-38 (A), PACAP-27 (B) and maxadilan (C) on PAC1-receptor-expressing Xenopus laevis oocytes (A to C). In MrgB3-receptor-expressing Xenopus laevis oocytes (D to F) PACAP-38 caused a change in current (D) while there was no change after perfusion with PACAP-27 (E) and maxadilan (F). The membrane potential was clamped to −70 mV. Scale bars represent 50 nA and 10 s.
Figure 6. (A) The PAC1-receptor antagonist PACAP(6–38; 10 μM) is as potent as PACAP-38 (10 μM) to induce degranulation of rat meningeal mast cells. Values are given as percentage of degranulated mast cells (****p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test), n = 4–7. (B) PACAP(6–38; 0.1 μM) is unable to induce currents in PAC1-receptor-expressing Xenopus laevis oocytes using two-electrode voltage-clamp (TEVC) after 1 min perfusion of ligand. However, PACAP(6–38) caused a significant inhibition of PACAP-38 induced responses (*p < 0.05, **p < 0.01 one-way ANOVA followed by Tukey’s multiple comparisons test), n = 8–13. (C) In MrgB3-receptor-expressing Xenopus laevis oocytes PACAP(6–38; 3 μM) was equipotent to PACAP-38 (3 μM) to elicit currents (ns, p = 0.2944, unpaired t-test), n = 21–27. All measurements were done at a holding potential of −70 mV. All values are given as mean ± SEM.
Figure 7. A correlation of 0.967 is obtained between the mean amount of degranulation induced by five PACAP isoforms (PACAP-38, PACAP-27, PACAP(6–38), maxadilan and M65), n = 5–11 in meningeal mast cells and their net charge at neutral pH. Calculations are performed using the Bachem Peptide Calculator (http://www.bachem.com/service-support/peptide-calculator/).
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