Calcium signals elicited by PAR agonists. A. Adult mouse neuron in which an increase in [Ca]i was elicited by a specific PAR2 activator peptide (PAR2-AP, SLIGRL, 100 μM), but not by thrombin (100 nM) which activates PAR1, 3 and 4. The neuron also expresses receptors for TRPA1 and TRPV1, as shown by its responses to the specific TRPA1 agonist mustard oil (MO, 100 μM) and the specific TRPV1 agonist capsaicin (1 μM).
B. Most PAR2-AP-responsive adult mouse neurons also responded to capsaicin and mustard oil but none responded to thrombin (n = 180 neurones). Staining of unfixed cells with fluorescently labelled IB4 (isolectin B4 from Griffonia simplicifolia coupled to Alexa 594, Molecular Probes) immediately after the calcium imaging experiment showed that most PAR2-AP responsive neurons were IB4-positive (grey bar). Similar results were obtained in neonatal rat neurons (88.6 ± 5.1% of cells responding to PAR2-AP also responded to capsaicin, and 82.5 ± 6.0% were IB4+).
C. Adult mouse neuron in which an increase in [Ca]i was elicited by thrombin (100 nM). This neuron also expresses the ion channels TRPA1 and TRPV1, as shown by its responses to mustard oil (MO, 100 μM) and capsaicin (1 μM). Cell was identified as a neuron on morphological grounds, confirmed by calcium increase observed in response to 25 mM KCl.
D. Around 25-33% of thrombin-responsive neurons (n = 455) also responded to capsaicin (1 μM), mustard oil (100 μM), the peptide Bv8 (100 nM) and bradykinin (1 μM) but none responded to PAR2-AP (100 μM). Final bar shows that no thrombin-responsive adult mouse neuron bound IB4.
E. Glial cell which responded with increase in [Ca]i to PAR1-AP (100 μM) and to PAR2-AP (100 μM). Cell was identified as a glial cell on morphological grounds, confirmed by absence of calcium increase in response to 25 mM KCl. In separate experiments, cells of this morphology were also identified by the glial-specific anti-S100 antibody (not shown).
F. Percentage of glial cells responding to thrombin (100 nM), PAR1-AP (100 μM) and PAR2-AP (100 μM). Deletion of PAR1 ablated responses to both thrombin and PAR1-AP (bars 4 and 5) while deletion of PAR2 was without effect on responses to thrombin and PAR1-AP (bars 6 and 7).