Activity of Protease-Activated Receptors in the Human Submucous Plexus Kerstin Mueller, Klaus Michel, Dagmar Krueger, Ihsan Ekin Demir, Güralp Onur Ceyhan, Florian Zeller, Martin E. Kreis, Michael Schemann  Gastroenterology  Volume 141, Issue 6, Pages 2088-2097.e1 (December 2011) DOI: 10.1053/j.gastro.2011.08.034 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Neural actions of PAR-APs in human and guinea pig submucous neurons recorded with Di-8-ANEPPS. (A) Images show outlines of Di-8-ANEPPS–stained neurons in human submucous ganglia. The traces to the right show representative responses to a 2-second spritz application (indicated by horizontal bars) of PAR1-AP, PAR2-AP, or PAR4-AP. The traces are from neurons marked by white arrows in the images. Each peak corresponds to one action potential. (B) PAR1-AP induced a strong spike discharge in the majority of human submucous neurons, whereas the PAR2-AP or PAR4-AP caused weaker activation in a lower proportion of neurons. In contrast, the PAR2-AP evoked the strongest activation in guinea pig submucous neurons, whereas the PAR1-AP and PAR4-AP evoked less spike discharge in smaller proportions of neurons. Symbols mark significant differences as explained in the figure. Numbers indicate numbers of tissues/ganglia/neurons studied. Gastroenterology 2011 141, 2088-2097.e1DOI: (10.1053/j.gastro.2011.08.034) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Effect of PAR-APs, thrombin, and tryptase on [Ca]i in human and guinea pig submucous neurons and glia. (A) Images show a human submucous ganglion when PAR1-AP–evoked [Ca]i was maximal in neurons and glia. Note the distinct morphologies of a neuron (arrow) and glia cell (arrowhead). Traces show representative [Ca]i signals after PAR1-AP spritz application (horizontals bars below the traces) in a submucous neuron and glia. In human submucous plexus, PAR1-AP and thrombin induced [Ca]i signals in (B) enteric neurons and (C) glia. Thrombin effects were inhibited by the PAR1 antagonist SCH79797. PAR2-AP, PAR4-AP, and tryptase evoked significantly smaller responses in a small number of cells. Note that the PAR2-AP–evoked responses are not affected by tissue treatment with serine protease inhibitor FUT-175. In guinea pig submucous plexus, PAR2-AP induced the strongest [Ca]i responses in (B) neurons and (C) glia. The PAR1-AP–induced [Ca]i increase in neurons and glia was significantly higher in the human submucous plexus. Symbols mark significant differences as explained in the figure. Numbers indicate numbers of tissues/ganglia/neurons or of tissues/ganglia/responding glia cells studied. Gastroenterology 2011 141, 2088-2097.e1DOI: (10.1053/j.gastro.2011.08.034) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 PAR1-AP–induced neuronal and glial activation is dose-dependently reduced by the PAR1 antagonist SCH79797. Doses of 100 nmol/L and 1 μmol/L SCH79797 significantly reduced while 10 μmol/L SCH79797 completely abolished the [Ca]i signal to 100 μmol/L PAR1-AP in both neurons and glia. Symbols mark significant differences to control PAR1-AP application. Numbers indicate numbers of tissues/ganglia/neurons or tissues/ganglia/responding glia studied. Gastroenterology 2011 141, 2088-2097.e1DOI: (10.1053/j.gastro.2011.08.034) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Effects of PAR-APs on mucosal ion secretion in the human intestine. PAR1-AP (10 μmol/L) induced a significantly higher response (ΔIsc) than PAR2-AP (10 μmol/L). The PAR4-AP (10 μmol/L) induced no response. The PAR1-AP–induced secretion was abolished by the nerve blocker TTX, while the PAR2-AP–evoked secretion was TTX insensitive. Symbols mark significant differences as explained in the figure. Gastroenterology 2011 141, 2088-2097.e1DOI: (10.1053/j.gastro.2011.08.034) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Response of macrophages to application of PAR2-AP in the human submucous plexus. On average, macrophages showed an increase in [Ca]i of 17.2% ΔF/F (9.4/35.2% ΔF/F) (8 tissues and 61 regions studied). (A) Representative trace showing the PAR2-AP (application marked by horizontal bar below the trace) induced increase in [Ca]i in a cell that was located outside a ganglion. The images illustrate the origin of the signal. (B) Fluo-4AM–stained cells outside a ganglion marked by white arrows. (C) The cells were identified as macrophages by CD68 immunoreactivity (mouse anti-CD68 antibody; 1:200; AbD Serotec, Düsseldorf, Germany). Staining was performed as described in Materials and Methods. Arrows mark the same cells as in panel B. (D) A PGP9.5 staining reveals neurons in the ganglion that is faintly stained in panels B and C. Gastroenterology 2011 141, 2088-2097.e1DOI: (10.1053/j.gastro.2011.08.034) Copyright © 2011 AGA Institute Terms and Conditions