Figure 1.
Compound 48/80 (c48/80) evoked activation of visceral afferents in vivo. A
Jejunal afferent discharge stimulated by c48/80 administered intraluminally. A second administration 5 min later evoked a similar afferent response but subsequent administrations are completely desensitized, despite similar mechanosensitivity during the luminal perfusions. B In sensitized animals intraluminal application of the antigen egg albumin (EA) evoked a response similar to that observed with c48/80 and which too rapidly desensitized so that subsequent antigen failed to evoke a response. C Following desensitization to c48/80, intraluminal antigen still evoked a response similar to that in naïve control animals. When the order of administration was reversed and the response to antigen was desensitized, c48/80 was still able to evoke a response D illustrates mean data from these cross-desensitization experiments. The left panel shows the afferent response to saline or antigen in sensitized animals that had received pretreatment with vehicle (saline, N = 11) or c48/80 (N = 5). The panel on the right shows similar data for the response to vehicle (saline) or c48/80 in naïve control animals (N = 9) or sensitized animals following desensitization to antigen (N = 6). The response to c48/80 was significantly augmented after desensitization to antigen (P<0.01).
Figure 2.
Compound 48/80 (c48/80) evoked Ca++ transients in primary cultured enteric neurons. A
shows two images of a Fluo-4 AM stained ganglion before c48/80 application and at the time of the maximal response. B illustrates the Ca++ signal of the neuron marked by an arrow in A. Two consecutive c48/80 applications (marked by the bars below the traces) evoked comparable responses. In this ganglion all 8 neurons responded to c48/80. C confirms that responses to c48/80 application were not caused by activation of mechanosensors because spritz application of Krebs solution using the same application parameters had no effect. D illustrates that c48/80 evoked [Ca++]i increase is concentration dependent.
Figure 3.
Compound 48/80 (c48/80) evoked spike discharge in primary cultured enteric neurons. A
Di-8-ANEPPS stained ganglion; the dye incorporates into the outer membrane revealing the outline of individual neuronal cell bodies. B illustrates action potential discharge in response to two consecutive c48/80 applications (marked by bars below the traces) from the neuron marked by white arrow in A. The traces show responses during three recording periods with non-recording periods of 6 s in between. In this ganglion 17 of 19 neurons responded to c48/80. C PGP9.5-positive cultured enteric neurons. Lack of c-kit immunoreactivity demonstrated lack of mast cells in the culture. D demonstrates that c48/80 application still evokes spikes in cultures treated with a combination of the H1 and H2 blockers pyrilamine (1 µM) and ranitidine (10 µM), respectively. E shows green PGP9.5-positive neurons in a whole mount preparation of the guinea pig submucous plexus (left panel) and red c-kit-positive round and smooth mast cells (some marked by white triangles in center panel), which are morphologically distinct from the spindle shaped interstitial cells of Cajal. The right panel shows the merged image of the two stainings.
Figure 4.
Compound 48/80 (c48/80) evoked Ca++ transients in isolated DRG and nodose ganglion cells in culture.
In both cases c48/80 evoked a calcium signal that was about 40% of the maximum response to the calcium ionophore, ionomycin. The response of nodose and DRG neurons to c48/80 was different in two respects, first in the proportion of cells responding and in the time course of the onset of the response. More nodose neurons responded than DRG and the onset of the response was quicker.