An acrolein-derivatized cAMP antiserum to study cAMP signaling and visualization in the enteric nervous system-implications for gut inflammation
Background & Aims: The general aim was to elucidate the role of cAMP signaling in intact neural circuits of the enteric nervous system in normal or inflamed gut. Forskolin binding, AC-ir and FIChR/cAMP recordings suggest AC/cAMP signaling occurs in a heterogeneous population of gut neurons. An acrolein-derivatized cAMP antiserum was developed and used for the visualization, quantitation, classification and polarity of gut neurons. We tested hypothesis 1 that cAMP signaling occurs in functional classes of neurons other than AH/Dogiel Type II intrinsic primary afferent neurons (IPANs). Hypothesis 2 tested if cAMP signaling in the two neural plexuses contributes to cAMP-dependent reflexes. Hypothesis 3 tested if amplification of the R/Gs/AC/cAMP-dependent pathway occurs in intestinal inflammation induced by Trichinella spiralis infection. Experimental Design: Forskolin stimulation was used for visualization of cAMP immunoreactivity, quantitation and functional classification of neurons according to their morphology and polarity. This technique was complimented by physiological, electrophysiological, molecular, immunochemical, ELISA or biochemical approaches in normal or T. spiralis infected jejunum. Results: Overall, 15-20% of myenteric and 60% of submucous neurons generate cAMP. Myenteric cAMP-visualized neurons had polarized projections for descending reflexes. Visualized cells were classified as IPANs with Dogiel II shape, descending myenteric interneurons (Dogiel I/filamentous), descending LM motor neurons (Dogiel I/simple), short-projection interneurons (Dogiel I/lamellar), VIP-ergic secretomotor neurons (filamentous), novel Dogiel I/lamellar or simple submucous neurons. Co-localization occurred with cAMPir in VIP but not NPY – secretomotor-neurons. Notable differences between the plexuses exist in cAMP/cGMP cross-talk, PDE IV activity, A1/A2aR cAMP coupling, polarity, proportions and numbers of each type. Synaptic blockade or neurosecretion studies indicated that cAMP contributes to synaptic communication. Acute inflammation causes AH cell hyper-excitability, elevates AC expression in calbindin-D28 and calretinin-positive neurons and amplification in ganglionic cAMP content in response to various stimulants. Inflammation, histamine or forskolin also induce phosphorylation of CREB that is blocked by cAMP-dependent PKA inhibition. H1/H2 histamine or COX 2 inhibitors attenuate AH hyper-excitability. Conclusion. The cAMP antiserum provided new insights into cAMP function in the enteric nervous system. Cyclic AMP signaling is involved in specific neural circuits, polysynaptic pathways, neurosecretion, motility reflexes, neuronal hyperexcitability in inflamed gut and neuronal plasticity.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:cyclic amp camp adenylate cyclase ac adenosine receptor ar
Date of Publication:01/01/2004