Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator (Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method using substrate-specific enzyme reactor-columns. Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine was detected in the surface epithelium of human bronchi (33 pmol/g), mouth (female 0.7 and male 8 pmol/sample), small and large intestine (800 and 16 pmol/g, respectively), gall bladder (12 pmol/g), vagina (6 pmol/sample), skin 1000 (pmol/g) and in pulmonary pleura (5 pmol/sample). Somewhat higher amounts of acetylcholine were found in rat tracheal and intestinal epithelium and in rat skin. The synthesizing enzyme choline acetyltransferase (ChAT) was demonstrated in human surface epithelium by immunohistochemistry and by Western blot analysis. Enzymatic ChAT activity was demonstrated in isolated epithelial cells of human bronchi and small intestine (3.5 and 28 nmol/mg protein/h, respectively). Applied acetylcholine (in nM concentrations) increased, whereas inhibition of ChAT activity by bromoacetylcholine (10 microM) reduced the growth of cultured human bronchial epithelial cells. Inhibition of cell growth occurred also in the presence of atropine (1 microM) together with (+/-)-tubocurarine (30 microM). In conclusion, the present experiments demonstrate a widespread existence of non-neuronal acetylcholine in surface cells of man. Non-neuronal acetylcholine may act as a local signalling molecule.

Soluble forms of the IL-6 receptor (sIL-6R) bind to the cytokine IL-6 with similar affinity as the membrane-bound IL-6R. IL-6⅐sIL-6R complexes initiate IL-6 trans-signaling via activation of the ubiquitously expressed membrane-bound ␤-receptor glycoprotein 130 (gp130). Inhibition of IL-6 trans-signaling has been shown to be favorable in numerous inflammatory diseases. Furthermore, different soluble forms of gp130 (sgp130) exist that, together with the sIL-6R, are thought to form a buffer for IL-6 in the blood. However, a functional role for the different sgp130 forms has not been described to date. Here we demonstrate that the metalloproteases ADAM10 and ADAM17 can produce sgp130 by ectodomain shedding of gp130, even though this mechanism only accounts for a minor proportion of sgp130 in the circulation. We further show that full-length sgp130 and the shorter forms sgp130-rheumatoid arthritis-associated peptide (RAPS) and sgp130-E10 are differentially expressed in a cell type-specific manner. Remarkably, full-length sgp130 is expressed by monocytes, but this expression is completely lost during differentiation into macrophages in vitro. Using genetically engineered murine pre-B cells that secrete different forms of sgp130, we found that these secreted sgp130 proteins are able to prevent trans-signaling-driven cell proliferation of the secreting cells, whereas conditioned supernatant from these cells failed to block IL-6 trans-signaling in other cells. Thus, our data suggest that the different sgp130 forms are released from cells into their immediate surroundings and appear to form cell-associated gradients to modulate their own susceptibility for IL-6 trans-signaling.IL-6 is a pleiotropic cytokine with pro-and anti-inflammatory functions (1-4). Classic signaling via the membranebound IL-6R 3 accounts for the regenerative properties of IL-6; trans-signaling via sIL-6R is rather pro-inflammatory (2, 4). In both cases, IL-6 binds initially to the (s)IL-6R, and the IL-6⅐(s)IL-6R complex recruits two signal-transducing gp130 ␤-receptors. This final IL-6⅐(s)IL-6R/gp130 complex formation initiates the activation of intracellular signaling pathways, among them Jak/STAT, PI3K, MAPK, and Src/Yes-associated protein (YAP) (5).Soluble forms of gp130 (sgp130) exist in human serum at concentrations of up to 400 ng/ml (6) and have been shown to act as natural inhibitors of IL-6 trans-signaling (7), although, at high concentrations, they can interfere with classic signaling as well (8). Interestingly, specific inhibition of trans-signaling with an Fc-dimerized version of sgp130 (sgp130Fc) has been shown to be beneficial in numerous inflammatory diseases and cancer (9, 10). sgp130Fc is currently in clinical development as a drug candidate to treat inflammatory bowel diseases (11).gp130 is a transmembrane protein with three N-terminal Iglike domains followed by three fibronectin-type III domains. sgp130 forms with molecular weights of 50, 90, and 110 kDa have been detected in human body fluids (6, 12, 13). Where and how these thre...

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Stored endogenous acetylcholine (ACh) and in vitro synthesis of [3H]ACh were measured in isolated, mucosa-intact and mucosa-denuded airways of rat, guinea pig, and humans. In addition, choline acetyltransferase (ChAT) activity and ACh content were measured in freshly isolated airway mucosa as well as in cultured epithelial cells of rat tracheas. Rat tracheas stored 25 nmol/g ACh, whereas guinea pig tracheas and human bronchi contained only 2-3 nmol/g ACh. When incubated with [3H]choline, the isolated airways of rat, guinea pig, and human synthesized significant amounts of [3H]ACh. In guinea pig and human airways, removal of the mucosa affected neither stored ACh nor in vitro synthesis of [3H]ACh. In rat tracheas, however, removal of the mucosa resulted in a 50% reduction of stored ACh. Freshly isolated mucosa wiped off from the luminal surface of rat tracheas contained large amounts of ACh (6.5 nmol/g airway), whereas in human mucosa (central bronchi) only small amounts of ACh were found. In enzymatically isolated mucosal cells of rat tracheas, a considerable ChAT activity (21 nmol.mg protein 1.h-1) was detected, blockable by bromoacetylcholine. Enzymatically isolated human mucosa contained a rather low ChAT-like activity (0.5 nmol.mg protein 1.h-1), not sensitive to bromoacetylcholine. In cultured epithelial cells of rat tracheas (4th-6th passage), neither ChAT activity nor ACh was detected. The large airways of rat, guinea pig, and humans contain considerable amounts of ACh, supporting histological evidence of a dense cholinergic innervation, particularly of rat tracheas. The mucosa of rat tracheas synthesizes and stores large amounts of ACh, whereas the low ChAT activity in human mucosa argues against the presence of cholinergic neurons able to synthesize and store ACh.

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