Characterization of Prejunctional Muscarinic Receptors: Effects on the Release of VIP and Functional Responses and Receptor Expression in the Ovine Submandibular Gland

In the in vivo experiments on anaesthetized sheep, it was presently examined whether muscarinic receptor antagonists with diverse selectivity affect the release of VIP in response to electrical stimulation of the parasympathetic chorda tympanic nerve differently, and if the changes in the release could be associated to altered secretory and vasodilator responses. The location of the muscarinic receptor subtypes was examined also. In the experiments, blood was collected out of the submandibular venous drainage before and during electrical stimulation of chorda tympani nerve in the absence and presence either of pirenzepine or methoctramine. While metchoctramine increased the output of protein, pirenzepine inhibited flow of saliva and increased protein output, vasodilatation, and VIP output. In morphological examinations, the inhibitory muscarinic M4 receptor occurred interacinarily in the gland. It is concluded that prejunctional muscarinic receptors, most likely of the M4 subtype, exert inhibitory modulation of the parasympathetic release of VIP in the ovine submandibular gland

Intestinal Epithelial Stem/Progenitor Cells Are Controlled by Mucosal Afferent Nerves

Background: The maintenance of the intestinal epithelium is of great importance for the survival of the organism. A possible nervous control of epithelial cell renewal was studied in rats and mice. Methods: Mucosal afferent nerves were stimulated by exposing the intestinal mucosa to capsaicin (1.6 mM), which stimulates intestinal external axons. Epithelial cell renewal was investigated in the jejunum by measuring intestinal thymidine kinase (TK) activity, intestinal H-3-thymidine incorporation into DNA, and the number of crypt cells labeled with BrdU. The influence of the external gut innervation was minimized by severing the periarterial nerves. Principal Findings: Luminal capsaicin increased all the studied variables, an effect nervously mediated to judge from inhibitory effects on TK activity or H-3-thymidine incorporation into DNA by exposing the mucosa to lidocaine (a local anesthetic) or by giving four different neurotransmitter receptor antagonists i.v. (muscarinic, nicotinic, neurokinin1 (NK1) or calcitonin gene related peptide (CGRP) receptors). After degeneration of the intestinal external nerves capsaicin did not increase TK activity, suggesting the involvement of an axon reflex. Intra-arterial infusion of Substance P (SP) or CGRP increased intestinal TK activity, a response abolished by muscarinic receptor blockade. Immunohistochemistry suggested presence of M3 and M5 muscarinic receptors on the intestinal stem/progenitor cells. We propose that the stem/progenitor cells are controlled by cholinergic nerves, which, in turn, are influenced by mucosal afferent neuron(s) releasing acetylcholine and/or SP and/or CGRP. In mice lacking the capsaicin receptor, thymidine incorporation into DNA and number of crypt cells labeled with BrdU was lower than in wild type animals suggesting that nerves are important also in the absence of luminal capsaicin, a conclusion also supported by the observation that atropine lowered thymidine incorporation into DNA by 60% in control rat segments. Conclusion: Enteric nerves are of importance in maintaining the intestinal epithelial barrier.Original Publication:Ove Lundgren, Mats Jodal, Madeleine Jansson, Anders T Ryberg and Lennart Svensson, Intestinal Epithelial Stem/Progenitor Cells Are Controlled by Mucosal Afferent Nerves, 2011, PLOS ONE, (6), 2, 16295.http://dx.doi.org/10.1371/journal.pone.0016295Copyright: Public Library of Science (PLoS)http://www.plos.org

On the Autonomic Control of Blood Flow and Secretion in Salivary Glands. Functional and morphological aspects on muscarinic receptor subtypes in different species.

Parasympathetic nervous activity is the principal stimulus for evoking fluid responses within salivary glands. Concomitantly to the onset of this response, the blood flow increases. The responses, in particular the vasodilatation, consist of an atropine-sensitive acetylcholine-mediated part and an atropine-resistant part mediated via non-adrenergic, non-cholinergic (NANC) transmitters. It has been generally agreed that the cholinergic effects are mediated by muscarinic M3 receptors. However, this view has been questioned, since most muscarinic receptors are expressed and muscarinic M1 receptors elicit functional effects in salivary glands. The distribution and function of the muscarinic receptors is not unravelled, neither according to secretion nor vasodilatation. The aim of this thesis has been to investigate the roles of different muscarinic subtypes in the control of blood flow and secretion in salivary glands. In the thesis, the expression of muscarinic receptors in salivary glands and related blood vessels was investigated using immunoblotting and/or immunohistochemistry. Furthermore the effects of muscarinic stimulation and blockade were investigated on isolated vessels, on the secretion of saliva, on glandular blood flow and vessel permeability. The thesis includes observations on rats, sheep and man. It is shown that M1 receptors contribute considerably, in addition to the functionally most significant M3 receptor, to the fluid secretory responses of rats and sheep. The M1 receptor is particularly apparent in seromucous and mucous glands, and of particular functional significance at low intense stimulation. Since the occurrence pattern was the same in human salivary glands, M1 receptors may be of significance in man also. Notably, in the human glands, inflammation increased the expression of muscarinic M5 receptors. In the arterial blood vessels muscarinic M1 receptors generally occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the smooth muscle. In venous endothelium muscarinic M1 and M4 receptors occurred, while M1 and/or M3 were expressed in the smooth muscle layer. Cholinergic stimulation generally caused arterial vasodilatation, which was mainly dependent on nitric oxide. The response was mediated by muscarinic M1 and possibly M5 receptors, in addition to the M3 receptor. The venous response included a contractile M1 mediated component that may preserve perfusion pressure during the secretory process. In tissue in close vicinity to the parenchymal tissues, M1 and in particular M4 receptors occurred. In the sheep, the increase of submandibular secretory and vasodilator responses to electrical stimulation of the parasympathetic nerve in the presence of muscarinic antagonists were explained by neuronal muscarinic M4 receptors. These receptors inhibited the release of transmitters as was shown for the NANC transmitter VIP. The role of muscarinic M5 receptors is unclear but may affect on the vascular response or more likely to be correlated to inflammation. In general, the expression pattern and functions of the muscarinic receptors subtypes showed resemblance in the examined species. All muscarinic receptors occur in the salivary glands. In seromucous/mucous glands, muscarinic M1 receptors contribute substantially to the secretory response. In the vasculature, the muscarinic receptor subtypes interact, possibly via autocrine mechanisms, for preserving the hemodynamics in the glands

Intestinal Epithelial Stem/Progenitor Cells Are Controlled by Mucosal Afferent Nerves

Background: The maintenance of the intestinal epithelium is of great importance for the survival of the organism. A possible nervous control of epithelial cell renewal was studied in rats and mice. Methods: Mucosal afferent nerves were stimulated by exposing the intestinal mucosa to capsaicin (1.6 mM), which stimulates intestinal external axons. Epithelial cell renewal was investigated in the jejunum by measuring intestinal thymidine kinase (TK) activity, intestinal H-3-thymidine incorporation into DNA, and the number of crypt cells labeled with BrdU. The influence of the external gut innervation was minimized by severing the periarterial nerves. Principal Findings: Luminal capsaicin increased all the studied variables, an effect nervously mediated to judge from inhibitory effects on TK activity or H-3-thymidine incorporation into DNA by exposing the mucosa to lidocaine (a local anesthetic) or by giving four different neurotransmitter receptor antagonists i.v. (muscarinic, nicotinic, neurokinin1 (NK1) or calcitonin gene related peptide (CGRP) receptors). After degeneration of the intestinal external nerves capsaicin did not increase TK activity, suggesting the involvement of an axon reflex. Intra-arterial infusion of Substance P (SP) or CGRP increased intestinal TK activity, a response abolished by muscarinic receptor blockade. Immunohistochemistry suggested presence of M3 and M5 muscarinic receptors on the intestinal stem/progenitor cells. We propose that the stem/progenitor cells are controlled by cholinergic nerves, which, in turn, are influenced by mucosal afferent neuron(s) releasing acetylcholine and/or SP and/or CGRP. In mice lacking the capsaicin receptor, thymidine incorporation into DNA and number of crypt cells labeled with BrdU was lower than in wild type animals suggesting that nerves are important also in the absence of luminal capsaicin, a conclusion also supported by the observation that atropine lowered thymidine incorporation into DNA by 60% in control rat segments. Conclusion: Enteric nerves are of importance in maintaining the intestinal epithelial barrier.Original Publication:Ove Lundgren, Mats Jodal, Madeleine Jansson, Anders T Ryberg and Lennart Svensson, Intestinal Epithelial Stem/Progenitor Cells Are Controlled by Mucosal Afferent Nerves, 2011, PLOS ONE, (6), 2, 16295.http://dx.doi.org/10.1371/journal.pone.0016295Copyright: Public Library of Science (PLoS)http://www.plos.org

Observations supporting the model of Figure 12.

<p>TS: this study.</p

Autoradiography of rat small intestine after giving tritiated thymidine i.v.

<p>The labeled compound (50 µCi ³H-methyl-thymidine) was administred 6 h prior to removing the intestine The left panel illustrates dark field microscopy of an autoradiogram showing radiolabeled cells (white spots) almost exclusively located in the epithelial layer of the crypts of Lieberkühn. To the right the same section is seen in light microscopy. Van Gieson.</p

Rat: BrdU labeled cells per crypt in intestinal segments exposed to a capsaicin or a control solution.

<p>BrdU (50 mg/kg bwt) was given i.v. 2 h prior to removal of the intestinal segments. The number of labeled cells in 255 crypts in control (n = 5) and in 249 crypts in capsaicin segments (n = 5) were counted. Asterisk indicates statistical significance. Mean ± s.e.m. <b>Mouse: BrdU labeled cells per crypt in wild type or capsaicin receptor knock-out mice.</b> Five capsaicin receptor knock-out mice (−/−; 225 crypts) and in five wild type mice (WT; 215 crypts) were investigated. BrdU (100 mg/kg b.wt.) was administered i.p. 2 h before removal of the small intestine. Asterisk indicates statistical significance. Mean ± s.e.m.</p

The effect of neurotransmitter receptor antagonists on capsaicin evoked increase of thymidine kinase activity.

<p>The receptor antagonists tested were: Sendide (NK1 receptor antagonist); 8–37 α-GGRP (CGRP receptor antagonist); atropine (muscarinic receptor antagonist). Thymidine kinase activity is expressed in per cent of activity measured in control segments (no capsaicin). N = 6 for each antagonist experiments. Asterisks indicate statistical significance compared to control. Mean ± s.e.m.</p