Acute denervation alters the epithelial response to adrenoceptor activation through an increase in alpha1-adrenoceptor expression on villus enterocytes

Abstract

Loss of sympathetic input due to intestinal denervation results in hypersensitivity and increased intestinal secretion. It is unknown whether denervation-induced alterations in intestinal epithelial physiology are the result of changes in adrenoceptors on enterocytes (ENTs). The purpose of this study was to examine adrenoceptor distribution and pharmacology on small intestinal ENTs following acute intestinal denervation. Lewis rats underwent small bowel transplantation (SBT) or sham operation and proximal small intestinal segments were harvested 1, 2 and 4 weeks postoperatively. Intestinal electrolyte movement was assessed using short-circuit current (Isc) measurements of stripped epithelial sheets following stimulation with phenylephrine (PE), an alpha(1)-adrenoceptor agonist. The presence of adrenoceptor subtypes on separated villus and crypt ENTs was assessed using flow cytometry. Alpha(1)-adrenoceptors were found on approximately 27% of jejunal villus ENTs, but not crypt ENTs, following acute extrinsic denervation. ENTs from the Lewis rat have few beta-adrenoceptors. Alpha(1)-adrenoceptor stimulation of acutely denervated intestinal epithelial sheets decreased Isc by -13.45%. This effect was mediated by a reduction in chloride (Cl(-)) secretion; the absence of Cl(-) reversed the Isc to +13.79%. In conclusion, loss of sympathetic innervation to the gastrointestinal epithelium causes acute upregulation of alpha(1)-adrenoceptors on villus ENTs, leading to inhibition of Cl(-) secretion at the villus tip. The increase in adrenoceptors may reflect a compensatory mechanism to combat the increased secretory state of the bowel due to the loss of the sympathetic innervation and tonic control over intestinal secretion.

Figure 1

Representative dot-plot profile of villus intestinal epithelial cells isolated from the proximal small intestine of the Lewis rat. The forward scatter of light (FSC-H) is a measure of increasing cell size and the side scatter of light (SSC-H) is a measure of increasing cell granularity (values are in arbitrary units). The box, ENT, represents enterocytes and all binding experiments were conducted from cells within this region. IEL: intraepithelial lymphocytes.

Figure 2

Percentage of (a) villus and (b) crypt ENTs isolated from the small intestine of Lewis rats exhibiting specific (displaceable) binding for α₁-adrenoceptor. (a) Villus ENTs from the denervated gut have a higher proportion of cells with α₁-adrenoceptors at week 2 compared with weeks 1 and 4 and at any time point in the sham (^*P<0.05). (b) Displaceable binding of the α₁-adrenoceptor probe BODIPY FL prazosin from crypt ENTs was low and not significantly different between the experimental groups at any time point. Results are expressed as mean±s.e.m., n=3–5 animals.

Figure 3

Phenylephrine induced α₁-adrenoceptor stimulation induces a significant decrease in Isc in denervated, but not sham-operated, jejunum at week 2. (a) Representative trace of the Isc response (μamp/cm²) to α₁-adrenoceptor stimulation by phenylephrine (PE) of epithelial sheets from denervated jejunum 2 weeks postoperative. Arrow indicates the time at which PE was added to the apical and basolateral surfaces. (b) Basal Isc values were not significantly different between control and denervated intestine. There was a significant decrease in Isc after administration of PE in the denervated gut at 2 weeks postoperative that was not observed in the innervated controls (ΔIsc=−4.2±2.75). ^*P<0.05 for both the difference from baseline at week 2 and for the change in Isc between the sham and transplant intestine at week 2. Results are expressed as mean±s.e.m., n=4.

Figure 4

Intestinal denervation does not alter sodium absorption. There was a significant increase in the Isc response upon addition of apical glucose to epithelial sheets from both (a) denervated and (b) sham-operated animals. ^*P<0.05 represents the difference from baseline. There was no significant difference between the two experimental groups at any time point. Results are expressed as mean±s.e.m., n=4–5.

Figure 5

Phenylephrine-induced α₁-adrenoceptor activation inhibits Cl⁻ secretion in the denervated small intestine. The PE-induced decrease in Isc of denervated gut is significantly reversed in low Cl⁻ buffer (^**P<0.001). The decrease in Isc in the denervated gut bathed in buffer 1 was significantly different than both controls groups bathed in either buffer (^*P<0.05). The PE-induced change in Isc of tissue from either naïve (controls) or sham-operated (Sham) controls was the same, regardless of the buffer. Results are expressed as mean±s.e.m., n=3–10.