Effects of α2-adrenoceptor stimulation on luminal alkalinisation and net fluid flux in rat duodenum

Abstract

The sympathetic nervous system is highly involved in the regulation of gastrointestinal functions such as luminal alkalinisation and fluid absorption. However, the exact mechanisms are not clear. This study aimed to delineate how α2-adrenergic receptor stimulation reduces duodenal luminal alkalinisation and induces net fluid absorption. This was tested by perfusing the duodenum of anesthetized rats with isotonic solutions devoid of Cl- and/or Na+, in the absence and presence of the α2-adrenoceptor agonist clonidine. The clonidine was also studied in rats treated with dimethylamiloride (a Na+/H+ exchange inhibitor), vasoactive intestinal peptide, and the nicotinic receptor antagonist hexamethonium. Clonidine reduced luminal alkalinisation and induced net fluid absorption. The Cl--free solution decreased luminal alkalinisation and abolished net fluid absorption, but did not prevent clonidine from doing so. Both the Na+-free solution and luminal dimethylamiloride increased luminal alkalinisation and abolished net fluid absorption, effects counteracted by clonidine. The NaCl-free solution (D-mannitol) did not affect luminal alkalinisation, but reduced net fluid absorption. Clonidine reduced luminal alkalinisation and induced net fluid absorption in rats perfused luminally with mannitol. However, clonidine did not affect the vasoactive intestinal peptide-induced increase in luminal alkalinisation or fluid secretion. Pre-treatment with hexamethonium abolished the effects of clonidine on luminal alkalinisation and net fluid flux. In summary, our in vivo experiments showed that clonidine-induced reduction in luminal alkalinisation and induction of net fluid absorption was unrelated to luminal Na+ and Cl-, or to apical Na+/H+ or Cl-/HCO3- exchangers. Instead, clonidine seems to exert its effects via suppression of nicotinic receptor-activated acetylcholine secretomotor neurons.

Fig 1. Experimental setups of the intestinal perfusion.

In all rat groups, the duodenum was initially perfused (0.4 mL/min) with isotonic saline (blue) for 30 min (stabilization period) followed by a 30 min period to assess basal values of luminal alkalinisation and net fluid flux. Each group was thereafter perfused with isotonic saline for 60, 105 or 120 min, or with a solution free from Na⁺ (light green), Cl^- (green), or NaCl (dark green). Each of these groups were tested alone, and after intravenous clonidine treatment. In one set of experiments, clonidine was also tested with or without intravenous VIP, and in another with or without luminal DMA, a Na⁺/H⁺ exchange inhibitor.

Fig 2. The effects of clonidine on mean arterial blood pressure, fluid flux and luminal alkalinisation.

Duodenum was perfused with isotonic saline for 90 min and clonidine was administered from 30 min as a constant i.v. infusion at a dose of 10 or 50 μg kg^-1 h^-1 (Fig 1). Shown are the mean (a) arterial blood pressure (MABP), (b) rate of luminal alkalinisation, (c) transepithelial net fluid flux, (d) net change in luminal alkalinisation in response to clonidine, (e) net change in net fluid flux and (f) net change in MABP in response to clonidine. Relationship between the (g) basal luminal alkalinisation and the clonidine-induced decrease in alkalinisation (y = -0.65x + 1.60, r² = 0.77, P<0.001), and (h) basal net fluid flux and the clonidine-induced change in net fluid flux (y = -0.70x - 1.26, r² = 0.85, P<0.001). Values are means ± SEM or box plots with all individual points, n = 12. Changes are presented as the mean of values at 80 and 90 min minus the mean of the three control values. ***P<0.001 compared with basal values.

Fig 3. The effects clonidine on net fluid flux and luminal alkalinisation in rat duodenum perfused with an isotonic Cl^--free solution.

Duodenum was perfused with isotonic saline for 30 min and subsequently with an isotonic Cl^--free Na₂SO₄ solution for 100 min (Fig 1). Effects on luminal alkalinisation (a and c) and transepithelial net fluid flux (b and d) in the absence and presence of i.v. infusion of clonidine at a dose of 10 μg kg^-1 h^-1. Net changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 110–130 min and 50–70 min (Na₂SO₄ plus clonidine vs. Na₂SO₄ alone). Values are means ± SEM or box plots with all individual points. *P<0.05, **P<0.01 and ***P<0.001 compared with basal values. ^ΦP<0.05 and ^ΦΦP<0.01 compared with values at 40–70 min. Fig (e) and (f); *P<0.05 and ***P<0.001 compared with values in animals treated with Na₂SO₄ alone.

Fig 4. The relationship between the changes in luminal alkalinisation in response to different luminal solutions and basal luminal alkalinisation.

The duodenum was perfused for 40 min with (a) Cl^--free Na₂SO₄, (b) Na⁺-free NMDG, (c) dimethylamiloride (DMA), (d) NaCl-free D-mannitol, or (e) Cl^--free Na₂SO₄ plus DMA. Each x-y value is the mean of three basal values before treatment and the mean of the two last values in response to treatment. Regression analysis: (a). y = -0.64x + 1.68, r² = 0.70, P<0.001, n = 14. (b) y = 0.21x + 1.55, r² = 0.12, P = 0.25, n = 13. (c). y = 0.40x + 1.32, r² = 0.19, P = 0.21, n = 10. (d). y = -0.52x + 2.08, r² = 0.86, P<0.001, n = 12. (e). y = -0.62x + 3.31, r² = 0.79, P = 0.001, n = 11.

Fig 5. The effects of clonidine on fluid flux and luminal alkalinisation in rat duodenum perfused with an isotonic Na⁺-free solution.

The duodenum was perfused with isotonic saline for 30 min, followed by an isotonic Na⁺-free NMDG chloride solution for 100 min (Fig 1). Effects on luminal alkalinisation (a and c) and transepithelial net fluid flux (b and d) in the absence and presence of i.v. infusion of clonidine at a dose of 10 μg kg^-1 h^-1. Net changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 110–130 min and 50–70 min (NMDG plus clonidine vs. NMDG alone). Values are means ± SEM or box plots with all individual points. *P<0.05 and **P<0.01 compared with basal values. ^ΦΦP<0.01 and ^ΦΦΦP<0.001 compared with values at 40–70 min. Fig (e) and (f); *P<0.05 and ***P<0.001 compared with values in animals treated with NMDG alone.

Fig 6. The effects of clonidine on fluid flux and luminal alkalinisation in the rat duodenum perfused with DMA.

Duodenum was perfused with isotonic saline for 30 min and subsequently with isotonic dimethylamiloride (DMA) solution (10⁻³ M) for 100 min (Fig 1). Effects on luminal alkalinisation (a and c) and transepithelial net fluid flux (b and d) in the absence and presence of i.v. infusion of clonidine at a dose of 10 μg kg^-1 h^-1. Net changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 110–130 min and 50–70 min (DMA plus clonidine vs DMA alone). Values are means ± SEM or box plots with all individual points. *P<0.05 and **P<0.01 compared with basal values. ^ΦP<0.05, ^ΦΦP<0.01 and ^ΦΦΦP<0.001 compared with values at 50–70 min. Fig (e) and (f); *P<0.05 and ***P<0.001 compared with values in animals treated with DMA alone.

Fig 7. The effects of clonidine on fluid flux and luminal alkalinisation in rat duodenum perfused with a NaCl-free solution.

Duodenum was perfused with isotonic saline for 30 min and then with an isotonic D-mannitol solution for 100 min (Fig 1). Effects on luminal alkalinisation (a and c) and transepithelial net fluid flux (b and d) were determined in the absence and presence of i.v. infusion of clonidine at a dose of 10 μg kg^-1 h^-1. Net changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 110–130 min and 50–70 min (D-mannitol plus clonidine vs. D-mannitol alone). Values are means ± SEM or box plots with all individual points. **P<0.01 and ***P<0.001 compared with basal values. ψP<0.05, ψψP<0.01 compared with values at 50–70 min. Fig (e) and (f); *P<0.05 and ***P<0.001 compared with values in animals treated with D-mannitol alone.

Fig 8. The effects of clonidine on fluid flux and luminal alkalinisation in rat duodenum perfused with a Cl^--free + DMA solution.

Duodenum was perfused with isotonic saline for 30 min and then an isotonic Na₂SO₄ plus dimethylamiloride (DMA) solution for 100 min (Fig 1). Effects on (a and c) luminal alkalinisation and (b and d) transepithelial net fluid flux were determined in the absence and presence of i.v. infusion of clonidine at a dose of 10 μg kg^-1 h^-1. Changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 110–130 min and 50–70 min (Na₂SO₄ plus DMA plus clonidine vs. Na₂SO₄ plus DMA alone). Values are means ± SEM or box plots with all individual points. Fig b) and d) *P<0.05 and **P<0.01 compared with values at 0–30 min. Fig (c) **P<0.01 compared with values at 40–70 min. Fig (c) ΦΦP<0.01 compared with compared with values at 50–70 min. Fig (e) and (f) **P<0.01 compared with animals treated with Na2SO4 plus DMA alone.

Fig 9. The effects of clonidine administered before and after treatment with vasoactive intestinal peptide (VIP) on fluid flux and luminal alkalinisation in rat duodenum.

Duodenum was perfused with isotonic saline for 150 min. Effects on (a and c) luminal alkalinisation and (b and d) transepithelial net fluid flux determined with (a and b) VIP (i.v. 15 μg kg^-1 h^-1) from 30 min and clonidine (i.v. 10 μg kg^-1 h^-1) from 90 min, or with (c and d) clonidine from 30 min and VIP from 90 min. The (e) increase in luminal alkalinisation and the (f) change in net fluid flux in response to VIP alone (mean 70–90 min minus 0–30 min) and in response to clonidine (mean 130–150 min minus 70–90 min). The (e) decrease in luminal alkalinisation and the (f) change in net fluid flux in response to clonidine alone (mean 70–90 min minus 0–30 min) and in response to VIP (mean 130–150 min minus 70–90 min). Values are means ± SEM or box plots with all individual points. Fig a-b. **P<0.01 and ***P<0.001 compared with basal values. ^φP<0.05 and ^φφP<0.01 compared with values at time points 70–90 min. Fig c-d. **P<0.01 and ***P<0.001 compared with values at time point 70–90. Fig e. *P<0.05 compared with VIP alone.

Fig 10. The effect of clonidine administered after treatment with a non-selective nicotinic receptor inhibitor (hexamethonium) on blood pressure, net fluid flux and luminal alkalinisation in the rat duodenum.

Duodenum was perfused with isotonic saline for 130 min with hexamethonium (i.v. 10 mg kg^-1 h^-1) from 30 min followed by clonidine (i.v. 10 μg kg^-1 h^-1) from 70 min (Fig 1). Effects on (a) mean arterial blood pressure, (b) transepithelial net fluid flux, and (c) luminal alkalinisation with. The (d) relationship between the basal luminal alkalinisation and the changes in luminal alkalinisation in response to hexamethonium compared to baseline (0–30 min). Changes in (e) luminal alkalinisation and (f) transepithelial net fluid flux between 100–130 and 40–70 min in animals treated with hexamethonium alone and hexamethonium plus clonidine. Values are means ± SEM or box plots with all individual points. **P<0.01 and ***P<0.001 compared with basal values. ^ψP<0.05 and ^ψψP<0.01 compared with values at time point 70–90.

Fig 11. A summary of the effects on duodenal fluid absorption and luminal alkalinisation in the villi and crypts.

Effects on rat duodenal fluid absorption and luminal alkalinisation of clonidine combined with sodium and/or chloride free luminal perfusates, luminal inhibition of the Na⁺/H⁺ exchanger (NHE3) with DMA, and intravenous administrations of the vasoactive intestinal peptide (VIP) or hexamethonium, a non-selective nicotinic receptor antagonist. DRA/PAT1—chloride anion exchanger, CaCC–calcium-activated chloride channels, CFTR—Cystic fibrosis transmembrane conductance regulator, Ach–acetylcholine, cAMP–Cyclic adenosine monophosphate, nAChR—Nicotinic acetylcholine receptor, alpha2—Alpha-2 adrenoceptor, VPAC1—Vasoactive intestinal polypeptide receptor 1, M3—Muscarinic M3 receptor.