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Modulatory effects of taurine on jejunal contractility

Taurine (2-aminoethanesulfonic acid) is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM) can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca2+ dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism.

The function of taurine in the cardiovascular system has been well studied (9)(10)(11). Oral supplementation of taurine is shown to reduce blood pressure and improve cardiac function in hypertensive subjects (12)(13)(14). Ex vivo studies have shown that contractile responses to norepinephrine (NE) and KCl in aortic rings are attenuated both in taurinetreated normal rats and diabetic rats compared with controls (15,16); NE-, KCl-, and adenosine-receptor-agonistinduced hypercontractility of the aorta are enhanced in taurine-depleted rats compared with the effects in control animals (17,18). Although most studies show that taurine induces inhibitory effects in precontracted vessels, some reports indicate that taurine at concentrations of 20-60 mM inhibits phenylephrine-induced contraction in normal arteries of rats without affecting the basal tone of the arteries (19). However, taurine further enhances the NE-or KCl-induced contraction of arteries in the insulin-resistant rat (20). The studies cited above indicate that the effects of taurine on vascular smooth muscle are still controversial and the associated mechanisms remain unclear.
The divergent effects of taurine on vascular smooth muscle attracted our interest. We proposed a bidirectional modulation of taurine on smooth muscle and noted that the effects of taurine on intestinal smooth muscle contraction are rarely reported. It is known that the contraction of intestinal smooth muscle is modulated by the enteric nervous system (ENS), which can control functions of the intestine even when it is completely separated from the central nervous system (CNS) (21). To characterize the effects of taurine on intestinal contractility and reveal the possible mechanism, isolated jejunal segments and 3 different pairs of low and high contractile states of the segments were established by modification of ionic concentrations or by inhibitory and stimulatory neurotransmitters.

Determination of jejunal contractility
All rats were treated according to the Guidelines for the Care and Use of Laboratory Animals of Dalian Medical University, and all experimental procedures were carried out in accordance with the Declaration of Helsinki. Healthy male Sprague-Dawley rats (certificate No. SCXK 2008-0002), weighing 180-220 g, were used for measurement of jejunal contractility, Rats were fasted for 24 h but allowed water before experiments.
Rats were sacrificed by cervical dislocation and the jejunum was carefully removed and kept in Krebs buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 , 1.2 mM  MgSO 4 , 4.2 mM NaHCO 3 , 2.5 mM CaCl 2 , 10 mM glucose, pH 7.4). The isolated jejunum was rinsed, clipped into segments (about 12 mm long), and the mesenteric border was removed. The segments were suspended in longitudinal orientation in 20-mL organ baths of warm (376C), aerated Krebs buffer. The distal end of each segment was tied to an organ holder, and the proximal end was secured with a silk thread to an isometric force transducer. A 1-g/cm resting tension was applied to each segment, which was equilibrated for about 1 h. Rhythmic spontaneous contractions developed in all preparations. The contractile responses of the isolated jejunal segment were recorded with a BL-420 physiological recording system (Chengdu Taimeng Software Co., Ltd., China).
The contractility of isolated jejunal segments in Krebs buffer was selected as the normal contractile state (NCS). Modified low-Ca 2+ + (1.25 mM), low-K + (2.5 mM), or adrenaline (5 mM) Krebs buffers were selected as low contractile states (LCS). Modified high-Ca 2+ + (5.0 mM), high-K + (10.0 mM), or carbachol (CBC, 1 mM) Krebs buffers were selected as high contractile states (HCS). To study the mechanisms of taurine-induced bidirectional modulation, the HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer and LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer were selected as the representative HCS and representative LCS, respectively. A sodium channel blocker (TTX, 0.3 mM for 3 min); a voltage-dependent L-type calcium channel blocker (verapamil, 1 mM for 3 min); an adrenergic areceptor antagonist (phentolamine, 1 mM for 3 min); an adrenergic b-receptor antagonist (propranolol, 1 mM for 3 min); a cholinergic M receptor antagonist (atropine, 1 mM for 3 min); a histamine H 1 -receptor antagonist (diphenhydramine, 10 mM for 3 min); a histamine H 2 -receptor antagonist (cimetidine, 10 mM for 3 min), and a nitric oxide (NO) synthase inhibitor [L-N G -nitroarginine (L-NNA), 300 mM for 3 min] were used in the assay. The contractile amplitude of isolated jejunal segments is reported as a percentage of the contractile amplitude in the NCS. The contractile amplitude at NCS was set to a relative value of 100%.

Measurement of myosin phosphorylation
Myosin and myosin light chain kinase (MLCK) used in the assay were purified from chicken gizzard smooth muscle as described previously (22). Myosin phosphorylation was carried out in a 20 mM Tris-HCl, pH 7.4, buffer containing 1 mM dithiothreitol (DTT), 5 mM MgCl 2 , 60 mM KCl, 0.1 mM CaCl 2 , 0.6 mM calmodulin, 4 mM myosin, and 2 mM ATP with or without taurine at 256C for 20 min. MLCK (2.0 and 0.02 mM) was used to phosphorylate MLC 20 , exerting a high-extent and low-extent of phosphorylation, respectively. The extent of 20-kDa myosin light chain (MLC 20 ) phosphorylation was measured by 10% glycerol polyacrylamide gel electrophoresis (PAGE) (23,24). A Gel-Pro Analyzer (Media Cybernetics, USA) was used to scan the density and size of phosphorylated MLC 20 . The extent of phosphorylated MLC 20 is reported as the percentage of phosphorylated MLC 20 in total MLC 20 . The extent of full mono-phosphorylation was set at 100% (control).

Statistical analysis
All data are reported as means±SE. Statistical significance was tested by one-way analysis of variance, followed by the Dunnett multiple comparisons test, using the SPSS software 13.0 (SPSS Inc., USA). Statistical significance was defined as P,0.05.

Results
Effects of taurine on the contractility of isolated jejunal segments Taurine (5-80 mM) enhanced the contractile amplitude of isolated jejunal segments in a concentration-dependent manner (Figure 1, Figure S1). The contractile amplitude in both HCS and LCS was statistically different from that of NCS (Figure 2, Figure S2). Taurine (5-80 mM) significantly enhanced the contractile amplitude of jejunal segments in all 3 LCS (P,0.05), and significantly decreased the amplitude in all 3 HCS (P,0.05, Figure 2, Figure S2). These data indicated that taurine induced bidirectional modulation of the spontaneous contractility of isolated jejunal segments.

Underlying mechanism of taurine-induced bidirectional modulation
In the presence of TTX, neither an inhibitory effect of taurine (10-60 mM) on the contractile amplitude of isolated jejunal segments in the HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer nor a stimulatory effect on the contractile amplitude in LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer were observed ( Figure 3). These data showed that TTX abolished bidirectional modulation of taurine on jejunal contractility.
The nonselective muscarinic receptor antagonist atropine blocked the stimulatory effect of taurine (10-60 mM) on the contractile amplitude of isolated jejunal segments in LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer. Neither the histamine H 1 -receptor antagonist diphenhydramine nor the H 2 -receptor antagonist cimetidine blocked taurine (10-60 mM)-induced stimulatory effects on the contractile amplitude in the LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer (Figure 4). In the HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer, b-adrenergic receptor antagonist propranolol, and NO synthase inhibitor L-NNA blocked the taurine (10-60 mM)-induced inhibitory effect on the contractile amplitude of isolated jejunal segments. However, the a-adrenergic receptor antagonist phentolamine did not block the taurine-induced (10-60 mM) inhibitory effects on the contractile amplitude in the HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer (Figure 4).
Pre-incubation of isolated jejunal segments with the Ca 2+ + channel blocker verapamil at NCS, HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer, and LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer abolished the effects of taurine (10-60 mM) on the contractile amplitude of isolated jejunal segments ( Figure 5). This suggests that verapamil blocked the effects of taurine-induced contractility of isolated jejunal segments.
No significant effects of taurine on myosin phosphorylation were observed at concentrations of 5-40 mM. Although the effects of 80 mM taurine were significant, both high-extent and low-extent of myosin phosphorylation were affected ( Figure 6). These data indicate that taurine did not modulate myosin phosphorylation bidirectionally.

Discussion
Taurine exerted stimulatory and inhibitory effects on the contractility of isolated jejunal segments in both low and high contractile states. Considering that the HCS and LCS were established under different assay conditions and by using agents with different mechanisms, the results suggest that taurine-induced bidirectional modulation on isolated rat jejunal segments depends on its contractile state. The evidence that taurine induced a bidirectional modulation on jejunal contractility depending on its contractile state indicates that taurine plays an important homeostatic role in intestinal function.
Ca 2+ + /CaM-dependent phosphorylation of myosin light chains by MLCK is generally considered to be the primary mechanism for regulating the contraction of smooth muscle. Nevertheless, the effects of taurine on phosphorylation of myosin from chicken gizzard were not consistent with its bidirectional effect on isolated segments of rat jejunum. We only observed a slight inhibition effect of taurine on both high-and low-extent phosphorylation of myosin from chicken gizzard. Considering that such a high intracellular concentration of taurine can hardly be achieved in the muscle, we believe that the inhibition of 80 mM taurine on myosin phosphorylation was due to the toxic effects induced by such high concentration. These results indicated that taurine might exert dual modulation not on myogenic contractions of intestinal smooth muscle directly, but by some other mechanisms, e.g., regulation of neurotransmitter or hormone release. However, due to species differences, we cannot completely exclude the possibility that taurine has a direct effect on phosphorylation of myosin light chains in rats.
The neuronal regulation of intestinal contraction involves the ENS, as well as extrinsic nerves (25); TTX is a blocker of neuronal conduction (26); taurine-induced modulation on  the contractility of isolated jejunal segments was blocked by TTX, suggesting that neural regulation of ENS is involved in taurine-induced effects.
Activation of muscarinic or histamine receptor increases intestinal contractility, and stimulation of a and b adrenoceptors inhibits intestinal contractility. Inhibition of intestinal contractility is also mediated by NO, a nonadrenergic, noncholinergic neurotransmitter that produces its effect by directly acting on smooth muscle and by indirectly inhibiting acetylcholine and substance P release (27,28). In LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer, atropine blocked the stimulatory effects of taurine on the contractility of isolated jejunal segments, but diphenhydramine and cimetidine did not, implicating that the stimulatory effects of taurine on the jejunal contractility are correlated with muscarinic receptor-linked stimulation. The results are in line with reports that found the effect of taurine (10 -6 -10 -1 mM) on acid secretion in the rat stomach was completely or partially inhibited by atropine (29). Although the doses of taurine used in our study were different from those in the above report, the difference might be due to the different target tissues of taurine. In HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer, propranolol and L-NNA abolished the inhibitory effects of taurine on the contractility of isolated jejunal segments, however, phentolamine did not modify the inhibitory effects of taurine, suggesting that the inhibitory effects of taurine are correlated with the activation of adrenergic b receptors, as well as NO synthase-linked relaxation mechanisms, rather than adrenergic a receptors. Consistent with our results, taurine has been reported to increase serum levels of NO, NO synthase, and reactive nitrogen oxide species in the rat model of hypertension (30) and in guinea pig spleen tissue (4). However, the possible mechanisms of taurine on NO synthase in our study and the above studies may differ, and remains uncertain in this study.
Smooth muscle contraction is initiated by an increase in cytosolic free Ca 2+ + , brought about either by release of Ca 2+ + from intracellular stores or by influx of Ca 2+ + through voltage-dependent Ca 2+ + channels (31). Our results indicated that verapamil, an L-type Ca 2+ + channel antagonist, blocked the effect of taurine on the contractility of isolated jejunal segments in NCS and HCS induced by high Ca 2+ + (5.0 mM) Krebs buffer and LCS induced by low Ca 2+ + (1.25 mM) Krebs buffer, suggesting that modulation of intestinal contractility by taurine is Ca 2+ + dependent and involves L-type Ca 2+ + channels. It has been demonstrated that taurine may exert a normalizing action on the [Ca 2+ + ] i by directly or indirectly modulating the activity of the voltage-dependent Ca 2+ + channels or other transmembrane ion channels and transporters in the cell organelles (16,(32)(33)(34). Such dual effects on [Ca 2+ + ] i may explain the bidirectional modulation of taurine on smooth muscle contraction.
Although the present research characterized a homeostatic modulation of taurine on contractility of intestinal smooth muscle, it should still be considered as preliminary. Further studies are needed to reveal the detailed mechanism, including the possible neurotransmitters involved and the details of cell signal transduction.

Supplementary Material
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