Intrinsic and selective activity of functionalized carbon nanotube/nanocellulose platforms against colon cancer cells.

Given their large surface area and versatile chemical reactivity, single-walled carbon nanotubes (SWCNTs) are regarded as the basis of new pharmacological complexes. In this study, SWCNTs are chemically functionalized with fluorescein, folic acid, and capecitabine, a drug that is commonly used against colorectal cancer. These functionalized SWCNTs are dispersed in water by taking advantage of their synergistic interaction with type-II nanocrystalline cellulose (II-NCC), and the resulting colloidal system is tested in vitro on both normal (differentiated) and cancerous (proliferative) human colon cells (Caco-2). The functionalized SWCNT/II-NCC hybrids show a higher activity than the reference (capecitabine) against the Caco-2 cancer cell line. However, this effect appears to be intrinsically associated with the SWCNT/II-NCC complex, particularly boosted by fluorescein, as the presence of capecitabine is not required. In addition, confocal microscopy fluorescence imaging using cell cultures highlights the enormous potential of this nanohybrid platform for colon cancer theranostics.

Differential properties and effects of fluorescent carbon nanoparticles towards intestinal theranostics.

Given the potential applications of fluorescent carbon nanoparticles in biomedicine, the relationship between their chemical structure, optical properties and biocompatibility has to be investigated in detail. In this work, different types of fluorescent carbon nanoparticles are synthesized by acid treatment, sonochemical treatment, electrochemical cleavage and polycondensation. The particle size ranges from 1 to 6 nm, depending on the synthesis method. Nanoparticles that were prepared by acid or sonochemical treatments from graphite keep a crystalline core and can be classified as graphene quantum dots. The electrochemically produced nanoparticles do not clearly show the graphene core, but it is made of heterogeneous aromatic structures with limited size. The polycondensation nanoparticles do not have CC double bonds. The type of functional groups on the carbon backbone and the optical properties, both absorbance and photoluminescence, strongly depend on the nanoparticle origin. The selected types of nanoparticles are compatible with human intestinal cells, while three of them also show activity against colon cancer cells. The widely different properties of the nanoparticle types need to be considered for their use as diagnosis markers and therapeutic vehicles, specifically in the digestive system.

Toll-like receptors 2 and 4 exert opposite effects on the contractile response induced by serotonin in mouse colon: role of serotonin receptors.

What is the central question of this study? The action of Toll-like receptors (TLRs) 2 and 4 on the motor response to serotonin in mouse colon has not previously been reported. What is the main finding and its importance? Toll-like receptors 2 and 4 modulate the serotonin-induced contractile response in mouse colon by modifying the expression of serotonin (5-HT) receptors. Alterations in 5-HT2A and 5-HT2C receptors explain the increase of the response to serotonin in TLR2(-/-) mice. Alterations in 5-HT2C and 5-HT4 receptors explain the suppression of the response to serotonin in TLR4(-/-) mice. The microbiota, through Toll-like receptors (TLRs), may regulate gastrointestinal motility by activating neuroendocrine mechanisms. We evaluated the influence of TLR2 and TLR4 in spontaneous contractions and in the serotonin (5-HT)-induced motor response in mouse colon, and assessed the 5-HT receptors involved. Muscle contractility studies to evaluate the intestinal spontaneous motility and the response to 5-HT were performed in the colon from wild-type (WT), TLR2(-/-) , TLR4(-/-) and TLR2/4 double knockout (DKO) mice. The 5-HT receptor mRNA expression was determined by real-time PCR. The amplitude and frequency of the spontaneous contractions of the colon were smaller in TLR4(-/-) and TLR2/4 DKO mice with respect to WT mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 100 µm 5-HT evoked a contractile response. The contractile response induced by 5-HT was significantly higher in TLR2(-/-) than in WT mice. In TLR4(-/-) mice, 5-HT did not evoke any contractile response. The mRNA expression of 5-HT2A was increased in TLR2(-/-) and TLR2/4 DKO mice. The 5-HT2C and 5-HT4 mRNA expressions were increased in TLR4(-/-) and TLR2/4 DKO mice. The 5-HT2C mRNA expression was diminished in TLR2(-/-) mice. The 5-HT3 mRNA expression was increased in TLR2(-/-) , TLR4(-/-) and TLR2/4 DKO mice. The 5-HT7 mRNA expression was diminished in TLR2/4 DKO mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 5-HT2 , 5-HT3 , 5-HT4 and 5-HT7 receptor antagonists reduced or blocked the contractile response evoked by 5-HT. We postulate that TLR2 and TLR4 modulate the serotonin contractile motor response in mouse colon in an opposing manner by modifying the expression of several serotonin receptors.

Toll-like receptors 2 and 4 modulate the contractile response induced by serotonin in mouse ileum: analysis of the serotonin receptors involved.

BACKGROUND: Microbiota through toll-like receptors (TLR) may regulate gastrointestinal motility by activating neuroendocrine mechanisms. We evaluated the influence of TLR2 and TLR4 in the spontaneous contractions and serotonin (5-HT)-induced motor response in mouse ileum, and the 5-HT receptors involved. METHODS: Muscle contractility studies to evaluate the spontaneous intestinal motility and the response to 5-HT were performed in the ileum from wild type (WT), TLR2(-/-), TLR4(-/-), and TLR2/4 double knockout (DKO) mice. 5-HT receptor expression was determined by real-time PCR. KEY RESULTS: The amplitude of spontaneous contractions in ileum was higher in TLR2(-/-), TLR4(-/-), and TLR2/4 DKO mice with respect to WT. 5-HT evoked concentration-dependent contractile responses in the ileum from TLR2(-/-) and TLR4(-/-) mice similar to WT. However, in ileum from TLR2/4 DKO, 5-HT did not induce any contractile response. Expression of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT3 receptors resulted increased in ileum from TLR4(-/-) and TLR2/4 DKO. Expression of the 5-HT4 receptor was diminished in TLR2(-/-) and TLR2/4 DKO. High levels of 5-HT7 receptor expression were found in TLR2/4 DKO but not in TLR2(-/-) or TLR4(-/-). In WT and TLR4(-/-), 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptor antagonists reduced the contractile response evoked by 5-HT. In TLR2(-/-) mice, 5-HT4 antagonist did not reduce the 5-HT response. In TLR2/4 DKO mice, only 5-HT4 and 5-HT7 receptor antagonists reduced the relaxing response induced by 5-HT. CONCLUSIONS & INFERENCES: TLR2 and TLR4 signaling may modulate the spontaneous contractions and the serotonin contractile response by acting on 5-HT2, 5-HT3, 5-HT4, and 5-HT7 receptors.

Mechanism of action of Trolox on duodenal contractility.

Trolox is a hydrophilic analogue of vitamin E. The aim of this work was to study the mechanism of action of Trolox on rabbit duodenal spontaneous motility and contractility. The duodenal contractility studies in vitro were carried out in an organ bath. Trolox (12 mM) reduced the amplitude and frequency of spontaneous contractions and the acetylcholine-induced contractions in the longitudinal and circular smooth muscle of rabbit duodenum. Quinine reverted the Trolox-induced (12 mM) reduction on the amplitude and frequency of spontaneous contractions in the longitudinal and circular muscle. Charibdotoxin and glibenclamide reverted only the amplitude of spontaneous contractions in circular muscle of the duodenum. The decrease of ACh-induced contractions evoked by Trolox 12 mM in the longitudinal and circular smooth muscle of the duodenum was antagonized by quinine in longitudinal and circular muscle and by Bay K8644, 1H-[1,2,4]oxadiazolo [4, 3-α]quinoxalin-1-one (ODQ) and nimesulide in circular muscle. We conclude that in the decrease of duodenal contractility induced by Trolox participate K(+) and Ca(2+) channels, adenylyl cyclase, guanylyl cyclase and cyclooxygenase-2.

Extracellular signal-regulated kinase (ERK) is involved in LPS-induced disturbances in intestinal motility.

BACKGROUND: Lipopolysaccharide (LPS) is a causative agent of sepsis. A relationship has been described between LPS, free radicals, and cyclooxygenase-2 (COX-2). Here, we investigate the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPK) in the effect of LPS on intestinal motility, oxidative stress status, and COX-2 expression. METHODS: Rabbits were injected with (i) saline, (ii) LPS, (iii) U0126, an ERK MAPK inhibitor, or (iv) U0126+LPS. Duodenal contractility was studied in an organ bath with acetylcholine, prostaglandin E(2), and KCl added. Neuromuscular function was assessed by electrical field stimulation (EFS). Neurotransmitter blockers were used to study the EFS-elicited contractile response. The formation of products of oxidative damage to proteins (carbonyls), lipids, [malondialdehyde (MDA), and 4-hydroxyalkenals (4-HDA)] was quantified in plasma and intestine. The protein expression of phospho-ERK (p-ERK), total ERK, and COX-2 in the intestine was measured by western blot, and p-ERK was localized by immunohistochemistry. KEY RESULTS: Acetylcholine, prostaglandin E(2), and KCl-induced contractions decreased with LPS. Electrical field stimulation induced a neurogenic contraction that was reduced by LPS. Lipopolysaccharide increased p-ERK and COX-2 expression and the levels of carbonyls and MDA+4-HDA. U0126 blocked the effect of LPS on acetylcholine, prostaglandin E(2), KCl, and EFS-induced contractions, the levels of carbonyls and MDA+4-HDA and p-ERK and COX-2 expression. Phospho-ERK was detected mostly in the neurons of the myenteric and submucosal ganglia. CONCLUSIONS & INFERENCES: We can suggest that ERK is involved in the mechanism of action of LPS in the intestine.

Role of potassium channels in rabbit intestinal motility disorders induced by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH).

Oxidative stress appears to play a role in the pathogenesis of several inflammatory gastrointestinal diseases. Changes in intestinal motility have been reported in different models of intestinal inflammation. The initiating factor of altered motility could be an alteration of gut redox status. The aim of this study was to investigate the effect of oxidative stress evoked by 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH) on the intestinal motility of rabbit duodenum and the possible contribution of different K(+) channels in mediating this response. Whole thickness segments of rabbit duodenum were suspended in the direction of the longitudinal or circular smooth muscle fibres in an organ bath to study the effects of AAPH alone, or in the presence of different K(+) channel blockers on the amplitude, frequency and tone of spontaneous contractions. In circular muscle, AAPH 20 mM induced a reduction of the amplitude, the frequency and tone of the spontaneous contractions. In longitudinal muscle, AAPH 10 mM induced a reduction of the amplitude and tone of the spontaneous contractions. The reduction of the amplitude and tone induced by AAPH was reverted by BaCl2 (1 mM) and TEA (5 mM). Charybdotoxin (100 nM) and iberiotoxin (100 nM) only reverted the reduction of the tone induced by AAPH. In conclusion, our results show that the peroxyl radicals released by AAPH reduced the amplitude and the tone of the spontaneous contractions of the longitudinal smooth muscle from rabbit small intestine. Inward rectifier and intermediate and large-conductance Ca(2+)-activated K(+) channels could be involved in these effects.

Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model.

BACKGROUND: Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen-activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E(2) and KCl-induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine. METHODS: Rabbits were injected with (i) saline, (ii) LPS, (iii) SB203580, a specific p38 MAPK inhibitor or (iv) SB203580 + LPS. Duodenal contractility was studied in an organ bath. SB203580 was also tested in vitro. The protein expression of p-p38 and total p38 was measured by Western blot and p-p38 was localized by immunohistochemistry. The formation of products of oxidative damage to proteins (carbonyls) and lipids (MDA+4-HDA) was quantified in intestine and plasma. KEY RESULTS: ACh, PGE(2) and KCl-induced contractions decreased with LPS. LPS increased phospho-p38 expression and the levels of carbonyls and MDA+4-HDA. SB203580 blocked the effect of LPS on the ACh, PGE(2) and KCl-induced contractions in vivo and in vitro and the levels of carbonyls and MDA+4-HDA. P-p38 was detected in neurons of the myenteric plexus and smooth muscle cells of duodenum. CONCLUSIONS & INFERENCES: Lipopolysaccharide decreases the duodenal contractility in rabbits and increases the production of free radicals. p38 MAPK is a mediator of these effects.

A downregulation of nNOS is associated to dysmotility evoked by lipopolysaccharide in rabbit duodenum.

Alterations in gastrointestinal motility have been reported in response to endotoxin. The effects of lipopolysaccharide (LPS) on motility have been attributed to several substances, including prostaglandins and nitric oxide. The aim of this study was to investigate the expression and the contribution of NOS and COX enzymes to the local effect of LPS on ACh-evoked contractions in rabbit duodenum. The ACh evoked contractions were inhibited by LPS in longitudinal and circular muscles of duodenum. L-NNA, aminoguanidine, ODQ, indomethacin, and NS-398 but not NPLA antagonized the inhibitory effect of LPS. Western blot analysis showed protein bands of 155, 130, 70 and 72 kDa for nNOS, iNOS, COX-1 and COX-2 respectively in rabbit duodenum. All of these isoforms were expressed constitutively and only the nNOS was reduced in the presence of LPS. Expression of nNOS, iNOS, COX-1 and COX-2 was detected by inmunohistochemistry in the smooth muscle layers and in the neurons of the myenteric ganglia of rabbit duodenum. In conclusion, LPS locally administered reduces the contractility of rabbit duodenum and a downregulation of nNOS is associated to this effect. The iNOS, COX-1 and COX-2 were expressed constitutively but their expression was not modified by LPS.

Intestinal secretory and absorptive function in Trichinella spiralis mouse model of postinfective gut dysfunction: role of bile acids.

OBJECTIVE: Observations showing that bile acid malabsorption is frequent in irritable bowel syndrome (IBS) suggest that alterations in bile acid-induced secretion and absorption could contribute to IBS-associated diarrhoea. The secretory response to bile acids, fluid transport and bile absorption was examined in intestinal tissues from a Trichinella spiralis mouse model of postinfectious gut dysfunction in vitro. Changes in the protein expression of apical sodium-dependent bile acid transporter (ASBT) were also measured. DESIGN: T. spiralis-infected mice were killed at 18 and 25 days postinfection. Jejunal, ileal, proximal and distal colon segments were exposed to taurodeoxycholic acid (TDCA) or cholic acid. Short circuit current (SCC) increases were determined. Tritiated taurocholic acid (3H-TCA) absorption was determined in everted jejunal and ileal sacs. ASBT protein expression was determined by Western blot analysis and immunohistochemistry. RESULTS: Basal SCC increased in ileum and distal colon at 18 and 25 days postinfection, respectively. Ileal SCC responses to TDCA and cholic acid were enhanced at 18 days postinfection. Distal colon SCC response to TDCA was raised at 18 days postinfection but was significantly reduced by 25 days. Ileal 3H-TCA uptake was significantly reduced at 18 and 25 days postinfection. Surprisingly, increased ASBT expression was observed in infected animals. CONCLUSIONS: In a T. spiralis model of postinfectious gut dysfunction, decreased bile absorption and enhanced secretion in response to bile acids was observed. Decreased absorption was not, however, caused by decreased ASBT as increased expression was observed. If similar events occur postinfection, the combined effects of these disturbances may contribute to some symptoms observed in postinfectious IBS patients.

Ca2+-activated K+ channels involved in duodenal dismotility induced by ethanol.

The purpose of this study was to investigate the role of K+ channels in duodenal dismotility induced by ethanol in vitro. The amplitude of spontaneous contractions was reduced by ethanol in longitudinal and circular muscle, while frequency did not change. Charybdotoxin antagonized ethanol-induced inhibition of the amplitude of spontaneous contractions. Ethanol decreased ACh-induced contractions and this effect was cancelled out by charybdotoxin. Ca2+-activated K+ channels may be involved in duodenal dismotility induced by ethanol.

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+channels. The aim of this study was to examine the types of K+ channels involved in the contractility of longitudinal smooth muscle of rabbit small intestinein vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions (AU)

La mayoría de las células excitables, incluyendo las células lisas gastrointestinales, expresan varios tipos de canales de K+. El objetivo de este estudio es examinar los tipos de canales de K+que están involucrados en la contractilidad del músculo liso longitudinal del intestino delgado de conejoin vitro. Las contracciones espontáneas y las producidas por KCl se redujeron por atropina, fentolamina, propranolol, suramina, tetrodotoxina e indometacina. La amplitud y tono de las contracciones espontáneas aumentaron por apamin, charybdotoxina, iberiotoxina, E4031, tetraetilamonio (TEA) y BaCl2, mientras que la frecuencia de las contracciones se redujo en presencia de apamin, charybdotoxina y TEA. La 4-aminopiridina aumentó el tono de las contracciones espontáneas y redujo la amplitud y frecuencia de las contracciones. La glibenclamida no modificó la amplitud, frecuencia y tono de las contracciones. Las contracciones producidas por el KCl aumentaron en presencia de E4031, no fueron modificadas por el apamin, glibenclamida, NS1619 o diazóxida y disminuyeron en presencia de la charybdotoxina, TEA, 4-aminopiridina o BaCl2. Estos resultados sugieren que los canales de K+ activados por Ca2+ de pequeña y gran conductancia, canales de K+ HERG canales de K+ rectificadores de entrada participan en las contracciones espontáneas del intestino delgado. Por otra parte, los canales de K+ voltaje-dependientes, canales de K+ HERG, canales de K+ rectificadores de entrada y canales de K+activados por Ca2+ de gran conductancia están implicados en las contracciones producidas por el KCl (AU)

The role of tyrosine kinase in prostaglandin E2 and vanadate-evoked contractions in rabbit duodenum in vitro.

Prostaglandin E2 (PGE2) can interact with at least four cell surface receptors (EP1-EP4) in smooth muscle, which evokes a variety of intracellular responses depending on the G protein to which the cell surface receptors are coupled. The activation of G protein-coupled receptors and receptor tyrosine kinases can lead to the phosphorylation of tyrosine residues of various cellular proteins. The aim of this study was to examine the role of tyrosine phosphorylation in PGE2, vanadate and carbachol-evoked contractions. PGE2, vanadate, and carbachol induced contractile motor responses in the longitudinal smooth muscle of rabbit duodenum. PGE2-evoked contractions decreased in the presence of genistein or tyrphostin B44. PGE2-evoked contractions increased in the presence of vanadate. Vanadate-evoked contractions decreased in the presence of genistein. In contrast, tyrphostin 47 increased the vanadate-evoked contractions. Vanadate-evoked contractions were reduced in the presence of Ca2+-free solutions, verapamil, or indomethacin. U-73122 decreased PGE2-evoked contractions. Carbachol-evoked contractions decreased in the presence of genistein, tyrphostin B44 or tyrphostin 47. Our results suggest that PGE2, vanadate or carbachol-evoked contractions are mediated by protein tyrosine phosphorylation. Protein tyrosine phosphorylation might cause an increase in calcium influx through voltage-dependent channels and the release of prostaglandins in the longitudinal smooth muscle of the rabbit duodenum.

K+ channels involved in contractility of rabbit small intestine.

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K' channels involved in the contractility of longitudinal smooth muscle of rabbit small intestine in vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.

The role of NO in the contractility of rabbit small intestine in vitro: effect of K+ channels.

Nitric oxide (NO) is an inhibitory neurotransmitter of intestinal smooth muscle cells. The aim of this study was to determine the role of NO in the contractility of rabbit small intestine smooth muscle in vitro. The amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of duodenum, jejunum and ileum were determined and the sodium nitroprusside (SNP), acetylcholine (ACh) and KCl responses were quantified. L-NAME, L-NNA, L-arginine and D-arginine did not affect the amplitude, frequency and tone of spontaneous contractions. ODQ (10(-6) M) increased the tone of spontaneous contractions of the types of tissues examined, and the amplitude in ileum, without modifying the frequency. SNP (10(-4) M) evoked relaxations that were not influenced by atropine (10(-6) M) plus guanethidine (10(-6) M), apamin (10(-8) M) or glybenclamide (10(-6) M), but were increased by TTX (10(-6) M) and verapamil (10(-7) M). SNP-induced relaxations were reduced by charybdotoxin (10(-8) M) and ODQ (10(-6) M). ODQ (10(-5) M) reduced ACh-induced contractions, but it did not influence KCl-evoked contractions. Those results suggest that NO modulates the spontaneous contractions of small intestine in rabbits. This effect is mediated by cGMP and Ca2+-dependent K+ channels of large conductance.

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.