Augmentation of the ascending component of the peristaltic reflex and substance P release by glial cell line-derived neurotrophic factor.

BACKGROUND: Glial cell line-derived neurotrophic factor (GDNF) is present in adult gut although its role in the mature enteric nervous system is not well defined. The aim of the present study was to examine the role of GDNF as neuromodulator of the ascending phase of the peristaltic reflex. METHODS: Colonic segments were prepared as flat sheets and placed in compartmented chambers so as to separate the sensory and motor limbs of the reflex. Ascending contraction was measured in the orad compartment and mucosal stroking stimuli (two to eight strokes) were applied in the caudad compartment. GDNF and substance P (SP) release were measured and the effects of GDNF and GDNF antibody on contraction and release were determined. Mice with reduced levels of GDNF (Gdnf(+/-)) and wild type littermates were also examined. KEY RESULTS: GDNF was released in a stimulus-dependent manner into the orad motor but not caudad sensory compartment. Addition of GDNF to the orad motor but not caudad sensory compartment augmented ascending contraction and SP release. Conversely, addition of GDNF antibody to the orad motor but not caudad sensory compartment reduced ascending contraction and SP release. Similarly, the ascending contraction and SP release into the orad motor compartment was reduced in Gdnf(+/-) mice as compared to wild type littermates. CONCLUSIONS & INFERENCES: The results suggest that endogenous GDNF is released during the ascending contraction component of the peristaltic reflex where it acts as a neuromodulator to augment SP release from motor neurons thereby augmenting contraction of circular muscle orad to the site of stimulation.

Differential changes in brain-derived neurotrophic factor and extracellular signal-regulated kinase in rat primary afferent pathways with colitis.

Brain-derived neurotrophic factor (BDNF) has been postulated to participate in inflammation-induced visceral hypersensitivity by modulating the sensitivity of visceral afferents through the activation of intracellular signalling pathways such as the extracellular signal-regulated kinase (ERK) pathway. In the current study, we assessed the expression levels of BDNF and phospho-ERK in lumbosacral dorsal root ganglia (DRG) and spinal cord before and during tri-nitrobenzene sulfonic acid (TNBS)-induced colitis in rats with real-time PCR, ELISA, western blot and immunohistochemical techniques. BDNF mRNA and protein levels were increased in L1 and S1 but not L6 DRG when compared with control (L1: two- to five-fold increases, P < 0.05; S1: two- to three-fold increases, P < 0.05); however, BDNF protein but not mRNA level was increased in L1 and S1 spinal cord when compared with control. In parallel, TNBS colitis significantly induced phospho-ERK1/2 expression in L1 (four- to five-fold, P < 0.05) and S1 (two- to three-fold, P < 0.05) but not in L6 spinal cord levels. Immunohistochemistry results showed that the increase in phospho-ERK1/2 expression occurred at the region of the superficial dorsal horn and grey commisure of the spinal cord. In contrast, there was no change in phospho-ERK5 in any level of the spinal cord examined during colitis. The regional and time-specific changes in the levels of BDNF mRNA, protein and phospho-ERK with colitis may be a result of increased transcription of BDNF in DRG and anterograde transport of BDNF from DRG to spinal cord where it activates intracellular signalling molecules such as ERK1/2.

Coupling of the insulin-like growth factor-I receptor tyrosine kinase to Gi2 in human intestinal smooth muscle: Gbetagamma -dependent mitogen-activated protein kinase activation and growth.

Endogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, G(i), which mediates G(beta)gamma-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of G(i2). G(beta)gamma subunits selectively mediated IGF-I-dependent MAP kinase activation; G(alpha)i2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase activity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of G(i)/G(o) activation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of G(i2) activation. These data suggest that IGF-I specifically activates G(i2), resulting in concurrent G(beta)gamma-dependent stimulation of MAP kinase activity and growth, and G(alpha)i2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression.

Motility disorders of the small intestine: new insights into old problems.

Motility of the gastrointestinal tract is responsible for the orderly movement of food, in an oral-to-aboral direction, and allows for the digestion and absorption of nutrients and water, and the elimination of indigestible material. This complex series of events results from the integrated activity of enteric nerves, extrinsic nerves, the intrinsic properties of smooth muscle, and gastrointestinal hormones. Abnormalities in any of these components or in their integration can result in dysmotility: increased transit, decreased transit, or nonpropulsive activity. This review outlines the current understanding of the causes, pathophysiology, diagnostic evaluation, and treatment of motility disorders of the small intestine.

Regulation of IGFBP-4 levels in human intestinal muscle by an IGF-I-activated, confluence-dependent protease.

Human intestinal smooth muscle cells in culture produce insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3), IGFBP-4, and IGFBP-5, which can modulate the effects of IGF-I on growth. This study examined the role of IGFBP-4 on IGF-I-induced growth and the mechanisms regulating IGFBP-4 levels. IGFBP-4 inhibited IGF-I-induced [(3)H]thymidine incorporation. IGFBP-4 mRNA levels were not altered by IGF-I. IGF-I caused a concentration-dependent activation of an endogenous IGFBP-4 protease, resulting in time-dependent degradation of intact IGFBP-4 into inactive fragments. Protease activity was measured in a cell-free assay using smooth muscle cell conditioned medium containing the IGFBP-4 protease. The protease was inhibited by EDTA and benzamidine. Protease activity was highest in proliferating cells and lowest in postconfluent cells. The role of endogenous IGF-I in regulating IGFBP-4 degradation was confirmed by the ability of an IGF-I antagonist to inhibit IGF-I-activated IGFBP-4 proteolysis in intact cells. We conclude that in human intestinal smooth muscle cells levels of secreted IGFBP-4 are determined by the confluence-dependent production of a cation-dependent serine protease that is activated by endogenous IGF-I.

Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes.

The role of protein kinase C (PKC) in sustained contraction was examined in intestinal circular and longitudinal muscle cells. Initial contraction induced by agonists (CCK-8 and neuromedin C) was abolished by 1) inhibitors of Ca(2+) mobilization (neomycin and dimethyleicosadienoic acid), 2) calmidazolium, and 3) myosin light chain (MLC) kinase (MLCK) inhibitor KT-5926. In contrast, sustained contraction was not affected by these inhibitors but was abolished by 1) the PKC inhibitors chelerythrine and calphostin C, 2) PKC-epsilon antibody, and 3) a pseudosubstrate PKC-epsilon inhibitor. GDPbetaS abolished both initial and sustained contraction, whereas a Galpha(q/11) antibody inhibited only initial contraction, implying that sustained contraction was dependent on activation of a distinct G protein. Sustained contraction induced by epidermal growth factor was inhibited by calphostin C, PKC-alpha,beta,gamma antibody, and a pseudosubstrate PKC-alpha inhibitor. Ca(2+) (0.4 microM) induced an initial contraction in permeabilized muscle cells that was blocked by calmodulin and MLCK inhibitors and a sustained contraction that was blocked by calphostin C and a PKC-alpha,beta,gamma antibody. Thus initial contraction induced by Ca(2+), agonists, and growth factors is mediated by MLCK, whereas sustained contraction is mediated by specific Ca(2+)-dependent and -independent PKC isozymes. G protein-coupled receptors are linked to PKC activation via distinct G proteins.

Endogenous IGF-I regulates IGF binding protein production in human intestinal smooth muscle cells.

Human intestinal smooth muscle in culture produces insulin-like growth factor (IGF)-I and IGF binding protein (IGFBP)-3, IGFBP-4, and IGFBP-5, which modulate the effects of IGF-I. This study examined the regulation of IGFBP production by endogenous IGF-I. R3-IGF-I, an agonist unaffected by IGFBPs, elicited concentration-dependent increase in growth, measured by [(3)H]thymidine incorporation, and production of IGFBP-3, IGFBP-4, and IGFBP-5, measured by Western blot. Antagonists of the IGF-I receptor, IGF-I Analog or monoclonal antibody 1H7, elicited concentration-dependent inhibition of growth and decrease in IGFBP-3, IGFBP-4, and IGFBP-5 production, implying that endogenous IGF-I stimulated growth and IGFBP production. R3-IGF-I-induced increase in IGFBP-3, IGFBP-4, and IGFBP-5 production was partially inhibited by a mitogen-activated protein (MAP) kinase or a phosphatidylinositol-3-kinase (PI 3-kinase) inhibitor and abolished by the combination. We conclude that endogenous IGF-I stimulates growth and IGFBP-3, IGFBP-4, and IGFBP-5 production in human intestinal smooth muscle cells. Regulation of IGFBP production by IGF-I is mediated by activation of distinct MAP kinase and PI 3-kinase pathways, the same pathways through which IGF-I stimulates growth.

IGFBP-3 and IGFBP-5 production by human intestinal muscle: reciprocal regulation by endogenous TGF-beta1.

Insulin-like growth factor-I (IGF-I)-mediated growth of cells can be modulated by specific IGF binding proteins (IGFBPs) that inhibit or augment IGF-I ligand-receptor interaction. IGFBP expression and production by human intestinal muscle cells in culture was characterized in rapidly growing cells (day 3 of culture), in confluent cells (day 7), and in postconfluent cells (day 14). RT-PCR analysis identified IGFBP-3, IGFBP-4, and IGFBP-5 mRNA during all three phases of growth. The production of IGFBP-3 and IGFBP-5 was regulated in reciprocal fashion. IGFBP-5 production was high on day 3 and decreased two- to fivefold by day 14, and IGFBP-3 production was low on day 3 and increased five- to eightfold by day 14. IGFBP-4 production remained constant. IGFBP-3 inhibited and IGFBP-5 augmented IGF-I-induced proliferation. IGFBP-3 and IGFBP-5 production was regulated in reciprocal fashion by transforming growth factor-beta1 (TGF-beta1). Immunoneutralization of endogenous TGF-beta1 decreased the production of IGFBP-3 and increased the production of IGFBP-5. Addition of exogenous recombinant human TGF-beta1 had the opposite effect. We conclude that the expression and time-dependent production of IGFBP-3, IGFBP-4, and IGFBP-5 and their regulation by endogenous TGF-beta1 represent mechanisms by which human intestinal muscle cells regulate autocrine IGF-I-mediated growth.

Selective expression of vasoactive intestinal peptide (VIP)2/pituitary adenylate cyclase-activating polypeptide (PACAP)3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells.

In both functional and radioligand binding studies of gastric smooth muscle from rabbit and guinea pig, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) show equal potency indicating that the receptor type is either a VIP1/PACAP2 or a VIP2/PACAP3 receptor. We have characterized the VIP/PACAP receptor expressed in freshly dispersed and cultured gastric and tenia coli smooth muscle cells of rabbit and guinea pig by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern analysis, and cloning of the first extracellular domain. Specific primers based on cDNA sequences for rat VIP1/PACAP2, VIP2/PACAP3 and PACAP1 receptors were designed spanning the first extracellular domain. A 275 base pair product corresponding to VIP2/PACAP3 receptor was amplified by RT-PCR in muscle cells from both species. No RT-PCR product was obtained with primers for VIP1/PACAP2 and PACAP1 receptors. The deduced amino acid sequences showed 90% similarity in rabbit and 77% in guinea pig to the sequence in rat. The location of the aspartate, tryptophan and glycine residues and all six N-terminal cysteines required for VIP binding were conserved. The sequence in guinea pig tenia coli differed from that in guinea pig stomach by two amino acid residues, Phe40 and Phe41. Northern analysis revealed a single 3.9 kilobase (kb) mRNA corresponding to VIP2/PACAP3 receptors in rabbit and a 2.1 kb mRNA in guinea pig gastric and tenia coli muscle cells. We conclude that only VIP2/PACAP3 receptors are expressed in smooth muscle cells of rabbit and guinea pig. The two amino acid difference in the sequence obtained from guinea pig tenia coli may reflect the distinct binding and functional properties of this tissue.

Expression of endothelial nitric oxide synthase in human and rabbit gastrointestinal smooth muscle cells.

The aim of this study was to identify the nitric oxide synthase (NOS) isoform expressed in freshly dispersed rabbit gastric smooth muscle cells and in cultured rabbit gastric, human intestinal, and guinea pig taenia coli smooth muscle cells. RT-PCR products of the predicted size (354 bp) were obtained with endothelial NOS (eNOS)-specific primers, but not neuronal NOS (nNOS)- or inducible NOS (iNOS)-specific primers, in all smooth muscle preparations except guinea pig taenia coli. Control RT-PCR studies showed absence of the endothelial markers, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor receptor (VEGFR), and the interstitial cell marker, c-kit, from cultures of smooth muscle cells. Cloning and sequence analysis showed that the predicted amino acid sequence (117 residues) in rabbit and human smooth muscle cells differed by only one residue from that of human eNOS. Northern blot analysis, using the PCR-generated and cloned eNOS cDNA from rabbits and humans as probes, demonstrated the expression of eNOS mRNA (4.4 kb) in both species. eNOS, but not nNOS or iNOS, transcripts were localized by in situ RT-PCR in single, freshly dispersed rabbit gastric smooth muscle cells; expression was evident in the majority of cells in each preparation. We conclude that eNOS is selectively expressed in rabbit gastric and human intestinal smooth muscle cells. The results confirm functional evidence for the existence of a constitutive NOS in smooth muscle cells of the gut in different species, except for guinea pig taenia coli.

IGF-I stimulates intestinal muscle cell growth by activating distinct PI 3-kinase and MAP kinase pathways.

Insulin-like growth factor I (IGF-I), acting via its cognate receptor, plays an autocrine role in the regulation of growth of intestinal muscle cells. In the present study the signaling pathways mediating the growth effects of IGF-I were characterized in cultured human intestinal smooth muscle cells. Growth induced by a maximally effective concentration of IGF-I (100 nM), measured as [3H]thymidine incorporation, was only partially inhibited by LY-294002 [phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor] or PD-98059 [mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor] (86 +/- 7% and 35 +/- 6% inhibition, respectively) alone but was abolished by the two combined (114 +/- 18% inhibition), implying the participation of both pathways. IGF-I elicited time- and concentration-dependent increases in PI 3-kinase activity. This effect was inhibited only by LY-294002 (89 +/- 12%). IGF-I elicited time- and concentration-dependent phosphorylation of p44/p42 MAP kinase and increased MAP kinase activity. These effects were inhibited only by PD-98059 (78 +/- 9% and 98 +/- 7%, respectively). We conclude that in human intestinal muscle cells IGF-I activates distinct PI 3-kinase and MAP kinase signaling pathways, which act in conjunction to mediate growth.

Synergistic regulation of NOS II expression by IL-1 beta and TNF-alpha in cultured rat colonic smooth muscle cells.

Interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured as L-[3H]citrulline production, was increased 24.3 +/- 6.9 pmol.min-1.mg protein-1 by 1 nM IL-1 beta and 3.2 +/- 1.1 pmol.min-1.mg protein-1 by 1 nM TNF-alpha, and increased synergistically by a combination of the two (51.8 +/- 7.3 pmol.min-1.mg protein-1). Measurement of NOS II activity as nitrite production yielded similar results: IL-1 beta, 27.2 +/- 1.2; TNF-alpha, 1.6 +/- 0.1; and IL-1 beta + TNF-alpha, 46.8 +/- 3.2 pmol.min-1.mg protein-1 above basal. LPS (10 micrograms/ml) had a small but significant effect at 48 h that was only additive with that of IL-1 beta. The increase in NOS II activity induced by IL-1 beta and TNF-alpha was inhibited 73-86% by transforming growth factor-beta 1 (TFG-beta 1). The NOS isoform induced by IL-1 beta and TNF-alpha was identified as NOS II by Western immunoblot analysis and confirmed by its 66-97% inhibition by 100 microM S-methylisothiourea, a selective NOS II inhibitor, and its Ca(2+)-independent activity. We conclude that the cytokines IL-1 beta and TNF-alpha act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-beta 1.

Longitudinal smooth muscle of the mammalian intestine. A model for Ca2+ signaling by cADPR.

Ca2+ mobilization in muscle cells from the circular muscle layer of the mammalian intestine is mediated by IP3-dependent Ca2+ release. Ca2+ mobilization in muscle from the adjacent longitudinal muscle layer involves a distinct, phosphoinositide-independent pathway. Receptors for contractile agonists in longitudinal muscle cells are coupled via a pertussis toxin-sensitive G protein to activation of PLA2 and formation of arachidonic acid (AA). The latter activates Cl- channels resulting in depolarization of the plasma membrane and opening of voltage-sensitive Ca2+ channels. Ca2+ influx via these channels induces Ca2+ release by activating sarcoplasmic ryanodine receptor/Ca2+ channels. The increase in [Ca2+]i activates membrane-bound ADP ribosyl cyclase, and the resultant formation of cADPR enhances Ca(2+)-induced Ca2+ release.

Autocrine regulation of growth in cultured human intestinal muscle by growth factors.

BACKGROUND & AIMS: Transforming growth factor (TGF)-alpha, insulin-like growth factor (IGF)-I, and TGF-beta 1 are expressed in vivo by intestinal smooth muscle. The aim of this study was to determine whether these growth factors were produced by human intestinal muscle cells in culture and to identify their roles in regulating growth. METHODS: Muscle cells were examined at various times in culture: during rapid growth (day 3), at confluence (day 7), and after confluence (day 14). Growth factor production was measured by radioimmunoassay or enzyme-linked immunosorbent assay. Growth was measured from [3H]thymidine incorporation. RESULTS: Production of pro-TGF-alpha and TGF-alpha (1550 +/- 100 and 1260 +/- 150 pg/mg protein, respectively) and free IGF-I (86.2 +/- 23.7 ng/mg protein) was highest during rapid growth and 3-40-fold lower later in culture. Production of soluble and latent TGF-beta 1 was highest in postconfluent cells (280 +/- 74 and 4320 +/- 610 pg/mg protein, respectively) and 4-7-fold lower earlier in culture. TGF-alpha and IGF-I caused concentration-dependent stimulation of growth in rapidly growing cells. TGF-beta 1 caused concentration-dependent inhibition of growth predominantly in postconfluent cells. Neutralizing antibodies to TGF-alpha or IGF-I inhibited growth and neutralizing antibody to TGF-beta augmented growth. CONCLUSIONS: Human intestinal muscle cells produce TGF-alpha, IGF-I, and TGF-beta 1 in a time-dependent reciprocal fashion that parallels their effects on growth.

Distinct 5-HT receptors mediate the peristaltic reflex induced by mucosal stimuli in human and guinea pig intestine.

BACKGROUND & AIMS: The role of 5-hydroxytryptamine (5-HT) in regulating the peristaltic reflex in humans is unknown. The neural pathways subserving peristalsis induced by mucosal stimulation were characterized in human jejunum and guinea pig colon. METHODS: Compartmented flat-sheet preparations that enable measurement of 5-HT and sensory transmitter release into one compartment and mechanical responses in adjacent compartments were used. RESULTS: Mucosal stimuli (2-8 brush strokes) caused concomitant release of 5-HT and calcitonin gene-related peptide (CGRP) into the compartment where stimulation was applied in both species; in contrast, muscle stretch caused release of CGRP only. CGRP release as well as ascending contraction and descending relaxation of circular muscle induced by mucosal stimulation were inhibited by a selective 5-HT4 antagonist in human jejunum and by selective 5-HT4 and 5-HT3 antagonists in guinea pig colon. The effects of the 5-HT3 and 5-HT4 antagonists in guinea pig colon were additive. A selective 5-HT1P antagonist mimicked the effect of the 5-HT4 antagonist. The CGRP antagonist human CGRP8-37 inhibited ascending and descending responses in both species. CONCLUSIONS: 5-HT released by mucosal stimulation initiates a peristaltic reflex by activating 5-HT4/5-HT1P receptors on sensory CGRP neurons in human intestine and 5-HT4/5-HT1P and 5-HT3 receptors in guinea pig colon.

5-HT released by mucosal stimuli initiates peristalsis by activating 5-HT4/5-HT1p receptors on sensory CGRP neurons.

The intestinal peristaltic reflex can be elicited by mucosal stimulation or circular muscle stretch. Muscle stretch activates extrinsic, whereas mucosal stimulation activates intrinsic calcitonin gene-related peptide (CGRP)-containing sensory neurons. The present study examined the role of 5-hydroxytryptamine (5-HT) in sensory transmission. A three-compartment preparation of rat colon was used that enables separate measurement of sensory transmitters and modulators. Mucosal stimuli (2-8 brush strokes) caused concurrent increase in 5-HT and CGRP release in proportion to the intensity of stimulation. Release of both 5-HT and CGRP occurred exclusively into the central compartment where the stimuli were applied. Exogenous 5-HT caused a concentration-dependent release of CGRP. Release of CGRP induced by exogenous 5-HT or mucosal stimulation was inhibited by selective 5-HT4 and 5-HT1p antagonists but was not affected by 5-HT1A, 5-HT2, and 5-HT3 antagonists. Ascending contraction and descending relaxation of circular muscle measured in the peripheral orad and caudad compartments, respectively, were also selectively inhibited by 5-HT4 and 5-HT1p antagonists added to the central but not peripheral compartments. In contrast, muscle stretch elicited CGRP but not 5-HT release; the ascending contraction and descending relaxation components of the peristaltic reflex induced by muscle stretch were not affected by 5-HT antagonists. We conclude that 5-HT released by mucosal stimulation initiates the peristaltic reflex by activating 5-HT4/5-HT1p receptors on sensory CGRP-containing neurons.

Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells.

BACKGROUND & AIMS: The type and function of 5-hydroxytryptamine (5HT) receptors on intestinal muscle cells in humans are not known. 5-HT receptors were characterized pharmacologically and by radioligand binding. METHODS: Contraction, relaxation, inositol 1,4,5-triphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) formation, and 5-HT binding were measured in dispersed muscle cells and in cells in which only one receptor type was preserved by selective receptor protection. RESULTS: 5-HT binding was completely inhibited by 5-HT and partially by 5-HT2A (ketanserin), 5-HT4 (SDZ-205,557), and 5-HT1p (N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide; 5-HTP-DP) receptor antagonists. 5-HT caused contraction that was inhibited by ketanserin and augmented by SDZ-205,557 and 5-HTP-DP. In the presence of ketanserin, 5-HT caused relaxation of cholecystokinin-contracted cells that was inhibited by SDZ-205,557 and 5-HTP-DP. 5-HT increased IP3, which was inhibited by ketanserin, and cAMP, which was inhibited by SDZ-205,557 and 5-HTP-DP. In cells with only 5-HT2A receptors, 5-HT caused contraction only, and residual binding was inhibited by ketanserin. In cells with only 5-HT4/5-HT1p receptors, 5-HT caused only relaxation and residual binding was inhibited by SDZ-205,557 and 5-HTP-DP. CONCLUSIONS: 5-HT2A receptors mediating contraction and 5-HT4 receptors mediating relaxation coexist on human intestinal muscle cells. The 5-HT4 receptors are closely similar or identical to 5-HT1p receptors.

Agonist-stimulated cyclic ADP ribose. Endogenous modulator of Ca(2+)-induced Ca2+ release in intestinal longitudinal muscle.

We have previously shown that agonist-induced Ca2+ mobilization in intestinal longitudinal muscle is mediated by ryanodine-sensitive, inositol 1,4,5-trisphosphate-insensitive sacroplasmic Ca2+ channels. Ca2+ release via these channels is triggered by agonist-stimulated Ca2+ influx and results in Ca(2+)-induced Ca2+ release. The present study examined whether cyclic ADP-ribose (cADPR) is synthesized in response to stimulation of longitudinal muscle by agonists and modulates the activity of Ca2+ release channels. Cyclic ADPR bound with high affinity to dispersed longitudinal muscle cells (IC50 1.9nM) and induced Ca2+ release (EC50 3.8 nM), increase in [Ca2+]i (EC50 2.0 nM), and contraction (EC50 1.1 nM); cADPR had no effect on circular muscle cells. The effects of cADPR were blocked by ruthenium red, dantrolene, and the specific antagonist, 8-amino-cADPR, and were augmented by caffeine but not affected by heparin. The binding of cADPR and its ability to stimulate Ca2+ release were dependent on the concentration of Ca2+. Cyclic ADPR was capable of stimulating Ca2+ release at subthreshold Ca2+ concentrations (25-100 nM) and of enhancing Ca(2+)-induced Ca2+ release. Longitudinal muscle extracts incubated with beta-NAD+ produced a time-dependent increase in Ca(2+)-mobilizing activity identified as authentic cADPR by blockade of Ca2+ release with 8-amino-cADPR and ruthenium red. Ca2+ mobilizing activity was increased by cholecystokinin octapeptide (CCK-8) in a concentration-dependent fashion. The increase induced by CCK-8 was suppressed by the CCK-A antagonist, L364,718, nifedipine, and guanyl-5'-yl thiophosphate. The study shows that ADP-ribosyl cyclase can be stimulated by agonists and that cADPR can act as an endogenous modulator of Ca(2+)-induced Ca2+ release.

Agonist-mediated activation of PLA2 initiates Ca2+ mobilization in intestinal longitudinal smooth muscle.

Recent studies have shown that Ca2+ mobilization in longitudinal muscle is initiated by inositol 1,4,5-trisphosphate (IP3)-independent Ca2+ influx that acts as a trigger for Ca(2+)-induced Ca2R release. The present study examined whether arachidonic acid (AA) acts as mediator of the initial Ca2+ influx. Cholecystokinin octapeptide caused transient concentration-dependent increase in AA release in dispersed intestinal longitudinal but not circular muscle cells followed by sustained increase in both muscle cell types. The initial increase in AA release coincided with the initial Ca2+ transient and muscle contraction: all three events were abolished by guanosine 5'-O-(2-thiodiphosphate), pertussis toxin (PTX), and the phospholipase A2 (PLA2) inhibitor, dimethyleicosadienoic acid, but were not affected by calphostin C or neomycin. Exogenous AA caused concentration-dependent contraction and increase in cytosolic free Ca2+ ([Ca2+]i) in longitudinal but not circular muscle cells; both events were abolished by Ca2+ channel blockers. Depletion of Ca2+ stores with thapsigargin attenuated with thapsigargin attenuated agonist- and AA-mediated increase in [Ca2+]i and contraction in longitudinal muscle cells: the residual [Ca2+]i increase (35%) and contraction (25%) reflected the component of Ca2+ influx. We conclude that AA released by agonist-mediated G protein-dependent PTX-sensitive activation of PLA2 mediates Ca2+ influx, which then triggers Ca(2+)-induced Ca2+ release. The process is independent of phosphatidylinositol hydrolysis and occurs exclusively in longitudinal smooth muscle, in which Ca2+ release channels are highly sensitive to Ca2+, ryanodine, and cyclic ADP-ribose and insensitive to IP3.

Therapy of gastrointestinal motility disorders: moving in the right direction.

Coordinated motility of the gastrointestinal tract is responsible for the orderly movement of food and digesta from the mouth to the rectum. This complex series of events results from the integrated activity of enteric nerves, extrinsic nerves, circulating hormones, and smooth muscle. Abnormalities in any of these components or in their integration can result in dysmotility. The important distinction between motility and the transit of luminal contents is emphasized by the recognition that disordered motility may manifest as increased or decreased transit or even as nonpropulsive activity. Similarly, altered motility or transit may or may not be accompanied by alterations in sensation. Ideally, treatment of motility disorders is directed at correction of the underlying pathophysiological defect. Although many of these disorders have been associated with characteristic patterns of abnormal motility, the pathological etiologies involved are poorly understood. Therapy in these cases must then be directed toward amelioration of clinical features and symptoms. The present therapeutic recommendations summarize current modalities in the medical treatment of motility disorders of the esophagus, stomach, small intestine, and colon. Emphasis is given to recent advances in our understanding of motility and to the use of the newer agents in our pharmacological armamentarium.