µ-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain.

Opiate analgesia in the spinal cord is impaired during neuropathic pain. We hypothesized that this is caused by a decrease in µ-opioid receptor inhibition of neurotransmitter release from primary afferents. To investigate this possibility, we measured substance P release in the spinal dorsal horn as neurokinin 1 receptor (NK1R) internalization in rats with chronic constriction injury (CCI) of the sciatic nerve. Noxious stimulation of the paw with CCI produced inconsistent NK1R internalization, suggesting that transmission of nociceptive signals by the injured nerve was variably impaired after CCI. This idea was supported by the fact that CCI produced only small changes in the ability of exogenous substance P to induce NK1R internalization or in the release of substance P evoked centrally from site of nerve injury. In subsequent experiments, NK1R internalization was induced in spinal cord slices by stimulating the dorsal root ipsilateral to CCI. We observed a complete loss of the inhibition of substance P release by the µ-opioid receptor agonist [D-Ala(2), NMe-Phe(4), Gly-ol(5)]-enkephalin (DAMGO) in CCI rats but not in sham-operated rats. In contrast, DAMGO still inhibited substance P release after inflammation of the hind paw with complete Freund's adjuvant and in naïve rats. This loss of inhibition was not due to µ-opioid receptor downregulation in primary afferents, because their colocalization with substance P was unchanged, both in dorsal root ganglion neurons and primary afferent fibers in the dorsal horn. In conclusion, nerve injury eliminates the inhibition of substance P release by µ-opioid receptors, probably by hindering their signaling mechanisms.

Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

Sex-dependent differences in the activity and modulation of N-methyl-d-aspartic acid receptors in rat dorsal root ganglia neurons.

Women have greater temporal summation of experimental pain stimuli and also have a higher propensity for developing chronic visceral pain conditions. Sex hormone-mediated regulation of N-methyl-d-aspartic acid receptors (NMDARs) in nociceptive pathways is a plausible mechanism that may underlie these phenomena. The aim of this study was to compare the effect of 17-beta-estradiol (E2) in modulation of NMDAR activity in adult male and female rat dorsal root ganglia (DRG) neurons. DRG neurons were collected from adult male or female rats and grown in short-term culture in steroid-free media. NMDAR currents were recorded on small to medium size neurons by whole cell patch clamp using rapid perfusion with saturating concentrations of N-methyl-d-aspartic acid and glycine in the absence of extracellular Mg(2+). We found that the average density of NMDAR currents was 2.8-fold larger in DRG neurons from female rats compared with male rats (P<0.0001). Addition of 100 nM E2 increased NMDAR currents 55+/-15% in female neurons, but only 19+/-7% in male neurons. Potentiation was maximal after 20-40 min and dose dependent with an apparent 50% excitatory concentration of 17-23 nM. This effect was mimicked by E2 conjugated to BSA and attenuated by pretreatment with the protein tyrosine kinase inhibitor lavendustin A (1 microM) or the estrogen receptor (ER) antagonist, ICI 182,780 (1 microM), strongly suggesting activation of a cell surface ER acting through a non-genomic mechanism involving protein tyrosine kinases to increase NMDAR currents. These results identify sex-based differences in both the basal expression and the regulation of the NMDARs in DRG neurons.

CRF2 receptor activation prevents colorectal distension induced visceral pain and spinal ERK1/2 phosphorylation in rats.

BACKGROUND AND AIMS: Activation of corticotropin releasing factor 1 (CRF1) receptors is involved in stress related responses and visceral pain, while activation of CRF2 receptors dampens the endocrine and some behavioural stress responses. We hypothesised that CRF2 receptor activation may influence visceral pain induced by colorectal distension (CRD) in conscious rats, and assessed the possible sites and mechanisms of action. METHODS: Male Sprague-Dawley rats were exposed to CRDs (60 mm Hg, 10 minutes twice, with a 10 minute rest interval). Visceromotor responses (VMR) were measured by electromyography or visual observation. Spinal (L6-S1) extracellular signal regulated kinase 1/2 (ERK 1/2) activation following in vivo CRD and CRF2 receptor gene expression in the T13-S1 dorsal root ganglia (DRG) and spinal cord were determined. Inferior splanchnic afferent (ISA) activity to CRD (0.4 ml, 20 seconds) was assessed by electrophysiological recording in an in vitro ISA nerve-inferior mesenteric artery (intra-arterial)-colorectal preparation. RESULTS: In controls, VMR to the second CRD was mean 31 (SEM 4)% higher than that of the first (p<0.05). The selective CRF2 agonist, human urocortin 2 (hUcn 2, at 10 and 20 microg/kg), injected intravenous after the first distension, prevented sensitisation and reduced the second response by 8 (1)% and 30 (5)% (p<0.05) compared with the first response, respectively. RT-PCR detected CRF2 receptor gene expression in the DRG and spinal cord. CRD (60 mm Hg for 10 minutes) induced phosphorylation of ERK 1/2 in neurones of lumbosacral laminae I and IIo and the response was dampened by intravenous hUcn 2. CRD, in vitro, induced robust ISA spike activity that was dose dependently blunted by hUcn 2 (1-3 microg, intra-arterially). The CRF2 receptor antagonist, astressin2-B (200 microg/kg subcutaneously or 20 microg intra-arterially) blocked the hUcn 2 inhibitory effects in vivo and in vitro. CONCLUSIONS: Peripheral injection of hUcn 2 blunts CRD induced visceral pain, colonic afferent, and spinal L6-S1 ERK 1/2 activity through CRF2 receptor activation in rats.

N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C.

N-methyl-D-aspartate (NMDA)receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1-3 days. [Ca2+]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 microM NMDA in the presence, but not the absence, of co-agonist (10 microM D-serine) caused transient [Ca2+]i responses (101+/-9 nM) and potentiated [Ca2+]i peak responses to subsequent mechanical stimulation more than two-fold (P < 0.001). NMDA-mediated potentiation of mechanically induced [Ca2+]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 microM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 microM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+]i responses nearly two-fold (P < 0.001), while longer exposure (>10 min) inhibited the [Ca2+]i transients by 44% (P < 0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2+ channels with 25 microM La3+ had no effect on mechanically induced [Ca2+]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+]i transients through a PKC-mediated process.

Neonatal maternal separation alters stress-induced responses to viscerosomatic nociceptive stimuli in rat.

This study investigated the combined effect of neonatal maternal separation and acute psychological stress on pain responses in adult rats. Long-Evans dams and their male pups were reared under two conditions: 1) 180 min daily maternal separation (MS180) on postnatal days 2-14 or 2) no handling or separation (NH). At 2 mo of age, visceromotor responses to graded intensities of phasic colorectal distension (10-80 mmHg) at baseline as well as following acute 60 min water avoidance stress (WA) were significantly higher in MS180 rats. Both groups showed similar stress-induced visceral hyperalgesia in the presence of naloxone (20 mg/kg ip). MS180 rats had smaller stress-induced cutaneous analgesia in the tail-flick test compared with NH rats, with a residual naloxone-resistant component. MS180 rats showed an enhanced fecal pellet output following WA or exposure to a novel environment. These data suggest that early life events predispose adult Long-Evans rats to develop visceral hyperalgesia, reduced somatic analgesia, and increased colonic motility in response to an acute psychological stressor, mimicking the cardinal features of irritable bowel syndrome.

Nitric oxide synthase inhibitors enhance mechanosensitive Ca(2+) influx in cultured dorsal root ganglion neurons.

Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca(2+) influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2-5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca(2+)](i) by fluorescent ratio imaging. In the presence of the NOS inhibitors L-NAME, TRIM, or 7-NI, but not the inactive analogue D-NAME, peak [Ca(2+)](i) transients to mechanical stimulation were increased more than 2-fold. Neither La(3+) (25 microM), an inhibitor of voltage activated Ca(2+) channels, or tetrodotoxin (TTX, 1 microM), a selective inhibitor of voltage-gated Na(+) channels, had an effect on mechanically activated [Ca(2+)](i) transients under control conditions. However, in the presence of L-NAME, both La(3+) and TTX partially blocked the [Ca(2+)](i) response. Addition of Gd(3+), a blocker of mechanosensitive cation channels and L-type Ca(2+) channels, at a concentration (100 microM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of L-NAME. The combination of either La(3+) or TTX with Gd(3+) caused near complete inhibition of mechanically stimulated [Ca(2+)](i) transients in the presence of L-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca(2+) influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca(2+) influx occurs through voltage-sensitive Ca(2+) channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na(+) and Ca(2+) channels.

Role of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral nociception in rats.

BACKGROUND & AIMS: N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS: Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS: Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.

Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cell apoptosis occurs continually without apparent permeability defects and is increased in response to intestinal inflammation. We hypothesized that increased, immune-mediated apoptosis during inflammation might result in barrier dysfunction of the epithelium. METHODS: T84 cells were cultured as a polarized monolayer and exposed to agonist antibody to Fas. Barrier function was assessed by transepithelial resistance and permeability measurements. Immunofluorescent staining was used to examine junctional protein expression. RESULTS: Fas expression is predominantly basolateral in polarized T84 monolayers. Basolateral cross-linking of the Fas receptor resulted in T84 cell apoptosis and a loss of 50% of the cells within 24 hours. Apoptosis was coincident with a decrease in transepithelial electrical resistance and increased flux of small but not large molecules. Preservation of barrier function was associated with dramatic rearrangement of tight junctions and desmosomal junctions in apoptotic monolayers. E-cadherin-mediated cell contact was maintained between intact cells in the monolayer, thus sealing gaps created by apoptotic cells. Apoptosis and barrier dysfunction could be prevented by caspase inhibition. CONCLUSIONS: Immune-mediated apoptosis of intestinal epithelial cells may contribute to the permeability defects associated with inflammatory conditions of the bowel, but the intestinal epithelium is remarkably resilient in the face of apoptosis.

Multiple isoforms of the ryanodine receptor are expressed in rat pancreatic acinar cells.

Regulation of cytosolic Ca(2+) is important for a variety of cell functions. The ryanodine receptor (RyR) is a Ca(2+) channel that conducts Ca(2+) from internal pools to the cytoplasm. To demonstrate the presence of the RyR in the pancreatic acinar cell, we performed reverse transcriptase (RT)-PCR, Western blot, immunocytochemistry and microscopic Ca(2+)-release measurements on these cells. RT-PCR showed the presence of mRNA for RyR isoforms 1, 2 and 3 in both rat pancreas and dispersed pancreatic acini. Furthermore, mRNA expression for RyR isoforms 1 and 2 was demonstrated by RT-PCR in individual pancreatic acinar cells selected under the microscope. Western-blot analysis of acinar cell immunoprecipitates, using antibodies against RyR1 and RyR2, showed a high-molecular-mass (>250 kDa) protein band that was much less intense when immunoprecipitated in the presence of RyR peptide. Functionally, permeablized acinar cells stimulated with the RyR activator, palmitoyl-CoA, released Ca(2+) from both basolateral and apical regions. These data show that pancreatic acinar cells express multiple isoforms of the RyR and that there are functional receptors throughout the cell.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation.

The mechanisms underlying intracellular Ca2+ waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+ concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+ response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]i responses involved generation of inositol 1,4, 5-trisphosphate and release of Ca2+ from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+ and partially on release of intracellular Ca2+ from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+ entry channels that are activated after mechanical stimulation but not by substance P.

Differential expression and localization of IGF-I and IGF binding proteins in inflamed rat colon.

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6-trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with 35S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.

Involvement of chloride channels in the receptormediated activation of longitudinal colonic muscle.

In gastrointestinal smooth muscle, intracellular Cl- is maintained at levels higher than its electrochemical equilibrium. Therefore, Cl- efflux through receptor-mediated opening of Cl- channels should result in membrane depolarization and may be sufficient to activate voltage-sensitive calcium channels (VSCCs). To determine the contribution of Cl- channels to receptor-mediated contraction of the longitudinal muscle layer of the rabbit distal colon, we studied the mechanical response of muscle strips to substance P, carbachol and potassium depolarization following the depletion of Cl- i, and in the presence of the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB). A 60-min incubation of tissues in a HEPES-buffered solution in which NaCl had been replaced by Na isethionate (or Na gluconate) in equimolar amounts resulted in disappearance of phasic contractions, and in a partially reversible reduction of the tonic response to substance P and carbachol, but not to KCl depolarization. When the agonist was applied to tissues in control solution, or to Cl-(-)depleted tissues in a solution in which Na+ was acutely replaced in equimolar amounts by N-methyl-D-glucosamine, the mechanical response to substance P and carbachol was almost abolished. Acute Na+ replacement alone without prior Cl- depletion did not abolish phasic contractions, but reduced the tonic response to substance P and carbachol. Similar to the effect of Cl- depletion, incubation of tissues in NPPB (6.6 x 10(-5) M) reduced the tonic response to substance P and carbachol, and abolished phasic contractions. These findings are consistent with a contribution of a Cl- channel to the receptor-mediated activation of colonic smooth muscle. In addition, the data suggest that transient Cl- channel mediated depolarizations may play a role in the generation of phasic contractions.

Increased expression of transforming growth factor alpha precursors in acute experimental colitis in rats.

BACKGROUND AND AIM: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha), members of the EGF family of growth factors, protect rat gastric and colonic mucosa against injury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was to evaluate the endogenously expressed ligand mediating the protective effect of EGF/TGF-alpha in vivo. METHODS: In an experimental model of trinitrobenzene sulphonic acid (TNBS)/ethanol induced colitis in rats EGF and TGF-alpha expression was evaluated using a ribonuclease protection assay, northern blot analysis, western blot analysis, and immunohistochemistry. RESULTS: TGF-alpha mRNA increased 3-4 times at 4-8 hours after induction of colitis and returned to control levels within 24 hours. TGF-alpha immunoreactive protein with a molecular size of about 28 kDa representing TGF-alpha precursors increased markedly after induction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-alpha protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the rat colon and no EGF protein was observed by immunohistochemistry or western blot analysis. CONCLUSIONS: TGF-alpha precursors are the main ligands for the EGF receptor in acute colitis. It is hypothesised that TGF-alpha precursors convey the biological activity of endogenous TGF-alpha peptides during mucosal defence and repair.

First intracellular loop of the human cholecystokinin-A receptor is essential for cyclic AMP signaling in transfected HEK-293 cells.

Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.

Expression of insulin-like growth factor I receptors and binding proteins by colonic smooth muscle cells.

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.

Acyl-coenzyme A causes Ca2+ release in pancreatic acinar cells.

The regulation of cytosolic Ca2+ is important for a variety of cell functions. One non-inositol 1,4,5-trisphosphate (IP3) compound that may regulate Ca2+ is palmitoyl-coenzyme A (CoA), a fatty acid-CoA that is reported to cause Ca2+ release from intracellular stores of oocytes, myocytes, and hepatocytes. To study the role of palmitoyl-CoA in the pancreatic acinar cell, rat pancreatic acini were isolated by collagenase digestion, permeablized with streptolysin O, and the release of Ca2+ from internal stores was measured with fura-2. Palmitoyl-CoA released Ca2+ from internal stores (EC50 = 14 microM). The palmitoyl-CoA-sensitive pool was distinct from, and overlapping with the IP3-sensitive Ca2+ pool. The effects of submaximal doses of IP3 or cyclic ADP-ribose plus palmitoyl-CoA were additive. Fatty acid-CoA derivatives with carbon chain lengths of 16-18 were the most potent and efficacious. Ryanodine and caffeine or elevated resting [Ca2+] sensitized the Ca2+ pool to the actions of palmitoyl-CoA. Fatty acid-CoA levels in pancreatic acini were measured by extraction with 2-propanol/acetonitrile, followed by separation and quantification using reverse phase high performance liquid chromatography, and were found to be 10.17 +/- 0.93 nmol/mg protein. These data suggest the presence of an IP3-insensitive palmitoyl-CoA-sensitive Ca2+ store in pancreatic acinar cells and suggest that palmitoyl-CoA may be needed for Ca2+-induced Ca2+ release.

Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats.

BACKGROUND & AIMS: Keratinocyte growth factor (KGF) is known to enhance tissue repair in the skin; however, its role in the gastrointestinal tract is largely unknown. The aim of this study was to evaluate the effects of exogenous KGF in an experimental model of colitis in rats. METHODS: KGF was administered before or after induction of colitis with 2,4,6-trinitrobenzenesulfonic acid/ethanol. In the first two study groups, KGF (5 mg/kg) was administered intraperitoneally 24 hours and 1 hour before induction of colitis; animals were killed 8 hours (n=10) and 1 week (n=10) after injury. In subsequent study groups, KGF or vehicle treatment was begun 24 hours after the induction of colitis at doses of 5 (n=20), 1 (n=10), and 0.1 (n=10) mg/kg intraperitoneally and continued once daily for 1 week. Colonic tissue samples were evaluated macroscopically and microscopically for mucosal injury and assayed for myeloperoxidase activity. RESULTS: Administration of KGF after but not before induction of colitis significantly ameliorated tissue damage. Macroscopic necrosis and microscopic ulcerations were reduced by 40%-50% at KGF doses of 1 and 5 mg/kg. CONCLUSIONS: Exogenous KGF has a key role in mucosal healing in an experimental model of colitis in rats.

Up-regulation of insulinlike growth factor I binding sites in experimental colitis in rats.

BACKGROUND/AIMS: The gastrointestinal tract is a major target of insulinlike growth factor (IGF) I. IGF-I binds to two different receptors and to binding proteins (IGFBPs), which act as carriers and mediators. This study investigated the regulation of IGF-I binding sites in rat colitis. METHODS: Colitis was induced by colonic instillation of 2,4,6-trinitrobenzenesulfonic acid in ethanol. IGF-I binding sites in colon sections were localized by incubation with 125I-IGF-I. The contribution of binding to the IGF-I receptor was estimated by competition with unlabeled IGF-I, IGF-II, and insulin. Colonic RNA was screened for IGFBPs by Northern hybridization. RESULTS: IGF-I binding sites were increased more than two-fold in the muscularis propria of inflamed colon as soon as 12 hours and up to 1 week after injury. Insulin could not displace this elevated level of binding, even though it could displace IGF-I from the mucosa and muscularis mucosa. Northern hybridization showed a 2-3-fold increase in IGFBP-4 and IGFBP-5 messenger RNA from inflamed colon. CONCLUSIONS: Experimental colitis in rats causes an increase in IGF-I binding to the muscularis propria, which represents increased levels of IGFBP-4 and IGFBP-5. These data suggest an important role for IGFBPs in modulating IGF effects during inflammation and tissue repair.