Post inflammatory damage to the enteric nervous system in diverticular disease and its relationship to symptoms.

Some patients with colonic diverticula suffer recurrent abdominal pain and exhibit visceral hypersensitivity, though the mechanism is unclear. Prior diverticulitis increases the risk of being symptomatic while experimental colitis in animals increases expression of neuropeptides within the enteric nervous system (ENS) which may mediate visceral hypersensitivity. Our aim was to determine the expression of neuropeptides within the ENS in diverticulitis (study 1) and in patients with symptomatic disease (study 2). Study 1 - Nerves in colonic resection specimens with either acute diverticulitis (AD, n = 16) or chronic diverticulitis (CD, n = 16) were assessed for neuropeptide expression recording % area staining with protein gene product (PGP9.5), substance P (SP), neuropeptide K (NPK), pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP) and galanin. Study 2 - Seventeen symptomatic and 15 asymptomatic patients with colonic diverticula underwent flexible sigmoidoscopy and multiple peridiverticular mucosal biopsies. Study 1- Neural tissue, as assessed by PGP staining was increased to a similar degree in circular muscle in both AD and CD. The CD specimens showed significant increases in the immunoreactivity of SP, NPK and galanin in both mucosal and circular muscle layer compared with controls. Study 2 - Mucosal histology was normal and PGP9.5 staining was similar between groups however patients with symptomatic diverticular disease demonstrated significantly higher levels of SP, NPK, VIP, PACAP and galanin within the mucosal plexus. Patients with symptomatic diverticular disease exhibit increased neuropeptides in mucosal biopsies which may reflect resolved prior inflammation, as it parallels the changes seen in acute and chronic diverticulitis.

Human islet amyloid polypeptide transgenic mice: in vivo and ex vivo models for the role of hIAPP in type 2 diabetes mellitus.

Human islet amyloid polypeptide (hIAPP), a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2). To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.

Ghrelin and insulin gene expression changes in streptozotocin-induced diabetic rats after rosiglitazone pretreatment.

The aim of the study was to evaluate the effect of rosiglitazone treatment on islet ghrelin and insulin gene expressions in streptozotocin (STZ)-induced diabetic rats. Animals were divided into four groups. 1. Intact controls. 2. Rosiglitazone-treated controls. 3. STZ-induced diabetes. 4. Rosiglitazone-treated diabetes. Rosiglitazone was given for 7 days at a dose of 20 mg/kg body weight. Ghrelin and insulin gene expressions were investigated by immunohistochemistry and in situ hybridization. There was no statistically significant difference in body weight between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats during the experimental period. Furthermore, there were no significant differences in blood glucose levels and insulin immunoreactive cell numbers between STZ-induced diabetic rats and rosiglitazone-treated diabetic rats. There was a tendency towards a reduction of ghrelin gene expression in diabetic animals compared with intact controls. We found, in addition, that ghrelin immunoreactive and ghrelin mRNA expressing cells were frequent in the epithelial lining of the ducts suggesting ductal epithelium might be the source of the regenerating islet ghrelin cells, as is known for other islet cells. The results show that short-term rosiglitazone pretreatment had no significant effect on ghrelin and insulin gene expressions.

Cutaneous innervation before and after one treatment period of acupuncture.

BACKGROUND: The effect of acupuncture on nociceptive pain is well documented, but effects on nociceptive itch have been contradictory. OBJECTIVES: To evaluate possible effects of acupuncture on the occurrence, distribution and function of sensory nerve fibres in human skin. METHODS: Ten subjects were treated by inserting 10 acupuncture needles subcutaneously at the upper lateral aspect of one buttock. The subjects were recruited from an acupuncture clinic and were undergoing specific acupuncture treatment for their disorders. The needles were stimulated (rotated to and fro) twice during the twice-weekly 25-min sessions over 5 weeks. Skin biopsies, diameter 3 mm, were taken before and 3-6 days after local acupuncture. Antibodies to the pan-neuronal marker protein gene product 9.5 (PGP 9.5), calcitonin gene-related peptide (CGRP), vanilloid receptor 1 (VR1) and mu- and delta-opioid receptors were employed to study sensory unmyelinated nerve fibres that transmit nociceptive pain and itch. A histamine prick test using planimetry was used to record experimental itch after acupuncture on the treated area and on the corresponding control skin, and a visual analogue scale was used to evaluate itch. RESULTS: The mean +/- SEM number of CGRP-immunoreactive nerve fibres per biopsy section was reduced from 36.0 +/- 3.3 to 21.3 +/- 4.0 (P = 0.05) after the treatment. PGP 9.5-immunoreactive nerve fibres were found both in the epidermis and in the subpapillary dermis. The mean +/- SEM total number of PGP 9.5-immunoreactive nerve fibres decreased from 249.8 +/- 16.7 to 211.8 +/- 12.0 (P = 0.03). The PGP 9.5-immunoreactive nerve fibres occurring in the dermis appeared more fragmented after the acupuncture compared with pretreatment. VR1 immunoreactivity was found both in the free nerve fibres and in kite-like formations, possibly mast cells, throughout the dermis, sometimes occurring around hair follicles. The mean +/- SEM number of VR1-immunoreactive elements was not significantly influenced by acupuncture, at 33.5 +/- 4.6 vs. 43.0 +/- 4.4 (P = 0.09). No immunoreactivity was found in the skin against mu- and delta-opioid receptors with the antibodies used in this study. Neither histamine-induced itch nor cutaneous responses were influenced by acupuncture. CONCLUSIONS: The present data indicate an effect of acupuncture on neuropathic itch but not histamine-mediated itch. Our findings support the opinion that the pain-relieving effects of acupuncture partly depend on its effect on the peripheral innervation.

CART knock out mice have impaired insulin secretion and glucose intolerance, altered beta cell morphology and increased body weight.

CART peptides are anorexigenic and are widely expressed in the central and peripheral nervous systems, as well as in endocrine cells in the pituitary, adrenal medulla and the pancreatic islets. To study the role of CART in islet function, we used CART null mutant mice (CART KO mice) and examined insulin secretion in vivo and in vitro, and expression of islet hormones and markers of beta-cell function using immunocytochemistry. We also studied CART expression in the normal pancreas. In addition, body weight development and food intake were documented. We found that in the normal mouse pancreas, CART was expressed in numerous pancreatic nerve fibers, both in the exocrine and endocrine portion of the gland. CART was also expressed in nerve cell bodies in the ganglia. Double immunostaining revealed expression in parasympathetic (vasoactive intestinal polypeptide (VIP)-containing) and in fewer sensory fibers (calcitonin gene-related peptide (CGRP)-containing). Although the expression of islet hormones appeared normal, CART KO islets displayed age dependent reduction of pancreatic duodenal homeobox 1 (PDX-1) and glucose transporter-2 (GLUT-2) immunoreactivity, indicating beta-cell dysfunction. Consistent with this, CART KO mice displayed impaired glucose-stimulated insulin secretion both in vivo after an intravenous glucose challenge and in vitro following incubation of isolated islets in the presence of glucose. The impaired insulin secretion in vivo was associated with impaired glucose elimination, and was apparent already in young mice with no difference in body weight. In addition, CART KO mice displayed increased body weight at the age of 40 weeks, without any difference in food intake. We conclude that CART is required for maintaining normal islet function in mice.

Ghrelin is expressed in a novel endocrine cell type in developing rat islets and inhibits insulin secretion from INS-1 (832/13) cells.

Ghrelin is produced mainly by endocrine cells in the stomach and is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). It also influences feeding behavior, metabolic regulation, and energy balance. It affects islet hormone secretion, and expression of ghrelin and GHS-R in the pancreas has been reported. In human islets, ghrelin expression is highest pre- and neonatally. We examined ghrelin and GHS-R in rat islets during development with immunocytochemistry and in situ hybridization. We also studied the effect of ghrelin on insulin secretion from INS-1 (832/13) cells and the expression of GHS-R in these cells. We found ghrelin expression in rat islet endocrine cells from mid-gestation to 1 month postnatally. Islet expression of GHS-R mRNA was detected from late fetal stages to adult. The onset of islet ghrelin expression preceded that of gastric ghrelin. Islet ghrelin cells constitute a separate and novel islet cell population throughout development. However, during a short perinatal period a minor subpopulation of the ghrelin cells co-expressed glucagon or pancreatic polypeptide. Markers for cell lineage, proliferation, and duct cells revealed that the ghrelin cells proliferate, originate from duct cells, and share lineage with glucagon cells. Ghrelin dose-dependently inhibited glucose-stimulated insulin secretion from INS-1 (832/13) cells, and GHS-R was detected in the cells. We conclude that ghrelin is expressed in a novel developmentally regulated endocrine islet cell type in the rat pancreas and that ghrelin inhibits glucose-stimulated insulin secretion via a direct effect on the beta-cell.

Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice.

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.

Immunolocalization of cholecystokinin-2 receptors in rat gastric mucosa.

BACKGROUND: Gastrin exerts trophic effects on the gastric mucosa by mechanisms not yet completely elucidated. Our aim was to localize the cholecystokinin-2 (CCK2) receptor in epithelial cells of foetal and adult rat stomachs in order to determine the cell types that are directly affected by gastrin. METHODS: Gastric tissue was subjected to indirect double immunofluorescence staining with antiserum against the C-terminal decapeptide of the CCK2 receptor and antibodies against 5' bromo-2-deoxyuridine, which had been injected into the rats I h before they were killed, the acid pump H,K-ATPase, the membrane-cytoskeletal linker ezrin, pepsin/pepsinogen or histidine decarboxylase. RESULTS: Undifferentiated foetal gastric epithelial cells expressed CCK2 receptors, whereas stem cells of adult gastric glands did not exhibit immunoreactivity. However, other epithelial cells in the progenitor zone of adult gastric glands did express CCK2 receptors. Some of these cells were faintly stained for H,K-ATPase; pepsin/pepsinogen was also detected in this region. Parietal cells in the isthmus/pit region of the glands contained ezrin, and some showed weak immunoreactivity for the CCK2 receptor. As expected, enterochromaffin-like cells also expressed CCK2 receptors. CONCLUSION: Our findings are consistent with the hypothesis that a CCK2 receptor mediates direct effects of gastrin on gastric epithelial cells during both stomach organogenesis and adult life.

The ghrelin cell: a novel developmentally regulated islet cell in the human pancreas.

OBJECTIVES: Ghrelin, an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), was recently identified in the stomach. Ghrelin is produced in a population of endocrine cells in the gastric mucosa, but expression in intestine, hypothalamus and testis has also been reported. Recent data indicate that ghrelin affects insulin secretion and plays a direct role in metabolic regulation and energy balance. On the basis of these findings, we decided to examine whether ghrelin is expressed in human pancreas. Specimens from fetal to adult human pancreas and stomach were studied by immunocytochemistry, for ghrelin and islet hormones, and in situ hybridisation, for ghrelin mRNA. RESULTS: We identified ghrelin expression in a separate population of islet cells in human fetal, neonatal, and adult pancreas. Pancreatic ghrelin cells were numerous from midgestation to early postnatally (10% of all endocrine cells). The cells were few, but regularly seen in adults as single cells at the islet periphery, in exocrine tissue, in ducts, and in pancreatic ganglia. Ghrelin cells did not express any of the known islet hormones. In fetuses, at midgestation, ghrelin cells in the pancreas clearly outnumbered those in the stomach. CONCLUSIONS: Ghrelin is expressed in a quite prominent endocrine cell population in human fetal pancreas, and ghrelin expression in the pancreas precedes by far that in the stomach. Pancreatic ghrelin cells remain in adult islets at lower numbers. Ghrelin is not co-expressed with any known islet hormone, and the ghrelin cells may therefore constitute a new islet cell type.

Neuropeptide expression in the human trigeminal nucleus caudalis and in the cervical spinal cord C1 and C2.

In migraine and other primary headaches there is a strong vascular component. Besides the trigeminovascular components some of the associated symptoms point to the involvement of brain stem regions. The central limb of the trigeminal vascular pathway is its projection to the trigeminal nucleus caudalis (TNC) and to the C1-C2 levels of the spinal cord. The aim of the present study was to demonstrate the occurrence of some neurotransmitters in these regions in man. In both the TNC and in the Rexed's laminae I and II of the dorsal horns at the C1 and C2 levels there were numerous substance P immunoreactive fibres. Fibres containing calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) were moderately dense in number. Fibres containing vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) were not seen in the TNC or at the C1 and C2 levels of the spinal cord.

The effect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on induced tone in isolated human airways and pulmonary artery.

Ro 25-1553 is a metabolically stable analogue of endogenous vasoactive intestinal polypeptide (VIP). This compound is a potent bronchodilator in vitro as well as in vivo. Moreover, Ro 25-1553 has been shown to be highly selective of the VPAC2 receptor. We assessed the effect of Ro 25-1553 on isolated human bronchi and pulmonary arteries in vitro. Macroscopically normal human airways and pulmonary arteries were obtained from patients undergoing surgery for lung cancer. The relaxing capability of Ro 25-1553 on bronchial and pulmonary artery tone was measured using standard techniques. Bronchial rings were pre-contracted with 0.1 mM histamine, and tone in pulmonary artery rings was induced with 10 microM PGF2alpha. Increasing concentrations of Ro 25-1553 within a range of 1 pM to 10 microM were added and isometric tension changes were recorded. Ro 25-1553 caused a concentration-dependent relaxation of airway and pulmonary artery preparations, with an EC50 of approximately 10 nM and a maximal relaxation of 70%-75% of the induced tone. The presence of VPAC2 receptors in the two tissues, though low in density, was confirmed by in situ hybridization, immunocytochemistry and ligand binding. These findings indicate that the VIP analogue Ro 25-1553 may be useful in the treatment of asthma and/or chronic obstructive pulmonary diseases.

Cutaneous field stimulation in the treatment of severe itch.

OBJECTIVE: To evaluate the efficacy of cutaneous field stimulation of C fibers for the treatment of itchy skin and its effect on peripheral nerve fibers as shown in skin biopsy specimens. DESIGN: We conducted an open-label uncontrolled study of 19 patients with itching. Each patient applied a flexible plate containing electrodes to the itchy area for 20 minutes at a time once daily for 5 weeks to stimulate nerve fibers with a constant current (0.8 mA). Skin biopsy specimens were collected before treatment and at the end of treatment and were immunostained for calcitonin gene-related peptide and protein gene product 9.5. SETTING: University hospital in Lund, Sweden. PATIENTS: Sixteen patients with nostalgia paresthetica or brachioradial pruritus and 3 with generalized itch. INTERVENTIONS: Cutaneous field stimulation and punch biopsies of the itchy skin. MAIN OUTCOME MEASURES: Visual analog scale for assessment of itching and counting the immunoreactive nerve fibers in 3-mm biopsy specimens. RESULTS: Patients with localized itching experienced a reduction in mean values on the visual analog scale (from 78% before treatment to 42% by the end of the fifth week). The number of protein gene product 9.5- immunoreactive nerve fibers in the epidermis was reduced by 40% by the end of treatment compared with baseline values. CONCLUSIONS: Cutaneous field stimulation is an effective alternative for the treatment of localized itching. The reduction in itching is accompanied by degeneration of the epidermal nerve fibers, as evidenced by the loss of protein gene product 9.5 immunoreactivity.

The method of administration of cholecystokinin determines the effects evoked in the pancreas.

Earlier studies have shown different effects on cell proliferation and weight characteristics by sulfated cholecystokinin-8 (CCK-8S) in the rat pancreas when the peptide has been administered continuously rather than intermittently. The aim of this study was as follows: (i) to compare the effect of continuous infusion and of intermittent injections of CCK-8S on cell proliferation, weight gain, and induction of apoptosis and (ii) to examine the effect of injections of CCK-8S on CCK-A receptor gene expression in the rat pancreas. Male Sprague-Dawley rats had subcutaneous continuous infusion of CCK-8S in a dose of 5 microg/kg/h or 1% bovine serum albumin (BSA) (vehicle) by implanted osmotic minipumps. The rats were killed after 4 days. Other rats were either injected subcutaneously only once or injected twice daily for 3 days with either 6 microg of CCK dissolved in 0.5 mL BSA or 0.5 mL BSA alone. The rats were killed 1, 3, 6, and 12 hours after the last injection. One hour before death they received 5-bromo-2-deoxyuridine (BrdU) intraperitoneally to localize and quantitate the cell proliferation. Plasma was collected for analysis of CCK. The pancreas was dissected and immunohistochemistry was performed for analysis of the expression of the apoptosis promoting protein bax and the apoptosis inhibiting protein bcl-2, and for BrdU and CCK-A receptor localization. In situ hybridization (ISH) was used for examination and semiquantification of CCK-A receptor mRNA expression. Continuous infusion of CCK-8S led to a sixfold increase in plasma CCK and a 40% increase in pancreatic weight without any effect on BrdU labeling. Immunohistochemistry revealed decreased tissue expression of bax but unaffected expression of bcl-2. Intermittent injections of CCK-8S led to hyper-CCK-emia with increased incorporation of BrdU, indicating increased cell proliferation but no increase in pancreatic weight. Immunohistochemistry showed increased expression of bax, whereas bcl-2 remained unchanged. Immunofluorescence and ISH for the CCK-A receptor and its gene expression, respectively, showed a lowest intensity at 3 hours after CCK-8S injections. The results indicate that decreased apoptosis could explain the increased pancreatic weight during continuous infusion of CCK-8S. An increased apoptosis could explain the lack of pancreatic weight gain upon intermittent injections of CCK-8S despite the stimulation of cell proliferation. Injections of CCK-8S only transiently decreased the tissue levels of its receptor.

Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been proposed to have a role in nociception. Here we have used the formalin test, thermal laser stimulation and mechanical von Frey stimulation to investigate possible alteration of PAC1-/- mice nociceptive behaviour. Our finding, that PAC1-/- mice have a substantial, 75% decrease in nociceptive response during the late phase, provides clear evidence that the specific PACAP-receptor PAC1 is involved in the mediation of nociceptive responses during chronic conditions such as inflammation. PAC1-/- mice had small or no changes in the response to mechanical and thermal laser stimulation. This suggests a limited, if any, involvement of PAC1 in nociception after short-lasting stimuli. Injury-induced changes in DRG neuropeptide expression were more pronounced in PAC1-/- mice, implying neuroregulatory functions of PAC1.

Beta cell adaptation to dexamethasone-induced insulin resistance in rats involves increased glucose responsiveness but not glucose effectiveness.

Islet beta cell adaptation to dexamethasone-induced insulin resistance was characterized with respect to glucose-stimulated insulin secretion and islet innervation. Male Sprague-Dawley rats were injected daily with dexamethasone (2 mg/kg for 12 days), which resulted in hyperinsulinemia and hyperglycemia compared with controls (which were injected with sodium chloride). Insulin secretion was characterized in collagenase-isolated islets. Islet innervation was examined by immunocytochemical analysis of tyrosine hydroxylase, neuropeptide Y (sympathetic nerves), and vasoactive intestinal polypeptide (cholinergic nerves). In islets isolated from the insulin-resistant animals, the insulin response to 3.3 or 8.3 mM glucose was three times greater during perifusion compared with controls (p < 0.001). Incubation of islets at 0 to 20 mM glucose revealed a marked leftward shift of the glucose dose-response relation after dexamethasone treatment (potency ratio, 1.78; p < 0.01), with no difference at 0 or 20 mM glucose. Thus, the potency but not the efficacy of glucose was increased. The number of islet nerves did not differ between dexamethasone-treated rats and controls. Dexamethasone-induced insulin resistance leads to adaptively increased glucose responsiveness of the islet beta cells, with increased potency, but not increased efficacy, of glucose to stimulate insulin secretion without any evidence of altered islet innervation.

Direct cytoplasmic CA(2+) responses to gastrin-releasing peptide in single beta cells.

The neuropeptide gastrin releasing peptide (GRP) stimulates insulin secretion and induces oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) in clonal insulinoma cells. It is not known whether GRP affects [Ca(2+)](cyt) in normal beta cells. We investigated, in single, normal, mouse islet beta cells, the effects of GRP on [Ca(2+)](cyt), by dual wavelength spectrophotofluorometry. Beta cells were identified by their typical response to glucose or tolbutamide. At 15 mM glucose, GRP (100 nM) evoked an immediate [Ca(2+)](cyt) transient to 423 +/- 48 nM compared to 126 +/- 18 nM before GRP (P < 0.001). After the initial transient, [Ca(2+)](cyt) exhibited either a sustained elevation or oscillations. At 3.3 mM glucose, in cells with a non-oscillating [Ca(2+)](cyt), GRP stimulated a prompt increase in [Ca(2+)](cyt) (from 60 +/- 6 to 285 +/- 30 nM, P = 0.024) followed by either a sustained increase in [Ca(2+)](cyt) or [Ca(2+)](cyt) oscillations. We conclude that GRP promptly elevates [Ca(2+)](cyt) by a direct action in normal mouse pancreatic beta cells.

Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk-1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk-1 receptor and showed an increased mRNA expression and immunoreactivity for flk-1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk-1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk-1-positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene-related peptide (CGRP). Small IB4-positive neurons were devoid of flk-1 immunoreactivity. Most flk-1-positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin-1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF-induced axonal outgrowth was blocked by the flk-1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.

Expression and motor effects of secretin in small and large intestine of the rat.

Immunocytochemistry and in situ hybridization revealed abundant secretin expressing cells on duodenal villi with a gradual decrease throughout the small intestines of the rat. They were absent in pancreas, stomach and colon. Secretin caused relaxation of rat intestinal longitudinal muscle in vitro. Studies on colon revealed that the secretin-evoked response was unaffected by apamin, tetrodotoxin, L-NAME, VIP or PACAP pretreatment; secretin itself caused desensitization. Addition of VIP or PACAP when the secretin-evoked relaxation was maximal evoked a further relaxation suggesting the presence of distinct receptors. Secretin causes relaxation via activation of secretin receptors located on the smooth muscle and not via any of the related VIP/PACAP receptors.

Cholecystokinin does not affect the pancreatic contents of epidermal growth factor or its receptor.

Cholecystokinin (CCK) is a hormone with well-known secretory and trophic effects on the pancreas. This also is true for epidermal growth factor (EGF), which acts in a paracrine and autocrine way. The aim was to study the influence of CCK on cell proliferation in rat pancreas with special reference to the expression of EGF, the EGF receptor, and phosphorylated tyrosine. Twenty-four male Sprague-Dawley rats received either one single injection, or injections twice daily for 3 days of 6 microg sulfated CCK-8 (CCK-8S) subcutaneously in the neck. The same number of rats received injections of 1% bovine serum albumin (BSA) in the same way. The rats were killed 1, 3, or 6 hours after the last injection. One hour before killing, they received 50 mg/kg of bromodeoxyuridine (BrdU) intraperitoneally. Plasma was collected for analysis of CCK. The pancreas was dissected, and in situ hybridization using a probe for EGF mRNA was performed for semiquantification of gene expression. Immunocytochemistry using antibodies against the EGF receptor and phosphotyrosine was performed to examine the expression of the proteins, and against BrdU for measuring the cell proliferation. A single injection of CCK-8S led to hyperCCKemia at 1 and 3 hours afterward. After 6 hours, plasma CCK had returned to the same levels as in control rats. The cell proliferation was unaffected. The rats that received CCK-8S injections for 3 days still had hyperCCKemia 6 hours after the last injection. The cell proliferation was increased by CCK, as indicated by the BrdU labeling. However, neither body weight nor pancreatic weight was affected. In controls, EGF was expressed all over the gland, but its receptor and phosphotyrosine were expressed only in ductal cells and in the islet cells of endocrine pancreas. There was no difference in the pancreatic staining of EGF, its receptor, or phosphotyrosine at the different time points studied. There was no difference in the staining of EGF and its receptor between CCK-8S- and BSA-treated animals, but phosphotyrosine staining was detectable in acinar cells after 3 days of CCK-8S injections. Thus CCK-8S causes hyperCCKemia with ensuing enhanced cell proliferation in rat pancreas. This effect on the cell proliferation seems to be a direct effect of CCK and not mediated by changes in the tissue levels of EGF or its receptor.