Expression of glial cell line-derived neurotrophic factor and its mRNA in the nigrostriatal pathway following MPTP treatment.

Striatal glial cell line-derived neurotrophic factor (GDNF) mRNA levels in both young (2-month old) and old (11-month old) C57BL/6J mice were quantified at 3, 7 and 21 days following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment. MPTP did not alter the expression of GDNF mRNA in these animals. Immunoreactive staining of GDNF in the substantia nigra and the striatum was also unchanged. In conclusion, MPTP-induced dopaminergic neurotoxicity does not elicit any changes in the expression of endogenous GDNF or its mRNA in the adult mouse brain.

Increase of low-affinity neurotrophin receptor p75 and growth-associated protein-43 immunoreactivities in the rat urinary bladder during experimentally induced nerve regeneration.

PURPOSE: Nerve regeneration in the urinary bladder after pelvic nerve plexus injury remains uncertain. The objectives were to establish a rat model of nerve regeneration in the bladder and to examine possible changes of low-affinity neurotrophin receptor p75 and growth-associated protein-43 (GAP-43) immunoreactivities during denervation and nerve regeneration. MATERIALS AND METHODS: Adult male rats were divided into 3 groups: controls, crush of the nerve bundles from the right major pelvic ganglion (MPG) to the bladder (nerve crush group) and removal of the right MPG (MPG removal group). Bladders were collected at 3, 7, 14, 30 and 60 days, and immunohistochemically stained for protein gene product 9.5 (PGP9.5: an axonal marker), p75 and GAP-43. RESULTS: In the nerve crush group, PGP9.5 positive nerves were decreased in number at 3 and 7 days, and then increased after 14 days in the muscle layer of the operated side. By 60 days, the density returned to control levels. However, MPG removal resulted in a decrease of the density of PGP9.5 positive nerves throughout the experimental periods. These findings indicate that nerve regeneration occurred in the nerve crush group. The density of p75 labeled fibers was significantly increased at 3 to 30 days postoperatively in the nerve crush group. p75 immunoreactivity showed smooth surface and cytoplasmic staining, indicating that Schwann cells were p75 positive. GAP-43 labeled fibers showed significantly greater density at 3 to 14 days postoperatively. Schwann cells were GAP-43 immunoreactive and, in particular, regenerating nerve fibers appeared to be GAP-43 positive at 14 days. CONCLUSION: The present study suggests that p75 and GAP-43 are involved in the mechanism(s) of nerve regeneration in the urinary bladder.

Increase of growth-associated protein-43 immunoreactivity following cyclophosphamide-induced cystitis in rats.

We examined the effect of inflammation on immunoreactivity of growth-associated protein (GAP-43) in the rat urinary bladder in which acute cystitis was induced with cyclophosphamide (CPA). Following CPA injection, the number of GAP-43 labeled nerves was significantly increased in the muscle layer. Immunoreactivity of PGP9.5, which was used as an axonal marker, was not augmented following CPA injection. Double fluorescence immunohistochemistry revealed that substance P immunoreactivity was present in most GAP-43 immunoreactive fibers (90.2%) in the inflamed bladder. Electron microscopic examination showed that GAP-43 immunoreactivity was localized on axons. Some GAP-43 positive axons showed degeneration. Possible significance of the increase of GAP-43 immunoreactive afferent nerve fibers in the muscle layer of acutely inflamed bladder was discussed.

Immuno-electron microscopic study of differential localization of motilin and serotonin in the rabbit duodenal epithelium.

A guinea pig antibody against rabbit motilin was generated to study the localization of motilin-containing cells in the rabbit small intestine with special reference to the co-localization of motilin and serotonin. A pre- and post-embedding technique for immuno-electron microscopy was used; duodenal sections were stained with either motilin or serotonin in the pre-embedding DAB-nickel reaction, followed by subsequent staining of ultrathin sections of positive cells with either motilin or serotonin in the post-embedding immunogold reaction. Samples were divided into four groups: 1) pre-motilin, post-motilin, 2) pre-motilin, post-serotonin, 3) pre-serotonin, post-serotonin, and 4) pre-serotonin, post-motilin. Motilin-containing cells in the rabbit duodenum were characterized by round granules (395.3 +/- 66.1 nm in diameter) with medium electron density, located basally or in the perinuclear cytoplasm. In contrast, serotonin-containing cells were characterized by round to pleomorphic secretory granules (344.5 +/- 90.5 nm in diameter with electron dense cores and prominent halos. In motilin-containing cells, massive aggregations of immunogold particles reacted to motilin occurred over secretory granules. A few immunogold particles scattered diffusely over the cytoplasm reacted to serotonin; however, this reaction appeared to be background staining because the density was not changed if the section was treated by preabsorption. In serotonin-containing cells, immunogold particles reacted to serotonin were aggregated over the secretory granules and a large number of gold particles were scattered diffusely at the extragranular cytoplasm; however, very few or no immunogold particles were observed within the cells which reacted to motilin. Results of the present study indicate that motilin and serotonin are not co-localized in the epithelial endocrine cells of the rabbit intestine.

Immunohistochemical localization of low-affinity nerve growth factor receptor in the enteric nervous system of adult rats.

The localization of low-affinity nerve growth factor receptor in the enteric nervous system of adult rats has been studied by immunohistochemistry using a monoclonal antibody (clone 192) against the rat receptor. Cryostat and whole-mount sections were stained. By light and confocal microscopy, positive staining in neural structures was found in every part of the gut. In the ganglionic plexus, dense staining was detected in the neuropil surrounding neuronal cell bodies that were themselves devoid of immunoreactivity. Immunoelectron microscopy revealed deposition of reaction products on the outer plasma membranes of both perikarya and processes of neuronal as well as glial cells. Such a selective localization of the receptor in the plasma membrane, but not the cytoplasm, suggests that the mechanisms of receptor-ligand interaction in the gut may differ from those in the brain, where internalization of the receptor is observed in cholinergic cells. The present study provides the morphological basis for future studies designed to elucidate the functional significance of this enteric nervous system receptor. Since it is found in both neuronal and glial cells, it is probably under the influence of a number of trophic factors, including nerve growth factor.

Effect of carbachol on vascular and luminal release of immunoreactive gastrin from isolated perfused rat duodenum.

The release of intestinal gastrin was examined using the isolated vascularly and luminally perfused rat duodenum. Results show that duodenal gastrin was released into both the intestinal lumen and circulation. The basal release of immunoreactive gastrin (IRG) into the lumen was 23.3 +/- 1.8 pg/min and that into the vasculature was 70.1 +/- 8.2 pg/min. The administration of 1 microM carbachol into the vascular perfusate resulted in a marked reduction of luminal release of IRG; however, vascular release of IRG was not affected. The carbachol-induced inhibition of luminal release of IRG was blocked by atropine but not by hexamethonium. These data suggest that only the luminal release of IRG from the rat duodenum, but not vascular release of IRG, is under the inhibitory control of the cholinergic muscarinic mechanism.

Increase of p75 immunoreactivity in rat urinary bladder following inflammation.

Previous studies suggest that NGF may function as a mediator of inflammatory pain. Here, we examined the effect of inflammation on expression of the low affinity neurotrophin receptor p75, using the model of cyclophosphamide-induced cystitis in rats. In control rats, p75-positive thick fibre bundles were scattered in the muscle layer. At 2 and 3 days after injection of cyclophosphamide, numbers of p75-positive fine fibres in the muscle layer were dramatically increased. Electron microscopy revealed that p75 immunoreactivity was localized on the surface of Schwann cells and at the sites where they were apposed to axons. Results show that p75 is up-regulated in inflamed tissues, suggesting that p75 may bind to and take up nerve growth factor (NGF), thus participating in NGF-induced hyperalgesia.

Altered glucose dependence of glucagon-like peptide I(7-36)-induced insulin secretion from the Zucker (fa/fa) rat pancreas.

In previous studies on the enteroinsular axis in Zucker rats, it was found that glucose-dependent insulinotropic polypeptide (GIP) levels were normal in obese animals, but the glucose threshold for the insulinotropic action of GIP in the perfused rat pancreas was reduced. Glucagon-like peptide I (GLP-I)(7-36) is also an important incretin, and in the current study, glucose, insulin, and immunoreactive (IR)-COOH-terminal GLP-I responses to oral glucose were compared in lean (Fa/?) and obese (fa/fa) rats. In addition, the concentration thresholds for stimulation and glucose dependence of perfused pancreases to GLP-I(7-36) were examined. Glucose responses to oral glucose were similar in fa/fa and Fa/? rats. Obese animals were hyperinsulinemic when fasting and after oral glucose. Significant increases in IR-GLP-I levels in response to glucose were only observed in fa/fa rats. Perfused pancreases from fa/fa rats hypersecreted insulin at all glucose concentrations. In the presence of 4.4 mmol/l glucose, GLP-I(7-36) increased insulin secretion in fa/fa pancreases approximately 25-fold, whereas there was only a 5-fold increase in Fa/? pancreases. Pancreases from fa/fa rats, perfused with a glucose gradient (2.8-11 mmol/l) in the presence of GLP-I(7-36), responded with an immediate increase in insulin secretion, i.e., at a glucose concentration of 2.8 mmol/l, whereas Fa/? pancreases required a minimum of 4.22 mmol/l glucose for stimulation. With high glucose (16.7 mmol/l), both fa/fa and Fa/? rat pancreases exhibited similar responsiveness to GLP-I(7-36), having thresholds of < 50 pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of capsaicin on release of substance P-like immunoreactivity from vascularly perfused rat duodenum.

The release of substance P-like immunoreactivity (SPLI) from the rat duodenum was investigated using an in situ vascularly perfused preparation. Results show that the basal release of SPLI was measurable and significantly enhanced by the administration of both 1,3, and 10 microM of capsaicin, indicating that SPLI is present in capsaicin-sensitive afferent nerve fibers. It is concluded that the present model may be useful for the study of the control of duodenal release of SPLI.

Presence and actions of vasoactive intestinal peptide in the isolated rabbit heart.

The objectives of these experiments were to localize vasoactive intestinal peptide immunoreactivity (VIP-IR) in the intracardiac ganglia of the interatrial septum of the rabbit heart and to examine the effects of vasoactive intestinal peptide (VIP) on the isolated perfused rabbit heart. Cell bodies of neurones containing VIP-IR were located in the interatrial septa of rabbit hearts by using immunocytochemistry. In addition, the effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) on the isolated rabbit heart were compared with those of VIP. Bolus injections of SP, NKA, VIP, and NKB caused dose-dependent decreases in the perfusion pressure of hearts perfused at constant flow with ED50 values of 2.73 +/- 0.10 fmol (n = 6), 0.18 +/- 0.01 pmol (n = 8), 93.75 +/- 1.88 pmol (n = 6), and 75.00 +/- 3.06 pmol (n = 6), respectively. The slope of the dose-response curve of VIP was much greater than those of SP and NKA, suggesting the presence of one receptor subtype for VIP and multiple receptor subtypes for the neurokinins on rabbit coronary vessels. Differences in the slopes of the dose-response curves and potency may reflect differences in the mechanism of vasodilatation. The maximal values of vasodilatation of all of the peptides did not differ. None of the peptides produced significant changes in heart rate, left ventricular pressure, or contractility. These results suggest that VIP found in the intracardiac neurones of the rabbit heart can mediate coronary vasodilatation.

Gastric inhibitory polypeptide and glucagon-like peptide-1(7-36) amide exert similar effects on somatostatin secretion but opposite effects on gastrin secretion from the rat stomach.

Previous studies on the isolated perfused stomach have shown that gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1(7-36) amide (GLP-1(7-36) amide) stimulate release of somatostatin (somatostatin-like immunoreactivity, SLI). GIP produced a paradoxical increase in gastrin secretion, whereas GLP-1(7-36) was inhibitory. In the current study, the actions of synthetic (sp) and native (np) porcine and synthetic human (sh) GIP, GLP-1(7-36), and GLP-1(7-37) on SLI and gastrin secretion were compared using a gradient perfusion of peptide. All peptides increased SLI secretion at a threshold concentration of approximately 50 pmol/L (p < 0.05). The initial rate of increase in response to spGIP (119 +/- 39 pg/min) was greater than with other forms of GIP or GLP-1. Maximal increases obtained with the two porcine peptides did not differ. Gastrin secretion was increased by concentrations of spGIP and npGIP similar to those increasing SLI secretion, but the maximal response to shGIP was lower. In contrast to GIP-induced increases, both GLP-1(7-36) and GLP-1(7-37) suppressed gastrin secretion. It is concluded that human and porcine GIP, GLP-1(7-36), and GLP-1(7-37) all stimulate SLI secretion but with different maximal effects, and GIP stimulates gastrin secretion whereas both forms of GLP-1 inhibit gastrin secretion.

Beta-funaltrexamine blockade of opioid-induced inhibition of somatostatin secretion from rat stomach.

Opioid peptides are potent inhibitors of gastric somatostatin secretion. In the current investigation the effect of mu-opioid receptor blockade on responses to [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO) was studied. Gastric inhibitory polypeptide (GIP; 1 nM) -stimulated secretion of immunoreactive somatostatin was almost completely inhibited by DAGO (1 microM). The mu-receptor antagonists, beta-funaltrexamine and naloxonazine, blocked the effect of DAGO. Pretreatment of rats with beta-funaltrexamine, 24 h prior to perfusion, reduced the percentage inhibition by DAGO from 88.6 +/- 5.2% to 50.7 +/- 9.3%. These studies support the involvement of mu-opioid inhibitory receptors in the regulation of gastric somatostatin secretion.

Effect of cholecystokinin and secretin on somatostatin release from cultured antral cells.

BACKGROUND: Both secretin and cholecystokinin (CCK) inhibit gastric acid secretion. However, their mode of action has yet to be determined. A newly developed primary culture of human antral epithelial cells has been used to examine the effect of secretin and cholecystokinin on somatostatin release. METHODS: Normal human antral epithelial cell cultures enriched for D cells were maintained in culture for 2 days before release studies. RESULTS: Native human secretin at 10(-8) mol/L stimulated somatostatin release threefold. Porcine secretin and the secretin analogs, Tyr10 human secretin, Tyr13 porcine secretin, and Tyr10,13 porcine secretin were equipotent to native human secretin. CCK stimulated somatostatin release with the greatest response (eight times basal) at 10(-7) mol/L. The response to CCK was inhibited in a competitive manner by the addition of the benzodiazepine analog, MK-329. Addition of secretin in the presence of 10(-8) mol/L CCK resulted in a potentiation of somatostatin release, with the greatest response at 10(6) mol/L secretin, resulting in a 12-fold increase above basal. CONCLUSIONS: The stimulation observed after the addition of CCK was the result of activation of the CCK-A receptor subtype. The secretin receptor resembles that of the pancreatic D cells and acts through increasing intracellular cyclic adenosine monophosphate levels. Finally, these data indicate that the inhibitory action of CCK and secretin on gastric acid secretion may result from a synergistic action on antral D cells to release somatostatin, which in turn decreases antral gastrin release.

Inhibitory actions of a somatostatin analog SMS 201-995 on endocrine secretion from the isolated perfused stomach and pancreas of the rat.

An analog of somatostatin, SMS 201-995 (SMS; Sandostatin), has been shown to have increased potency in the inhibition of hormone secretion in vivo. In this study, the effect of SMS on the secretion of immunoreactive insulin, gastrin and gastric somatostatin was examined in the rat using in situ vascularly perfused organ preparations. The analog was found to inhibit both basal somatostatin and gastrin secretion from the perfused stomach in a concentration-dependent manner. SMS (9.4 x 10(-8) mol/l) also inhibited somatostatin release stimulated by gastric inhibitory polypeptide (GIP), but did not suppress gastrin release under the same conditions; an opposite effect was obtained when l-isoproterenol was used to stimulate somatostatin release. In the perfused pancreas both SS-14 (6.1 x 10(-9) mol/l) and SMS (9.4 x 10(-9) mol/l) inhibited the GIP (2 x 10(-9) mol/l) or acetylcholine (1 x 10(-6) mol/l), but not 17.8 x 10(-9) mol/l glucose-stimulated insulin secretion. Insulin secretion stimulated by 17.8 x 10(-3) mol/l glucose was unaffected by either peptide at this concentration. These results show that SMS is a potent inhibitor of gastric and pancreatic endocrine secretion. However, the inhibitory effect of SMS on gastric endocrine secretion was uncoupled in the presence of different secretagogues, suggesting that the action of SMS was dependent on the activation of different mechanisms or pathways in the stomach. In the pancreas, SMS appears to be acting on the beta-cell via a similar mechanism as somatostatin.

Changes in somatostatin-like immunoreactivity in lungs from perinatal guinea pigs and the effects of somatostatin-14 on lung liquid production.

Somatostatin-like immunoreactivity was measured by radioimmunoassay with a monoclonal antibody in lungs from perinatal guinea pigs (62 +/- 2 days of gestation). Fetuses delivered by Caesarean section and dissected before breathing showed 4748 +/- 758 pg/lung (n = 25). Fetuses allowed to breathe (neonates) showed marked increases in activity: 7629 +/- 1355 pg/lung (n = 12) after breathing 30 seconds, and 10729 +/- 1064 pg/lung (n = 6) after breathing 3 minutes (2.3-fold increase, P < 0.005). Values then declined (5203 +/- 1050 pg/lung (n = 9) at 30 minutes; 1458 +/- 105 pg/lung (n = 4) at 60 minutes). Changes were similar in pg/g wet tissue. HPLC characterized the immunoreactive peptides as somatostatin-14 (SS-14) and somatostatin-28 (SS-28) in both fetuses and neonates (n = 11). SS-28 made up only 13.7 +/- 1.7% of the activity; this percentage did not change with breathing. The effects of synthetic SS-14 on lung liquid production were investigated in in vitro lungs from 42 fetal guinea pigs. All 21 preparations immersed in 10(-5)-10(-7) M SS-14 during the middle hour of 3 h incubations reduced production, often approaching zero after treatment (rates, ml/kg body weight per h, succeeding hours: 10(-5) M (n = 9), 3.09 +/- 0.68, 0.93 +/- 0.39, -0.05 +/- 0.60 (fall significant during and after treatment, P < 0.025-0.005); 10(-6) M (n = 6), 3.06 +/- 0.68, 1.29 +/- 0.58, 0.36 +/- 0.38 (P < 0.05-0.005); 10(-7) M (n = 6), 1.96 +/- 0.66, 1.11 +/- 0.34, 0.64 +/- 0.28 (P < 0.05-0.025).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum.

The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumen was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SLI from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about 1 during basal release but increased to approximately 2 during carbachol stimulation.

Cholinergic stimulation of neuropeptide Y secretion from the isolated perfused rat stomach.

Neuropeptide Y (NPY) is present in both extrinsic sympathetic adrenergic nerve terminals and intrinsic nerves of the gastrointestinal (GI) tract. Based on this localization a number of functions have been attributed to GI NPY including regulation of blood flow, intestinal fluid and electrolyte transport, and motility. There is nothing currently known, however, about the regulation of its secretion from GI nerves. The effect of cholinergic agonists and antagonists on secretion of NPY immunoreactivity (NPY-IR) from the isolated perfused rat stomach was investigated in the present study. Perfusate samples were extracted and concentrated on SepPak cartridges. Basal levels of NPY-IR varied between 98 and 147 fmol/min. Release was stimulated by high potassium concentrations (50 mM) and acetylcholine (ACh; 1 microM). ACh-induced secretion was unaffected by atropine, but inhibited by hexamethonium. Further evidence for a nicotinic component in the regulation of NPY-IR secretion was obtained by the finding of hexamethonium-induced reduction in basal secretion and stimulation of secretion by 1,1-dimethyl-4-phenyl-piperazinium (DMPP). In conclusion, cholinergic agonists and antagonists can modulate gastric NPY-IR secretion, and the cholinergic stimulatory effects are probably mediated via nicotinic receptor stimulation at the level of the intrinsic ganglia.

Stimulus-specific inhibition of insulin release from rat pancreas by both rat and porcine galanin.

The effect of the neuropeptide galanin on insulin and somatostatin secretion in the rat was studied under various conditions. In the perfused rat pancreas, insulin secretion stimulated by arginine, but not cholecystokinin-8 (CCK-8) or acetylcholine (ACh) was inhibited by both rat and porcine galanin, whereas ACh-stimulated somatostatin release was inhibited by rat but not porcine galanin. Neither arginine nor CCK-8 significantly altered somatostatin secretion and galanin was without effect under those conditions. Gastric inhibitory polypeptide-stimulated insulin release from cultured mixtures of purified rat beta- and non-beta-cells was inhibited by rat and porcine galanin in a concentration-dependent and equipotent manner. The results suggest that the inhibitory effect of galanin on insulin and somatostatin secretion may be stimulus-specific and species-specific.

Muscarinic regulation of somatostatin release from primary cultures of human antral epithelial cells.

A newly developed, primary culture of human antral epithelial cells has been utilized to examine the effect of parasympathomimetics on somatostatin release. The cholinergic agonists, carbachol and methacholine, stimulated somatostatin secretion in a concentration-dependent manner. Maximal release in response to carbachol was observed at 0.1 mmol/l. Methacholine was 10 times more potent with a significant release being observed at 1 mumol/l, maximal secretion was observed at 10 mumol/l. Somatostatin release, stimulated by the mixed nicotinic and muscarinic agonist, carbachol, was attenuated by the addition of atropine at 0.1 mumol/l but was unaffected by the same concentration of pirenzepine. Methacholine-stimulated release was attenuated by addition of 0.1 mumol/l atropine and unaffected by the same concentration of pirenzepine. The response to methacholine was reversed by the addition of 0.1 mumol/l 4-diphenylacetoxy-n-methylpiperidine methiodide (4-DAMP) and attenuated by 1 nmol/l 4-DAMP indicating that the effect was mediated by an M3 receptor. In conclusion, human antral D cells are stimulated by parasympathomimetics acting at an M3 receptor.

Galanin inhibition of cholecystokinin-8-induced increase in [Ca2+]i in individual rat pancreatic B-cells.

The intracellular free Ca2+ ion concentration [( Ca2+]i) was measured in individual rat pancreatic B-cells loaded with fura-2. The cells were prepared by enzymatic digestion and fluorescence-activated cell sorting. The resting concentration of [Ca2+]i in B-cells was 126.3 +/- 3.1 nM in the presence of 4.4 mM glucose. Addition of cholecystokinin-8 (CCK-8) resulted in rapid and transient rises in [Ca2+]i. Perifusion of B-cells with galanin attenuated the amplitude and duration of CCK-8-induced [Ca2+]i changes and this inhibitory effect was concentration-dependent and reversible. Perifusion of B-cells with nifedipine, a voltage-sensitive Ca2+ channel blocker, reduced the duration of the [Ca2+]i increase induced by CCK-8, indicating that the Ca2+ entry from the extracellular space was, at least in part, involved in CCK-8-induced increases in [Ca2+]i.