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K A Sharkey

Publications and source records attributed to K A Sharkey.

At least 73 records · Page 4Linked to original sources
PubMed1997

Induction of nitric oxide synthase in rat gastric smooth muscle preparations.

The induction in vitro of inducible nitric oxide synthase (iNOS) in intact gastric circular (CM) and longitudinal (LM) smooth muscle preparations was evaluated 1) pharmacologically, by the appearance of 1 mM L-arginine (L-Arg)-induced relaxation in a precontracted tissue; 2) biochemically, according to the appearance of iNOS mRNA using a reverse transcriptase-polymerase chain reaction; and 3) immunohistochemically, using an iNOS-specific antibody. Functionally, iNOS induction affected the contractile properties of the CM but not the LM preparation. The time course of iNOS induction monitored pharmacologically paralleled exactly the appearance of iNOS mRNA. The relaxant response to L-Arg in iNOS-induced CM tissues was blocked by the iNOS inhibitor aminoguanidine and by the guanylyl cyclase inhibitor LY-83583. The addition of oxyhemoglobin to the organ bath also attenuated the relaxant response, but tetrodotoxin had no effect. The transcriptional inhibitor actinomycin D completely blocked iNOS induction as assessed by both pharmacological and biochemical criteria. In iNOS-induced preparations the iNOS immunoreactivity was not detected in the smooth muscle elements but was localized in a layer of macrophage-related cells that were in apposition to the CM smooth muscle elements. We conclude that the spontaneous induction of iNOS in rat gastric tissue can affect the pharmacomechanical reactivity of the CM elements and that this regulation of the CM contractility is due to the induction of iNOS in a set of macrophage-related cells that are closely apposed to the CM elements so that they selectively affect only the contractility of the CM preparation.

Animals↗
PubMed1996

Exacerbation of inflammation-associated colonic injury in rat through inhibition of cyclooxygenase-2.

Cyclooxygenase type 1 is constitutively expressed and accounts for synthesis of prostaglandins in the normal gastrointestinal tract. Cyclooxygenase-2 is expressed at sites of inflammation. Selective inhibitors of cyclooxygenase-2 have been suggested to spare gastrointestinal prostaglandin synthesis, and therefore lack the ulcerogenic effects associated with standard nonsteroidal antiinflammatory drugs. However, the effects of cyclooxygenase-2 inhibitors on inflamed gastrointestinal mucosa have not been examined. We examined cyclooxygenase-2 mRNA and protein expression before and after induction of colitis in the rat, the contribution of cyclooxygenase-2 to colonic prostaglandin synthesis during colitis and the effects of selective inhibitors of cyclooxygenase-2 on colonic injury in this model. Cyclooxygenase-2 mRNA expression increased three to sixfold during the period 24 h to 1 wk after induction of colitis, with marked increases in cyclooxygenase-2 protein expression in the lamina propria and muscularis of the colon during colitis. Cyclooxygenase-1 expression (mRNA and protein) was not affected by the induction of colitis. The prostaglandins produced during colitis were largely derived from cyclooxygenase-2. Treatment with selective cyclooxygenase-2 inhibitors resulted in exacerbation of colitis, with perforation occurring when the compounds were administered for a week. These studies demonstrate that suppression of cyclooxygenase-2 can result in exacerbation of inflammation-associated colonic injury.

Animals↗
PubMed1996

Secretion and serotonin release in the isolated rat lacrimal gland: the effects of substance P and calcitonin gene-related peptide.

A close anatomical relationship between nerves containing substance P and calcitonin gene-related peptide (CGRP) and mast cells containing serotonin has been demonstrated in the rat lacrimal gland. This study investigates the potential for peptidergic regulation of lacrimal mast cells by examining the actions of substance P, CGRP and serotonin on protein and peroxidase secretion from isolated lacrimal segments and on substance P and CGRP to release serotonin from the lacrimal mast cells. Substance P, CGRP and serotonin evoked marked increases in total protein and peroxidase from the lacrimal. Sodium cromoglycate, a mast cell stabilizer, significantly reduced or blocked the secretory responses elicited by these agonists. Chromatographic analysis using electrochemical detection revealed that substance P, but not CGRP, augmented the release of serotonin from the gland. The substance P evoked peroxidase secretion and serotonin release was blocked by CGRP and by sodium cromoglycate. These results support a role for mast cells in the regulation of lacrimal secretion and suggest a novel regulatory interaction between substance P and CGRP in the control of lacrimal function through a neuro-immune interaction.

Animals↗
PubMed1996

Immunohistochemically-defined subtypes of neurons in the inferior mesenteric ganglion of the guinea-pig.

The distribution of somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), tyrosine hydroxylase (a marker of noradrenergic neurons, NA) and nitric oxide synthase-immunoreactivity (NOS-IR) was examined in the inferior mesenteric ganglion of guinea pigs with double- and triple-labelling immunohistochemistry. About 75% of neurons identified were NA/SOM, almost 20% were NA/NPY and the remainder consisted of small groups of NA/- (1-5%), NA/NPY/SOM (2-5%) and VIP (1-2%) neurons. VIP neurons contained NPY-IR, usually contained SOM-IR and were surrounded by dense pericellular baskets of SP fibres. NOS-IR was found in a small proportion of neurons colocalized with VIP but both NOS- and VIP-IR were also found alone in some neurons. Some NOS reactive varicose fibres throughout the ganglia also contained VIP-IR but much of the NOS- and VIP-IR appeared to be localized in discrete varicosities. SOM-IR was also detectable in TH fibres within myenteric ganglia of the distal colon. We conclude that the subtypes of neurons in the inferior mesenteric ganglion share some properties with other sympathetic and abdominal ganglia but they exist in distinct proportions and may make dissimilar projections along the length of the gut.

Animals↗
PubMed1996

Hepatic mucosal mast cell hyperplasia in rats with secondary biliary cirrhosis.

Mast cells have been shown to play a role in many chronic inflammatory and fibrotic disorders. However, their possible contribution to the pathological changes that occur in liver cirrhosis is unknown. To explore this, we examined whether changes in hepatic mast cell number and mediator content were associated with fibrotic changes in experimental biliary cirrhosis. Rats were studied 7, 14, or 21 days after bile duct resection (BDR). Hepatic mast cells were identified by histochemical and immunohistochemical stains. Rat mast cell protease II (RMCP-II), a marker of mast cell degranulation, was measured in liver by enzyme-linked immunosorbent assay. Hepatic collagen deposition was assessed by Sirius Red F3BA staining. In day 21 BDR rats, there was a one- to twofold increase (P < .001) in the number of hepatic mast cells, but this was not observed in day 7 or 14 BDR rats. Mild fibrotic changes were noted in BDR rat livers as early as 7 days after induction of cholestasis. Significant expansion and organization of fibrous tissue had occurred in day 14 BDR rats which progressed to bridging fibrosis by day 21. Liver RMCP-II levels were decreased by 50 percent (P < .05) and mast cell degranulation was apparent as shown by histamine immunostaining. These results suggest that hepatic mast cell hyperplasia and degranulation occur during prolonged cholestasis in the rat. Although these changes do not correlate with the onset of hepatic fibrosis, they do occur at a time during which there is significant deposition and organization extracellular matrix elements. Hepatic mast cells, by releasing profibrogenic mediators, may contribute to fibrotic changes in biliary cirrhosis.

Animals↗
PubMed1996

The dorsal vagal complex of the ferret: anatomical and immunohistochemical studies.

To further the understanding of gastrointestinal function in this species, and in particular to advance our own work concerning central emetic pathways, the cytoarchitecture and the distribution of eight neurochemicals were studied in the ferret dorsal vagal complex (DVC; area postrema, nucleus of the solitary tract [nTS] and dorsal motor nucleus of the vagus). The cytoarchitectural features of this region in the ferret were similar to those seen in other species; however, the ferret possesses a particularly large and distinct subnucleus gelatinosus of the nTS. Dense calcitonin gene-related peptide-immunoreactivity was found in the gelatinous, interstitial and commissural subnuclei of the nTS, with lesser amounts in other regions of the DVC. Enkephalin-immunoreactivity of varying densities was found throughout the DVC. Moderate to dense galanin-immunoreactivity was observed throughout the DVC, with the exception of the subnucleus gelatinosus of the nTS, from which it was virtually absent. Dense neuropeptide Y-immunoreactivity was observed in the subnucleus gelatinosus and interstitial subnucleus, with moderate staining in other regions of the DVC. Neurotensin immunoreactivity was very sparse or absent. Immunoreactivity for serotonin was sparsely distributed throughout the DVC. Moderate somatostatin-immunoreactivity was observed over a large portion of the DVC, but was virtually absent from the gelatinosus and interstitial subnuclei. Substance P immunoreactivity was observed throughout the DVC and was particularly dense in the dorsal/dorsolateral subnucleus and the dorsal aspects of the medial and commissural subnuclei. In terms of its cytoarchitecture the DVC of the ferret is more similar to the cat than the rat, especially with regard to the area postrema and the subnucleus gelatinosus of the nTS. The distribution of neuroactive substances was largely similar to other species; however, differences were present particularly in patterns of immunoreactivity for enkephalin, serotonin, neuropeptide Y and somatostatin.

Animals↗
PubMed1996

Role of 5-HT in cholera toxin-induced mucin secretion in the rat small intestine.

We examined the role of 5-hydroxytryptamine (5-HT) in cholera toxin (CT)-induced mucin secretion in the proximal and distal regions of the rat small intestine. Neither the 5-HT2 receptor antagonist ketanserin nor the cyclooxygenase inhibitor indomethacin was capable of inhibiting choleraic mucin secretion. However, in the presence of the mixed 5-HT3/4 receptor antagonist tropisetron at doses that block both receptor subtypes, the secretory response was reduced to baseline levels in the proximal and distal small intestine. The selective 5-HT3 receptor antagonist ondansetron had no significant effect. These findings suggest that choleraic mucin secretion is mediated primarily through the activation of a 5-HT4-like receptor. Mucin secretion in response to the exogenous application of 5-HT occurs via two pathways: one is mediated by a 5-HT4-like receptor and is capsaicin sensitive but tetrodotoxin (TTX) insensitive, and one lacks the capsaicin-sensitive 5-HT4-mediated response but is TTX sensitive. Both converge on a common pathway that is cholinergic. No significant differences were observed between proximal and distal intestinal segments.

Animals↗
PubMed1996

Control of exocrine secretion in the guinea-pig pancreas by histamine H3 receptors.

The aim of this study was to investigate the possible involvement of the histamine H3 receptor in control of exocrine pancreatic secretion from the guinea-pig. In in vitro experiments, the H3 receptor agonist (R)-alpha-methylhistamine (0.01-10 microM) elicited a concentration-dependent decrease in the release of alpha-amylase. (R)-alpha-Methylhistamine concentrations above 10 microM evoked a concentration-dependent increase in alpha-amylase secretion. Application of mepyramine (1 microM) partially blocked this increase. The H3 receptor antagonist thioperanide (1 microM) blocked the effects of (R)-alpha-methylhistamine below 10 microM. Histamine and (R)-alpha-methylhistamine attenuated both protein release elicited during electrical-field stimulation and the release of tritiated choline, and these effects were reversed by thioperamide. In an in vivo study, (R)-alpha-methylhistamine increased juice secretion and total protein content of the juice by 40%. Histamine H1 and H2 receptor antagonists blocked this increase and uncovered an attenuation of the secretory parameters (juice flow 28%, total protein content 44%). This attenuation was blocked by thioperamide. These observations suggest that stimulation of the histamine H3 receptor in the pancreas results in a decreased fluid and enzyme release by inhibition of acetylcholine release from intrinsic pancreatic nerves.

Animals↗
PubMed1995

Nitric oxide synthase in tiger salamander retina.

Previous studies have indicated that nitric oxide, a labile freely diffusible biological messenger synthesized by nitric oxide synthase, may modulate light transduction and signal transmission in the retina. In the present work, the large size of retinal cells in tiger salamander (Ambystoma tigrinum) allowed the utilization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and nitric oxide synthase immunocytochemistry to delineate the cell-specific intracellular localization of nitric oxide synthase. NADPH-diaphorase activity was highly concentrated in the outer retina, in rod and cone inner segment ellipsoids, and between and adjacent to the photoreceptor cell bodies in the outer nuclear layer. Examination of enzymatically isolated retinal cells indicated that outer nuclear layer NADPH-diaphorase activity was localized to the distal processes of the retinal glial (Müller) cells and to putative bipolar cell Landolt clubs. Less intense NADPH-diaphorase activity was seen in the photoreceptor inner segment myoid region, in a small number of inner nuclear layer cells, in cap-like configurations at the distal poles of cells in the ganglion cell layer and surrounding ganglion cell layer somata, and in punctate form within both plexiform layers, the pigment epithelium, and the optic nerve. Nitric oxide synthase-like immunoreactivity was similarly localized, but was also concentrated along a thin sublamina centered within the inner plexiform layer. The potential for nitric oxide generation at multiple retinal sites suggests that this molecule may play a number of roles in the processing of visual information in the retina.

Animals↗
PubMed1995

The source and action of histamine in the isolated guinea-pig gallbladder.

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H1-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H1-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H2-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H2-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H3-receptor agonist, (R)-alpha-methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10(-10) - 10(-7) M) indicating little pre-synaptic H3 activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H1 and H2 but not H3 receptors.

Animals↗
PubMed1995

Impaired vasodilatory responses in the gastric microcirculation of anesthetized rats with secondary biliary cirrhosis.

BACKGROUND/AIMS: The increased susceptibility of the stomach to injury observed in portal hypertension may be related to a defect in the hyperemic response to luminal irritants. The aim of this study was to evaluate the components that mediate this hyperemic response in a rat model of cirrhosis and portal hypertensive gastropathy. METHODS: Cirrhosis was induced by bile duct ligation, whereas controls underwent sham operation. Gastric blood flow responses to topical application of acid, capsaicin, nitrovasodilators, misoprostol, 8-bromo-cyclic guanosine monophosphate, and 8-bromo-cyclic adenosine monophosphate were measured by laser Doppler flowmetry using an ex vivo gastric chamber preparation. Calcitonin gene-related peptide immunoreactivity was used as an index of the anatomic integrity of the sensory afferent neurons of the stomach. RESULTS: Blood flow responses to acid, capsaicin, nitrovasodilators, and 8-bromo-cyclic guanosine monophosphate were significantly depressed in cirrhotic rats, whereas they were augmented after topical application of misoprostol and 8-bromo-cyclic adenosine monophosphate. Calcitonin gene-related peptide immunoreactivity was similar in the stomachs of cirrhotic and control rats. CONCLUSIONS: Gastric vasodilation after stimulation of sensory afferent neurons is impaired in cirrhotic rats despite the normal anatomic distribution of these nerves. This effect seemed to be related to a depressed response of the gastric microcirculation to cyclic guanosine monophosphate-dependent vasodilators. This alteration may contribute to the increased susceptibility to gastric ulceration in cirrhotics.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Evidence for CGRP accumulation and activity in experimental neuromas.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and widely distributed neuropeptide that may participate in the injury response of peripheral nerve. We examined evidence for the presence of CGRP immunoreactivity (IR) and its activity in experimental neuromas of Sprague-Dawley rats created by sectioning the midsciatic nerve with resection of 2-3 cm of its distal portion and branches. CGRP activity was evaluated by measuring local blood flow in neuromas using hydrogen polarography and laser-Doppler flowmetry. At all time points studied after nerve section (24 h, 48 h, 7 days, 14 days) there was a rise in local blood flow in the neuroma stumps. At 48 h the hyperemia was maximum but was reversed by topical application of human CGRP(8--37), a specific CGRP-receptor antagonist. CGRP presence was evaluated by immunohistochemistry and radioimmunoassay (RIA). At 24 and 48 h, CGRP IR was intense and distributed in a globular and diffuse pattern apparently not confined to discrete axonlike profiles. At 7 and 14 days, CGRP IR remained prominent and was associated with disorganized axonlike profiles, sometimes directed in a circumferential pattern around the outside of the neuroma. RIA confirmed rises in CGRP content at 24 and 48 h that accompanied the changes in local blood flow and altered distribution of CGRP IR. CGRP accumulates in a time-related fashion within experimental neuromas, where it induces among other possible actions prominent local vasodilatation. CGRP may be important in the regenerative milieu of injured nerves.

Animals↗
PubMed1994

Accumulation and degranulation of mast cells in experimental neuromas.

Mast cell accumulation and degranulation occur within the endoneurium of injured peripheral nerves. We investigated the time course of mast cell accumulation and degranulation in experimental neuromas of the sciatic nerve in rats. Mast cell accumulation and degranulation were significant only after the first week following neuroma creation and were prominent at 14 days within the neuroma stump. Mast cell degranulation could account for microvascular changes within neuromas after the first week following injury.

Animals↗
PubMed1994

c-Fos- and JunB-immunoreactivities in the enteric nervous system of the guinea-pig ileum.

We examined the expression of c-Fos and JunB in four immunohistochemical subtypes of enteric neurones in the guinea-pig ileum. In whole mount preparations of the myenteric and submucous plexuses from isolated segments incubated in normal Krebs' solution, increased numbers of many cells expressed visible c-Fos- and JunB-immunoreactivities. These increases may have been associated with the process of isolation and/or incubation conditions. Depolarizing stimulation by veratridine or 50 mM K+ induced further increases of neuronal c-Fos and JunB expression with no obvious subtype preference. This probably reflected a non-specific activation of most enteric neurones by these stimuli and supports the idea that expression of c-Fos and JunB in most or all enteric neurones may be a useful determinant of activation.

Animals↗
PubMed1994

The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig.

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal antibody, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.

Animals↗
PubMed1994

Ionizing radiation reduces neurally evoked electrolyte transport in rat ileum through a mast cell-dependent mechanism.

BACKGROUND/AIMS: Mechanisms of neuroimmune regulation of intestinal electrolyte transport under pathophysiological conditions are unclear. This study investigated the effect of ionizing radiation on ileal electrolyte transport. METHODS: Rats were exposed to 10 Gy gamma-radiation and were killed 2, 24, and 48 hours later. Ileal segments were either mounted in Ussing chambers and exposed to electrical field stimulation, prostaglandin E2, leukotriene D4, or theophylline, or they were assayed for biochemical indices of inflammation. Other segments were processed for routine histological screening, mast cell counts, or immunohistochemical analysis of the distribution of vasoactive intestinal polypeptide or substance P immunoreactivity. RESULTS: Basal short-circuit current was unchanged 2, 24, or 48 hours postirradiation. However, there was a reduction of tissue responsiveness to electrical field stimulation, prostaglandin E2, and theophylline but not to leukotriene D4. Decreased responsiveness at 2-hours postirradiation was blocked by pretreatment with the H1 antagonist pyrilamine. Tissue myeloperoxidase activity and 5-hydroxytryptamine content were not altered postirradiation, but tissue histamine and mucosal mast cells were significantly reduced at 24 and 48 hours. There were no significant changes in villus-crypt architecture until 48 hours postirradiation. There was no significant alteration in the distribution of immunoreactive vasoactive intestinal polypeptide or substance P. CONCLUSIONS: Ionizing radiation reduced the transport response to neural stimulation. The effect correlated temporally with decreased mast cells and histamine, suggesting a functional role for previously reported mast cell-nerve interactions.

Animals↗
PubMed1994

Innervation and mast cells of the rat exorbital lacrimal gland: the effects of age.

The distribution of nerves and mast cells was studied in the lacrimal glands of 3-5-, 14- and 24-month-old rats, using light microscopic histochemical and immunohistochemical techniques. In 14-month and, to a greater extent, in 24-month-old rats there were signs of chronic inflammation and patchy destruction of acinar, ductal and vascular tissue. The glands of the three different age groups contained acetylcholinesterase (AChE), vasoactive intestinal polypeptide (VIP)-, neuropeptide Y (NPY)-, calcitonin gene-related peptide (CGRP)-, tyrosine hydroxylase-, substance P- and the phosphoprotein B-50-immunoreactive nerves. B-50-immunoreactive nerves were distributed around acini, blood vessels and ducts, in a similar manner to VIP and AChE. Substance P- and CGRP-immunoreactive nerves were sparsely distributed in interlobular connective tissue and around ducts and blood vessels. Tyrosine hydroxylase- and NPY-containing nerves were found around blood vessels. The 3-5- and 14-month-old rats had a similar pattern of innervation, however, by 24 months there was a reduction in the number and intensity of immunoreactive nerves. The loss of nerves was particularly associated with damage to the gland. Mast cells were also found in the lacrimal, mostly associated with neurovascular tissue. These could be histochemically labelled with alcian blue/safranin or toluidine blue and were immunohistochemically labelled with histamine and serotonin. Substance P-, CGRP-, VIP- and NPY-immunoreactive nerves were found apposed to mast cells. A large increase in mast cells was observed in 24-month compared to 3-5-month-old rats and these were found throughout the acinar tissue. These results show that a decrease in innervation and also chronic inflammation, with mast cell infiltration, occurs in aged rats. These findings may be contributing factors to reduced tear output in aging.

Acetylcholinesterase↗