Transit time flow measurement: experimental validation and comparison of three different systems.

BACKGROUND: Bloodflow measurements are of major clinical importance for quality control in vascular surgery. They allow detection of low-flow situations which may influence outcome adversely. The purpose of the present study was to validate three different flow systems for measuring absolute blood flow. METHODS: Measurements were performed in an experimental flow model using arteries and veins and blood or saline at two different temperatures. As a reference method true flow was measured by volume sampling. RESULTS: Correlation coefficients between transit time flow and true flow measurements ranged between 0.71 and 0.92. Systematic overestimation and underestimation of transit time flow were observed, but after second-order correction all correlations were excellent, ranging from 0.93 to 0.95 irrespective of flow medium and temperature. CONCLUSIONS: Transit time flow measurements are exact and reproducible. Second-order correction yields good accuracy and high precision, with minimal differences among the three systems evaluated.

Failed coronary artery bypass anastomosis detected by intraoperative coronary flow measurement.

OBJECTIVES: To assess intraoperative flow of arterial and venous coronary grafts after myocardial revascularization which may allow early detection of low flow situations, especially during minimally invasive coronary bypass surgery (MIDCAB), and lead to immediate correction of technical problems. METHODS: In two patients with severe and diffuse multi-vessel disease the left internal mammary artery (IMA) was connected to the left anterior descending artery (LAD). During reperfusion, the flow was measured in the IMA and vein grafts using a transit time flow meter. RESULTS: In both cases the IMA showed only a systolic pendulating flow curve with a mean flow of 0-1 ml/min and a high resistance. Manual IMA assessment revealed an adequate pulsation. Both distal IMA anastomoses were re-explored on cardiopulmonary bypass yielding an initial flow of 7 and 14 ml/min, respectively. After treatment with papaverine/adenosine the IMA flow increased from 7 to 26 ml/min (coronary flow reserve (CFR) = 3.7) and from 14 to 46 ml/min (CFR = 3.3), respectively. CONCLUSION: Intraoperative flow assessment of IMA and venous bypass grafts can be recommended to monitor flow; especially during MIDCAB procedures.

Transit-time flow measurement for detection of early graft failure during myocardial revascularization.

BACKGROUND: A low-flow situation in arterial and venous grafts has been associated with high rates of perioperative infarction and mortality. This study was designed to look at intraoperative graft flow and resistance in patients with coronary artery disease. METHODS: Coronary artery bypass graft flow was measured in 46 patients. Transit-time flow was used for coronary flow measurements at rest as well as after maximal vasodilation with adenosine infusion. RESULTS: Forty-three of the 46 patients showed normal internal mammary artery graft flow (>20 mL/min); 3 patients had no or minimal graft flow. Redoing the graft anastomosis in these 3 patients resulted in normalization of graft flow. The mean flow increased significantly after correction from 0.5 +/- 0.7 mL/min to 15.7 +/- 9.6 mL/min (p < 0.02). Conversely, vascular resistance decreased significantly from 138 +/- 10 to 4.8 +/- 1.8 Ohmv (p < 0.0001), as did the pulsatility index (from 146.9 +/- 95.7 to 3.4 +/- 1.8; p < 0.001). After correction, coronary flow reserve was 2.5 +/- 1.1. CONCLUSIONS: Measurements of intraoperative flow and resistance as well as derived variables allow assessment of early graft function and thus help prevent graft failure and reduce perioperative infarction. Transit-time volume flow might be a simple tool for quality control in coronary bypass procedures.

Galanin inhibits glucagon-like peptide-1 secretion through pertussis toxin-sensitive G protein and ATP-dependent potassium channels in rat ileal L-cells.

The neuropeptide galanin is widely distributed in the gastrointestinal tract and exerts several inhibitory effects, especially on intestinal motility and on insulin release from pancreatic beta-cells. The presence of galanin fibres not only in the myenteric and submucosal plexus but also in the mucosa, prompted us to investigate the regulatory role of galanin, and its mechanism of action, on the secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). Rat ileal cells were dispersed through mechanical vibration followed by moderate exposure to hyaluronidase, DNase I and EDTA, and enriched for L-cells by counterflow elutriation. A 6- to 7-fold enrichment in GLP-1 cell content was registered after elutriation, as compared with the crude cell preparation (929 +/- 81 vs 138 +/- 14 fmol/10(6) cells). L-cells then accounted for 4-5% of the total cell population. Bombesin induced a time-(15-240 min) and dose- (0.1 nM-1 microM) dependent release of GLP-1. Glucose-dependent insulinotropic peptide (GIP, 100 nM), forskolin (10 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA, 1 microM) each stimulated GLP-1 secretion over a 1-h incubation period. Galanin (0.01-100 nM) induced a dose-dependent inhibition of bombesin- and of GIP-stimulated GLP-1 release (mean inhibition of 90% with 100 nM galanin). Galanin also dose-dependently inhibited forskolin-induced GLP-1 secretion (74% of inhibition with 100 nM galanin), but not TPA-stimulated hormone release. Pretreatment of cells with 200 ng/ml pertussis toxin for 3 h, or incubation with the ATP-sensitive K+ channel blocker disopyramide (200 microM), prevented the inhibition by galanin of bombesin- and GIP-stimulated GLP-1 secretion. These studies indicate that intestinal secretion of GLP-1 is negatively controlled by galanin, that acts through receptors coupled to pertussis toxin-sensitive G protein and involves ATP-dependent K+ channels.

Expression of SNARE proteins in enteroendocrine cell lines and functional role of tetanus toxin-sensitive proteins in cholecystokinin release.

In neurons, synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin-1, synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)-2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines, including lines of endocrine and intestinal origin, by Western blotting and/or immunocytochemistry. The three core complex proteins were detected in the enteroendocrine, cholecystokinin (CCK)-secreting, cell lines STC-1 and GLUTag, and in the endocrine non-intestinal cell lines CA-77 and HIT-T15. In contrast, SNAP-25 and syntaxin-1 were undetected in the intestinal non-endocrine cell lines IEC-6, HT-29 and Caco-2, whereas a slight expression of VAMP-2 was documented in IEC-6 and HT-29 cells. Co-immunoprecipitation experiments indicated that syntaxin-1, SNAP-25 and VAMP-2 were present in a complex similar to that identified in brain. In the STC-1 cell line, treatment of streptolysin-O-permeabilized cells with tetanus toxin (Tetx) selectively cleaved VAMP-2 and VAMP-3/cellubrevin, and simultaneously abolished Ca2+-induced CCK secretion (IC50 approximately 12 nM). These results show that endocrine cell lines of intestinal origin express syntaxin-1, SNAP-25 and VAMP-2, and suggest a key role for a Tetx-sensitive protein (for example VAMP-2 and/or VAMP-3) in the CCK secretion by STC-1 cells.

Colonic mucin discharge by a cholinergic agonist, prostaglandins, and peptide YY in the isolated vascularly perfused rat colon.

UNLABELLED: The model of the isolated, vascularly perfused rat colon was assessed in the present study to investigate the nervous, hormonal, and local/paracrine pathways involved in colonic mucin secretion. A colonic loop was perfused via the superior mesenteric artery with a Krebs-Henseleit buffer containing 25% washed bovine erythrocytes at a rate of 2.5 ml/min. After a 10-min control period, each compound to be tested was infused intra-arterially for 30 min. Tissue samples from the proximal and midsegments of the perfused rat colon were then fixed and stained for mucus cell count. Intra-arterial administration of bethanechol evoked a concentration-dependent decrease in the number of stained mucus cells per crypt section over the range 2 x 10(-6) to 2 x 10(-4) M: 16.6 +/- 1.4 stained mucus cells per crypt in the midportion of the perfused rat colon (n = 5) with bethanechol 2 x 10(-4) M versus 28.8 +/- 1.5 for controls (n = 6). After infusion of 1.25 and 2.5 microM 16,16-dimethyl prostaglandin E2 (dmPGE2), the number of stained mucus cells per crypt section was significantly reduced: 21.6 +/- 0.6 (n = 6) and 20.6 +/- 1.4 (n = 7), respectively. An increase in the number of cavitated mucus cells was also observed (22.1 +/- 6.7 and 38.5 +/- 4.1% of cavitated mucus cells in the midsegment of the perfused rat colon with 1.25 and 2.5 microM dmPGE2, respectively, vs. 12.3 +/- 4.1% for controls). In contrast, prostaglandin F2alpha did not significantly affect mucus discharge from colonic cells. Peptide YY (10(-10), 10(-9) and 10(-8) M) induced a dose-dependent increase in the percentage of cavitated mucus cells (16.7 +/- 2.8, 23.1 +/- 4.2, and 31.2 +/- 3.4% of cavitated mucus cells in the midsegment, respectively). The proximal and midsegments of the perfused rat colon were equally sensitive to each secretagogue. CONCLUSION: In the isolated, vascularly perfused rat colon, mucus cells strongly respond to the well-known mucin secretagogues, bethanechol and dmPGE2. This approach has already led to the identification of a novel stimulant of mucin secretion: peptide YY. Our ex vivo model, in which goblet cells are submitted to well-defined luminal and blood-borne stimuli is, therefore, reliable to investigate the nervous, hormonal, and local/paracrine pathways involved in the colonic mucin secretion.

Metabolic and drug distribution studies do not support direct inhibitory effects of metformin on intestinal glucose absorption.

In an attempt to clarify the question of an involvement of the inhibition of intestinal glucose absorption in the mechanism of action of Metformin, we used several experimental approaches: 1 glucose/lactate measurement in rat portal blood in vivo and 2 in the venous effluent of an isolated perfused rat intestinal segment; 3 metabolism of freshly isolated enterocytes in vitro and tissue distribution of 3H-labeled Metformin was investigated both in vivo and in vitro. Metformin applied intraluminally had no significant effect on portal glycaemia after a glucose load, but lactate increased, whereas in vivo only a high Metformin dosage reduced portal glucose appearance significantly. Although high Metformin concentrations were found in gut biopsies, precise histological analysis in the isolated intestine revealed that it was absent from enterocytes; however the drug accumulated in villous lacteals. Intrarterially applied Metformin decreased glucose absorption in the isolated perfused ileo-jejunal segment. These data suggested that vascular Metformin boosted intestinal anaerobic glucose metabolism. Biochemical measurements performed on freshly isolated enterocytes showed that even high Metformin levels did not interfere with cell respiration or with Na+/K+ ATPase activity. Thus, our data agree with other recent reports, suggesting that even at nontherapeutic concentrations Metformin has no relevant inhibitory effect on intestinal glucose absorption. The data are discussed in the frame of previous divergent observations. The results suggest however that Metformin of vascular origin stimulates glucose consumption by the intestine, which then increases lactate output from the gut.

Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line.

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.

[Secretion of peptide YY and truncated glucagon-like peptide 1 by isolated intestinal cells in rats]. / Sécrétion du peptide YY et du peptide glucagon-like 1 tronqué par les cellules intestinales isolées de rat.

In order to study the cellular mechanisms involved in peptide YY (PYY) and truncated glucagon-like peptide 1 (TGLP1) release, a model of rat intestinal cells dispersed with collagenase/EDTA and enriched for L-cells by counterflow elutriation was developed. Elutriation significantly increased in the harvested cells the concentration of PYY (828 +/- 97 vs 151 +/- 16 fmol/10(6) cells) and TGLP1 (1,094 +/- 109 vs 167 +/- 20 fmol/10(6) cells), and brought the contribution of L-cells to 4-5% of the total cell population. Forskolin (1-10 microM) and dibutyryl cyclic AMP (dbcAMP, 1-5 mM) increased over an 1-h period PYY and TGLP1 secretion, with a maximal rate at 5 microM forskolin (232% and 250% of basal, respectively) and at 5 mM dbcAMP (347% and 234% of basal, respectively). Furthermore, 3-isobutylmethyl xanthine (IBMX, 1 mM) increased PYY (226% of basal) and TGLP1 (198% of basal) secretion. A combination of both 10 microM forskolin and 1 mM IBMX stimulated in an additive manner PYY (389% of basal) and TGLP1 (393% of basal) secretion. TPA (12-0-tetradecanoylphorbol-13-acetate, 0.1-1 microM) dose-dependently increased the secretion of PYY and TGLP1 (maximal release at 328% and 326%, respectively), whereas 4 alpha-phorbol was ineffective. Ionomycin (1-5 microM) and thapsigargin (0.1-5 microM) produced a dose-dependent increase in PYY and TGLP1 release (272% and 337% of basal for 5 microM ionomycin; 342% and 339% of basal for 5 microM thapsigargin, respectively). At gel chromatography, the immunoreactive PYY and TGLP1 material in cell extracts and in release medium co-eluted with the respective synthetic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

PAP, a pancreatic secretory protein induced during acute pancreatitis, is expressed in rat intestine.

The pancreatitis-associated protein (PAP) is a lectin-related secretory protein present in small amounts in the rat pancreas and rapidly overexpressed during the acute phase of pancreatitis. We demonstrate in this report that PAP is also expressed in rat intestine. A cDNA library from rat jejunum was probed with pancreatic PAP cDNA. The inserts of the selected recombinant clones corresponded to a transcript whose nucleotide sequence was identical to that of pancreatic PAP mRNA. The transcript was detected in duodenum, jejunum, ileum, and colon. A protein with same molecular mass (16 kDa) and pI (8.2) as pancreatic PAP was actually immunodetected in ileum homogenate after separation by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intestinal PAP was immunolocalized to the epithelial cells of the lower part of the villi. The protein accounted respectively for 0.02, 0.05, and 0.1% of soluble proteins in duodenum, jejunum, and ileum homogenates, as measured by enzyme-linked immunosorbent assay, and could not be detected in stomach and colon. Influence of fasting and feeding on PAP mRNA concentration was analyzed in ileum. Concentration decreased by 81 and 94% after animals were fasted for 24 and 48 h, respectively. Feeding restored the initial content within 6 h. On the other hand, intestinal PAP mRNA concentration was not altered during acute pancreatitis.

Variation of gastric lipase secretion in the Heidenhain pouch of the cat.

In the cat, gastric lipase secretion was equally but weakly stimulated by pentagastrin, a major stimulant of acid secretion, and by carbamylcholine, a major stimulant of pepsin secretion. Lipase was also stimulated by fresh liver, which induces a large blood gastrin release and not by canned food, which is a poor gastrin releaser. Lipase output always preceded that of acid an pepsin. Lipase was not correlated with acid and pepsin secretion while acid and pepsin were well correlated during all stimulations but not in basal state. Lipase is co-localized with pepsin in the chief cells but is also present in pepsin-free cells, the mucus surface cells of the fundus and the antrum. The distribution of lipase explains the lack of correlation between pepsin and lipase as already mentioned. However, our data show that lipase secretion is under the control of gastric stimulants and might play a role in the gastric initiation of pancreatic meal lipolysis.

Galanin in porcine vagal sensory nerves: immunohistochemical and immunochemical study.

In this work, the presence of galanin was examined by immunohistochemistry, radioimmunoassay and high performance liquid chromatography (HPLC) in porcine nodose ganglia, mainly constituted of cell bodies from the vagal sensory neurons. Galanin-like immunoreactivity (Gal-LI) was revealed in 10 to 15% of the total cell bodies by the indirect immunofluorescent technique of Coons. For comparison, a positive staining was revealed in a few cell bodies of the submucous plexus and in fibers located in the different layers of the ileum. The extractable Gal-LI content in nodose ganglia was 7.2 +/- 0.8 pmol/g wet tissue, which represents a concentration about nine times lower than that found in the ileum. HPLC of extractable material revealed a predominant peak which coeluted with the synthetic peptide. We propose that, in pigs, galanin may play a role in the transmission of visceral information through the vagal afferences.

Neuromedin-N is not released with neurotensin from rat ileum.

Neuromedin-N (NN) and neurotensin (NT) were shown recently to be encoded in the same precursor molecule. Colocalization and corelease of ileal NT and NN have not yet been demonstrated and were investigated in the rat using antisera that separately recognized intact NT and NN in ileal extracts. Immunofluorescence labeling of full thickness ileal wall revealed that NN-positive fluorescence was only found in the N-cells. However, only 50% of the N-cells also contained NN-like immunoreactivity (NN-LI). This was associated with a level of extractable NN that was 5-fold lower than that of NT. Corelease of NN- and NT-LI was investigated with the isolated, vascularly perfused jejunoileum model by using various substances that were described as potent stimulants of NT release in vivo. Luminal infusion of mixed nutrients, oleic acid (100 mM), glucose (5%), and taurocholic acid (1%) induced a well sustained release of NT, with plateau secretion of about 200%, 120%, 300%, and 700% above basal, respectively. Vascular bombesin (10(-7) M) and carbachol (10(-5) M) provoked a biphasic release of NT, consisting of a transient rise (approximately 600% above basal) followed by a less pronounced but sustained response. HPLC analysis of portal effluent revealed that 70-80% of NT-LI was intact NT. NN-LI was not coreleased with NT even upon vascular coinfusion of phenanthroline, which markedly protected exogenously infused NN. The coexistence but lack of corelease to any significant degree of NN with NT suggests different fates of these two precursor-related peptides within the ileal mucosa.

Immunocytochemical study of peptidergic structures in Brunner's glands.

Nervous and endocrine peptidergic structures in human Brunner's glands were studied by immunofluorescence. Endocrine cells storing immunoreactive components respectively similar to somatostatin 14, the amino-terminal portion (1-14) of somatostatin 28, gastrin-cholecystokinin, and peptide YY were distributed throughout the acini. Peptidergic nerve structures contained materials immunologically related to vasoactive intestinal peptide, peptide histidine methionine, substance P, neuropeptide Y, and gastrin-releasing peptide. The latter peptide was detected in discrete fibers running into the acini but within no cell body in the submucosa. All other neuropeptides were stored in fibers, isolated or grouped in bundles, and in perikarya of submucosal ganglia close to the acini. No immunoreactive structures were detected using antisera directed against pancreatic polypeptide, secretin, motilin, neurotensin, or calcitonin gene-related peptide. The results suggest that several regulatory peptides may be involved in the control of Brunner's glands in humans.