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Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.

Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.

Adenosine Triphosphate↗

Stability of lingual lipase in vivo: studies of the iodinated enzyme in the rat stomach and duodenum.

In order to examine the stability of purified lingual lipase (EC 3.1.1.3) in vivo under fasted and fed conditions, we iodinated rat lingual lipase to a specific activity of 5-10 microCi/micrograms protein with 125I-labelled Bolton-Hunter reagent. Labelled lingual lipase in either saline or Intralipid was administered via an intragastric or intraduodenal cannula. Control labelled lipase preparations were incubated at 37 degrees C. Serial aliquots of gastric or intestinal contents were aspirated and subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography, and quantitated by densitometric scanning. In all experiments, the amount of lingual lipase in both stomach and duodenum was significantly reduced (P less than 0.05 vs. controls) at 15 and 60 min after administration. In either the presence or absence of Intralipid, lingual lipase was more stable in the stomach than in the duodenum (P less than 0.05). In conclusion, lingual lipase was more stable in the stomach than in the duodenum, and lipid emulsion did not appreciably affect the stability of the enzyme in either location of the upper digestive tract of the rat.

Animals↗

Inhibition of rat liver and duodenum soluble catechol-O-methyltransferase by a tight-binding inhibitor OR-462.

The inhibition kinetics of rat liver and duodenum soluble catechol-O-methyltransferase (COMT) with a disubstituted catechol OR-462 was studied. After preincubation of the enzyme and inhibitor in the presence of magnesium and S-adenyosylmethionine, an inhibition about thirty times greater than that without preincubation was observed. Reversible tight-binding inhibition was demonstrated with Ki values of 0.7 nM and 1.0 nM for liver and duodenum enzyme, respectively. Km values of 53.4 microM and 56.9 microM for substrate 3,4-dihydroxybenzoic acid and 23.0 microM and 17.5 microM for S-adenosylmethionine were calculated for liver and duodenum enzyme, respectively. A catalytic number of 24/min for liver soluble COMT was calculated.

Animals↗

Induction of vitamin D 24-hydroxylase messenger RNA and activity by 22-oxacalcitriol in mouse kidney and duodenum. Possible role in decrease of plasma 1 alpha,25-dihydroxyvitamin D3.

The synthetic analog of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], 22-oxacalcitriol (OCT), retains most of the properties of 1,25(OH)2D3 but exhibits much less hypercalcemic action than the parent compound. The effects of OCT on plasma calcium, phosphorus, and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] concentrations were examined in mice. Administration of a single dose (24 pmol/g body wt, i.p.) of OCT had no effect on plasma calcium for up to 48 hr, significantly increased plasma phosphorus at 4 and 8 hr and significantly reduced the concentration of 1,25(OH)2D in plasma between 4 and 48 hr. Both OCT and 1,25(OH)2D3 at 24 pmol/g body wt (i.p.) induced a single, 3.4-kb mRNA encoding vitamin D 24-hydroxylase (24-OHase), the cytochrome P450 enzyme responsible for 1,25(OH)2D3 degradation, in kidney and duodenum. The OCT-induced increase in 24-OHase mRNA and an increase in enzyme activity were marked at 2 and 4 hr in both tissues. In kidney, mRNA abundance had decreased by 8 hr but remained above basal values for up to 30 hr; activity remained relatively high for up to 48 hr. In duodenum, 24-OHase mRNA abundance returned virtually to control values by 8 hr after OCT treatment; activity remained at nearly maximal levels for up to 30 hr but was decreased at 48 hr. The effects of OCT and 1,25(OH)2D3 on 24-OHase mRNA abundance and enzyme activity were dose-dependent in kidney and duodenum. Whereas the dose-response relations for the effects of both compounds on 24-OHase mRNA were similar, OCT was slightly more potent than 1,25(OH)2D3 in stimulating 24-OHase activity in both tissues. These results suggest that the OCT-induced decrease in plasma 1,25(OH)2D3 is attributable, at least in part, to an increased degradation of 1,25(OH)2D3, which results from an increase in 24-OHase abundance and enzyme activity.

Animals↗

Effect of intracerebroventricular administration of thyrotropin-releasing hormone upon the electroenteromyogram of rat duodenum.

Electroenteromyographic activity (EMG) of the duodenum was recorded in pentobarbital-anesthetized rats. TRH intraventricularly administered to rats produced changes in EMG such as increased amplitude, decreased frequency of slow waves and the association of bursts of spike potentials with nearly every cycle of the basal electric rhythm (BER). The effect was selectively prompt and marked in the EMG of proximal duodenum. The response was abolished by vagotomy or atropine injection and no response was elicited in the neonatally 6-OHDA-treated rat. Hypophysectomy, cord-transection or acute i.v. injection of 6-OHDA did not block the response. In the brain, TRH seems to stimulate the neuronal system controlling the vagus efferents involved in the regulation of the duodenal enteric nervous system which in turn modulates the myogenic excitability of the duodenum.

Animals↗

Postnatal change in receptivity for methionine5-enkephalin in rat duodenum: transition from neurogenic to myogenic receptivity.

The study concerned the postnatal ontogenesis of the response to methionine5-enkephalin (ENK) in the duodenum of developing rats. Sustained relaxation was produced by ENK in the isolated duodenum of 8 day old rats. The response was blocked by tetrodotoxin or naloxone. The response increased by day 18, decreased thereafter and was extinguished after 40 days. On the other hand, a tetrodotoxin resistant response to ENK, transient relaxation, appeared at day 20 and was augmented thereafter. The development of neurogenic receptivity for ENK preceded the appearance of myogenic receptivity in the rat duodenum.

Aging↗

Capsaicin activates neurogenic non-adrenergic non-cholinergic relaxations of the isolated rat duodenum.

Capsaicin induces a transient relaxation of the isolated rat duodenum in the presence of atropine plus guanethidine. The relaxant effect of capsaicin was not observed in vitro in the duodenum of capsaicin-pretreated animals. Capsaicin-induced relaxation was antagonized by tetrodotoxin or cold storage (24 h at 4 degrees C). It is concluded that capsaicin produces a neurogenic relaxation of the rat duodenum by activating an intramural non-adrenergic non-cholinergic mechanism which inhibits intestinal motility.

Animals↗

Identification, sequence analysis, and characterization of cDNA clones encoding two granzyme-like serine proteinases from rat duodenum.

Clones of cDNA encoding two serine proteinases were isolated from a cDNA library prepared from rat duodenum mRNA. The deduced amino acid sequences consisted of 248 residues and possessed a high level of homology to one another and to the sequences of granzymes, cathepsin G, and mast cell proteases I and II. Analysis of the enzymes' primary structures allowed the identification of the catalytic amino acid triad and the prediction of the substrate specificity. Northern blotting experiments showed that while one of these proteinases is expressed only in duodenum, the other enzyme is present in duodenum, lung, and spleen. It is supposed that these proteinases may play an important role in the function of an organism's defence systems.

Amino Acid Sequence↗

Cloning of cDNA for granzyme-like protein III, a novel serine proteinase from rat duodenum.

We previously reported cloning of cDNAs which encode two granzyme-like serine proteinases (GLP I and GLP II) from rat duodenum. In this paper we present the cDNA sequence for a novel member of the granzyme-like protein family from rat duodenum, GLP III. The amino acid sequence deduced from the cDNA consists of 248 residues and shows 88.2% identity to GLP I and 50.6% identity to GLP II. Comparison of the amino acid sequence of GLP III with sequences of related proteinases reveals the location of the catalytic amino acid triad and enables the prediction of the substrate specificity. Despite close similarity to GLP I, GLP III is expected to demonstrate different substrate specificity due to a substitution of the Arg residue by Glu at the critical position. Northern blot analysis demonstrates that the GLP III transcript is present only in duodenum.

Amino Acid Sequence↗

Intraluminal pH in the stomach, duodenum, and proximal jejunum in normal subjects and patients with exocrine pancreatic insufficiency.

In situ pH was measured simultaneously with microelectrodes in the stomach, duodenal bulb, midduodenum, duodenojejunal junction, and proximal jejunum. Fourteen healthy subjects and 8 patients with exocrine pancreatic insufficiency were studied under fasting conditions and for 3 h after a standard liquid meal. The luminal pH gradient was steepest in the proximal 10 cm of the duodenum, where acidity was reduced from pH 2 to pH 5 in the fasting state and from pH 1.7 to pH 4.3 in the second and third postprandial hour. Acidity was further reduced in the distal duodenum to a pH between 5 and 6 at the duodenojejunal junction. The frequent wide and rapid pH fluctuations seen in the duodenal bulb were gradually reduced along the duodenum and became rare in the jejunum. In patients with pancreatic insufficiency, duodenal or jejunal acidity did not differ significantly from the controls, with the exception of the single 10-min period occurring 70-80 min after the meal when duodenal bulb pH was 2.1 as compared with 3.1 in the normal subjects (p less than 0.05). All patients, including 2 patients with a very high duodenal acidity, demonstrated a duodenal pH gradient as steep as that found in the normal subjects, indicating sources of bicarbonate other than the pancreas.

Adult↗

Role of the duodenum in the control of canine gastrointestinal motility.

This study was designed to determine the role of the duodenum in controlling the interdigestive migrating motor complex of the canine stomach and small intestine. The motility patterns of 4 dogs were studied before and after resection of the entire duodenum with reimplantation of bile and pancreatic ducts. Before duodenectomy, plasma concentrations of motilin and pancreatic polypeptide varied cyclically with the migrating motor complex. After duodenectomy, the migrating motor complex was abolished in the stomach in 3 of the 4 dogs during fasting. The other dog demonstrated what appeared to be an intermittent cyclic increase in gastric contractile activity, but with markedly abnormal characteristics and at irregular intervals. The jejunal migrating motor complex continued to cycle after duodenectomy in all 4 dogs, but the mean period was shorter than before duodenectomy (83 min vs. 147 min, p less than 0.01). Duodenectomy abolished the cyclic variations of plasma motilin and pancreatic polypeptide and lowered the concentration of both peptides during all phases. This study suggests that the duodenum plays an important role in the initiation of the gastric migrating motor complex and in the coordination of interdigestive gastrointestinal motor activity, possibly by the release of motilin.

Animals↗

Mixed triglyceride breath test: a noninvasive test of pancreatic lipase activity in the duodenum.

A synthetic "mixed" triglyceride (1,3-distearoyl,2[13C]octanoyl glycerol), having a medium-chain fatty acid in the 2 position, was evaluated as a substrate for an exocrine pancreatic function test by comparing the 13CO2 breath excretion with the lipase output in the duodenum in 25 normal subjects, 29 patients with pancreatic disease, and 22 patients with steatorrhea of nonpancreatic origin. Excellent correlation was found in normal subjects and patients with pancreatic disease (r = 0.89) between lipase output in the duodenum and the 6-h cumulative 13CO2 excretion in breath, indicating that the mixed triglyceride breath test is an excellent noninvasive test of pancreatic lipase activity in the duodenum. As a test of exocrine pancreatic insufficiency, it has a sensitivity of 0.89 and a specificity of 0.81.

Adult↗

Phosphatidylcholines as mediators of adaptive cytoprotection of the rat duodenum.

BACKGROUND/AIMS: Surfactant phospholipids impede diffusion of acid through the gastric mucus, but their relevance in the defense of the duodenum against luminal acid is not known. METHODS: Duodenal resistance to acid was tested in anesthetized rats by instillation of HCl using a tube implanted in the proximal duodenum. The effects of a detergent (Brij 35; Sigma, St. Louis, MO) and a lipid mixture flushed through the luminal surface on duodenal resistance to acid were studied. The lipid content in the mucus and the effects of acid, prostaglandins, and indomethacin on the lipid layer were also analyzed. RESULTS: Instillation of 100 mumol HCl or 5 micrograms/kg 16,16-dimethyl prostaglandin E2 increased resistance to acid, preventing duodenal lesions induced by 500 mumol HCl. However, 100 mumol HCl or 16,16-dimethyl prostaglandin E2 did not prevent lesions induced by 500 mumol HCl in rats undergoing perfusions with 5% Brij 35. Indomethacin suppressed acid-induced protection. A mixture of tripalmitin and dipalmitoyl-phosphatidylcholine protected against 500 mumol HCl, and the effect was also observed in rats receiving indomethacin. Finally, 100 mumol HCl increased the phosphatidylcholine content in the duodenal mucus but not in rats receiving 5% Brij 35 or indomethacin. CONCLUSIONS: Surface-active phospholipids are critical for adaptive cytoprotection to acid in the rat duodenum.

16,16-Dimethylprostaglandin E2↗

Changes in muscarinic acetylcholine receptors in the isolated duodenum from repeatedly cold-stressed rats and the effect of neurotropin.

In rats repeatedly cold-stressed by specific alternation of rhythm in environmental temperature (SART-stressed rats), the contractile response to acetylcholine (ACh) of the isolated duodenum was remarkably decreased, whereas the contractile responses to K+, Ba2+ and Ca2+ were comparable to those in non-stressed rats. The amount of [3H]quinuclidinyl benzilate in the duodenum of SART-stressed rats was about 50% of that in non-stressed rats, but the KD value remained unchanged. Long-term administration of hexamethonium prevented the changes in SART-stressed rats. The daily treatment with Neurotropin, an extract isolated from inflamed rabbit dermis inoculated with vaccinia virus, dose-dependently prevented the changes in SART-stressed rats. However, Neurotropin had no effect on the ACh-induced decrease in muscarinic ACh receptor (m-ACh.R) in cultured vas deferens of guinea pig. These results suggest that down-regulation of m-ACh.R in duodenum by SART stress may be associated with enhanced activity in the parasympathetic center. Moreover, Neurotropin is thought to prevent the down-regulation of m-ACh.R throughout the central nervous system.

Acetylcholine↗

Involvement of endogenous nitric oxide in non-adrenergic, non-cholinergic contraction elicited by [Met5]-enkephalin in rat isolated duodenum.

To investigate the possible neuromodulatory role of nitric oxide (NO) in the gastrointestinal tract, an examination was made of the effects of NG-nitro-L-arginine (L-NOARG), an inhibitor of NO synthase, on the intestinal response to [Met5]-enkephalin (ENK) by recording the mechanical activity of the isolated duodenum from rats. [Met5]-enkephalin elicited a biphasic response of the duodenum, i.e. transient relaxation followed by contraction. The relaxation induced by ENK was blocked by naloxone, an opioid receptor antagonist, but not by tetrodotoxin (TTX). The contractile response of the duodenum to ENK was blocked by TTX but not by naloxone. The contractile response was not affected by hyoscine, a muscarinic antagonist, or guanethidine, an adrenergic neuron blocking agent, indicating mediation by non-adrenergic, non-cholinergic (NANC) nerves. The contractile but not the relaxant response to ENK was blocked by L- but not D-NOARG. The contractile response was also inhibited by methylene blue, an inhibitor of both NO synthase and guanylate cyclase, and by indomethacin, a cyclooxygenase inhibitor. Thus, endogenous NO and prostaglandins are involved in the contractile response to ENK. Endogenous NO may modulate the release of excitatory NANC transmitters via a prejunctional mechanism.

Amino Acid Oxidoreductases↗

Altered responses to calcium and trifluoperazine in K+-depolarized duodenum from alloxan diabetic rats.

Contractile responses to calcium were examined in the K+-depolarized duodenum from normal and alloxan-diabetic rats. In addition, verapamil and trifluoperazine which are well-known calcium channel blocker and calmodulin inhibitor respectively were used as tools in order to approach to the mechanism of changes resulting from diabetes. Decreased contractile responses to calcium were observed in the alloxan diabetic rat duodenum compared to normals. Trifluoperazine-induced non-competitive inhibition was significantly affected depending on diabetes, while verapamil-induced competitive inhibition was not changed. The non-competitive inhibition affinity constant for trifluoperazine was significantly elevated in the K+-depolarized duodenum from alloxan-diabetic rats. On the basis of findings obtained in this study, possible mechanism of the effect of experimental diabetes on the calcium-induced contraction is discussed.

Animals↗

The actions of neurokinins and substance P in canine pylorus, antrum and duodenum.

Analogues highly selective for receptors for substance P [beta-Ala4,Sar9,Met(02)11]-SP(4-11), for neurokinin A, [Nle10]-NKA(4-10), and for neurokinin B, [beta-Asp4,MePhe7]-NKB(4-10), were administered intraarterially before and after atropine or tetrodotoxin, to characterize the locations on nerve and muscle of the different receptor subtypes in the canine antrum, pylorus and duodenum. Circular muscle strips from each region were also studied in vitro. The NK-2 receptors in the antrum and the pylorus were located postsynaptically on smooth muscle. The NK-3 receptors, on the other hand, were located on neuronal sites in the antrum and duodenum. NK-1 receptors were located on neuronal and nonneuronal sites in the antrum, pylorus and duodenum. Only nonneural receptors could be activated in vitro.

Animals↗

Low tissue gastrin content in the ovine distal duodenum is associated with increased percentage of G34.

1. In adult sheep and in lambs, over 95% of gastrin in the abomasal antrum was G17 with small amounts of G34 and lesser amounts of Component I. 2. Low gastrin concentration in the proximal duodenum was associated with a reduced percentage of G17. 3. The proportion of G34 increased progressively down the duodenum from a mean of 7% proximally to 47% in the most distal segment, and correlated negatively in any segment with the gastrin content. 4. In both the antrum and proximal duodenum, 60-70% of the G17 was in the sulphated form. 5. The gastroepiploic venous serum contained less G17 and more G34 than the tissues and up to 19% G14.

Aging↗