Rat Intestinal Homogenates Research Articles

1 alpha, 25-Dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in vitro synthesis by human decidua and placenta.

The biologically active metabolite of vitamin D, 1α, 25-dihydroxyvitamin D (1α, 25(OH)2D), is synthesised by successive hydroxylation of vitamin D in the liver and kidney1. The latter organ is the only known site of 1α-hydroxylation of 25-hydroxyvitamin D (25-OH-D)2 and is also a major site of production of 24,25-dihydroxyvitamin D (24,25(OH)2D), a metabolite involved in bone ossification3. However, 24-hydroxylation of 25-OH-D may also occur in rat intestinal homogenates and in cartilage cells4,5. Accumulating data show that, during pregnancy, 1α, 25(OH)2D increases in the maternal circulation6 but is absent or present only in small amounts in the fetus7,8. In comparison, 24,25(OH)2D accumulates in fetal tissues, particularly in the skeleton8. Little is known, however, about the independent metabolism of vitamin D in the fetoplacental unit. It has been reported9 that the nephrectomised pregnant rat can synthesise 1α, 25(OH)2D3 and 24,25(OH)2D3 and that the fetoplacental unit is the most likely site of production of such metabolites. Those data suggested that there may be independent vitamin D metabolism in the fetoplacental unit. We now report in vitro synthesis of 24,25(OH)2D3 by human placenta and of 1α,25(OH)2D3 and 24,25(OH)2D3 by human decidua. The human decidua is a development of the endometrium that is unique to pregnancy, originating from both epithelial and stromal cells and intimately attached to fetal structures, that is, the placenta and chorion.

Ornithine synthesis from glutamate in rat intestinal mucosa homogenates: Evidence for the reduction of glutamate to γ-glutamyl semialdehyde

For 25 years, glutamate has been considered to be the source of the carbon and nitrogen atoms of ornithine and proline synthesized in mammalian tissues. However, the first enzyme reaction for these syntheses, namely the reduction of glutamate to γ-glutamyl semialdehyde, has not been observed in homogenates of mammalian tissues. These studies present evidence for this reaction in rat intestinal mucosa homogenates incubated with [U-14C]glutamate. The 14C-labeled ornithine was separated from other radioactive chemicals by ion-exchange chromatography, and was specifically identified by its capacity to serve as a substrate for ornithine transcarbamylase. The synthesis of ornithine required ATP, Mg2+ and NADPH in addition to glutamate.

Phosphopyridoxal cyclic compounds with histamine and histidine. 6: The formation of phosphopyridoxal cyclic compounds with histamine and histidine in the presence of biological material.

We have studied the dynamics of cyclic compound formation between histamine or histidine and pyridoxal 5'-phosphate (Hi-PLP or His-PLP) in incubates of rat gastric mucosa histidine decarboxylase (HD), rat intestinal diamine oxidase (DAO) or homogenates of either rat liver, intestine or gastric mucosa. For gastric mucosa HD, liver and gastric mucosa homogenates, the rate of cyclization was slightly decreased; however, the rate was significantly inhibited with intestinal DAO or intestinal homogenate. Binding of PLP by tissue components was measured; free PLP was bound abundantly by rat intestinal DAO and by rat intestinal homogenate. A possible mechanism by which intestinal tissues inhibit cyclic compound formation is discussed.

Phosphopyridoxal cyclic compounds with histamine and histidine. 7: The properties of pyridoxal and phosphopyridoxal cyclic compounds with histamine and histidine.

Cyclic compounds synthesized from histamine (Hi) or histidine (His) with pyridoxal (PL)--[Hi-PL, His-PL] and Hi or His with pyridoxal 5'-phosphate (PLP)--[Hi-PLP, His-PLP] were tested for stability in buffer, acid and base solutions, crude homogenates of various tissues and in the presence of enzymes which metabolize Hi or His. The cyclic pyridoxal compounds were stable in all experimental conditions, whereas phosphopyridoxal cyclic products were degraded by rat intestinal DAO and rat intestine homogenate, apparently enzymatically. In acidic and basic solutions changes in migration velocity and u.v. absorption spectrum occur. The characteristic fluorescence of these cyclic compounds is described.

Rat intestinal 25-hydroxyvitamin D3- and 1alpha,25-dihydroxyvitamin D3-24-hydroxylase.

The 24-hydroxylation of 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 occurs in vitro in rat intestinal homogenates. 1alpha,24,25-Trihydroxyvitamin D3 obtained by incubating 1alpha,25-dihydroxyvitamin D3 with rat intestinal homogenates was isolated and then identified by co-chromatography of the biologically generated material with synthetic 1alpha,24R,25-trihydroxyvitamin D3 on a high pressure liquid chromatography system and by ultraviolet spectroscopy, periodate cleavage, and mass spectroscopy. 24,25-Dihydroxyvitamin D3 obtained by incubating 25-hydroxyvitamin D3 with intestinal homogenates was identified by co-chromatography of the biologically generated material with authentic 24R,25-dihydroxyvitamin D3 on a high pressure liquid chromatography system and by periodate cleavage. Tissue distribution studies indicate that the process occurs in rat intestinal homogenates but not in rat muscle, liver, or fetal calvaria homogenates. Nephrectomy does not diminish the production of 1alpha,24,25-trihydroxyvitamin D3 in vivo while it reduces markedly, but does not eliminate, production of 24,25-dihydroxyvitamin D3.

Lipolytic activities against trioctanoin and monoolein in rat intestinal mucosa

The hydrolysis of trioctanoin and of monoolein by rat intestinal homogenates, catalyzed by the previously described "trioctanoin hydrolase" and "monoglyceride lipase" [Ref. 6 and 11], was shown to be entirely due to the action of the intestinal glycerol-ester hydrolase [Ref. 1 and 2] and to contaminating pancreatic lipase firmly bound to the intestinal cells. These conclusions are supported by experiments based on the chromatographic behaviour of the different lipolytic activities, the use of an immunoserum directed against rat pancreatic juice and the relief of bile salt inhibitions by pancreatic colipase. The results are in favour of the existence of a single lipolytic enzyme in the rat intestinal mucosa, i.e. glycerol-ester hydrolase, active against short- and medium chain mono-, di- and triglycerides and against long chain monoglycerides.

Phospholipases de l'intestin de rat : mode d'action

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.

Mechanism of intestinal absorption and brain uptake of L-5-hydroxytryptophan in rats, as compared to those of L-3,4-dihydroxyphenylalanine.

L-5-Hydroxytryptophan (5-HTP) and L-3, 4-dihydroxyphenylalanine (DOPA) were comparatively investigated of their mechanisms of intestinal absorption, metabolism in the intestine and liver tissues and mechanisms of passage through the blood-brain barriers, by means of in vitro and/or in situ techniques using rat tissues. The corresponding D-isomers were also investigated for comparison. L-5-HTP, but not the D-isomer, was found to be absorbed from the intestine by an active transport mechanism in the same way as L-DOPA. In vitro studies using rat brain cortex slices proved that both L-5-HTP and L-DOPA, but not their D-isomers, are taken up by the brain tissues by an active transport mechanism which is saturable, energy-dependent and competitive. There was no substantial difference between L-5-HTP and L-DOPA in the rate of intestinal absorption and that of brain uptake. It was indicated, however, that L-5-HTP was decarboxylated in the intestine to a much less extent than L-DOPA during the absorption and the decarboxylation activity of rat intestine and liver homogenates was found to be about four and seven times higher for L-DOPA than for L-5-HTP, respectively. The latter results suggest that the oral dose of L-5-HTP can be much more easily transferred into the circulating blood without being suffered from decarboxylation in the peripheral organs than L-DOPA, resulting in a more efficient penetration of L-5-HTP into the brain as a serotonine precursor.

Inhibition of cholesterol absorption by (-)N-[alpha-phenyl-beta-(p-tolyl)ethyl] linoleamide in rats.

The effect of (-)N-[alpha-phenyl-beta-(p-tolyl)ethyl] linoleamide (PTLA) on intestinal absorption of cholesterol was studied in rats. Oral administration of 15 mg PTLA to rats resulted in a significant (P less than 0.05) decrease in the radioactivity in serum and liver 4 hr after administration of labeled cholesterol. The effect of PTLA was greater on the absorption of cholesteryl oleate as compared with free cholesterol. The rate of hydrolysis of cholesteryl oleate in mucosal homogenates of rat intestine was decreased with PTLA, suggesting that the inhibition of cholesterol absorption by PTLA is related to its effect on cholesteryl ester hydrolysis in the intestine.

A rapid and simple radioassay for inosinic acid: Pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10 −5 m allopurinol (4-hydroxypyrazolo [3,4- d]pyrimidine) into the reaction mixture, the [8- 14C]hypoxanthine (Hx) is converted only to [8- 14C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase. Using [8- 14C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 m m ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8- 14C]IMP or [8- 14C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value. Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.

Phospholipid exchange proteins in rat intestine.

The 105,000 g supernatant and pH 5.1 supernatant fractions from rat intestinal homogenates stimulate phosphatidylcholine exchange between [32P] phosphatidylcholine liposomes and beef heart mitochondria. This active fraction shows the characteristics of a protein. Isoelectric focusing of the intestinal pH 5.1 fraction shows two peaks of phosphatidylcholine exchange activity: one at an acidic pH (4.5-5.3), the other in a basic pH range (8-9). The second peak of activity appears to be a new phospholipid exchange protein. The anatomic distribution of phosphatidylcholine exchange activity in intestine has been investigated. Expressed per mg of protein, phosphatidylcholine exchange activity is higher in mucosa than in the intestinal wall. No significant differences have been found between villi and crypts cells or between jejunal and ileal villi. Futhermore, exchange activity per mg of protein in mucosa is unaffected by fasting or by feeding a high fat or high cholesterol diet. This suggests that phospholipid exchange activity in the absorptive cells is not a rate limiting step in the process of fat absorption.

Indentification of an intestinal sodium and calcium-dependent phosphatase stimulated by parathyroid hormone.

Previous reports suggest that the site of the energy-dependent intestinal calcium transport against an electropotential and concentration gradient is located along the basal-lateral membrane of the mucosal cell. Accordingly, basal-lateral membranes were prepared from rat intestinal homogenates in order to identify the enzyme mediating this step in the transport process. An alkaline phosphatase was delineated which utilized ATP as a substrate and was dependent on both Na(-) and Ca(++) with optimum enzyme activity at 200 mM and 0.04 mM, respectively. Furthermore, the activity of the enzyme was demonstrated to decrease with the advance in age of the animal and to decrease with removal of the parathyroid glands, consistent with a decreased rate of (45)Ca release from mucosal cells under the same experimental conditions. Calcium binding to basal-lateral membrane fragments was also sodium dependent and enhanced by the prior administration of parathyroid extract. The consistent correlation between the rate of calcium transport across the basal-lateral membrane of the mucosal cell and the activity of this Na, Ca-dependent phosphatase under a variety of experimental conditions suggest that this enzyme may mediate the parathyroid hormone-sensitive active transport of calcium across the intestine.

Role of Bile Acids in the Activation of Trypsinogen by Enterokinase: Disturbance of Trypsinogen Activation in Patients with Intrahepatic Biliary Atresia

The effect of bile acids on the activation of trypsinogen by the soluble fraction of rat intestinal homogenates containing enterokinase (EC 3.4.4.8) was studied. Glycodeoxycholate, 2.5 mm, caused a 5.8-fold increase in the velocity of the activation process at low trypsinogen concentrations and a 2-fold increase of the maximal velocity of trypsinogen activation by enterokinase. Sodium taurocholate had a similar effect. The same concentration of glycodeoxycholate caused a decrease of the Michaelis constant for the enterokinase-catalyzed trypsinogen activation: Km was 0.09 mm in the absence of bile acids and fell to 0.015 mm in their presence. The total amount of trypsin formed from a known amount of trypsinogen was also augmented in the presence of bile acids. A similar result was obtained when human pancreatic juice was used as the source of trypsinogen and human small intestinal homogenates as the source of enterokinase. In three children with biliary atresia and no bile acids in the duodenal contents, abnormally low trypsin activities were found before and after stimulation of pancreatic function with pancreozymin-cholecystokinin and secretin. Chymotrypsin activity, however, was normal. Evidence was obtained that trypsinogen activation by enterokinase is incomplete in the absence of bile acids. It is concluded that in the rat and in man bile acids are important for a rapid and complete trypsinogen activation by enterokinase.

Action of bradykinin potentiating factor (BPF) and dimercaprol (BAL) on the responses to bradykinin of isolated preparations of rat intestines.

BPF and BAL inhibited kininase activity of homogenates of rat intestine. However, BFP potentiated and BAL inhibited the contractions induced by bradykinin on rat isolated duodenum (low calcium solution) and terminal ileum (normal calcium solution). Neither BPF nor BAL affects the relaxation induced by bradykinin of rat duodenum bathed in normal Tyrode. These results suggest that two different types of pharmacological receptor are involved in the action of bradykinin on rat intestine, and that other factors besides the inhibition of agonist destruction participate in the mechanism of potentiation of kinin action by BPF.

Degradation of selected cephalosporins by homogenates of the alimentary tract of rats.

Summary In-vitro tests with homogenates of rat intestine showed that cephaloridine, cephalexin and 7-(thienyl-2'-acetamido)-3-methylceph-3-em-4-carboxylic acid were degraded more quickly in the caecum and rectum than in other parts of the alimentary tract; even after 4 hr at 37°C at least 57 per cent. of the low dose of cephalosporin used remained intact in other portions of intestine. The extent of breakdown in the caecum varied with the structure of the cephalosporin. Cephalexin was more stable than cephaloridine to biological degradation. Different parts of rat intestine were assayed for β-lactamase and acylase activity and the effect of proteolytic enzymes on the cephalosporins was studied. The only degrading activity found was due to β-lactamase-producing bacteria in the caecum.

Effects of whole-body irradiation and radio-mimetic chemicals on the acid RNAse and phosphodiesterase levels of rat intestinal homogenate.

Effects of whole-body irradiation and radio-mimetic chemicals on the acid RNAse and phosphodiesterase levels of rat intestinal homogenate.

DISTRIBUTION OF A "MUTAROTASE" ACTIVITY IN RAT TISSUES AND POSSIBLE FUNCTION IN ACTIVE TRANSPORT OF SUGARS.

Homogenates of rat kidney, liver, intestine, stomach, lung, spleen, heart, brain, diaphragm and skeletal muscle were prepared both in EDTA and phosphate buffers. “Mutarotase” (EC 5.1.3.3) and lactate dehydrogenase (EC 1.1.1.27) activity were measured on the supernatant solutions following centrifugation. Mutarotase activity was highest in those tissues capable of active sugar transport. Enzyme from 1 g kidney cortex converted 380 mg of α to β-glucose per min. There was no correlation between either mutarotase, lactate dehydrogenase activity or Q -glucose values for the tissues, suggesting that enzyme is not involved merely as a component of normal glucose metabolism. Kidney, liver and small-intestinal homogenates were separated into nuclear, mitochondrial, microsomal and supernatant fractions in 0.2 M sucrose. Mutarotase activity was concentrated almost exclusively in supernatant fraction. Results are discussed in terms of a function for mutarotase protein as the hypothetical sugar “carrier” molecule of active transport theory.

Phenylbutazone (butazolidin) in gout

1. 1. The therapeutic efficacy of phenylbutazone was evaluated in 200 patients with acute gout or chronic gouty arthritis. Major improvement or complete remission was achieved in 84 per cent of these patients. Males responded more favorably than did females. Acute gout was more amenable to phenylbutazone therapy than was chronic gouty arthritis. 2. 2. Among 408 patients with painful musculoskeletal disorders other than gout, the beneficial effect of phenylbutazone was appreciably less in those patients who had elevated serum uric acid than in those with normal uric acid levels. This suggests that the characteristic diminution of serum uric acid promoted by phenylbutazone may not be the most important pharmacologic action of the drug. 3. 3. It is our impression that intramuscular administration effects a more rapid remission of acute gout than does oral phenylbutazone. 4. 4. Maintenance therapy in chronic gouty arthritis with phenylbutazone, 100 to 600 mg. daily, greatly reduced the attack rate, severity and duration of acute exacerbations. This control was exerted even in the presence of serum uric acid levels which had again risen to pretreatment magnitude. In no case were tophi observed to increase or decrease in size. 5. 5. Seventy-one toxic side effects occurred in fifty-two (26 per cent) of the 200 gouty patients. In fourteen patients (7 per cent) toxicity was severe enough to warrant discontinuance of the drug. The most common untoward actions were edema and nausea. There was no case of agranulocytosis or activation of peptic ulcer among these patients. The age of the patient did not appear to influence the incidence of toxicity. Undesirable effects were less severe in gout than in other painful musculoskeletal disorders and usually occurred early in the course of treatment. 6. 6. In homogenates of rat liver and intestine, phenylbutazone did not inactivate xanthine oxidase or adenosine deaminase. The drug was found to inhibit the growth of yeast.