Differential antagonism of platelet activating factor-induced neutrophilia and gastric hemorrhage in the rat.

Intravenous injection of platelet activating factor (PAF) in rats produced hypotension, neutrophilia, gastric congestion, and sloughing of the gastric epithelium. The congestion was quantified by measuring hemoglobin in the gastric mucosa. Other lesions were quantified by scores of gross pathology and histopathology. PAF-induced changes in neutrophil levels were prevented by pretreatment with the PAF-antagonist Ro24-4736, but not by the PAF-antagonist CV-3988. Both PAF antagonists reduced the hypotension, the amount of hemoglobin in the gastric mucosa, and the PAF-induced gastric pathology. These results suggest that PAF receptors involved in PAF-induced neutrophil mobilization respond differently from PAF receptors in the gastrointestinal and cardiovascular systems.

Platelet activating factor as a proinflammatory mediator in acetic-induced colitis in the rat.

Platelet activating factor (PAF) induces neutrophilia and produces a variety of gastrointestinal lesions. The role of PAF as a proinflammatory mediator was examined by measuring the production of PAF and the efficacy of the PAF antagonists WEB 2086 and Ro 24-0238 in acetic acid (HOAc)-induced colitis. PAF levels within the colonic mucosa, as measured by radioimmunoassay, increased from 259 +/- 119 ng/mg in control tissue, to 616 +/- 266 ng/mg in HOAc inflamed tissue. The accumulation of neutrophils within the mucosa was decreased by 53 +/- 10% by pretreatment with 3 mg/kg WEB 2086, and by 43 +/- 11% by 3 mg/kg Ro 24-0238. PAF-induced neutrophilia had no effect on the severity of HOAc-induced colitis, therefore, PAF my be involved in sustaining the chronic inflammation of colitis.

Evaluation of an interleukin-1 receptor antagonist in the rat acetic acid-induced colitis model.

The anti-inflammatory activity of the IL-1 receptor antagonist, IL-1ra, was evaluated in the acetic acid (HOAc)-induced model of colitis in rats. Animals treated with 10 mg/kg IL-1ra or vehicle were evaluated for general health, acute phase response, and colonic in flammation 24 hours after the initiation of inflammation. A significant decrease in the accumulation of neutrophils in the colonic mucosa as measured by myeloperoxidase activity was seen in animals with HOAc induced colitis that were treated intraperitoneally with IL-1ra when compared to animals with colitis that had been treated with vehicle. IL-1ra also reduced colonic necrosis measured grossly, although there was no effect on the histology IL-1ra had a modest effect on the HOAc-induced acute phase response, as indicated by changes in the serum iron, albumin and transferrin, but the results were not statistically significant. The number of circulating erythrocytes and neutrophils was significantly increased in animals with HOAc-induced colitis and treated with IL-1ra, suggesting that IL-1ra under these experimental conditions inhibited the migration of neutrophils to the injured colon and also the overall intestinal necrosis in the colon as assessed by gross pathology. IL-1ra may be useful as an intestinal anti-inflammatory agent.

Temperature regulation of microtiter plates for enzyme assays.

To facilitate the use of microtiter plates as vessels for enzyme assays, an incubator has been designed to maintain the wells of the microtiter plates at the appropriate temperature. The temperature variation within a single well was +/- 0.02 degrees (standard error of the mean) at 25 degrees C and +/- 0.02 degrees at 37 degrees C. The temperature variation was the same for internal and peripheral wells within the plates, although the internal wells were approximately 0.14 degrees C warmer than the outer wells at 25 degrees C and 0.68 degrees C cooler at 37 degrees C. The overall uncertainty (95% confidence interval) of the well temperature in a typical plate was +/- 0.4 degrees C at 25 degrees C and +/- 0.7 degrees C at 37 degrees C. This uncertainty is realistic for routine enzyme determinations, as opposed to precise studies. The incubator was designed to allow access to the plates from above so that additions could be made during the incubation. To demonstrate the suitability of microtiter plates and the incubator for enzyme determinations, this method was used to measure the activity of myeloperoxidase and alpha-naphthylbutyryl esterase.

Quantification of ethanol-induced gastric mucosal injury by transmission densitometry.

A method to objectively quantify the extent of ethanol-induced gastric lesions has been developed. The method utilizes a transmission densitometer to measure the optical density of the photographic negative of the stomach mucosa. Tissues from ethanol- and untreated animals are compared with tissues from animals pretreated with prostanoids before ethanol; this method permits a reproducible and objective evaluation of mucosal protection. We demonstrate that the optical density is proportional to subjective score and damage, and that the densitometry method differentiates between the protective effect of different doses of 16,16-dimethyl prostaglandin E2 and natural prostaglandin E2.

Amiloride-sensitive salt and fluid absorption in small intestine of sodium-depleted rats.

Secondary hyperaldosteronism produced by Na+ depletion was associated with increases in salt and fluid absorption in both the small intestine and the distal colon but not in the cecum and the proximal colon. Because these changes had not been documented for the small intestine, this study focused on the regulation of this tissue. Increased NaCl and water absorption was expressed in vitro by increases in short-circuit current and transepithelial potential and in vivo by increased fluid absorption and a decreased luminal content of Na+ and water. For example, the short-circuit current in the ileum of Na+-depleted rats was 2-fold that of adrenalectomized and 1.3-fold that of adrenal-intact control animals. The short-circuit current was inhibitable 24 +/- 14% by micromolar concentrations of amiloride in Na+-deficient animals compared with 1 +/- 3% in control animals. Similarly, ileal fluid absorption in vivo was 2.3-fold higher in Na+-deficient relative to control animals. The additional fluid absorption was sensitive to 50 microM amiloride, whereas amiloride had no effect in control animals. Furthermore, the Na+ content of the chyme from the ileum of Na+-deficient animals was about half that of controls. These results suggest that mineralocorticoids can induce the amiloride-sensitive Na+ transporter in the small intestine and that this type of epithelial salt transport can become a major pathway for salt retention by the small intestine.

Regulation of amiloride-sensitive electrogenic sodium transport in the rat colon by steroid hormones.

The role of steroids in the regulation of colonic sodium transport was examined by infusing steroids into adrenalectomized (ADX) rats and evaluating the short-circuit current (ISC) in vitro. Amiloride-sensitive ISC was induced by aldosterone and corticosterone with half-maximal doses (ED50) of 2 and 260 micrograms X kg-1 X h-1), respectively. Synthetic glucocorticoids such as methylprednisolone (33 mg/kg) and dexamethasone (ED50 = 30 micrograms X kg-1 X h-1) were also effective. Supramaximal doses of aldosterone (7.5 times ED50) for 24 h increased the total ISC (7-fold), the amiloride-sensitive ISC (366-fold), and the conductance (2-fold), as well as the potassium-stimulated phosphatase activity (2-fold) (reported previously). Compared with aldosterone, supramaximal doses of dexamethasone (4 times ED50) produced greater increases in the total ISC (15-fold) and the amiloride-sensitive ISC (674-fold). In contrast to aldosterone, dexamethasone also increased the amiloride-insensitive ISC (3-fold). Glucocorticoid action was not mediated by insulin since the ISC from diabetic ADX rats was increased by dexamethasone to a similar extent (11-fold) as in nondiabetic rats. Estradiol, progesterone, and testosterone did not stimulate the colonic ISC of ADX rats. The ED50 values of corticosterone and aldosterone, measured in terms of amiloride-sensitive sodium transport, produced serum levels that were slightly above those of unstressed, adrenal-intact animals and thus must be considered physiological. It is concluded that at physiological levels both steroids may mediate amiloride-sensitive sodium transport in the rat colon. However, as judged from changes in serum steroid levels, aldosterone is the physiological regulator of elevated sodium absorption in sodium deficiency.

Quantification of pepsin A activity in canine and rat gastric juice with the chromogenic substrate azocoll.

The activity concentration of pepsin may be quantified by using azocoll as a chromogenic substrate. The measured enzyme activity is constant between pH 1.2 and 3.4 and is proportional (r = 0.61) to the activity measured with hemoglobin as substrate. The activity of purified porcine pepsin is inhibited by pepstatin A with an apparent Ki of 115 nmol/L. The azocoll method is useful for measuring changes in pepsin secretion in response to pharmacological agents. For example, pepsin activity of canine gastric juice is decreased by 80% after in vivo administration of 0.5 mg of the synthetic trimethyl prostanoid Ro 22-6923 per kilogram of body weight. The method is sufficiently sensitive to measure the pepsin activity in 0.2 microL of canine gastric juice with a CV of approximately 10%, is simpler than the hemoglobin-substrate methods, and the substrate is commercially available.

Increased toxicity of oral antibiotics in sodium-deficient rats.

The toxicity of an oral antibiotic mixture used to decontaminate the gastrointestinal tract of experimental animals was compared in rats with a normal sodium intake to rats on a sodium-deficient diet. Sodium-depleted rats are quite sensitive to the oral antibiotic mixture. The antibiotic mixture was nephrotoxic, resulting in necrosis of the proximal tubules. Therefore, since the parenteral administration of antibiotics also produced necrosis of the proximal tubules, the mechanism of antibiotic toxicity in sodium-deficient rats is not influenced by the route of antibiotic administration.

A sugar and electrolyte drinking solution for long-term maintenance of adrenalectomized rats.

Adrenalectomized rats maintained on a diet containing the normal concentration of electrolytes and given physiological saline to drink had low growth rates and abnormal serum electrolytes. The presence of 29 mM (10 g/liter) sucrose in the drinking solution stimulated the animals to drink. The enhanced drinking resulted in an increase in the growth rate and improved the animals electrolyte status. This treatment was used to maintain adrenalectomized rats for more than 16 weeks and adrenalectomized-diabetic rats for 8 weeks.

Inhibition of amiloride-sensitive sodium conductance by indoleamines.

To examine a possible role of indoleamines in the regulation of epithelial sodium absorption, the effect of serotonin (5-hydroxytryptamine) and several derivatives on electrolyte transport was measured in vitro in the baboon bronchus and in the trachea and colon of sodium-deficient rats. Serotonin, melatonin (N-acetyl-5-hydroxytryptamine), and harmaline (1-methyl-7-methoxy-3,4-dihydro-beta-carboline) inhibited sodium transport in all three preparations in a similar manner to the natriuretic agent amiloride. In all three epithelia, sodium absorption via the amiloride-sensitive pathway constitutes a substantial portion of total electrolyte transport, measured as the amiloride-sensitive short-circuit current. Thus 25 microM amiloride inhibited the short-circuit current 21% in the rat trachea, 63% in the baboon bronchus, and 90% in the rat colon. Serotonin, melatonin, and harmaline inhibited the amiloride-sensitive portion of the short-circuit current from the luminal side of the epithelium. The inhibition was rapid, requiring only seconds, and maximal inhibition by serotonin was identical to that by amiloride. When sodium was omitted from the luminal solution, the short-circuit current was reduced a similar amount, suggesting that sodium absorption was being inhibited by both amiloride and the indoles. The IC50 value for amiloride was 50 nM in the baboon bronchus and 500 nM in the rat colon. In contrast, the IC50 value for serotonin was 0.4 mM in the baboon bronchus and 8 mM in the rat colon. These results, together with the wide distribution of amine-precursor-uptake-and-decarboxylation (APUD) cells in the respiratory and intestinal tract, suggest that certain indoleamines could play a role as local regulators of fluid and electrolyte transport. For example, in the airways, indoleamines may be one of the factors involved in regulation of the depth of the periciliary fluid layer.

Saliva from patients with cystic fibrosis inhibits amiloride-sensitive sodium transport.

A factor is present in the saliva and sweat from patients with cystic fibrosis (CF) which inhibits the reabsorption of sodium in the ducts of the salivary and sweat glands. Inasmuch as the physiology of sodium absorption is similar in salivary ducts and the distal colon, we have examined the sensitivity of the sodium absorption in the rat colon to saliva from CF patients. Sodium absorption by the rat colon, estimated as the shortcircuit current, was inhibited by saliva from both patients with CF and normal volunteers. However, only with CF saliva was the inhibition consistently proportional to the saliva concentration. Furthermore, the inhibitory activity of CF saliva was greater than the inhibition observed with saliva from age- and sex-matched controls (percentage of inhibition: CF = 20.1 +/- 2.2, and controls = 14.9 +/- 2.1; P < 0.02), and the inhibition of the colonic shortcircuit current was proportional to the activity measured by the ductal retrograde perfusion assay with the rat parotid gland (linear correlation coefficient = 0.634; P < 0.005). This latter assay is an accepted assay for CF factor activity. We conclude that the CF factor present in saliva probably interacts in a reversible manner with the amiloride-sensitive sodium transport system which is present in all sodium scavenging epithelia. The rat colon is a promising assay system for CF factor activity because the electrical measurements permit a rapid quantitative estimate of activity and a single piece of tissue can be used to measure the activity of several saliva samples.

Evidence against a specific effect of serum from patients with cystic fibrosis on sodium-dependent glucose transport in the rat jejunum.

Sera from patients with cystic fibrosis of the pancreas (CF) and normal human sera were assayed for the ability to inhibit sodium-dependent glucose transport in rat brush-border membrane vesicles. Fresh CF and age- and sex-matched control sera were both inhibitory when compared to physiologic saline. The inhibition by CF serum was 44 +/- 13% (mean +/- SD) at a final serum concentration of 6.7%, 67 +/- 34% at 10% serum, and 68 +/- 28% at 20% serum. The ratio of the inhibition of CF sera compared to that of control sera was 1.00, 0.78, and 0.93 at 6.7, 10, and 20% serum concentrations, respectively. Although a slightly greater inhibition by CF serum was observed at a concentration of 10%, this is probably not significant because no difference could be detected at a concentration of 20% serum. Glucose transport in the presence of serum was sensitive to phlorizin indicating that the residual glucose transport was proceeding by the sodium-dependent glucose transport system. These findings suggest that CF serum does not specifically inhibit the sodium-dependent glucose transport system. The intravesicular space accessible to glucose was reduced in the presence of CF or control serum. Fresh CF serum was 1.4 times more effective than fresh control serum (P less than 0.01). The presence of substantial vesicle-shrinking activity in control serum indicates that this activity cannot be considered specific for CF.

Methadone induced lysis of mammalian cells.

Methadone induced lysis of human erythrocytes and mouse leukemic cells was studied. The cells lyse without prior swelling that is a necessary step of colloid osmotic lysis. Methadone is accumulated by both cell types, and is widely distributed intracellurly in mouse leukemic cells. The maximum lytic rate is roughly proportional to the amount of methadone uptake and the Q10 for lysis is equal to the Q10 for methadone partitioning between octanol and water. It is concluded that the cells lyse as a result of a non-specific disruption of the plasma membrane.

The metabolism of methadone by cultured mammalian cells.

Rat hepatoma tissue culture cells and mouse leukemic cells were found to metabolize [1-3H] methadone to at least 2 unidentified radioactive compounds. These results suggest that cultured cells may be useful models for studying methadone metabolism by specific cell types.