Experimental Biology and Medicine

The effect of indomethacin and other NSAID on renal prostaglandin synthesis and metabolism was studied in nondiuretic rats prepared for acute experimentation. Thirty minutes after the administration of a 2 mg/kg iv dose of indomethacin, the concentration of prostaglandin in renal venous blood as determined by bioassay was reduced 69%. In addition, conversion of radiolabeled arachidonic acid to prostaglandin E₂ in vitro by the renal papillae of indomethacin pretreated rats was inhibited 97%.

Pretreatment with indomethacin also inhibited renal cortical-medullary prostaglandin E₂ 9-ketoreductase activity by 61%. NAD⁺-dependent 15-hydroxy-prostaglandin dehydrogenase activity was diminished 46%; however, this inhibition was not statistically significant. NADP⁺-dependent 15-hydroxy-prostaglandin dehydrogenase activity was unaffected by pretreatment. It is concluded that indomethacin alters net renal prostaglandin production by inhibiting both prostaglandin synthesis and its metabolism.

Calcium transport across the duodenum and ileum was measured by an in vivo ligated loop technique in Mg depleted rats and rats pair fed a magnesium adequate diet. Intestinal Ca transport and tibial ⁴⁷Ca uptake were consistently decreased in magnesium depletion. Analysis of Ca fluxes, carried out by in situ perfusion, showed a significant decrease in passive diffusion, with less consistent effects on the saturable transport component. Both bone and plasma showed markedly decreased Mg concentration. Tibia Ca levels were slightly but significantly increased and plasma levels were either normal or slightly, but significantly elevated. The basis for the decrease in Ca transport of Mg depleted rats observed in this investigation is not clear. The data suggest a general alteration in mucosal membrane transport rather than a specific effect on Ca transport per se.

The anti-idiotypic response of BALB/c mice to myeloma protein of BALB/c origin (purified Protein-315 from a plasmacytoma MOPC-315) was analyzed for its antigen-binding capacity and susceptibility to inhibition by excess hapten (DNP-lysine). The results showed that the anti-idiotypic antibody that is sensitive to inhibition by hapten could be detected for at least 3 months after completion of the primary course of immunization. Following a single booster injection, there was an increase of the antigen-binding capacity and the susceptibility of these post-boosting antisera to inhibition by hapten was markedly reduced (from more than 80% to less than 10% under the assay system employed). However, the hapten inhibition value gradually returned toward the preboosting level within a few weeks. The data obtained suggest that the change in the hapten inhibition value after boosting is associated with increased affinity rather than a shift in specificity.

Prostaglandin synthetase inhibitors, indomethacin and tolmetin, blocked furosemide-induced increase in renin secretion whether the furosemide was given intravenously or into the renal artery. Tolmetin and indomethacin did not affect the natriuretic, kaliuretic or calciuretic response to furosemide. Therefore, blockade of furosemide-in-duced renin release does not appear to require an alteration in sodium or calcium load at the macula densa. Thus, the site of action of prostaglandin synthetase inhibitors on renin release is probably subsequent to the signal perceived by the macula densa.

A sensitive index of in vitro cell damage has been used to investigate the possibility that HLA B27-positive fibroblasts are peculiarly susceptible to those infectious agents incriminated in the seronegative spondyloarthropathies. No evidence for differential susceptibility related to the presence or absence of the B27 antigen could be demonstrated.

Ingestion of 0.8 g/kg ethanol in 1 hr by normal man caused significant increases in both serum PTH and plasma CT concentrations, with peak values of 139% of baseline at 2 hr for PTH and of 138% at 3 hr for CT. Serum Ca did not change during the period of observation. Incubation of bovine parathyroid slices in 1.25 mM Ca Eagle media with 0.05% or 0.3% ethanol caused significant increases in PTH secretion to 122% and 166% of baseline respectively. Therefore: (1) in vitro, ethanol can be demonstrated to directly stimulate PTH secretion, (2) in vivo, ethanol ingestion induces an increase in PTH without detectable hypocalcemia, suggesting (a) prompt PTH secretion and action to compensate for a hypocalcemic effect of ethanol, so that actual hypocalcemia is not detectable, and/or (b) direct parathyroid stimulation. Though the exact mechanisms are unclear, the data indicate that ethanol, in amounts often ingested by social drinkers, increases both PTH and CT secretion, and therefore may modify Ca homeostasis.

Alkaline phosphatase activity was measured in hormonally stimulated pancreatic juice from six dogs and in pancreatic fistula fluid from a human subject. Isoenzyme characterization studies, based on different susceptibilities to urea and l-phenylalanine inhibition and to heat inactivation indicated similarities between canine and human pancreatic secretory alkaline phosphatase. Compared to intestinal alkaline phosphatase, the pancreatic isoenzyme was much more sensitive to heat inactivation and urea inhibition but much more resistant to l-phenylalanine inhibition. The electrophoretic mobility of the enzyme present in human pancreatic juice was different from that of human hepatic, bone, or intestinal alkaline phosphatase.

Thymopoietin, ubiquitin, and serum thymic factor, all of which induce T cell markers on lymphocytes, have been evaluated for their capacity to induce thymus-de-pendent activities in vivo. Multiple treatments over a period of weeks failed to restore either resistance to line l_b leukemia or responses to T cell mitogens in T cell-deficient C58 mice. The findings suggest that these substances are ineffective in inducing thymus-dependent im-munocompetence that is meaningful in the intact animal.

Human hepatitis A virus, partially purified from the liver of a rufiventer marmoset infected with CR326 strain virus, was inactivated with formalin and was shown to be highly potent in stimulating homologous antibody in marmosets when administered subcutaneously at bi-weekly intervals in eight divided doses. The vaccine was shown to prevent hepatitis A in all marmosets when challenged with live hepatitis A virus in a controlled study.

The acute effects of 24R,25-DHCC, lα,24R,25-THCC, and lα-HCC on the renal handling of phosphate, calcium, and sodium were evaluated in the TPTX dog which had been mildly volume expanded and infused with vasopression to establish a phosphaturia. Both lα-HCC and lα,24R,25-THCC when given intravenously in a dosage of 0.625 μg produced a significant decrease in urinary phosphate excretion. Percentage phosphate excretion decreased by 30 and 28%, respectively (P < 0.05, < 0.01). Since there was no alteration in renal hemodynamics or in the filtered load of this ion, the data suggest a direct action of these compounds on renal tubular transport mechanisms. No change in the urinary excretion of either calcium or sodium was observed following the administration of the two vitamin D₃ derivatives. 24R,25-DHCC was without effect on the renal handling of all three ions.

When previous experimental findings regarding the renal actions of 25-HCC and lα,25-DHCC are considered, the data suggest that the 1-hydroxyl grouping is required for the metabolites of vitamin D to influence phosphate transport at the renal tubular level. It appears that a sterically unhindered 25-hydroxyl group is necessary in order for the vitamin D₃ derivatives to act on the reabsorption of either calcium or sodium.

Intraventricular injection of 5HT (4 and 20 μg) in unanesthetized, unrestrained male rats fitted with permanent intrajugular cannulas for withdrawal of blood samples caused a dose-related elevation of plasma GH levels. Similar effects were also observed following intraventricular injection of the serotonin receptor agonist, quipazine. The GH-releasing effect of both drugs was abolished by a serotonin receptor blocker, methysergide. It is concluded that activation of the central serotoninergic system stimulates GH secretion in the rat.

To determine the effects of acute and chronic ethanol ingestion on the absorption of lead, experiments were performed using an in vivo isolated gut loop technique. Acute administration of 50% ethanol significantly reduced the absorption of lead at concentrations of 1 and 10 μg of Pb/ml. This effect appears to be independent of lead solubility in alcohol and is associated with structural changes in intestinal mucosa, suggesting toxicity. Absorption of a single dose of lead was also diminished in animals chronically exposed to ethanol. Elimination of a single intravenous dose of lead was not affected by chronic alcohol ingestion. These findings suggest that the clinically reported synergism of lead toxicity and ethanol is related not to increased lead absorption or diminished lead excretion but to nutritional deficiencies and increased lead exposure among some alcoholics.

Light scatter (LS) differences among cells of rabbit marrow was studied by flow analysis using a Coulter two-parameter cell sorter. A preliminary fractionation of the marrow into samples enriched with cells of varying degrees of maturation was accomplished in Ficoll/Hypaque density gradients. Subsequent study of each of these cell samples in flow analysis demonstrated unique LS profiles which distinguished erythrocytes from nucleated erythroid precursors and granulocyte precursors. The combined separation procedures made it possible to sort fractions of erythroid precursors with as high as 90% erythroid cells, two-thirds of which were nucleated precursors.

Using croton oil-induced rat ear edema, hydrocortisone and salbutamol show anti-inflammatory activity when applied topically. Both drugs act to some extent even when applied after the inflammation reaction is in progress. Both drugs are also active when applied to a shaven area of the back, a site remote from the ear inflammation. Salbutamol acts by a different mechanism than anti-inflammatory steroids. The advantages of salbutamol are discussed and it appears to be a useful adjunct in the treatment of inflammatory dermatoses.

Interrelationship between the effects of chronic ethanol ingestion and T₄ treatment on O₂ consumption by rat liver slices and isolated mitochondria was investigated. The data showed that ethanol influence on O₂ consumption by liver slices was dependent on the available oxidizable substrate as it was decreased when estimated in media containing glucose but increased in media containing succinate as oxidizable substrate. The respiration of thyrotoxic rat liver slices was altered by ethanol in a manner similar to that observed with euthyroid rat liver slices. Whereas ethanol ingestion enhanced succinate-supported respiration of euthyroid and thyrotoxic rat liver slices, it produced a similar effect in isolated mitochondria of thyrotoxic rat livers but not of euthyroid rat livers.

In order to examine a possible role of phosphodiesterase in mediation of the action of LH and FSH upon granulosa cell cAMP levels, porcine (GC) from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) follicles were incubated with human FSH (hFSH) and LH (hLH) for 30 and 60 min in the absence or presence of 3-isobutyl 1-methylxanthine (MIX), a potent phosphodiesterase inhibitor. Subsequently, the intracellular and incubation medium cAMP contents were determined by a protein binding assay. During a 30-min incubation, 10 μg of hFSH alone brought about an 11-fold, 5-fold, and 2-fold increase in intracellular cAMP accumulation and a 16-fold, approximately 4-fold, and 4-fold increase in incubation medium cAMP levels in GC from small, medium, and large follicles, respectively. Addition of 0.2 va.M MIX exerted a nonsignificant (P > 0.05) potentiating effect upon hFSH stimulation of intracellular cAMP accumulation in cells obtained from the three types of follicles. In the case of cells obtained from small and medium follicles, addition of 0.2 mM MIX in the presence of 10 μg/ml hFSH or 1.0 μg/ml hLH led to 41 to 69% potentiation (p < 0.001) of the effect of the FSH and LH upon cAMP accumulation in the incubation medium. This was evident after a 30-and 60-min incubation period. In the case of cells obtained from large follicles, addition of 0.2 mM MIX had a nonsignificant potentiating effect (p > 0.05) on either hFSH or hLH stimulation of cAMP accumulation in the incubation medium.

It may be concluded that probably there are low levels of intracellular phosphodiesterase in porcine granulosa cells and that gonadotropins act to stimulate the generation of cAMP rather than alter the rate of destruction of cAMP. The findings support the existence of an extracellular phosphodiesterase which may act to regulate or modulate the extracellular levels of cyclic AMP.

In dogs with gastric and pancreatic fistulas the potency of porcine big gastrin (G34-II) relative to human little gastrin (G17-1) was not significantly different for stimulation of gastric acid and pancreatic protein secretion.

Five subcellular fractions were isolated from guinea pig lung homogenates by differential centrifugation. These fractions were defined biochemically by the analysis of 18 enzymes representing different subcellular compartments. Succinate dehydrogenase and cytochrome oxidase distributed with the heavy mitochondrial fraction, while N-acetyl-β-glucosaminidase, acid p-nitrophenylphos-phatase, cathepsins A, Bl, and D, dipeptidyl-peptidases I and II, and elastolytic esterase distributed with the light mitochondrial fraction. Alkaline p-nitrophenylphosphatase, glucose 6-phosphatase, dipeptidylpeptidase IV, neutral protease, and alkaline protease all demonstrated a “microsomal” enrichment. In the cytosol were found lactate dehydrogenase, dipeptidylpeptidase III, and a dipeptidase. The lung subcellular fractions were heterogeneous with cross-contamination between the heavy mitochondrial, light mitochondrial, and “microsomal” fractions. The enzyme distributions noted were similar to those found in other tissues.

In this work we have examined the daily fluctuations of circulating hPP in normal individuals subjected to a conventional meal schedule (breakfast, lunch, and dinner) as well as during food deprivation for 84 hr. In addition, we have tested the effect of ingestion of a low-calorie, fiber-rich salad as well as 500 ml of tap water on hPP secretion.

Ingestion of each meal was followed by a sustained hPP elevation. Between meals, circulating hPP did not return to basal values. Both the vegetable meal and the water load evoked hPP release, suggesting that the hPP response to food intake is partially a nonspecific effect. In the fasted group, plasma hPP rose significantly 24 hr after the last meal and persisted elevated for the remainder of the experimental period. Moreover, in this condition hPP showed circadian variations, with higher values in the late evening than in the preceding and subsequent morning.

Since pancreatic polypeptide is suspected to possess gastrointestinal functions, its elevation in plasma throughout the daytime in conditions of normal feeding may be thought to exert a tonic influence on some digestive process. On this basis, the increase of hPP during prolonged fasting appears paradoxical and, indeed, the explanation of this phenomenon awaits a better knowledge of the biological activity of this peptide.

Intrarenal infusion of acetylcholine, bradykinin, eledoisin, and PGE₂ increased renal blood flow to a similar degree. Sodium and potassium excretion were similarly affected by each vasodilator. Renin secretion increased following PGE₂ but was unaffected by acetylcholine, bradykinin, or eledoisin. It is suggested that PGE₂ increases renin secretion by a direct effect on the juxtaglomerular apparatus.

The cancer chemotherapeutic and antiviral agent 9-β-D-arabinofuranosyladenine (ara-A) was examined for potential oncogenicity, using a serial line of Fischer rat embryo cells, which was previously shown to be a sensitive and accurate indicator of chemicals carcinogenic for rodents. We report here that at the concentrations tested, ara-A was not a transforming agent. Further, ara-A protected the cells from transformation induced by the known carcinogen, 3-methylcholan-threne.

Serum PRL and hepatic PRL receptors were measured in intact and OVX mice and OVX mice given several doses of EB, OVX significantly increased PRL binding in the liver of female mice, and EB reduced receptors to intact or below intact levels. It was concluded that estrogen decreases PRL receptors in the liver of female mice. This is a striking contrast to the stimulatory effect of estrogen on hepatic PRL receptors in male and female rats. EB elevated serum PRL in OVX mice, but since other investigators reported that PRL does not alter hepatic PRL receptors in female mice, it appears likely that estrogen reduced PRL binding sites by a direct effect on the liver. However, an indirect effect cannot be excluded. In male mice, estrogen increased PRL receptors in the liver as in male rats.

The present data demonstrate important species differences between female rats and female mice in estrogenic control of hepatic PRL receptors. Moreover, the inhibitory effect of estrogen in female mice, and its stimulatory action in male mice, suggest that the response of hepatic PRL receptors to estrogen may be sex dependent in different species. The mechanisms of action by which these effects are mediated remain to be clarified.

The numbers of pluripotent stem cells (CFU-S) and of the more differentiated granulocyte/macrophage elements in diffusion chambers (DCs) implanted into the peritoneal cavities of radio-⁸⁹Sr-marrow-ablated mice are increased as compared to those in DCs implanted into cold-⁸⁸ Sr-mar-row-ablated mice. These findings suggest that there is a systemic humoral response capable of stimulating hemopoiesis even in mice with aplastic marrows and whose hemopoiesis is localized to their spleens. The magnitude of this response and the promptness with which the response is manifest in DC growth suggests that marrow aplasia induced by ⁸⁹Sr provides a stronger stimulus for proliferation of cells in DCs than does either cyclophosphamide or lethal external whole-body irradiation.

Injection of epinephrine to 250-to 300-g rats (0.3 mg/day for 2 weeks, followed by 0.6 mg/day for another 3 weeks) progressively increased the serum PTH and CT, whereas administration of approximately 40 mg of propranolol daily, in drinking water, progressively decreased the serum levels of both these hormones in comparison to control animals. The studies indicate that, similar to the shortterm effects observed in previous studies, long-term modification of β-adrenergic stimuli can affect PTH and CT secretions.

The authors thank Mrs Barbara Lovett for her secreterial assistance.

This study assessed the effect of alcohol, given as single increasing doses or chronically, on regional cerebral acetylcholine concentration. In the acute studies in both rats and mice, brain acetylcholine rose significantly, but modestly, at higher blood ethanol concentrations. This effect was most consistent in the corpus striatum. At low blood alcohol levels, when brain acetylcholine levels were unaltered, the utilization rate of acetylcholine decreased in all brain areas and this was statistically significant in the cortex and midbrain. By contrast, in rats exposed to chronic oral ethanol intake but studied when blood alcohol was normal, brain acetylcholine was unaltered. These data are most consistent with the concept that alcohol directly depresses neuronal function resulting in decreased release (utilization) of acetylcholine and at high alcohol concentrations induces a modest accumulation of acetylcholine in brain.

Compared to chicks fed a diet containing corn, those fed a diet containing rye showed significantly lower growth that was ameliorated by antibiotic supplement to the diet. Adhesion of aerobes (excluding coliforms) and anaerobes to the intestinal wall was indicated in the chicks fed the diet containing rye which was reduced by penicillin supplementation. There was no mortality in chicks fed diets containing corn or rye, whereas the poorest growth and a very high mortality resulted in chicks fed diets containing raw beans. Such adverse effects were alleviated by dietary antibiotic supplement. High numbers of aerobes, mainly coliforms, were found adhered to the mucosal wall of the chicks fed diets containing raw beans, and E. coli and S. fecalis organisms were isolated from their kidneys. Autoclaving the beans greatly improved growth, reduced mortality, and caused no adhesion of intestinal aerobes to the mucosal wall. A penicillin supplement to the diet resulted in further improvement of growth and reduction of mortality.

The vascular response to norepinephrine is expressed in terms of a resistance ratio which is defined as the ratio of the vascular resistance seen 1.5 min after norepinephrine administration to that seen before norepinephrine administration. The injection of 1 μg/kg of norepinephrine to near-term sheep significantly increased the vascular resistance of the placenta to a ratio of 2.27 ± 0.52 (mean ± SEM; N = 7). Pretreatment with 20 jug of prostaglandin E₂ per minute significantly decreased the placental response to norepinephrine to a resistance ratio of 1.47 ± 0.21 which was 65% of the untreated response (N = 7). Pretreatment with 10 mg/kg indomethacin significantly increased the placental response to norepinephrine from a resistance ratio of 2.25 ± 0.70 to 2.71 ± 0.84, which is 120% of the untreated value (N = 6). Prostaglandin E₂ attenuated the placental vascular response to norepinephrine and indomethacin potentiated this response.

Duodenal CaBP levels and the efficiency of ⁴⁷Ca absorption by the duodenal segment of ovulating Japanese quail were determined as a function of eggshell formation. No differences in these parameters were noted in quail in which eggs were being calcified and in quail with no egg in the calcification stage. Thus, there is a correlation between the efficiency of Ca absorption and the level of vitamin D-dependent intestinal calcium-binding protein in this physiological state in Japanese quail.

Following in vivo treatment of rats with a nonlethal dose of cycloheximide (2 mg/kg body wt), analysis of the newly synthesized liver mitochondrial polypeptides by SDS-PAGE system showed: (i) sublethal levels of cycloheximide did transiently suppress synthesis and/or incorporation of large cytoplasmically synthesized proteins into mitochondria; (ii) synthesis and/or incorporation of this material was stimulated during the recovery phase. The differential labeling patterns of these mitochondrial proteins observed in vivo during cycloheximide treatment substantiate the cooperative nature of the cytoribosomal protein synthetic system to the formation of functional mitochondrion observed with mammalian cells in culture.

Fig. 3. Radioactivity profiles of soluble proteins extracted with acidic buffers from control and cycloheximidetreated (2 and 24 hr) mitochondria. For details see the legends to Figs. 1 and 2.

Fig. 4. Radioactivity profiles of soluble proteins extracted with alkaline buffers from control and cycloheximide-treated (2 and 24 hr) mitochondria. For details see the legends to Figs. 1 and 2.

Glutaminase-γ-glutamyltrans-ferase contributes some 30% of the ammonia produced from glutamine by mildly acidotic rat kidneys. The enzyme is localized in the cytosol and its inhibition results in an ammonia produced per glutamine extracted ratio of 2.0. The results are therefore consistent with a dual glutamine-utilizing system, one cytoplasmic and the other mitochondrial, in the functioning rat kidney.

Latex particles (2 μm in diameter) accumulated in intestinal Peyer's patches and mesenteric lymph nodes of mice given latex suspensions as drinking fluid. After a 61-day period of latex feeding, the particles were also present in villi adjacent to Peyer's patches; they were not seen, however, after only 3 days of latex feeding. The amount of latex in Peyer's patches 74 days after the termination of latex feeding was much less than the amount present 14 days after the termination of feeding. It is suggested that migratory macrophages take up latex particles within Peyer's patches and subsequently move out of the patch to mesenteric nodes and villi. Some free particles may also be transported out of Peyer's patches to mesenteric nodes and villi through open lymphatic channels. The observations support the contention that Peyer's patches “sample” intestinal contents and they suggest a mechanism for the elimination of accumulated inert particulate matter from these lymphoid structures.

Vitamin D metabolism was studied in pregnant, D-deficient rats and their fetuses. D-depleted, pregnant rats were supplemented with [³H]25OHD₃ on the 19th day of pregnancy. The distribution and metabolism of radiolabeled D metabolites was different in maternal and fetal blood, kidneys, and small intestine. 24,25(OH)₂D₃ was the predominant dihydroxylated D metabolite in the fetus, whereas 1,25(OH)₂D₃ was the predominant dihydroxylated D metabolite in the mother. The ratio of 24,25(OH)₂D₃:1,25-(OH)₂D₃ was 12-fold greater in fetal plasma than maternal plasma. Maternal nephrectomy reduced the metabolism of [³H]25OHD₃ to 24,25(OH)₂D₃ (43%) and 1,25(OH)₂D₃ (75%). However, plasma levels of these two metabolites were unchanged in the fetuses of these animals when compared with levels observed in fetuses from mothers with intact kidneys. These results suggest the possibility of independent control of 25OHD₃ metabolism by the feto-placental unit and raise questions as to the possible role of 24,25(OH)₂D₃ in fetal development.

In vivo rates of fatty acid synthesis in liver, removable adipose tissue, and mammary gland were obtained in mice. Liver contributed 60 to 65%, and adipose tissue 35 to 40% of the fatty acids synthesized in virgin mice fed a high-carbohydrate diet. Mice in the 18th and 19th day of gestation synthesized less than half the quantity of fatty acids synthesized in virgin mice, even though the pregnant mice consumed more food than the virgin mice. Rates of fatty acid synthesis were elevated more than threefold in lactating mice and 71 to 83% of the fatty acid synthesis occurred in the mammary gland of the lactating mice. Fasting for 12 hr or consumption of a high-fat diet for 5 days depressed rates of fatty acid synthesis in all three tissues examined. Removal of the pups for 12 hr decreased the rate of fatty acid synthesis in mammary gland and increased the rate in adipose tissue of lactating mice.

S-Adenosylhomocysteine synthase was present in extracts prepared from six lines of rat hepatoma. There was no apparent correlation between the specific activity of this enzyme and any of the other biological properties of the tumors. The presence of the hepatoma did not affect the activity of adenosylhomocysteine synthase in livers of host animals. Hepatic enzyme activity in both host and control rats showed an adaptive increase to an increase in dietary protein. In contrast, dietary protein failed to affect the specific activity of adenosylhomocysteine synthase in five hepatomas. Paradoxically, enzyme activity in hepatoma 7787 declined when the host rats were fed a high-protein ration.

The results obtained in this study show that a progressive depression in the splenic T-cell population occurs in P. berghei-infected mice and that T-cell migration is abnormal also. Since the thymus and lymph nodes involute in P. berghei-infected mice (10), it is likely that the total T-cell pool is depleted in the infected mouse. The decreased ability of transplanted thymus cells to seed into the infected spleen and the decreased T-cell population may indicate that infected mice have an environment hostile to T-cell viability.