Experimental Biology and Medicine

Blood contains more of the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA), than does cerebrospinal fluid. However, the source and physiologic importance of this compound in the peripheral circulation are unknown. We have found that blood drawn from the portal vein of normal rabbits contains twice the concentration of GABA as that drawn simultaneously from the abdominal aorta. Also, human-derived colonic bacteria grown anaerobically secreted a large amount of GABA equivalents into culture media when measured by a radioreceptor technique. The production of inhibitory neuroactive substances, such as GABA, by colonic bacteria may play a role in the pathogenesis of certain disease states, for example, hepatic encephalopathy.

The effect of bile acids on β-glucuronidase activity was studied with centrifuged and dialyzed urines as the source of enzyme and phenolphthalein glucuronide as the substrate. The enzyme activity was measured at 56° in acetate buffer pH 5.2. All unconjugated bile acids tested showed no effect on the enzyme activity, primarily due to their low solubility in water. The glycine and taurine conjugates of cholic acid also showed no significant influence on the enzyme. Conjugated deoxycholic and chenodeoxycholic acids, on the other hand, were profoundly inhibitory to the enzyme within the physiological range of concentration in bile. Kinetic studies revealed that taurine conjugates of these two bile acids were pure competitive inhibitors of the enzyme for the substrate; whereas, their glycine conjugates exhibited a mixed competitive and noncompetitive inhibition. Since both urine and bile enzymes have an identical narrow pH activity curve, we conclude that pH and bile acids are two major determinants of β-glucuronidase activity in bile and their alteration may predispose to bilirubin pigment gallstone formation.

Three experiments were conducted to investigate the effects of various levels of dietary phenylalanine (Phe) and tyrosine (Tyr) on weight gain, feed conversion, serum Tyr and Phe, and hepatic Phe hydroxylase and Phe-pyruvate transaminase activities in Sprague-Dawley rats. In general, the results indicated that these animals were not able to obtain Tyr from dietary Phe with 100% efficiency when the animals were fed diets devoid of Tyr. At or above the calculated requirement level of Phe (0.8% and greater), this phenomenon might be explained by substrate inhibition of Phe hydroxylase. Serum Tyr appeared to be a better indicator of the amount of dietary Phe and Tyr consumed than was serum Phe.

Radioimmunoassay (RIA) methodology for purposes of monitoring immune response of female mice to injection with porcine zonae pellucidae was investigated. Titers of antibodies prepared against zonae pellucidae and, as measured by RIA, were compared to those obtained by the two frequently used zona antibody titration methods of (i) antibody produced precipitation layer formation on the zona surface (PPT titer), and (ii) indirect immunofluorescence (IF titer). RIA 30% binding and endpoint titers produced comparable immune profiles which indicated rapid rise in anti-zona activity occurring during the first 4 weeks following immunization, peak titer achieved between approximately 5 and 8 weeks followed by a very gradual decline in titer. Anti-zona titers remained at significantly high levels at 225 days following injection. RIA endpoint titers were approximately 200 times greater than the IF titers which, in turn, were 32-64 times greater than PPT titers. These data emphasize the greater sensitivity of the RIA over existing methodology for titrating zona antibodies and the feasibility for using RIA procedures for monitoring immune response following injection with zona material.

The increase in plasma renin activity after angiotensin I-converting enzyme inhibition by Captopril (SQ 14225) is well documented. In a study of the mechanism of Captopril-induced renin release, conscious rats were treated with Captopril (100 mg/kg per day) in their drinking water. Plasma renin activity (PRA) rose sixfold to a plateau after 5 days. At the end of 5 days of treatment with Captopril, rats carrying Alzet osmotic minipumps which infused angiotensin II (ANG II) at 200 ng/kg per min intraperitoneally had a slightly elevated PRA, as opposed to a greatly suppressed one, in ANG II-infused controls (P < 0.05). Urinary prostaglandin ((PG)E₂) daily excretion was unchanged by dietary Captopril. Indomethacin (5 mg/kg) injected subcutaneously significantly reduced plasma PGE₂ after 90 min, whereas PRA in Captopril-treated rats was unchanged after indomethacin injection. Results were similar in ANG II-infused Captopril-treated rats. Propranolol (10 mg/kg) injected intraperitoneally significantly reduced PRA 45 min later, but the PRA of ANG II-infused rats was not lower after propranolol injection under Captopril treatment than in similar rats not infused with ANG II. Indomethacin did not potentiate the effects of propranolol or propranolol plus ANG II on PRA of Captopril-treated rats. In conclusion, Captopril increased renin secretion by β-adrenergically mediated activation of the sympathetic nervous system and by interruption of the short feeback loop of ANG II. PGs did not appear to be involved. Other factors may be operating, since propranolol plus ANG II did not normalize PRA.

Dietary-induced zinc deficiency results in an impaired cell-mediated immune response to non-H2 allogenic tumor cells in mice. Animals maintained on a zinc-deficient diet for as little as 2 weeks develop a severe impairment in their ability to generate a cytotoxic response in the face of tumor challenge. This impairment is totally reversible by returning zinc-deficient mice to normal dietary zinc intake. Such animals now demonstrate a normal cytotoxic response to tumor challenge. If mice are treated with toxic doses of dietary zinc, a similar impairment of the cell-mediated cytotoxic response occurs. This suggests that either a deficiency or toxic level of zinc impairs the immune response to allogeneic tumor cells.

Human lymphoid cell cultures produced an interferon-like activity in the absence of inducers. Using actinomycin D, cycloheximide, and other metabolic inhibitors, we have shown that the cellular requirement for de novo RNA synthesis is not a requisite for interferon induction. We show here that the production of an interferon-like protein may be triggered in cultured lymphoid cells by substances capable of suppressing DNA-dependent RNA synthesis. Our data suggest that a rapidly turning over regulatory mechanism controls the production of an interferon-like molecule in lymphoid cells. In fact, the induction by inhibitors of RNA synthesis and the requirement for protein synthesis for production, but not for induction, suggests a preformed messenger RNA for an interferon-like molecule whose translation is normally prevented by a rapidly turning over mechanism which requires ongoing RNA synthesis. Experiments with neutralizing antibody for interferon suggest a new antigenic type of immune interferon or a slightly different molecule with lowered affinity for antibody. The very existence, however, of such a rapidly activated regulation of interferon production infers an important role in lymphocyte response and immune regulation.

Furosemide (1 mg/kg bolus injection) caused a transient decrease of pentagastrin-stimulated gastric acid secretion of 29 ± 4% in six dogs with gastric fistulas, whereas the same injection, followed by a continuous infusion of 1 mg/kg-hr, caused a sustained mean reduction of 40 ± 4%. A bolus injection of 5 mg/kg caused no further reduction in acid secretion. The relative decreases in C1^- and volume outputs closely paralleled the reduction in acid secretion.

Sucrose polyester (SPE) is a nonabsorbable fat which has been shown to lower plasma cholesterol concentrations when incorporated into the diet of human volunteers. This study was designed to assess the long-term (15 months) effect of oral administration of SPE on plasma cholesterol concentrations, body weights, and health of African green monkeys, a species similar to man in its lipid metabolism. All animals were fed for 2 months a high-cholesterol-containing diet to induce hypercholesterolemia. To simulate therapeutic intervention, dietary cholesterol was then decreased substantially and the effects of high or low (40 or 25%) fat content and presence or absence of SPE in the diet (as about 10% of diet) were assessed. There were four groups of animals: Group I (low fat-no SPE), Group II (high fat-no SPE), Group III (low fat-SPE), and Group IV (high fat-SPE). SPE administration resulted in 36% (high fat-SPE) and 40% (low fat-SPE) reductions in mean plasma cholesterol concentrations compared to 23% (high fat-no SPE) and 19% (low fat-no SPE) mean reductions in groups not fed SPE. The effect of SPE was statistically significant (P < 0.003). The effect of SPE was maximal in those animals with a high or intermediate plasma cholesterol response to dietary cholesterol and minimal or nonexistent in animals with a low plasma cholesterol response to dietary cholesterol. No significant differences between groups could be detected in body weight gain or in animal health as determined by the frequency of clinical illness and serial clinicopathologic observations.

The effects of several central acting drugs on prolactin (PRL) secretion were compared in young (3-4 months) and old (18-19 months) Sprague-Dawley male rats. Administration of the catecholamine synthesis inhibitor, methyl-DOPA, or the dopamine receptor blocker, haloperidol, produced a significantly greater increase in plasma PRL in the old than in the young rats. Stimulation of the serotonergic system by quipazine or enhancement of opioid activity by morphine produced a significantly greater rise in plasma PRL in the young than in the old male rats. Administration of the specific opiate antagonist, naloxone, reduced plasma PRL levels more in old than in young male rats. but this difference was not significant. Measurement of pituitary content and concentration of PRL revealed that both were significantly greater in old than in young male rats. These results suggest that hypothalamic dopamine continues to be an important inhibitor of PRL release in old male rats, whereas serotonin and the opiates become relatively less effective as stimulators of PRL release.

A plaque-type variant of mouse adenovirus, strain FL was isolated using L-cell monolayer cultures for the plaque assay. Intraperitoneal inoculation of this variant strain was observed to cause death accompanied by hemorrhagic lungs and interstitial pneumonia in adult C3H/HeN mice. Suckling mice developed patchy interstitial pneumonia after intranasal inoculation. Lethal disease was associated with infectivity (LD₅₀ = 5.0 × 10⁶ PFU) and the virus was detected in the lungs by electron microscopy. This plaque-type variant may provide a useful model that heretofore was unavailable for the study of adenovirus pathogenesis in pulmonary disease.

A clinical syndrome has been recognized previously in horses, characterized by hypercalcemia, hypophosphatemia and renal failure. The accumulated evidence suggested that hypercalcemia was a secondary effect of renal failure rather than the primary cause of renal disease. To test this hypothesis, the metabolic response following bilateral nephrectomy was investigated in five ponies. In contrast to the response observed in other mammalian species, nephrectomy in ponies resulted in significant hypercalcemia and hypophosphatemia. Forty-eight hours following nephrectomy, serum calcium had increased from a mean control value of 12.2 ± 0.4 to 17.6 ± 0.7 mg/dl and serum inorganic phosphorus had decreased from a control value of 3.2 ± 0.5 to 1.5 ± 0.3 mg/dl. These changes persisted until the time of death (mean survival time 7.2 ± 3.2 days) and suggest that the equine kidney plays a unique role in calcium homeostasis.

Anestrous Wistar-Furth rats bearing implants of the Furth MtTW15 tumor and diestrous tumor-free rats were ovariectomized and serum luteinizing hormone (LH), follicle-stimulating hormone (FSH) concentrations, and hypothalamic luteinizing hormone-releasing hormone (LHRH) content, were studied. The increase of LH and FSH, after ovariectomy, was significantly greater in tumor-free rats than in rats bearing prolactin secreting tumor implants. The hypothalamic LHRH content of the intact tumor-bearing rats was greater (P < 0.01) than that of tumor-free rats. Furthermore, the depletion of hypothalamic LHRH content was greater in tumor-free rats than in tumor-bearing rats. These results suggest that this prolactin and growth-hormone-secreting tumor prevents gonadotropin release by reducing LHRH release from the hypothalamus.

A monoclonal antibody to the sea nettle, Chrysaora quinquecirrha, venom mouse lethal factor was prepared using the crude venom as the antigen. Cloned hybridomas were selected by an enzyme-linked immunosorbent assay (ELISA) using partially purified lethal factor as the antigen. Ascites fluid from the cloned hybridoma-bearing mouse had an ELISA titer of 12.800 and the capability to neutralize the lethal activity (2 LD₅₀) of the intravenous injection of crude venom into mice after overnight incubation.

The effects of Escherichia coli endotoxin administration on the activities of key glycolytic enzymes in normal and alloxan-diabetic dog hearts were studied and the results were correlated with changes in plasma glucose concentrations. Normal dogs had a fasting plasma glucose concentration of 100 mg%, which was increased to 130 mg% 1 hr posten-dotoxin, then followed by a progressive decrease at 2 hr and thereafter to hypoglycemic levels (68 mg% at 4 hr). Diabetic dogs had a fasting plasma glucose concentration of 360 mg%, which was decreased to and maintained at 250 mg% at 2 and 4 hr following endotoxin injection. Hexokinase activity was not significantly different between the normal and diabetic dogs (P = 0.09). After endotoxin injection hexokinase activity tended to increase at 2 to 4 hr (P = 0.056) but it did not respond differently between the two groups, normal versus diabetic (P = 0.29). Phosphofructokinase activity was not different between normal and diabetic dogs (P = 0.49). After endotoxin injection phosphofructokinase activity was depressed at 2 hr but returned nearly to the control value at 4 hr (P = 0.48); there was no indication that this response with time differed between normal and diabetic dogs (P = 0.64). In normal dogs, pyruvate kinase activity was increased by 152.8% at 2 hr but then returned to the control value 4 hr after endotoxin injection. In alloxan diabetes, pyruvate kinase activity was increased by 216.8% at 2 hr and then further increased to 425.4% at 4 hr following endotoxin injection. Postendotoxic pyruvate kinase activity in diabetic dogs was significantly higher than that in normal dogs (P < 0.01) and also the change in pyruvate kinase activity with time differed significantly between the two groups (P < 0.01). Since the transport of glucose across myocardial cells has been found to be unaltered by endotoxin, it is concluded that the increase in pyruvate kinase activity is responsible at least in part for the augmented glycolysis of the myocardium during endotoxic shock.

The purpose of this study was to determine selenium concentration in erythro-cytes and plasma and to compare these values to the measured erythrocyte glutathione-peroxidase activity for a university and a long-term intravenous hyperalimentation (IVH) population. In addition, we wished to determine if these chronic IVH patients were at risk for selenium deficiency. There was a positive correlation (r = 0.67) between erythrocyte selenium levels and erythrocyte glutathione-peroxidase activities in the university population. The patients receiving IVH had significantly lower mean selenium levels in erythro-cytes and plasma and mean glutathione-peroxidase activities than the values from the university population. No correlation existed between erythrocyte selenium levels and erythrocyte glutathione-peroxidase activities in this IVH patient population. These chronic IVH patients when compared with the university population appear to be at risk for selenium deficiency.

Several phenolic compounds were examined for their ability to inhibit in vitro antibody production and human and mouse cell clone formation. Those compounds (gallic acid, nordihydroguaiaretic acid, methylparaben, and propyl gallate) which inhibited antibody production also suppressed clone formation. These effects occurred well below toxic concentrations. Other closely related compounds (p-hydroxybenzoic acid, shikimic acid, and vanillic acid) were without either effect. The data suggest that certain phenolic compounds, present either as normal constituents of food or as additives, may alter various cellular functions at or below concentrations found in the gut.

The observation that the cytochemical patterns of certain enzymes represent a stable marker for the identification of blood cells and resist functional stimuli as well as malignant transformation prompted the study of cell-specific polymorphism of acid phosphatase (EC 3.1.3.2) in human blood cells. Normal and leukemic human T and B lymphocytes as well as normal and leukemic blood monocytes and human alveolar macrophages as a functional derivative of blood monocytes were separated and subjected to purity control. Lysosomes. collected after cell cavitation, were solubilized and the particle-free supernatant was subjected to a direct assay of acid phosphatase using p-nitrophenylphosphate. Aliquot samples were analyzed by thin-layer isoelectric focusing and the banding of the acid phosphatase was recorded on gels using naphthol AS-BI phosphate after having demonstrated that on a quantitative term a positive correlation exists between the results with both substrates. The banding patterns proved to be highly cell specific as far as blood cells were concerned. The cell specificity of the acid phosphatase polymorphism defied functional as well as neoplastic transformation allowing for a clear recognition and classification of the individual cell lines. The cell-specific polymorphism of lysosomal enzymes seems to bear the potentiality of a further biochemical cell marker as exemplified by the results obtained with acid phosphatase.

Isolated rat adrenocortical cells were incubated with bovine parathyroid hormone (PTH) in the absence and presence of ACTH and CaCl₂. PTH had no effect on corticosterone production. but CaCl₂ induced steroidogenesis in the absence or presence of ACTH. Maximal corticosterone production in the absence of ACTH occurred with 1.25 mM CaCl₂ and in the presence of ACTH with 5.00 mM CaCl₂. These results show that PTH does not directly affect acute corticosterone production by isolated adrenocortical cells. They also show that ACTH raises the maximal steroidogenesis inducible by CaCl₂.

The content of free and esterified cholesterol in epididymal and subcutaneous adipocytes was measured during starvation in adult male rats in order to test the hypothesis that adipose tissue cholesterol esters are mobilized concomitantly with free cholesterol and triglyceride during starvation. Plasma and liver lipid concentrations were also determined as a function of time during food deprivation. Plasma triglyceride decreased significantly within 72 hr and liver triglyceride increased significantly after 144 hr of starvation. Plasma total cholesterol also decreased due to starvation, but only after 144 hr. No significant alteration in hepatic cholesterol was observed at any time. In epididymal adipose cells, free cholesterol was mobilized during fasting while the cholesterol ester pool remained constant. In marked contrast, cells from the subcutaneous depot showed no consistent changes in either free or esterified cholesterol despite the fact that cells lost about 55% of their triglyceride pool. These data provide another example of the metabolic heterogeneity of the various adipose tissue beds. Although neither cholesterol ester turnover nor hydrolase activity was directly measured, the results appear to be inconsistent with the hypothesis that an adipose tissue cholesterol ester hydrolase plays a role in the hydrolysis of stored esters during acute starvation, and further suggest that the turnover of cholesterol and cholesterol esters is probably different in adipose tissue.

We examined the influence of temperature on the absorption of inhaled ¹²⁵I-bovine serum albumin (BSA) and ¹²⁵I^- into the pulmonary circulation in isolated rabbit lungs. The lungs were insufflated for 15 min with an aerosol containing either ¹²⁵I-BSA or Na ¹²⁵I. The inhaled dose was the amount deposited in the lungs distal to the main bronchi. Blood samples were obtained from the perfusion circuit for 4.5 hr following insufflation. When ¹²⁵I-BSA was inhaled, the ¹²⁵I appeared in the blood in trichloroacetic acid (TCA)-soluble and TCA-precipitable fractions. At 38°, the TCA-precipitable and TCA-soluble fractions appeared in the blood at rates of about 1.4 and 1.9% of the inhaled dose per hour, respectively. Cooling to 12° reduced these rates to 0.25 and 0.12% per hour, respectively. The initial rate of ¹²⁵I^- absorption was much more rapid (4%/min) and no temperature dependence was detected. These results indicate that the rates of absorption and breakdown of inhaled albumin were markedly temperature dependent, while the absorption of the low molecular weight ¹²⁵I was not.

Migration and chemotaxis of human polymorphonuclear leukocytes (PMN) under agarose was studied in the presence and absence of various proteins. Contrary to previous reports, chemotaxis could be elicited in a protein-free medium using both complement-derived factors and fMet - Leu - Phe. Cell movement was critically dependent on the choice of agarose and buffer system. Addition of human serum or albumin produced marked chemokinesis while gelatin had no effect. These results indicate that media protein is a methodological requirement of some in vitro test systems but not a physiological requisite for PMN chemotaxis. The described procedures provide a valuable control for studying protein effects on cell migration as well as a means of avoiding unwanted cell stimulation and inactivation of test substances by media protein. This should facilitate studying chemokinetic and chemotactic materials, helper factors, and pharmacological agents. Agarose types were found to differ in the occurrence of a concentration dependent inhibition of locomotion. This suggests that restriction of cell movement by agarose may influence migration of PMN with abnormalities possibly unrelated to locomotion (e.g., cell swelling and decreased deformability induced by pharmacological agents or disease). Conditions are described which should minimize or enhance detection of such phenomena.

Direct measurement of glomerular capillary pressure, without removing cortical tissue from the micropuncture site, was the objective of this study. The measurement was accomplished by random micropuncture below the kidney surface with simultaneous recording of pressure at the tip of the micropuncture pipet. Glomerular capillary pressure was identified by a stable, pulsatile tracing synchronous with renal artery pressure or blood flow. In 50 micropunctures in 16 dogs glomerular capillary pressure was 56 ± 3 mm Hg. Mean arterial pressure averaged 127 ± 3 mm Hg. The conclusion that pulsatile pressure recorded during random micropuncture resulted from impalement of glomerular capillaries is supported by experiments in Munich Wistar rats. In 14 rats there was no significant difference between pressure measured either in superficial glomeruli (46 ± 1 mm Hg) or during random micropuncture (47 ± 1 mm Hg).

Groups of rats were studied between the ages of 36 and 43 days. Phenobarbital was injected at noon in order to remove the source of endogenous LH-RH. Beginning at 1400 hr, blood samples were obtained at 1/2-hr intervals during the 3-hr period when LH-RH was infused at 50 ng/hr through a double-lumen jugular cannula implanted 1-4 days earlier. Vaginal opening occurred at proestrus or estrus on Days 41-43. The peak level of circulating LH following LH-RH was significantly greater at age 42 than at 36, 38, 40, or 43, Responses at other ages were not statistically different from one another. However, five of the seven animals studied at 42 days were in proestrus, and the peak LH response was most significantly correlated with the stage of the estrous cycle: that observed at proestrus was significantly greater than those of diestrous, estrous, or anestrous (prepubertal) animals. These results fail to suggest an essential role of altered pituitary responsiveness in the onset of puberty. Rather, in agreement with the results of previous investigators, they reflect events at the initial proestrus, indicating that cyclic mechanisms have already been set in motion.

The effect of cupric ion on amino acid incorporation into protein by isolatec rat liver mitochondria was investigated. In confirmation of previous observations, the incorporation was stimulated as much as 80% by the addition of 2 to 10 μM CuCl₂ when the mitochondria were incubated in the medium of Buchanan et al. The stimulation was maximal at 6 μM Cu²⁺. By contrast, cupric ion at comparable concentrations showed no significant effect on the incorporation when the mitochondria were incubated in the medium of Haldar and Freeman. Experiments involving omission or addition of the components which made the two media different showed that the stimulatory effect of cupric ion depended upon the presence of 50 mM Tris-HCl. Additional experiments with Tris-HCl and sucrose demonstrated that the extent of stimulation depended on the osmolarity of the incubation medium and that cupric ion stimulated the incorporation by preventing the inhibition caused by the hyperosmolarity of the medium.

The effects of in vivo alterations in local calcium concentration on small vessel responses to norepinephrine were obtained for a first order arteriole and venule in the cremaster muscle of normotensive and one-kidney, one-clip Goldblatt hypertensive rats. The cremaster with intact circulation and innervation was suspended in a modified Krebs solution which contained either 1.3, 2.6, or 5.1 mM CaCl₂. Closed-circuit television microscopy was used to measure vessel diameters. The resting lumenal diameters of first-order arterioles in hypertensive rats were 31% smaller than lumenal diameters of the corresponding arterioles in normotensive rats. Concentration-response curves showed that the norepinephrine sensitivity (pD ₂) of first-order arterioles in normotensive rats was significantly increased by a change in bath [Ca²⁺] from 1.3 to 2.6 mM; however, the norepinephrine sensitivity of comparable arterioles in hypertensive rats was not affected by this change in bath [Ca²⁺]. These data indicate that mechanisms for calcium modulation of vasoconstriction are attenuated for large arterioles in the cremaster muscle of one-kidney, one-clip Goldblatt hypertensive rats.

The utilization of exogenous oxaloacetate (OAA) was studied in isolated rat liver parenchymal cells. Phenylpyruvate and α-cyanohydroxycinnamate, which inhibit pyruvate carboxylase (EC 6.4.1.1) and pyruvate transport across the mitochondrial membrane, respectively, were equally effective in decreasing glucose formation from pyruvate and OAA. Additionally, the specific activity of glucose formed in cells incubated with sodium [¹⁴C]bicarbonate and either pyruvate or OAA was the same. The specific activity of glucose formed from asparagine plus sodium [¹⁴C]bicarbonate was approximately 25% of that observed when pyruvate or OAA served as the carbon sources. The results suggest that OAA is decarboxylated to pyruvate prior to its metabolism.