Experimental Biology and Medicine

The role of CSF in the production of activated phagocytes during infection is summarized in Figure 2. Resident macrophages and immigrating granulocytes serve in the first line of host defenses against microbial invasion. The phagocytes kill some of the invading microorganisms and the macrophages process and present microbial antigens to antigen-specific T lymphocytes. IL-1 secreted by a number of cell types, including macrophages, promotes clonal expansion and the activation of sensitized T lymphocytes, which in turn produce a variety of cytokines, including CSF. These cytokines, in combination with the microbial products released during infection, stimulate the proliferation and differentiation of bone marrow progenitor cells and the increased formation of circulating white blood cells. In response to chemotactic factors, the phagocytes in the circulation adhere to endothelial cells adjacent to the inflammatory site, undergo diapedesis, and migrate to the site of infection. These phagocytes, activated by CSF and other cytokines, exhibit increased antimicrobial activity. Thus, the CSF play a central role in phagocyte production, differentiation, and activation.

The genes encoding the CSF in humans and in mice have been cloned and expressed as recombinant material. The recombinant CSF exhibit essentially the same biological activity as do the native proteins. Moreover, many of the activities expressed in vitro have also been observed in vivo. The results of animal experiments and clinical trials confirm the role of CSF in phagocyte production and the augmentation of host defenses. While cytokines are currently being evaluated on an individual basis, therapies in the future may involve a combination of cytokines used in an effort to control host defenses precisely and to provide more effective treatment. It has been shown, for example, that murine IL-3 and M-CSF induce the cycling of progenitor cells when used together at doses that are individually ineffective (148).

PHT has multiple effects in adult humans and animals, and there is no reason to assume that it will not have multiple effects in embryos and fetuses. Although one of the first associations between anticonvulsant therapy and an adverse development effect in humans was noted in 1964 (104), the mechanism(s) whereby these adverse effects occur has thus far eluded research efforts. In this review, I have focused on three possible mechanisms. Overall, the evidence does not appear to implicate folate deficiency in PHT-induced embryotoxicity. A role for glucocorticoids or interaction between PHT and the glucocorticoid receptor has not been ruled out. However, a significant amount of work remains to be done to examine the involvement of the arachidonic acid cascade in PHT-induced embryotoxicity in vivo. The bulk of the experimental evidence would seem to favor a role for the generation of a reactive intermediate and its subsequent binding to embryonic macromolecules. This metabolite(s) has not been identified. Additionally, the association between covalent binding of metabolites and embryotoxicity remains simply an association; a causal relationship has not been established. Much work remains to be done to determine whether any of these possibilities, some other possibility, or a combination of several mechanisms will explain the adverse developmental effects of this very important, therapeutically useful anticonvulsant.

The author would like to thank Drs. Ruth Billings and Daniel M. Sheehan for their helpful discussions and critical reviews of the manuscript.

There are many reports in the literature that indicate that the adrenal cortex is essential to the normal renal response to a metabolic acidosis. A frequent way of introducing an acute or chronic metabolic acidosis is to administer ammonium chloride. The renal response to such an acidosis is an increase in H⁺ excretion and an increase in NH₄ ⁺ production and excretion.

Goth (1), in summarizing the therapeutic effects of NH₄Cl, notes that its natriuretic effect is associated with a “delay of several hours to days” before the kidney develops fully its capacity to form ammonia. The emphasis on this delayed action has served to cause researchers to pay little attention to the lesser, but prompt, response of the body to increases in both NH₄ ⁺ and H⁺ excretion within 2 hr or less.

There is good evidence, cited below, that indicates that this early response, as well as the later response, is influenced by the adrenal cortex in some manner. We have reviewed the literature on this subject with these questions in mind.

1. After administration of an acidifying substance such as NH₄Cl, are one or more adrenal cortical hormones released that have a specific effect on the kidney to result in either increased NH₄ ⁺ excretion (ammoniuretic effect) or increased H⁺ excretion (protonuretic effect), or both?

2. Is the delay in maximal NH₄ ⁺ and H⁺ excretion due to:

a. different substance(s) excreted by the adrenal cortex that are involved in affecting the kidney? or

b. a delay in the kidney responding to the ammoniuretic and protonuretic factors that initiate the acute effect?

Adrenal Gland and Control of NH₄ ⁺ Excretion

In the following section, we present some early literature describing the functional renal changes in metabolic acidosis. We then present some of the studies on possible cellular mechanisms which might produce an increase in NH₄ ⁺ excretion, and H⁺ excretion.

The pineal gland functions as a neurohumoral transducer, accepting photic information from the retinae and converting this neural message into chemical mediators, the best documented of which is melatonin (N-acetyl-5-methoxytryptamine) (1). The onset of darkness causes information to travel from the suprachiasmatic nuclei of the hypothalamus via the superior cervical ganglia to postganglionic sympathetic nerves that end in the pineal, where they release norepinephrine. The resultant stimulation of β-adrenergic receptors, possibly augmented by α-adrenergic stimulation, leads to an increased activity of N-acetyltransferase (NAT) (2), the rate-limiting enzyme involved in synthesis of melatonin (3). Serotonin, derived from dietary tryptophan taken up by pinealocytes from the blood, is N-acetylated by NAT, which is subsequently O-methylated by hydroxyindole-O-methyltransferase (HIOMT) to form melatonin, which is then released into the general circulation. Being a small lipophilic molecule, melatonin is generally considered to be released rapidly and passively following synthesis, although there is some evidence that short-term storage of melatonin by the pineal gland might occur (4). Melatonin's lipophilicity also ensures that it distributes widely to target tissues, which, therefore, receive photoperiodic information and can subsequently act with appropriate physiological responses (5).

The multiplicity of extracellular and intracellular events that interact to produce the melatonin message are not fully understood, although certain aspects have been reasonably well characterized. For example, it is well-known that β-adrenergic stimulation leads to activation of adenylate cyclase, and that the resultant increase in CAMP stimulates mRNA synthesis, leading to increased NAT activity (3). The α-adrenergic augmentation of this process results from activation of protein kinase C. A number of stimulatory/regulatory processes involved in melatonin production have been associated with calcium, and the following discussion serves to review current understanding of the relationship between calcium and pineal gland function.

The purpose of this investigation was to assess the actions of two avian prolactins (PRL) on rat Nb2 lymphoma cell proliferation. Various turkey and chicken PRL preparations stimulated the proliferation of rat Nb2 cells. The ability of avian PRL preparations to influence the behavior of rat Nb2 lymphoma cells provides a useful method for monitoring the PRL-like actions of native and recombinant avian PRL and suggests a potential role of PRL in modulating the avian immune system.

Active renin is a heterogeneous enzyme that can be separated into multiple forms with high-resolution isoelectric focusing. The isoelectric heterogeneity may result from differences in glycosylation between the different forms. In order to determine the relationship between active renin heterogeneity and differences in composition or attachment of oligosaccharides, two separate experiments were performed: (i) Tunicamycin, which interferes with normal glycosylation processing, increased the proportion of relatively basic renin forms secreted into the incubation media by rat renal cortical slices, (ii) Endoglycosidase F, which enzymatically removes carbohydrate from some classes of glycoprotein, similarly increased the proportion of relatively basic forms when incubated with active human recombinant renin. In addition, further studies with inhibitors of human renin activity revealed that the heterogeneous renin forms were similarly inhibited by two separate renin inhibitors. These results are consistent with the hypothesis that renin isoelectric heterogeneity is due in part to differences in carbohydrate moiety attachment and that the heterogeneity of renin does not influence access of direct renin inhibitors to the active site of renin.

Bile acids are one of the components of the gastric contents capable of disrupting the mucosal barrier to diffusion. The mechanism by which bile acids can damage the gastric epithelium is not completely understood. Several studies have emphasized mucosal lipid solubilization by bile acids in the pathogenesis of mucosal injury. Bile acid entry into gastric mucosal cells may be a critical and early step in the genesis of mucosal injury, but this possibility has not yet been investigated. The present study was designed to explore the interaction of bile acids with dispersed gastric mucosal cells isolated from the rabbit and guinea pig stomach. Results showed that both glycocholic and deoxycholic acid rapidly associated with the gastric cells and reached a steady state concentration by 30 min. Glycocholic acid accumulated in the cells to a concentration approximately eight times greater than that in the surrounding medium. The amount of bile acid associated with the cells was greater at an acidic than at a neutral pH, and was a function of the concentration of both the cells and the bile acid. The process did not require cellular energy, was nonsaturable, and was not species specific. Experiments with ⁸⁶Rb, a cytoplasmic marker, revealed that approximately one half of the cellular glycocholic acid was associated with the cytoplasmic compartment and the rest with the membranes. These findings are consistent with a combination of intracellular entrapment of the bile acids due to intracellular ionization and bile acid binding to cellular membrane components being the mechanisms by which bile acids accumulate in cells. Acid-driven bile acid accumulation may explain how relatively low luminal concentrations of bile acid can be damaging to the gastrointestinal mucosa.

To study biochemical changes in cerebrospinal fluid (CSF), we developed a reliable technique for repeated collection of CSF in anesthetized strain 13 guinea pigs. The animal's head was mounted in a stereotaxic instrument with ventral tilt at 30°, and cisternal puncture was made with an L-shaped, 23-gauge needle through the shaved skin. Clear CSF was collected in a 1-ml syringe surrounded by crushed ice. Each collection procedure lasted for 3 min, and three consecutive collections produced about 0.2 ml of CSF. Sampling was repeated at 3-hr intervals. With intravenous saline infusion (10 ml/kg hr), a total volume of 0.6–1.0 ml of CSF was collected over 6 to 12 hr. Animals maintained a mean blood pressure, heart rate, and minute volume, with few changes during CSF sampling for the entire collection.

3-Phenylacetylamino-2,6-piperidinedione (A10), an amino acid analog, has been reported to possess antineoplastic activity against certain neoplastic tissues. The antimitogenic properties of A10 were studied by determining its effect on prolactin (PRL)- and interleukin 2 (IL-2)-stimulated mitogenic responses in the rat Nb2 lymphoma cell line. The addition of A10 (1–12 mM) to PRL (0.4 ng/ml)-stimulated cells inhibited growth in a dose-dependent manner. DNA synthesis patterns studied by thymidine incorporation demonstrated that A10 was significantly inhibitory (25% at 20 hr; 50% at 40 hr, P < 0.01). IL-2 stimulation of mitogenesis was also sensitive to A10 inhibition. The inhibition of PRL stimulated mitogenesis was reversible when A10 was removed after 24 hr of culture and A10 showed no toxicity in a chromium release assay. These data suggest that A10 effects may be cytostatic, rather than cytotoxic.

Mammalian and avian growth hormones (GH) (pituitary derived or biosynthetic) exert two effects on chicken adipose tissue explants in vitro. They (i) increase the basal rate of glycerol release a lipolytic effect) and (ii) inhibit glucagon-stimulated glycerol release (an antilipolytic effect). The ability of lower vertebrate GH preparations to exert lipolytic and antilipolytic effects was examined and biological activity was compared to differences in amino-acid residue sequences and to predicted structure. Irrespective of species origin (blue shark, sturgeon, bonito, yellow tail, salmon, bullfrog, sea turtle), all lower vertebrate GH preparations showed very weak (<5% the potency of bovine GH), if any, lipolytic activity, but retained strong antilipolytic activity. The present data indicate that the structural requirements for lipolytic and antilipolytic activities of GH differ in chicken adipose tissue. Despite the high sequence homology (88%) between chicken and sea turtle GH, the latter preparation did not stimulate lipolysis. It is suggested that Pro¹³², conserved only in lipolytically active GH species (human, bovine, and chicken), represents a major determinant of lipolytic activity in chicken adipose tissue. The structural determinants for antilipolytic activity may comprise any or all of residues 3, 17, 64, 108, 109, and 152.

A soluble form of transferrin receptor has been detected in human serum and has been shown recently to be a truncated form of the intact membrane bound receptor. Mechanisms governing the release of transferrin receptor by cells are poorly understood and could be better defined by tissue culture. The present investigation was undertaken to characterize the transferrin receptor released by K562 erythroleukemic cells. In contrast with maturing sheep reticulocytes, which have been shown to release transferrin receptor in small vesicles termed exosomes, we demonstrated, with a monoclonal enzyme-linked immunoassay, that less than 30% of the transferrin receptor released by K562 cells in log phase growth was in a particulate form. The relative amounts of soluble and particulate receptor released to the supernatant did not change significantly during 48 hr of incubation. Soluble receptor was purified by immunoaffinity chromatography. On polyacrylamide gel electrophoresis, its mobility was the same (85 kDa) as that of the truncated monomeric form recently identified in human serum. Further evidence that serum and soluble receptors released by K562 cells are identical was provided by amino acid sequence analysis, which demonstrated that 16 of the first 19 residues of the N-terminal sequence of soluble K562 receptor are homologous with the serum receptor. The remaining three were not identifiable. K562 cells provide a useful in vitro model for studying the production of membrane-bound and soluble forms of released transferrin receptor.

The ability of interferons (IFN) to exert a systemic effect following their oral administration was evaluated. One systemic effect of parenteral interferon administration has been shown to be a suppression of the number of peripheral white blood cells both in man and in mouse models. Using the mouse model of peripheral white blood cell suppression, the relative systemic effects of orally and subcutaneously administered interferons were determined. Murine IFN-β, murine IFN-γ and cross-reactive recombinant human IFN-αA/D were examined. The oral administrations of each of the three interferons were found to cause a dose-dependent suppression of the peripheral white blood cell counts. Significant levels of suppression were seen with as little as 5 units/day of murine IFN-β and with 500 units/day of recombinant human IFN-αA/D and murine IFN-γ. The dose-response curves obtained with orally administered interferons were much more shallow than those obtained with subcutaneously administered interferons. The results demonstrate that oral administration of interferons can provide a significant systemic effect. Further, the results support the possibility that the oral administration of interferons may have therapeutic potential.

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, α-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on α-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.

Mouse peritoneal leukocyte lysophospholipase (LPL) activity was studied to determine whether or not noninfectious agents cause increased enzyme activity and whether neutrophils have LPL activity. In the first study, mice infected with Ascaris suum, a known inducer of LPL activity, were given intraperitoneal injections of proteose peptone, thioglycolate, bovine albumin, paraffin, glycogen, or A. suum whole worm extract (WWE). Cell populations collected from mice injected with A. suum WWE, proteose peptone, thioglycolate, or bovine albumin contained increased numbers of neutrophils and eosinophils. These cell populations had increased LPL activity when treated, in vitro, with either A. suum WWE, zymosan-activated complement, or with the agent they were induced with. However, the LPL activity of the different cell populations did not respond to all treatments in the same way. In a second study, A. suum-infected or noninfected mice were given intraperitoneal injections of paraffin, thioglycolate, glycogen, or A. suum WWE. Enriched cell populations containing either lymphocytes or macrophages, from infected or noninfected mice, did not have increased LPL activity following in vitro stimulation with A. suum WWE, zymosan-activated complement, or with the agent they were induced with. Enriched neutrophil populations from infected or noninfected mice had increased LPL activity following in vitro treatment with A. suum WWE or zymosan-activated complement. Results demonstrate that the LPL activity of peritoneal leukocytes can be induced by noninfectious agents and that neutrophils have increased LPL activity following in vitro stimulation.

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immuno-neutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.

Four miniature Brahman calves born in 1988 and 1989, along with four contemporary sex-matched Brahman control calves, were used in experiments to determine circulating concentrations of insulin-like growth factor I (IGF-I), growth hormone (GH), insulin, triiodothyronine, and thyroxine, and plasma glucose response to insulin challenge. The effect of plane of nutrition on plasma concentrations of IGF-I and insulin was also determined and a clinical screen of blood chemistries was conducted to determine effects of calf type. Plasma IGF-I was six times higher in control calves compared with miniature calves (209.0 vs 35.0 ng/ml; P = 0.001). However, miniature calves had mean plasma GH about six times higher (37.8 vs 6.2 ng/ml; P = 0.004) and had twice as many secretory episodes (9 vs 4.5; P = 0.005) over an 8-hr sampling period. Plasma concentrations of triiodothyronine (2.54 vs 1.80 ng/ml) and thyroxine (88.8 vs 56.2 ng/ml) were higher in control compared with miniature calves (P = 0.001), but concentrations of triiodothyronine and thyroxine in both calf types were within normal ranges. Although miniature calves displayed similar plasma glucose concentrations to controls, hypoglycemic response to insulin challenge tended to be greater in miniature calves. Nutritional regulation of circulating IGF-I appeared to be intact in miniature as well as control calves, as evidenced by a reduction in plasma IGF-I concentration following a decrease in plane of nutrition, and a subsequent increase in plasma IGF-I concentration following realimentation. Serum urea nitrogen was lower (P = 0.02) in control compared with miniature calves. These data describe a miniature condition in Brahman cattle that is manifested by apparently normal proportioned growth but small stature, and that is associated most notably with abnormally low circulating concentrations of IGF-I in the presence of paradoxically high circulating concentrations of GH.

The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 μg of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 μg of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-β may be present in this acid/ethanol extract of skeletal muscle.

The objective of this study was to determine the effects of thyrotropin-releasing hormone (TRH) and bromocriptine on plasma levels of biologically active prolactin in ovariectomized, diethylstilbestrol (DES)-treated rats. Female Long-Evans and Holtzman rats were ovariectomized and each was given a subcutaneous implant of diethylstilbestrol (DES). One week later, groups of DES-treated rats were fitted with indwelling intra-atrial catheters, and 2 days later blood samples were withdrawn before and at 1, 2, 5, 10, and 20 min after intravenous administration of TRH (250, 500, or 1000 ng/rat). Blood samples were obtained from other groups at 4 weeks of DES treatment by orbital sinus puncture under ether anesthesia before and at 30,60, and 120 min after bromocriptine administration (2.5 mg/rat sc). Plasma was assayed for prolactin by conventional radioimmunoassay (RIA) and by Nb2 lymphoma bioassay (BA). Holtzman rats released significantly more prolactin following TRH than did Long-Evans rats when the RIA was used to measure prolactin. However, when the BA was used to assay prolactin in the same samples, the Long-Evans rats released more prolactin than did the Holtzman rats. In addition, the ratio of the BA to RIA values was significantly increased in both strains following TRH, but the greatest increase was observed in the Long-Evans rats, in which the ratio was 4.5 at the peak of the TRH-induced rise in plasma prolactin. Gel filtration chromatography of plasma obtained at 5 min after TRH treatment in Long-Evans rats revealed large molecular forms of prolactin with BA to RIA ratios of 4–5. In addition, monomeric prolactin had a BA to RIA ratio of 2. Bromocriptine treatment reduced prolactin levels in both strains, but the effect was more rapid in Holtzman than in Long-Evans rats.

This study was conducted to determine the plasma levels of prolactin in prepubertal and young, postpubertal, proestrus rats of mammary tumor-susceptible (Sprague-Dawley) and tumor-resistant (Long-Evans) strains using a sensitive bioassay-Nb2 lymphoma cell replication. Prepubertal Long-Evans rats had significantly higher levels of prolactin than did Holtzman Sprague-Dawley rats of the same age. Likewise, Long-Evans rats secreted significantly more prolactin into the blood on the afternoon and evening of proestrus than did Holtzman rats. Finally, ovariectomized Long-Evans rats released more prolactin into the blood at 1 day, but not at 8 or 15 days, of treatment with diethylstilbestrol. Prolactin levels determined by conventional radioimmunoassay and by bioassay were similar except on the afternoon of proestrus, when, in both strains of rats, the bioassay to radioimmunoassay ratio increased significantly above 1.0 during the late evening. In addition, the ratio was significantly less than 1.0 in the early and late afternoon in the Holtzman rats, but not Long-Evans rats. These data indicate that a strain of rats that is resistant to experimentally induced mammary cancer has higher prolactin levels in the blood than does a strain that is susceptible to mammary cancer at a time when mammary gland growth is rapid. Furthermore, there are times during the proestrus prolactin surge when the bioassay yielded higher and lower values of prolactin than radioimmunoassay of the same samples, suggesting functional heterogeneity of prolactin that may impact on mammary gland or other target tissue function.

To compare the intrinsic permeability properties of the small intestine in adult (average body wt 300 g) and 25- to 27-day-old (average body wt 50 g) male rats, the uptake rates of cycloleucine and of cadmium were measured in intestinal segments isolated in situ with their blood supply intact. Uptake rates were expressed on the basis of that of ethanol, a solute whose absorption depends primarily on the size, rather than the composition, of the available surface area and on the presence of unstirred layers. These layers may be concluded to affect movement of cycloleucine, cadmium, and ethanol to the same extent. The ratio of uptake rates, therefore, provides in arbitrary units a measure of the intrinsic permeability of the luminal surface area to cadmium and to cycloleucine. On this basis, no developmental change in cycloleucine permeability could be detected. In contrast, the rate of cadmium uptake relative to that for ethanol decreased with age by about 50%. Possible mechanisms are discussed for this significant change in the intrinsic cadmium permeability of the jejunum in post-“closure” animals.

Various 6-adrenergic agonists were found to inhibit rates of protein degradation and net protein breakdown in isolated chick extensor digitorum communis (EDC) and atrial muscles. Rates of protein synthesis were not altered by these compounds. The 3-agonist cimaterol inhibited rates of protein degradation in EDC muscles incubated with or without amino acids and insulin. Cimaterol also inhibited the increased proteolysis induced by injury to muscle or by incubating muscles at body temperature (42°C) versus 37^CC. Thus, 3-agonists may help promote skeletal muscle accretion in vivo even under conditions of severe negative nitrogen balance by slowing muscle proteolysis.