Experimental Biology and Medicine

An attempt is made in this review to summarize new findings that propose the direct involvement of two fetal proteins (i.e, fetuin and α-fetoprotein) in fatty acid transport and delivery to cells. The presence of these proteins at a high plasma concentration in fetal life and the critical need for fatty acids during development strongly support this hypothesis. Tissue and cell specificities involved in the uptake of these fetal proteins and the mechanism of fatty acid delivery awaits further research.

Until the 1920s, it was assumed that the sole gonadal hormones modulating anterior pituitary glandular secretions were lipid-soluble entities called steroids. Experimental evidence then surfaced during the ensuing decade that demonstrated that the testis also contained a hydrophilic material that might also influence pituitary secretory activity (1, 2). The name “inhibin” was given to this novel substance in view of its ability to inhibit hypertrophy of pituitary cells in castrated rats (2). Work in later years evoked the concept that inhibin could be an important regulator of follicle-stimulating hormone (FSH) secretion (3, 4). Thus, as a consequence of these landmark studies, investigators began a search to elucidate the chemical nature of the inhibins.

Although inhibin was discovered as a potential secretory product of the testis, it was almost 45 years later that a nonsteroidal FSH suppressor was demonstrated in the ovarian follicular fluid of cows (5). Ironically, it was the ovarian follicular fluid from which chemists eventually isolated what was most likely the putative inhibin molecule described in the 1920s and 30s (6–9). Chemical characterization of the purified FSH-suppressing material revealed that inhibin is a 31-to 32-kDa heterodimer consisting of two distinct polypeptide chains, α and β. In pig follicular fluid, two inhibin heterodimers, A and B, were isolated and found to consist of a common α-subunit chain linked by disulphide bridges to a β_A- or β_B-subunit chain, respectively. Both inhibins were essentially equipotent in suppressing FSH secretion in the dispersed anterior pituitary cell bioassay system (6).

At long last, the elusive inhibin had been proven a true chemical entity—a major breakthrough in reproductive endocrinology. However, little did anyone know that the gonads produced other FSH-regulatory polypeptide factors besides inhibin, and that the ability of these other factors to modulate FSH secretion could be only the tip of the iceberg with regard to the multi-functional properties of these factors, thereby extending their importance to other areas of biology.

Evidence implicating prolactin (PRL) and growth hormone (GH) in the regulation of the immune system has been reviewed. Hypophysectomized animals have deficiencies in both cell-mediated and humoral immunological functions and either PRL or GH corrects these deficiencies. Animals administered bromocryptine, a drug that specifically blocks PRL release, have impaired immune responses similar to hypophysectomized animals, and again both PRL and GH correct these deficiencies. Genetically dwarf animals, which lack both PRL and GH, are also immunocompromised, and once again PRL and GH can correct the deficiencies. In dwarf animals, however, fewer studies have examined PRL actions. In growth-deficient children, immune function is not dramatically altered and basal secretion of GH has been reported. Very few clinical studies have examined whether PRL secretion is also deficient, and this may explain why a clear loss in immune function is not evident in growth-deficient children. In a number of species, including man, both PRL and GH stimulate thymic function and increase the secretion of thymulin, a thymic hormone. No studies, however, have reported on the effects of PRL and GH on other thymic hormones. A number of studies have reported in vitro effects of PRL and GH on cells involved with immunity, and the presence of high-affinity PRL and GH receptors have been observed on a number of these cells. The action of GH on the proliferative response of cells involved with immunity in vitro appears to be mediated by the production of insulin-like growth factor I. The effect of PRL on insulinlike growth factor I production by these ceils has not been examined. One of the most consistent findings from in vitro studies is that prolactin antisera blocked a number of immune reactions. This led to the discovery that cells involved with immunity appear capable of producing PRL and GH, but the physiological significance of these observations have not been explored. There is a great need to identify the cell types responding to PRL and GH and this should be a goal of future investigations. There is also a need for investigators to be aware that both PRL and GH are involved in the regulation of the immune system and to design experiments to elucidate where each functions in the maturation cascade of cells involved with immunity. From the evidence available, it is apparent that PRL and GH have an important function in the immune system and future investigations should be directed toward elucidating their site(s) of action.

The chronological analysis of anomalies that emerge within the first postnatal week in fa/fa rats strongly suggests that increased lipid storage leading to obesity in Zucker rats is a consequence of a defect in energy expenditure. In the search for an unifying hypothesis that might explain the etiology of this genetic obesity, an alteration in the nervous system appears the most likely to agree with the monogenic transmittance of the syndrome. Such an alteration could cause a general defect in the peripheral organs, which are sympathetically innervated, and explain the decreased thermogenesis in BAT of fa/fa rats and in other organs like skeletal muscles (via decreased futile cycles activity; 150). It may also explain the decrease in the peripheral utilization of triglycerides and glucose, principally by skeletal muscles and BAT, which results in excess circulating levels of these substrates that could then be shunted for storage in WAT.

The hyperinsulinemia, which progressively develops in preobese rats from 10 days onward, could be explained either by an increased parasympathetic nervous system activity (8) or by a vagal sensitization of pancreatic insulin secretion to the increased levels of circulating glucose (105). This latter idea is consistent with a cause and effect dependency between a decreased sympathetic activity (possibly responsible for increasing plasma glucose levels) and the emergence of hyperinsulinemia.

Hyperphagia, the date of emergence of which cannot be separated from that of hyperinsulinemia, could be a consequence of the latter (78), and/or a defect in the hypothalamic control of food intake (1 5 1).

When hyperphagia and hyperinsulinemia develop, the anomalies in adipose tissue accretion get worse and a cascade development of new alterations develops in other organs (liver, muscles, brain, etc.). These alterations may give rise to a new dynamic metabolic and hormonal re-equilibration. As a result, anomalies present in older fa/fa rats may only be a consequence of earlier re-equilibrations and should be analyzed accordingly

The etiology of the genetic obesity syndrome of Zucker rats appears to result from a disturbance in the regulation of the sympathetic nervous system, whose main and first expression is a decreased thermogenesis. The primary genetic defect(s) of fa/fa rats probably resides in discrete regions of the hypothalamus or at a higher level of regulation, i.e., in the central nervous system.

With the development of CD diet L-489E 14SE, it is possible to maintain prodction colonies of BALB/c mice under GF conditions with totally defined nutritional intake after weaning. These mice are exposed to a minimum of antigenic stimulation and thus provide a unique model in which all exogenous factors may be controlled.

Administration of 50, 250, and 1,250 ng/kg iv of recombinant bovine tumor necrosis factor-α (RBTNF) did not affect basal plasma concentrations of growth hormone (GH) or thyroid-stimulating hormone in male calves. However, when administered 30 min before challenge with 1 μg/kg iv of thyrotropin-releasing hormone (TRH), 250 ng/kg of RBTNF increased the subsequent incremental GH response. At 1,250 ng/kg of RBTNF, GH response to TRH was significantly blunted. For each dose of RBTNF administered, the incremental change in plasma thyroid-stimulating hormone following TRH was not significantly different from control. To examine direct effects of RBTNF on pituitary function, fresh bovine pituitaries were sliced into 1-mm cubes and incubated with 0 or 10^–8, 10^–9, or 10^–10 M RBTNF. Additional cultures were treated with 10^–8 or 10^–9 M GH-releasing factor or 10^–8 M TRH and 0 or 10^–8 M RBTNF. Media GH increased in cultures with 10^–10 M RBTNF and declined linearly as RBTNF concentration increased. RBTNF blocked GH release from GH-releasing factor- and TRH-challenged pituitary slices. Membranes prepared from homogenized bovine pituitaries had specific saturable binding characteristics for monomeric ¹²⁵I-RBTNF. Membranes treated with 4 M MgCI₂ for 10 min and washed free of Mg²⁺ produced Scatchard plots fit to a two-site model (high affinity site K_d = 6.6 nM), while Scatchards of non-Mg²⁺-treated membranes fit a single site (K_d = 8.9 nM). Polyacrylamide gel electrophoresis separation of ¹²⁵I-RBTNF cross-linked pituitary membranes showed specific binding of monomeric ¹²⁵I-RBTNF to protein components ranging in molecular weight from 19,000 to 77,000. The data suggest that RBTNF has modulatory effects on the regulation of GH secretion acting directly at the pituitary through specific receptors.

We have reported a paradoxical plasma atrial natriuretic factor (ANF) decline following prolonged high salt intake that was attributed to an increased tissue uptake of circulating ANF, leading to its augmented distribution volume (V_ss) and metabolic clearance rate (MCR) as compared with control rats on a standard diet. To explore this phenomenon further, we evaluated possible chronic salt-loading-induced changes in ANF clearance (C-ANF) receptors, which appear to play a major role in ANF removal from the circulation. We studied changes in plasma [¹²⁵I]ANF(1–28) and its pharmacokinetics after preoccupation of C-ANF receptors by its specific ligand, C-ANF(4–23), in high-salt-treated rats and their controls. Following C-ANF(4–23) administration, we detected significantly higher circulating [¹²⁵I]ANF levels throughout the study period (8 min) in high-salt-fed rats compared with the controls (280–470% vs 100–215% increase of basal values, P < 0.05). C-ANF(4–23) infusion caused a significantly greater decrease of the metabolic clearance rate and distribution volume of [¹²⁵I]ANF in high-salt-fed rats than in control animals (74 ± 6% vs 41 ± 6% and 75 ± 4% vs 50 ± 5% of basal values, respectively; P < 0.05). These data suggest that a prolonged high salt diet may increase the availability of C-ANF receptors and, through this mechanism, may negatively modulate plasma ANF concentrations. C-ANF receptors may thus fulfill a regulatory function on circulating ANF during prolonged salt loading in rats.

In this study, we evaluated the use of mouse preimplantation embryos as a model to study zinc deficiency-induced abnormal development. In Experiment 1, the effect of culture medium Zn concentrations on blastocyst development was studied. Preimplantation embryos (2 and 4 cells) obtained from superovulated females developed normally in media containing 0.7–30 μM Zn for up to 72 hr; higher levels of medium Zn resulted in abnormal development. In Experiment 2A, females were fed diets containing 50 (+Zn) or 0.4 (-Zn) μg Zn/g (760 vs 6 nmol/g, respectively) from 1 day before to 1 day after mating (3 days total). Preimplantation embryos were removed from the dams and cultured for 72 hr in 0.7 μM Zn medium. Embryos from the –Zn dams were morphologically normal at time zero; however, over the 72-hr period, these embryos tended to develop at a slower rate than controls, although compaction and cavitation frequency were similar. By the end of the 72-hr culture period, embryos from –Zn dams had significantly fewer cells than did embryos from control dams. In Experiment 2B, an extended period of maternal Zn deprivation (6 days) was used to investigate the potential for further impairment of in vitro preimplantation embryo development observed in Experiment 2A. Results from this experiment were consistent with those from Experiment 2A, in addition to providing evidence that the developmental progress of embryos obtained from mice fed Zn-deficient diets for 6 days was significantly impaired. In Experiment 3, the potential for supplemental Zn in culture medium to overcome the impairment in development due to maternal Zn deficiency was investigated. Embryos from female mice subjected to the same dietary regimen described in Experiment 2A were cultured to the blastocyst stage in medium containing Zn at a concentration of either 0.7 or 7.7 μM. Medium Zn supplementation did not improve development of embryos from dams fed Zn-deficient diets. In summary, embryos from mice fed –Zn diets for a 3-or 6-day period encompassing oocyte maturation and fertilization exhibited impaired development in vitro. This impairment was not overcome by medium Zn supplementation.

Malonyl-CoA, the inhibitor of carnitine palmitoyl transferase I, has been examined in this study in the muscle and liver of diabetic rats. Male Sprague-Dawley rats were rendered diabetic with streptozotocin (6 mg/100 g body wt). The gastrocnemius/plantaris muscles and liver samples were frozen at liquid nitrogen temperature. Muscle malonyl-CoA was 1.8 ± 0.2 pmol/mg in control rats and 1.5 ± 0.2 pmol/mg in the diabetic rats. This difference was not statistically significant. Liver malonyl-CoA of control rats was 8.6 ± 0.8 pmol/mg, in comparison to 4.3 ± 0.6 pmol/mg in diabetic rats. In the liver, high concentrations of malonyl-CoA inhibit fatty acid oxidation and ketogenesis. Failure of malonyl-CoA to decline in muscle in the diabetic may be responsible in part for the diversion of fatty acids to the liver, thereby enhancing hepatic fatty acid oxidation and ketogenesis.

Platelet-activating factor (PAF) is a powerful vasodilator with important effects on kidney function. It has been suggested that the renal effects of PAF are mediated by thromboxane A₂ (TxA₂). We examined the effect of PAF on renal function in sham-operated rats and rats that had undergone unilateral release of bilateral ureteral obstruction (BUO) of 24-hr duration, a condition in which the synthesis of TxA₂ is increased. To eliminate systemic hemodynamic changes, PAF was infused directly into the left renal artery using the lowest dose that affected renal function (2.3 × 10^–13 moles/min). Infusion of PAF significantly decreased the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in normal rats and rats with BUO. Normal (shamoperated) rats pretreated with an inhibitor of TxA₂ synthesis also had a significant decrease in GFR after administration of PAF (ERPF also decreased, but not significantly). Rats with BUO pretreated with an inhibitor of TxA₂ synthesis had significantly greater basal GFR and ERPF (increases of 72 and 171%, respectively) than untreated BUO rats. Administration of PAF to the former group further increased GFR and ERPF (by 37 and 39%, respectively; P < 0.001). The role of endogenous PAF was evaluated by administering a specific PAF receptor antagonist. Sham-operated rats pretreated with high doses of the PAF receptor antagonist had significantly higher mean arterial pressure values than normal untreated rats, and had no decrease in GFR and ERPF during PAF infusion. Rats with BUO pretreated with the PAF antagonist had a significant, dose-dependent decrease in basal GFR and ERPF. These data suggest that endogenous PAF has a vasodilatory role in obstructive nephropathy. No significant differences in eicos-anoid excretion in the urine corrected per GFR were observed during infusion of PAF in any of the groups examined. In BUO rats with intact TxA₂ synthesis, exogenous administration of PAF decreased renal function, presumably through further increases in the production of TxA₂. However, when TxA₂ production was inhibited, PAF administration increased GFR and ERPF, presumably due to its unopposed vasodilatory properties. The data suggest an important role of PAF in the hemodynamic changes seen in obstructive nephropathy.

The effect of acute alcohol infusion on the established suckling-induced prolactin surge in lactating rats was examined. Dams were implanted with an atrial catheter on Day 6 of lactation and blood sampling was done on Day 10. Following the separation of litters from dams for a 6-hr period, a baseline blood sample was removed via a catheter extension. Pups were weighed and returned to dams. Subsequent blood samples were obtained 10, 30, and 60 min after initiation of suckling. Dams were then infused with alcohol doses of 0, 0.5, 1.0, 2.0, or 2.5 g/kg body wt. Infusion (0.1 ml/min) was completed in approximately 30 min. Additional blood samples were obtained 10, 30, 60, and 120 min after the termination of infusion. In a separate group of rats, pups were removed from the dam after the first 60 min of suckling and additional blood samples were obtained 40, 70, 90, and 150 min after removal of pups (corresponding to 10-, 30-, 60-, and 120-min samples for rats infused with various alcohol doses). Alcohol, when administered after the establishment of suckling-induced prolactin surge and resulting in blood alcohol levels equal to or greater than legal human intoxication levels, inhibited prolactin release. However, continued suckling for an extended period (120 min in the present study) overcame this inhibitory effect, even when the blood alcohol level was comparable to (2.0 g/kg group) or greater than (2.5 g/kg group) the human legal intoxication level. Furthermore, in rats with established prolactin surges, the patterns of prolactin decline that followed alcohol administration or pup removal were comparable, indicating that similar mechanism(s) may be involved.

To investigate possible gender differences in the response of hepatic fatty acids and cytosolic fatty acid-binding capacity to ethanol consumption, both female and male rats (41 days of age) were pair fed liquid diets (with a littermate of the same sex) for 28 days. The diets contained 36% of energy either as ethanol or as additional carbohydrate. After ethanol feeding, the hepatic concentration of fatty acids increased 155% in females (P < 0.01), whereas there was only a trend for an increase (22%) in males. This was associated with a much smaller increase of cytosolic fatty acid-binding capacity in females (58%) than in males (161%). Whereas the ethanol-induced increase in fatty acid-binding capacity provided an ample excess of binding sites for the fatty acids in the males, the increase in females was barely sufficient for the binding of the large increase of fatty acids produced by ethanol in the females. The cytosolic protein responsible for this binding, the liver fatty acid-binding protein of the cytosol (L-FABP_c), also promotes esterification of the fatty acids. In keeping with the postulated role of this protein, the ethanol-induced increases in hepatic triacylglycerols, phospholipids, and cholesterol esters were smaller in females than in males. The gender difference in cholesterol esters was associated with parallel changes in acyl-CoA transferase activity. A possible implication of the relatively small and most likely inadequate increase in liver fatty acid-binding capacity and fatty acid esterification during alcohol consumption in the females is that under these circumstances the risk for development of a potentially deleterious accumulation of fatty acids in the liver is increased, thereby contributing to the enhanced vulnerability of females to alcohol-induced hepatotoxicity.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA In CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with ¹²⁵I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Incubations of rat anterior pituitary cells with transforming growth factor (TGF)-β1 for 48 hr suppressed the secretion of basal prolactin (PRL) in a dose-dependent manner (ED₅₀, 100 pg/ml). Activin, a gonadal hormone processing cysteine distribution similar to TGFβ, also suppressed basal PRL secretion, but it was less effective (ED₅₀, 4 ng/ml). Treatment with TGFβ1 significantly suppressed basal PRL secretion from the pituitary after 24 hr and up to 72 hr of incubation. TGFβ1 also inhibited thyrotropin-releasing hormone-mediated PRL secretion and activin inhibited thyrotropin-releasing hormone-mediated PRL secretion slightly, but significantly. In addition, we also measured the secretion of growth hormone by cultured pituitary cells treated with TGFβ1 or activin for 24 to 72 hr. TGFβ1 and activin showed an opposite effect on growth hormone secretion; TGFβ stimulated and activin inhibited basal secretion of growth hormone. These results suggest that TGFβ1 is a potent inhibitor of basal secretion of PRL by the pituitary, and both TGFβ1 and activin play a multifunctional role in basal secretion of pituitary hormones.

Previous studies showed that molecules of in vitro-cultured primary turkey kidney cells bound to 23-, 40-, and 60- to 65-kDa antigens of sodium dodecyl sulfate-solubilized sporozoites of Eimeria adenoeides. Similar binding to antigens of three other species of avian Eimeria, E. tenella, E. acervulina, and E. meleagrimitis, is now reported. Strips containing the most avidly bound sporozoite antigen (~40 kDa) were excised from the sodium dodecyl sulfate-polyacrylamide gels on which E. adenoeides antigens had been electrophoretically separated. The strips were homogenized and injected into mice to produce hybridoma cell lines. Twelve cell lines secreting monoclonal antibodies (McAb) that reacted with E. adenoeides sporozoites were detected. One of these McAb, H11C3, reacted with structures in the anterior tip of sporozoites of E. adenoeides and five other species of avian Eimeria. When included in the inoculation medium, this McAb significantly inhibited invasion of cultured kidney cells by sporozoites of E. adenoeides and E. tenella. In contrast, when the sporozoites were pretreated with McAb H11C3 and then washed free of the antibody, no inhibition of invasion was observed.

Cell swelling produced by a variety of techniques is a potent stimulus intensity-related inducer of an immediate secretory burst of thyroid-stimulating hormone (TSH) and prolaction (PRL) secretion from anterior pituitary cells. A 2-min “square wave” exposure to either hyposmolarity or isotonic urea induced stimulus intensity-correlated TSH and PRL secretory bursts peaking within 3 min, but the PRL zenith occurred 1 min later than that of TSH. With continuous exposure to these stimuli, TSH secretion rapidly decreased and remained only slightly above the unstimulated rate after 5 min. PRL secretion fell to and remained below the unstimulated level after 10 min. After stopping the stimulus, another secretory burst (“off” response) occurred with PRL, but not with TSH. A progressive “ramp” increase in stimulus intensity over 18 min induced a corresponding gradual increase in TSH secretion; there was a progressive depression, rather than increase, in PRL secretion during the stimulus ramp, with an off response secretory burst when the stimulus was discontinued. Removal of extracellular Ca²⁺ or addition of verapamil to the medium did not alter the dynamics of hyposmolarity-induced TSH secretion, but markedly altered those of PRL secretion; there was no off response PRL secretion and a hyposmolar ramp induced a corresponding gradual increase in PRL secretion, with a return to baseline after removing the stimulus. The dramatic qualitative differences in the response of the thyrotroph and lactotroph may reflect differences between the cell types in the size of secretory vesicles, membrane potential, the mechanism of exocytosis, and/or the role of Ca²⁺ influx across the plasmalemma.

The effect of trace elements lithium, selenium, and zinc on interleukin 2 (IL-2) production by peripheral blood mononuclear cells (PBMC), MLA144, and Jurkat cell lines has been studied. Lithium markedly enhanced IL-2 production by MLA144 and PBMC, but not by Jurkat. Selenium could only enhance IL-2 production by MLA144, whereas in none of these three systems was IL-2 production altered by zinc. The enhancing effect of lithium on IL-2 production showed some differences from that of tetradecanoylphorbol acetate (TPA) in the following aspects: (i) TPA could reverse the inhibitory effect of anti-CD2 monoclonal antibody on IL-2 production, whereas lithium could not; and (ii) lithium was unable to synergistically induce IL-2 production with anti-CD3 monoclonal antibody as TPA did. The effect of lithium on IL-2 production was in the early phase of lymphocyte activation. The addition of cholera toxin or theophylline to phytohemagglutinin-stimulated PBMC culture suppressed IL-2 production. However, IL-2 production could be restored by lithium. There was a corresponding increase in cAMP in cholera toxin or theophylline-treated PBMC, which could be reversed by lithium. Therefore, lithium restores IL-2 production via a decrease in cAMP.

The infusion of certain amino acids, such as serine, alanine, and proline (SAP), has been shown to increase the glomerular filtration rate, whereas branched chain amino acids (BCAA) leucine, isoleucine, and valine fail to modify the glomerular filtration rate. It has been suggested that this effect of amino acids on the glomerular filtration rate is mediated by the action of the hormone glomerulopressin. The purpose of this work was to study the action of SAP and BCAA on glomerulopressin production. Livers isolated from rats were perfused with (i) Krebs-Ringer-Bicarbonate, (ii) SAP, or (iii) BCAA. Results indicate that glomerulopressin production is stimulated by SAP, but inhibited by BCAA.

We reported previously that a transient occlusion followed by reperfusion of the portal vein and the hepatic artery of the rat significantly decreased the transhepatic transport of a cholephilic compound, and that this decrease was prevented by pretreating animals with poly(styrene co-maleic acid butyl ester)-conjugated superoxide dismutase (SM-SOD). To elucidate the mechanism for oxidative injury of the liver and the site for the generation of superoxide radicals, the effect of a portosystemic bypass on the liver function was examined in the rat whose hepatic vessels were temporarily occluded. A portosystemic bypass inhibited the reperfusion-induced decrease in hepatic transport of bromosulfophthalein as effectively as did SM-SOD. Kinetic analysis using ¹²⁵I-labeled albumin revealed that the permeability of the small intestine markedly increased after a transient occlusion. The increase in intestinal permeability was also inhibited either by SM-SOD or by the portosystemic bypass. Xanthine oxidase activity in portal plasma markedly increased during occlusion and reperfusion, while it remained within normal ranges in the bypassed group. Thus, superoxide radical, and/or its metabolite(s), might play a critical role in increasing the intestinal permeability and in the pathogenesis of reperfusion-induced liver injury.

Two experiments were conducted to determine the effect of free fatty acids (FFA) and glucose treatment on growth hormone (GH) and luteinizing hormone secretion in the pig. In Experiment (Exp) 1, 15 prepuberal gilts received an intravenous infusion of FFA (n = 5; 3 ml of 10% Liposyn II/kg), glucose (n = 5; 1 g/kg), or saline (n = 5; 3 ml of 0.9%/kg). Jugular blood samples were collected every 15 min for 2 hr before and 3 hr after intravenous infusion of saline, FFA, and glucose. Synthetic [Ala¹⁵]-h growth hormone-releasing factor-(1–29)NH₂ (1 μg/kg) and gonadotropin-releasing hormone (0.2 μg/kg) were administered 30 min after infusion (Time 0 = infusion). In Exp 2, eight prepuberal gilts received either FFA (n = 4) or saline (n = 4) as described in Exp 1, except that treatments were given every hour over a 10-hr period. Blood samples were collected every 15 min from 1 hr before to 10 hr after the start of FFA or saline infusion. In Exp 1, the peak GH response to growth hormone-releasing factor was delayed by 45 min (P < 0.01) by glucose treatment and suppressed (P < 0.01) by FFA treatment. The luteinizing hormone response to gonadotroph-releasing hormone was suppressed (P < 0.03) by glucose and enhanced (P < 0.03) by FFA. In Exp 2, the number of GH pulses was increased (P < 0.05) by FFA infusion and GH concentrations were positively correlated (r = 0.58, P < 0.0003) with FFA concentrations, while luteinizing hormone pulse amplitude was greater (P < 0.01) in FFA gilts than in saline gilts. These results indicate that FFA are more effective modulators of GH secretion than acute hyperglycemia, while metabolic status can alter pituitary responsiveness to gonadotropin-releasing hormone.

To validate that growth hormone (GH) and growth hormone-releasing hormone (GHRH) can be produced by leukocytes, we have assessed the presence of GH and GHRH-related mRNA in leukocyte cultures by reverse transcription and the polymerase chain reaction. A sample of the polymerase chain reactions were size-fractionated by electrophoresis in a 0.8% agarose gel and examined with ultraviolet light after ethidium bromide staining. Single major DNA bands corresponding in length to the distance between the 5'ends of the two GH and GHRH specific primers, 603 base pairs and 260 base pairs, respectively, were obtained. The DNA bands hybridized specifically to GH- and GHRH-specific probes after Southern transfer to nitrocellulose. The identity of the GH polymerase chain reaction material was confirmed by restriction enzyme analysis. The results showed that GH and GHRH gene expression occurs in mononuclear leukocytes and support the idea that these neuroendocrine hormones may be common signal molecules between the immune and neuroendocrine systems. [P.S.E.B.M. 1991, Vol 198]

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-IIe-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-IIe-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.

Recent work revealed that mice in which platelet function was inhibited by acetylsalicylic acid (ASA) treatment showed evidence of increased platelet production. It was proposed that poorly functioning platelets gave rise to elevated thrombocytopoiesis by causing the release and action of thrombopoietin. However, direct evidence is lacking. Therefore, in the work reported here, plasma from mice treated with ASA was injected into normal recipient mice in an attempt to document the existence of the humoral factor. Compared with control mice given normal plasma, the injection of mice with plasma from ASA-treated mice resulted in increased thrombocytopoiesis, as evidenced by significant increases in the percentage of ³⁵S incorporation into platelets, larger platelet size, and elevated megakaryocyte precursor cells (the small acetylcholinesterase-positive cell). For a positive control, additional mice were treated with plasma from animals made thrombocytopenic by an injection of antiplatelet serum. These mice also showed significant increases in thrombocytopoiesis. The results support the hypothesis that platelet production in ASA-treated mice is elevated by release and action of thrombopoietin.