Experimental Biology and Medicine

This article provides an update of a minireview published in 1996 (Abdel-Latif AA. Proc Soc Exp Biol Med 211:163–177, 1996), the purpose of which was to examine in nonvascular smooth muscle the biochemical and functional cross talk between the sympathetic nervous system, which governs the formation of cAMP and muscle relaxation, and the parasympathetic nervous system, which governs the generation of IP₃ and diacylglycerol, from the polyphosphoinositides, Ca²⁺ mobilization, and contraction. This review examines further evidence, both from nonvascular and vascular smooth muscle, for cross talk between the cyclic nucleotides, cAMP and cGMP via their respective protein kinases, and the Ca²⁺-dependent- and Ca²⁺-independent-signaling pathways involved in agonist-induced contraction. These include the IP₃-Ca²⁺-CaM- myosin light chain kinase (MLCK) pathway and the Ca²⁺-independent pathways, including protein kinase C-, MAP kinase-, and Rho-kinase. In addition, MLC phosphorylation and contraction can also be increased by a decrease in myosin phosphatase activity. A summary of the cross talk between the cyclic nucleotides and these signaling pathways was presented. In smooth muscle, there are several targets for cyclic nucleotide Inhibition and consequent relaxation, including the receptor, G proteins, phospholipase C-β1–4 Isoforms, IP₃ receptor, Ca²⁺ mobilization, MLCK, MAP kinase, Rho-kinase, and myosin phosphatase. While significant progress has been made in the past four years on this cross talk, the precise mechanisms underlying the biochemical basis for the cyclic nucleotide inhibition of Ca²⁺ mobilization and consequently muscle contraction remain to be established. Although it is well established that second-messenger cross talk plays an important role in smooth muscle relaxation, the many sources which exist in smooth muscle for Ca²⁺ mobilization, coupled with the multiple signaling pathways involved in agonist-induced contraction, contribute appreciably to the difficulties found by many investigators in identifying the targets for cyclic nucleotide inhibition and consequent relaxation. Better methodology and more novel interdisciplinary approaches are required for elucidating the mechanism(s) of cAMP- and cGMP-inhibition of smooth muscle contraction.

Increasing evidence suggests that superantigens play a role in Immune-mediated diseases. Superantigens are potent activators of CD4^* T cells, causing rapid and massive proliferation of cells and cytokine production. This characteristic of superantigens can be exploited in diseases where strong immunologic responses are required, such as in the B16F10 animal model of melanoma. Superantigen administration is able to significantly enhance Ineffective anti-tumor Immune responses, resulting in potent and long-lived protective anti-tumor immunity. However, superantigens are more well-known for the role they play in diseases. Studies using an animal model for neurologic demy-elinatlng diseases such as multiple sclerosis show that superantigens can induce severe relapses and activate auto-reactive T cells not involved in the Initial bout of disease. This may also involve epitope spreading of disease. Superantigens have also been implicated in acute diseases such as food poisoning and TSS, and in chronic diseases such as psoriasis and rheumatoid arthritis. Viral superantigens are also involved in the disease process, including superantigens derived from human Immunodeficiency virus and mouse mammary tumor virus. Finally, immunotherapies that ameliorate the role played by superantigens in disease are discussed.

Eosinophilie Myalgia Syndrome is a hypereosinophilic disorder that appears to result from the ingestion of the dietary supplement L-tryptophan by susceptible individuals. It is unclear if this disease results from tryptophan, contaminants found in tryptophan, individual predisposition (such as immune status and allergies), or some combination of effects. To evaluate effects of L-tryptophan on eosinophil migration, guinea pigs were compared with or without supplemental tryptophan (0. g/kg/day), with or without immune sensitization, and with or without immune challenge. Eosinophil counts were obtained from bone marrow, blood, lung, and bronchial alveolar lavage fluid (BAL). Lung cells were obtained to measure eotaxin concentrations in supernates and lysates with or without antigen and calcium ionophore challenge using direct ELISA. Skin biopsies were taken from both non-injected and antigen injection sites. The tryptophan supplemented, antigen-sensitized/antigen-challenged guinea pigs showed a significant decrease in blood eosinophils, compared to control (cellulose) supplemented antigen-sensitized/antigen-challenged guinea pigs [(0.086 ± 0.023) × 10⁶ vs (0.147 ± 0.021) × 10⁶ eoslnophils/ml recovered, respectively] with a significant increase in BAL eosinophils [(0.052 ± 0.008) × 10⁶ vs (0.033 ± 0.005) × 10^s eosinophils/ml recovered, respectively]. Unchallenged lung cell lysates from tryptophan-supplemented guinea pigs contained significantly less eotaxin compared to cellulose-supplemented guinea pigs regardless of whether they were sensitized (0.006 ± 0.002 vs 0.027 ± 0.008 ng/10⁶ cells, respectively). No differences were observed in skin biopsies between cellulose and tryptophan groups. These results suggest that L-tryptophan-supplemented guinea pigs have altered eotaxin regulation, a potential mechanism by which human overconsumption of tryptophan dietary supplements could lead to hypereosinophilic disorders in susceptible individuals.

Experimentally naive male Sprague Dawley rats (weighing 85–110 g) were used to examine the role of inducible nitric oxide synthase (iNOS) in cocaine-induced kindling. Repeated administration of cocaine (45 mg/kg, ip) to Sprague Dawley male rats for 7 consecutive days produced a progressive increase in the Convulsive responsiveness and death. Pretreatment with iNOS inhibitors, L-N⁶-(1-iminoethyl)lysine (NIL; 10 mg/kg, ip) and (–)-^ePigalloocatechin gallate (EGCG; 10 mg/kg, ip) 30 min before cocaine (45 mg/kg, ip) administration for 7 days attenuated the development of cocaine kindling and blocked cocaine-induced death. Results of NMDA receptor binding assay in the hippocampus showed a significant increase in the affinity without changes in the density in animals treated with cocaine, but there were no changes in these parameters in the cortex. Pretreatment with NIL or EGCG prior to cocaine administration abolished the cocaine-induced effect in the NMDA receptor affinity in the hippocampus. These results suggest that iNOS induction followed by an increase of NMDA receptor affinity in the hippocampus after repeated exposure to cocaine may participate in the process of the development of cocaine kindling.

The absorption, remodeling, and delivery of dietary lipids by intestinal cells are part of a complex multi-step process, the dynamics of which is influenced by the lipid composition of the diet and the physiological state of enterocytes. Emerging data indicate that, among the parameters known to modulate the cell functionality, the internal oxidative balance plays a pivotal role. In this study, we analyzed the effects of varying redox equilibria on the way in which the intestinal Caco-2 cell line utilize an otogenous lipid source such as oleic acid. Firstly, we manipulated the intracellular levels of soluble thiols (glutathione), and the amount of cell-associated products of lipid peroxidation, commonly regarded as two critical parameters characterizing the redox profile of the cells. Two different perturbants having opposite effects on the cell's redox profile were used: the pro-oxidizing agent CuSO₄ (2.5 and 10 µM) and the antioxidant and thiol supplier N-acetylcysteine (NAC, 2.5 and 5 mM). The influence of these mild but critical manipulations on the incorporation of oleate (50 and 500 µM) into cholesterol, triacylglycerol, end phospholipid was then evaluated. We found that the emerging pro-oxidant condition induced by CuSO₄ pre-exposure was associated with a significant up-regulation of phospholipid synthesis, while minor modifications were detected in that of triacylgiycerols. Conversely, when a more reducing state was induced by NAC pre-treatment, there was a significant down-regulation of triacylglycerol synthesis, with minor modifications in that of phospholipids. In addition, the incorporation of oleic acid in the cholesteryl ester fraction appeared to be unmodified under all the redox conditions reported. On the whole, these results indicate that the pre-existing internal redox potential of the enterocytes is a critical factor that is able to differentially modulate lipid synthesis at the Intestinal level. Thus, the adoption of a strategy designed to control/buffer the antioxidant capacity of the gastrointestinal tract could have important consequences for the modulation of lipid balance in the body.

Perinatal copper (Cu) deficiency was studied by offering pregnant Sprague Dawley rats a basal diet low in copper, 0.44 mg/kg, and drinking water containing 0 (–Cu) or 20 (+Cu) mg Cu/L as CuSO₄ starting at day 7 of gestation and continuing throughout lactation. To investigate dopamine-β-monooxygenase (DBM) and tyrosine monooxygenase (TM) in adrenal gland and brain, offspring were weaned at Day 21 to treatments of their respective dams for 9 days. Offspring, 30 days old, of Cu-deficient (–Cu) dams were smaller, anemic, and had biochemical features characteristic of severe Cu deficiency. Adrenal DBM enzyme activity of 30-day-old –Cu rats was 40% higher than Cu-adequate (+Cu) rats and DBM protein levels, estimated by Western immunoblot, were 45% higher. Adrenal DBM mRNA levels of –Cu rats were 108% higher than +Cu rats. Adrenal TM protein levels of –Cu rats were 39% higher than +Cu rats. Hypothalamus DBM activity was significantly higher in –Cu than +Cu rats but no reproducible changes in DBM or TM Protein levels could be detected by Western immunoblots. Diet history did not impact adrenal gland or hypothalamus levels of actin as detected on reblotted membranes. However, activity of the cuproenzyme Cu, Zn-superoxide dismutase was 50% lower and 30% lower, respectively, in extracts from rat adrenal gland and hypothalamus of –Cu than +Cu rats, indicating altered Cu status in the tissues studied. These data suggest that Cu deficiency is associated with increased formation of DBM and TM protein levels in adrenal gland. Further research will be required to determine the chemical signal responsible for this induction and if DBM or TM protein levels change in Other tissues.

Dehydroepiandrosterone (DHEA) is a precursor of sex hormones in mammals. Dietary DHEA serves to prevent or inhibit various diseases and also lengthens life spans of animals. Moreover, dietary DHEA inhibits food intake in certain strains of mice. We administered DHEA (0.45% w/w of food) to C57BL/6 (B6) and (B6 x DBA/2)F1 (BDF1) mice for 5 weeks. Food intake was inhibited in both strains of mice during the first week. Thereafter, B6, but not BDF1, mice consumed less food. Because hypothalamic serotonin and/or dopamine regulate appetite, satiety and other behaviors, the hypothesis tested was that hypothalamic concentration of serotonin, dopamine and/or their metabolites are affected differentially in B6 and BDF1 mice fed DHEA. In another study, mice were fed the AIN-76A diet with or without DHEA for 1 and 7 days or were pair-fed to DHEA-fed mice for 7 days. On Day 1 of DHEA feeding (acute effects) hypothalamic levels of serotonin, dopamine, and metabolites were unchanged in B6 mice, but levels of dopamine were increased and levels of dopamine metabolites were decreased in BDF1 mice. On Day 7 of DHEA feeding, levels of serotonin were increased in BDF1 but not B6 mice. On Day 7 of pair-feeding there were decreased levels of hypothalamic dopamine metabolites in BDF1 but not B6 mice. Paraventricular nuclei of BDF1 mice had decreased levels of serotonin but not of dopamine in all groups. Serum levels of DHEA and its metabolite, 5-androstene-3β,17β-diol, correlated significantly only with serotonin concentrations in BDF1 mice. The salient findings of these experiments are that DHEA inhibits food intake to a greater extent in B6 than in BDF1 mice. However, alterations of hypothalamic neurotransmitters were greater in BDF1 than in B6 mice. Because BDF1 and B6 mice share B6 genes, relevant gene(s) derived from DBA/2 mice might mediate the different responses detected.

Effects of a xenoblotic estrogen, bisphenol A (BPA), on reproductive functions were investigated using adult male rats. BPA was dissolved into sesame oil and injected sc every day (1 mg/rat) for 14 days. Animals were killed by decapitation after the final administration of BPA, and the trunk blood, pituitary, and testes were collected. Plasma concentrations of prolactin were dramatically Increased and pituitary contents of prolactin were slightly increased in the BPA group compared to the control group. Plasma concentrations of testosterone were decreased and plasma concentrations of LH were increased in BPA-treated rats compared to control rats. Testicular contents of inhibin were decreased in BPA-treated rats compared to control rats, although plasma concentrations of inhibin were not changed after administration of BPA. The testicular response to hCG for progesterone and testosterone release was decreased in BPA-treated rats. Administration of BPA did not change the pituitary response to luteinizing hormone-releasing hormone (LH-RH) in castrated male rats treated with testosterone. Male sexual behavior also was not changed as a result of BPA treatment. These results suggest that BPA directly inhibits testicular functions and the increased level of plasma LH is probably due to a reduction in the negative feedback regulation by testosterone. The testis is probably a more sensitive site for BPA action than the hypothalamus–pitultary axis.

Copper (Cu) deficiency suppresses macrophage activities in animals and humans. Our previous studies indicated that the Induction of Cu deficiency in differentiated U937 monocytic cells impairs respiratory burst and bactericidal activities and lipopolysaccharide-mediated secretion of inflammatory mediators. The current investigation examined the roles of Cu in the monocytic differentiation process. Human U937 promonocytic cells were exposed to a high affinity Cu chelator (5 µM 2,3,2-tetraamine [tet]) for 24 hr before inducing differentiation by treatment with 1,25-dihydroxyvitamin D₃ plus Interferon-γ (DI). This procedure decreased cell Cu by 55% without compromising cellular Zn, Fe, or general metabolic activities. Lower Cu status significantly attenuated the expression of maturation markers Mac-1 (CD11b), ICAM-1 (CD54), and LPS-R (CD14). This change was associated with a marked suppression in respiratory burst activity and killing of Salmonella. To examine if the adverse effect of inadequate Cu on the DI-induced differentiation represented a more general defect, U937 cells were treated with phorbol 12-myrlstate 13-acetate (PMA). Lower Cu status also suppressed PMA-mediated differentiation of U937 cells. Supplemental Cu, but not Zn or Fe, blocked the tet-induced declines in cell Cu, expression of maturation markers, and respiratory burst and bactericidal activities. These results demonstrate that Cu is essential for the monocytic differentiation process that contributes to the competency of the host's defense system.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially Inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca²⁺ flux, postulated to be a consequence of the release of ATP-dependent Ca²⁺ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for Its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca²⁺ flux and decrease ATP levels in Infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.