Proceedings of the Society for Experimental Biology and Medicine最新文献

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.

Colonies of house mice reach maximum population density in 120-180 days, irrespective of cage size and initial number of colonizing animals. Reproduction ceases because the females become aggressive and unreceptive to mating. The aggressive behavior is correlated with elevated levels of testosterone (T) and corticosterone (B) (Chapman et al., Phys Behav 64:529-533, 1998). In two of seven strains of mice, females developed ovarian lesions. The occurrence of the lesion in one strain was correlated with the age of the animal and duration of the study. In the second strain, cage size was the determining factor. Lesioned ovaries weighed significantly more than nonlesioned ovaries. The lesion consisted of accumulations of luteal membrane and organelle fragments, and other cellular debris, suggestive of incomplete and prolonged luteolysis. Electron microscopic (EM) analyses revealed the presence of deposits of permanganate-resistant congophilic amyloid fibrils in the intima and smooth muscle cells of luteal thecal arteries. Population females had thymus glands and uteri that weighed significantly less than the same organs from females housed in the breeding colony, whereas the adrenal glands from the population females weighed significantly more. It is proposed that the female aggression is due to high levels of T. It is also proposed that the high levels of B suppress the immune cells involved in normal luteolysis and contribute to the incomplete and prolonged luteolysis.

Previously, we have found that acetaldehyde, a volatile component of cigarette smoke, stimulates the protein kinase C (PKC) pathway and inhibits ciliary motility. A "smokeless" cigarette (Eclipse) now exists in which most of the tobacco is not burned, reducing the pyrolyzed components in the extract. We hypothesized that acetaldehyde is a component of cigarette smoke that activates PKC in the airway epithelial cell, and therefore the Eclipse cigarette would not activate epithelial cell PKC. In this study, bovine bronchial epithelial cells (BBEC) were incubated with cigarette smoke extract (CSE) or Eclipse smoke extract (ESE). We found that PKC activity was significantly higher in cells exposed to 5% CSE than cells exposed to 5% ESE or media. When acetaldehyde levels of both extracts were measured by gas chromatography, CSE was found to have 15-20 times greater concentration (microM) of acetaldehyde than ESE. When BBEC were treated with 5% CSE, ciliary beating was further decreased from baseline levels. This decrease in ciliary beating was not observed in cells treated with ESE, suggesting that acetaldehyde contained in CSE slows cilia. These results suggest that volatile components such as acetaldehyde in cigarette smoke may inhibit ciliary motility via a PKC-dependent mechanism.

Many advances have been made in the last few years concerning our understanding of the receptors and ligands composing the cannabinoid system. Likewise, the science surrounding cytokine biology has advanced enabling us to measure these proteins more precisely as well as understand and interpret the meaning of changes in their levels. Scientists wishing to study the health consequences of smoking marijuana as well as understand the possible role of endogenous cannabimimetic ligands in immune regulation have continued to study the influence of these substances on the regulation and development of the cytokine network. Research has shown that two major cannabinoid receptor subtypes exist and that subtype 1 (CB1) is expressed primarily in the brain whereas subtype 2 (CB2) is expressed primarily in the periphery. A variety of ligands for these receptors based on the cannabinoid structure have been synthesized and studied as well as low affinity compounds, noncannabinoid ligands, and endogenous ligands derived from fatty acid eicosanoids. Highly selective receptor antagonists have also been introduced and studied. Synthetic, low affinity ligands such as (+)-HU-211 and DMH-11C have been shown to cause anti-inflammatory effects possibly through inhibiting the production and action of TNF-alpha and other acute phase cytokines. In addition, suppression of TNF and other cytokines such as GM-CSF, IL-6, IFNgamma, and IL-12 has also been seen following exposure to high affinity and psychoactive ligands such as marijuana and THC. However, some of these ligands have also been shown to increase rather than decrease interleukins such as IL-1, IL-4, IL-10, and IL-6, cytokines such as TNF-alpha, and chemokines such as IL-8, MIP-1, and RANTES. The endogenous ligand, anandamide, has been shown in culture to either suppress the proliferation response to prolactin or enhance the response to cytokines such as IL-3 and IL-6. This eicosanoid has also been shown to increase the production of interleukins and other cytokines. Cannabinoid receptors have been shown to be involved in some but not all of these effects. It is clear that psychoactive and nonpsychoactive compounds have demonstrated effects in vivo and in vitro on the production and function of a variety of cytokines. Depending upon the model system, these effects are often conflicting, and the involvement of cannabinoid receptors is unclear. However, enough evidence exists to suggest that the cannabinoid system significantly impacts the functioning of the cytokine network, and this association may provide clues to the mechanisms of certain immune diseases and form the basis for new immunotherapies.

Ionizing radiation is classified as a potent carcinogen, and its injury to living cells is, to a large extent, due to oxidative stress. The molecule most often reported to be damaged by ionizing radiation is DNA. Hydroxyl radicals (*OH), considered the most damaging of all free radicals generated in organisms, are often responsible for DNA damage caused by ionizing radiation. Melatonin, N-acetyl-5-methoxytryptamine, is a well-known antioxidant that protects DNA, lipids, and proteins from free-radical damage. The indoleamine manifests its antioxidative properties by stimulating the activities of antioxidant enzymes and scavenging free radicals directly or indirectly. Among known antioxidants, melatonin is a highly effective scavenger of *OH. Melatonin is distributed ubiquitously in organisms and, as far as is known, in all cellular compartments, and it quickly passes through all biological membranes. The protective effects of melatonin against oxidative stress caused by ionizing radiation have been documented in in vitro and in vivo studies in different species and in in vitro experiments that used human tissues, as well as when melatonin was given to humans and then tissues collected and subjected to ionizing radiation. The radioprotective effects of melatonin against cellular damage caused by oxidative stress and its low toxicity make this molecule a potential supplement in the treatment or co-treatment in situations where the effects of ionizing radiation are to be minimized.

A dissociation between plasma luteinizing hormone (LH) and testosterone (T) appears to exist during exposure to altered gravity. The pulsatile nature of LH release and the diurnal variability of T secretion may mask or bias the effects of altered gravity on the pituitary-gonadal axis when analyzing plasma concentrations. Therefore, we examined the relationship between the excretion of urinary LH and T in male Sprague-Dawley rats during exposure to increased gravity upon return to Earth following a 14-day spaceflight (n = 6) and by 12 days of centrifugation at 2g (n = 8). Excreted LH and T were elevated on the first 3 days postflight. Excreted T was elevated between Days 1 and 8 of centrifugation; however, excreted LH was reduced on Days 2 and 3 compared with control animals. Excreted LH and T were significantly correlated (R = 0.731 and 0.706, respectively) in postspaceflight and centrifuged animals. Correlation curves had similar slopes (0.0213 and 0.023, respectively), but different y-intercepts (-1.43 and 3.32, respectively). The sustained increase in excreted T during centrifugation suggests that the pituitary-gonadal axis in postspaceflight animals may adapt quicker to increased gravity. The upward shift in the correlation curve exhibited by the centrifuged animals suggests that the sensitivity of LH-induced T release is increased in these animals. The previous dissociation between plasma LH and T during altered gravity was not observed in the present study in which excreted LH and T were measured.

This laboratory has previously described a method of preventing air-space enlargement in experimental pulmonary emphysema using aerosolized hyaluronan (HA). Although it was found that HA preferentially binds to elastic fibers (which undergo breakdown by elastases in emphysema), it remains to be shown that such attachment actually prevents damage to the fibers. In the current study, cell-free radiolabeled extracellular matrices, derived from rat pleural mesothelial cells, were used to test the ability of low molecular weight ( approximately 100 kDa) streptococcal HA to prevent elastolysis. Coating the matrices with HA significantly decreased elastolysis (P<0.05) induced by porcine pancreatic elastase (43%), human neutrophil elastase (53%), and human macrophage metalloelastase (80%). Concomitant in vivo studies examined the ability of an aerosol preparation of the streptococcal HA to prevent experimental emphysema induced by intratracheal administration of porcine pancreatic elastase. As seen with earlier studies involving bovine tracheal HA, a single aerosol exposure significantly decreased elastase-induced airspace enlargement, as measured by the mean linear intercept (107.5 vs 89.6 microm; P < 0. 05). Furthermore, repeated exposure to the HA aerosol for 1 month did not reveal any morphological changes in the lung. The results provide further evidence that aerosolized HA may be an effective means of preventing pulmonary emphysema and perhaps other lung diseases that involve elastic fiber injury.

The purpose of this study was to assess the participation of the atrial natriuretic peptide (ANP)-cGMP system in electrolyte and volume handling of cholestatic rats submitted to an acute oral sodium load. Cholestasis was induced by ligation and section of the common bile duct (n = 51). Control rats were sham operated (n = 56). Three weeks after surgery, 24-hr urinary volume, sodium, potassium, cGMP and creatinine excretion were measured. Three days later, animals received 10 mmol/kg NaCl (1 M) by gavage, and urinary excretion was measured for 6 hr. In parallel groups of rats, plasma volume, electrolytes and ANP concentration, extracellular fluid volume (ECFV), and renal medullary ANP-induced cGMP production were determined in basal conditions or 1 hr after oral sodium overload. As compared with controls, cholestatic rats had a larger ECFV and higher plasma ANP (67.2 +/- 5.2 vs 39.7 +/- 3.5 pg/ml), but lower hematocrit and blood volume, and were hyponatremic. Cholestatic rats showed higher basal excretion of sodium, potassium, and volume than controls, but equal urinary cGMP. After the NaCl overload, cholestatic rats showed a reduced sodium excretion but equal urinary cGMP. One hr after sodium overload, both groups showed hypernatremia, but whereas in control rats ECFV and ANP increased (50.7 +/- 4.1 pg/ml), in cholestatic rats ECFV was unchanged, and plasma volume and ANP were reduced (37.5 +/- 5.8 pg/ml). ANP-induced cGMP production in renal medulla was similar in cholestatic and control nonloaded rats (14.2 +/- 5.2 vs 13.4 +/- 2.6 fmol/min/mg). One hr after the load, medullary cGMP production rose significantly in both groups, without difference between them (20.6 +/- 3.1 vs 22.7 +/- 1. 7 fmol/min/mg). We conclude that the blunted excretion of an acute oral sodium load in cholestatic rats is associated with lower plasma ANP due to differences in body fluid distribution and cannot be explained by renal refractoriness to ANP.

Blood plasma was incubated with 50 mM AAPH [2, 2'-azobis-(2-amidinopropane) hydrochloride] in the absence or presence of catechins (5-100 microM). Lipid oxidation was evaluated by measuring the formation of 2-thiobarbituric acid reactive substances (TBARS). The concentration of alpha-tocopherol (AT), beta-carotene (BC), ascorbic acid (AA), and catechins was determined by reverse phase high performance liquid chromatography (HPLC) with electrochemical detection. All the assayed catechins inhibited plasma TBARS formation. Based on the calculated IC50, the order of effectiveness was: epicatechin gallate (ECG) > epigallocatechin gallate (EGCG) > epigallocatechin (EGC) > epicatechin (EC) > catechin (C). Catechins protected plasma AT and BC from AAPH-mediated oxidation. The order of effectiveness for AT protection was ECG > EGCG > EC = C > EGC; and for BC protection, the order was EGCG > ECG > EGC > > EC > C. The addition of catechins modified the kinetics of TBARS formation and AT depletion, but the rate of AA depletion was not affected. Catechin oxidation did not start until the complete depletion of AA, and it preceded AT depletion. These results indicate that catechins are effective antioxidants in human blood plasma, delaying the lipid oxidation and depletion of endogenous lipid-soluble antioxidants (AT and BC).