Retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells.

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Abstract

The regulated expression of cell adhesion molecules (CAM) on endothelial cells is central to the pathogenesis of various inflammatory processes. Retinoic acid and synthetic derivatives have been demonstrated to exert antiinflammatory effects in cutaneous diseases. To determine modes of retinoid action in the modulation of inflammatory responses, we explored effects of all-trans-retinoic acid (t-RA) on the TNFalpha-induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in cultured human dermal microvascular endothelial cells. Pretreatment with t-RA specifically prevented TNFalpha-induced VCAM-1 expression, but not ICAM-1 and E-selectin induction. t-RA significantly reduced VCAM-1-dependent T cell binding to TNFalpha-treated human dermal microvascular endothelial cells as well. This differential modulation of TNFalpha-induced CAM expression by t-RA was reflected at steady state mRNA levels and in nuclear run-on studies. In transcriptional activation studies, the TNFalpha-mediated activation of the human VCAM-1 promoter was inhibited after t-RA treatment, while the ICAM-1 promoter activation was unaffected, indicating that the selective inhibition of CAM expression is regulated in part at the level of gene transcription. Furthermore, the transcriptional inhibition by t-RA appears to be mediated by its effects upon the activation of NF-kappaB-dependent complex formation. Analysis of protein-DNA binding assays revealed marked inhibition of specific NF-kappaB-dependent binding to the tandem NF-KB sites of the VCAM-1 promoter, but not to the functional NF-kappaB motif of the ICAM-1 promoter. The specific inhibition of cytokine-mediated VCAM-1 gene expression in vitro may provide a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo.

Authors

J Gille, L L Paxton, T J Lawley, S W Caughman, R A Swerlick

The exchange between proglycogen and macroglycogen and the metabolic role of the protein-rich glycogen in rat skeletal muscle.

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Abstract

The aim of this study is to determine if proglycogen and macroglycogen are kinetically related in rat skeletal muscle. Eight groups of anesthetized fasted rats (seven hepatic-occluded and one nonoccluded) were intravenously infused with [3-3H]glucose at a rate of 1.7 microCi x min(-1) for 20 min. At the end of infusion, hindlimb muscles were excised and rapidly frozen in liquid nitrogen. Proglycogen was extracted by precipitation in 10% TCA; and macroglycogen as a part of total glycogen by precipitation in 20% KOH-65% ethanol. Along with the tracer, the occluded rats were also infused with: saline (group 1); insulin at rates ranging from 5 to 50 mU x min(-1) (groups 2 to 5); and insulin at a rate of 10 mU x min(-1) plus glucose at rates of 10.2 and 20.4 micromol x min(-1), respectively (groups 6 and 7). The infusion regimens resulted in up to 30-fold difference in whole-body glucose utilization among the rats. In the rats infused with saline and insulin at a rate of 5 mU x min(-1), [3H]glucose was found to be exclusively incorporated into proglycogen. Incorporation into macroglycogen was found in the rats infused with insulin at rates > 10 mU x min(-1). Supplementary glucose infusion increased the synthesis of [3H]proglycogen (four- to sixfold), and equilibrated the two extractable forms of glycogen in the insulin-infused rats. In the saline-infused nonoccluded rats, only proglycogen was found to be labeled. In conclusion, our data indicate that in the intact and hepatic-occluded rats, proglycogen in the skeletal muscles may undergo synthesis and degradation of its own more readily than exchange between itself and depot macroglycogen.

Authors

M Huang, C Lee, R Lin, R Chen

A cholecystokinin-releasing factor mediates ethanol-induced stimulation of rat pancreatic secretion.

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Abstract

The mechanisms by which short-term ethanol administration alters pancreatic exocrine function are unknown. We have evaluated the effects of ethanol administration on pancreatic secretion of digestive enzymes. In our studies, anesthetized as well as conscious rats were given ethanol at a rate sufficient to cause the blood ethanol concentration to reach levels associated with clinical intoxication. Ethanol was administered over a 2-h period during which blood ethanol levels remained stably elevated. We report that intravenous administration of ethanol results in a transient increase in pancreatic amylase output and plasma cholecystokinin (CCK) levels. The ethanol-induced increase in amylase output can be completely inhibited by the CCK-A receptor antagonist L-364,718 and partially inhibited by the muscarinic cholinergic antagonist atropine. The ethanol-induced rise in amylase output can be completely prevented by instillation of trypsin into the duodenum or by lavage of the duodenum with saline during ethanol administration. Furthermore, the intraduodenal activity of a CCK-releasing factor is increased by infusion of ethanol. These studies indicate that administration of ethanol causes rat pancreatic exocrine secretion to increase. This phenomenon is mediated by a trypsin-sensitive CCK-releasing factor which is present within the duodenal lumen. These observations lead us to speculate that repeated CCK-mediated ethanol-induced stimulation of pancreatic digestive enzyme secretion may play a role in the events which link ethanol abuse to the development of pancreatic injury.

Authors

A K Saluja, L Lu, Y Yamaguchi, B Hofbauer, M Rünzi, R Dawra, M Bhatia, M L Steer

Activation of large conductance potassium channels inhibits the afferent and efferent function of airway sensory nerves in the guinea pig.

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Abstract

Sensory nerves play an important role in airway disease by mediating central reflexes such as cough, and local axon reflexes resulting in the peripheral release of neuropeptides. We have tested whether the benzimidazolone compound, NS1619, an opener of large conductance calcium-activated potassium (BK Ca) channels, inhibits the activity of sensory fibers, and central and local airway reflexes in guinea pig airways. In in vitro single fiber recording experiments, NS1619 applied to identified receptive fields in the trachea inhibited the firing of A(delta)-fibers evoked by hypertonic saline and distilled water, and bradykinin-evoked firing of C-fibers. Electrically evoked nonadrenergic noncholinergic contractions of isolated bronchi mediated by the release of neurokinin A (NKA) from C-fibers, but not those elicited by exogenous NKA, were inhibited by NS1619. These effects of NS1619 were prevented by iberiotoxin, a selective blocker of BK Ca channels. In conscious guinea pigs, cough evoked by aerosolized citric acid was also inhibited by NS1619. These data show that BK Ca channel activation inhibits sensory nerve activity, resulting in a reduction of both afferent and efferent function. BK Ca channel openers may therefore be of potential benefit in reducing neurogenic inflammation and central reflexes seen during inflammatory conditions of the airways, and may represent a new class of antitussive drug.

Authors

A J Fox, P J Barnes, P Venkatesan, M G Belvisi

Alternative expression of platelet glycoprotein Ib(beta) mRNA from an adjacent 5' gene with an imperfect polyadenylation signal sequence.

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Abstract

Glycoprotein (GP) Ib is a major component of the platelet membrane receptor for von Willebrand factor, designated the GP Ib-IX-V complex. GP Ib is composed of two subunits (GP Ib(alpha) and GP Ib(beta)) each synthesized from separate genes. The 206 amino acid precursor of GP Ib(beta) is synthesized from a 1.0-kb mRNA expressed by megakaryocytes and was originally characterized from cDNA clones of human erythroleukemic (HEL) cell mRNA, a cell line exhibiting megakaryocytic-like properties. The cell line CHRF-288-11 also exhibits megakaryocytic-like properties, but synthesizes two related GP Ib(beta) mRNA species of 3.5 and 1.0 kb. We performed cDNA cloning experiments to identify the origin of the 3.5-kb transcript and determine its relationship to the 1.0-kb GP Ib(beta) mRNA found in megakaryocytes, platelets, and HEL cells. Our cloning experiments demonstrate that the longer transcript results from a nonconsensus polyadenylation recognition sequence, 5'AACAAT3', within a separate gene located upstream to the platelet GP Ib(beta) gene. In the absence of normal polyadenylation the more 5' gene uses the polyadenylation site within its 3' neighbor, the platelet GP Ib(beta) gene. This newly identified 5' gene contains an open reading frame encoding 369 amino acids with a high degree of sequence similarity to an expanding family of GTP-binding proteins.

Authors

B Zieger, Y Hashimoto, J Ware

Neutrophil margination, sequestration, and emigration in the lungs of L-selectin-deficient mice.

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Abstract

These studies tested the hypothesis that L-selectin plays a role in neutrophil traffic in the lungs, particularly in neutrophil margination, sequestration, and emigration, using L-selectin-deficient mice. No defect in neutrophil margination within either capillaries or arterioles and venules was observed in uninflamed lungs of L-selectin-deficient mice. The initial rapid sequestration of neutrophils within the pulmonary capillaries 1 min after intravascular injection of complement fragments was not prevented. In contrast, L-selectin did contribute to the prolonged neutrophil sequestration (> or = 5 min). Interestingly, neutrophil accumulation within noncapillary microvessels required L-selectin at both 1 and 5 min after complement injection. During bacterial pneumonias, L-selectin played a role in neutrophil accumulation within noncapillary microvessels in response to either Escherichia coli or Streptococcus pneumoniae and within capillaries in response to E. coli but not S. pneumoniae. However, L-selectin was not required for emigration of neutrophils or edema in response to either organism. These studies demonstrate a role for L-selectin in the prolonged sequestration of neutrophils in response to intravascular complement fragments, in the intracapillary accumulation of neutrophils during E. coli-induced pneumonia, and in the accumulation of neutrophils within noncapillary microvessels when induced by either intravascular complement fragments or

Authors

N A Doyle, S D Bhagwan, B B Meek, G J Kutkoski, D A Steeber, T F Tedder, C M Doerschuk

Differentiation of glucose toxicity from beta cell exhaustion during the evolution of defective insulin gene expression in the pancreatic islet cell line, HIT-T15.

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Abstract

Chronic exposure of HIT-T15 cells to supraphysiologic glucose concentration diminishes insulin gene expression and decreased binding of two critical insulin gene transcription factors, STF-1 and RIPE-3b1 activator. To distinguish whether these changes are caused by glucose toxicity or beta cell exhaustion, HIT-T15 cells grown from passage 75 through 99 in media containing 11.1 mM glucose were switched to 0.8 mM glucose at passage 100. They regained binding of STF-1 and RIPE-3b1 activator and had a partial but minimal return of insulin mRNA expression. In a second study, inclusion of somatostatin in the media-containing 11.1 mM glucose inhibited insulin secretion; however, despite this protection against beta cell exhaustion, dramatic decreases in insulin gene expression, STF-1 and RIPE-3b1 binding, and insulin gene promoter activity still occurred. These data indicate that the glucotoxic effects caused by chronic exposure to supraphysiologic concentration of glucose are only minimally reversible and that they are not due simply to beta cell exhaustion. These observations carry with them the clinical implication that Type II diabetic patients who remain hyperglycemic for prolonged periods may have secondary glucose toxic effects on the beta cell that could lead to defective insulin gene expression and worsening of hyperglycemia.

Authors

A Moran, H J Zhang, L K Olson, J S Harmon, V Poitout, R P Robertson

Nitric oxide (NO) modulates the neurogenic control of blood pressure in rats with chronic renal failure (CRF).

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Abstract

Increased sympathetic nervous system (SNS) activity plays a role in the genesis of hypertension in rats with chronic renal failure (CRF). Because nitric oxide (NO) modulates the activity of the SNS, a deficit of NO synthesis could be responsible for the increased SNS activity in these animals. In the present study, we evaluated the effects of L-arginine and L-NAME on blood pressure and SNS activity-in Sprague Dawley 5/6 nephrectomized or sham-operated rats. SNS activity was determined by measuring norepinephrine turnover rate in several brain nuclei involved in the regulation of blood pressure. In the same brain nuclei, we measured NO content and nitric oxide synthase (NOS) gene expression by semiquantitative measurements of NOS mRNA reverse transcription polymerase chain reaction. In CRF rats, norepinephrine turnover rate was increased in the posterior hypothalamic nuclei, locus coeruleus, paraventricular nuclei, and the rostral ventral medulla, whereas NOS mRNA gene expression and NO2/NO3 content were increased in all brain nuclei tested. L-NAME increased blood pressure and NE turnover rate in several brain nuclei of both control and 5/6 nephrectomized rats. In CRF rats, a significant relationship was present between the percent increment in NOS mRNA gene expression related to the renal failure, and the percent increase in norepinephrine turnover rate caused by L-NAME. This suggests that endogenous NO may partially inhibit the activity of the SNS in brain nuclei involved in the neurogenic regulation of blood pressure, and this inhibition is enhanced in CRF rats. In summary, the increase in SNS activity in the posterior hypothalamic nuclei and in the locus coeruleus of CRF rats is partially mitigated by increased local expression of NOS m-RNA.

Authors

S Ye, S Nosrati, V M Campese

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

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Abstract

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.

Authors

G Tiao, S Hobler, J J Wang, T A Meyer, F A Luchette, J E Fischer, P O Hasselgren

Shared gamma(c) subunit within the human interleukin-7 receptor complex. A molecular basis for the pathogenesis of X-linked severe combined immunodeficiency.

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Abstract

Genetic evidence suggests that mutations in the gamma(c) receptor subunit cause X-linked severe combined immunodeficiency (X-SCID). The gamma(c) subunit can be employed in receptor complexes for IL-2, -4, -7, -9, and -15, and the multiple signaling defects that would result from a defective gamma(c) chain in these receptors are proposed to cause the severe phenotype of X-SCID patients. Interestingly, gene disruption of either IL-7 or the IL-7 receptor (IL-7R) alpha subunit in mice leads to immunological defects that are similar to human X-SCID. These observations suggest the functional importance of gamma(c) in the IL-7R complex. In the present study, structure/function analyses of the IL-7R complex using a chimeric receptor system demonstrated that gamma(c) is indeed critical for IL-7R function. Nonetheless, only a limited portion of the cytoplasmic domain of gamma(c) is necessary for IL-7R signal transduction. Furthermore, replacement of the gamma(c) cytoplasmic domain by a severely truncated erythropoeitin receptor does not affect measured IL-7R signaling events. These findings support a model in which gamma(c) serves primarily to activate signal transduction by the IL-7R complex, while IL-7R alpha determines specific signaling events through its association with cytoplasmic signaling molecules. Finally, these studies are consistent with the hypothesis that the molecular pathogenesis of X-SCID is due primarily to gamma(c)-mediated defects in the IL-7/IL-7R system.

Authors

S Y Lai, J Molden, M A Goldsmith