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Human ether-a-go-go-related gene (HERG, Kv11.1, KCNH2) voltage-gated [K.sup.+] channels dominate cardiac action potential repolarization. In addition, HERG channels play a role in neuronal and smooth cell excitability as well as cancer pathology. Extracellular pH ([pH.sub.o]) is modified during myocardial ischemia, inflammation, and respiratory alkalosis, so understanding the response of HERG channels to changes in pH is of clinical significance. The relationship between [pH.sub.o] and HERG channel gating appears complex. Acidification has previously been reported to speed, slow, or have no effect on activation. We therefore undertook comprehensive analysis of the effect of [pH.sub.o] on HERG channel activation. HERG channels have unique and complex activation gating characteristics with both voltage-sensitive and voltage-insensitive steps in the activation pathway. Acidosis decreased the activation rate, suppressed peak current, and altered the sigmoidicity of gating near threshold potentials. At positive voltages, where the voltage-insensitive transition is rate limiting, [pH.sub.o] modified the voltage-insensitive step with a pKa similar to that of histidine. Hill coefficient analysis was incompatible with a coefficient of 1 but was well described by a Hill coefficient of 4. We derived a [pH.sub.o]-sensitive term for a five-state Markov model of HERG channel gating. This model demonstrates the mechanism of [pH.sub.o] sensitivity in HERG channel activation. Our experimental data and mathematical model demonstrate that the [pH.sub.o] sensitivity of HERG channel activation is dominated by the [pH.sub.o] sensitivity of the voltage-insensitive step, in a fashion that is compatible with the presence of at least one proton-binding site on each subunit of the channel tetramer. voltage-gated channels; potassium channels; gating kinetics; Kv11.1; human ether-a-go-go gene doi: 10.1152/ajpheart.01246.2009.

The transcription factor carbohydrate response element-binding protein (ChREBP) mediates insulin-independent, glucose-stimulated gene expression of multiple liver enzymes responsible for converting excess carbohydrate to fatty acids for long-term storage. To investigate ChREBP's role in the development of obesity and obesity-associated metabolic dysregulation, ChREBP-deficient mice were intercrossed with ob/ob mice. As a result of deficient leptin expression, ob/ob mice overeat, become obese and resistant to insulin, and display marked elevations in hepatic lipogenesis, gluconeogenesis, and plasma glucose and triglycerides, mRNA expression of all hepatic lipogenic enzymes was significantly lower in ob/ob-ChREB[P.sup.-/-] than in ob/ob mice, resulting in decreased hepatic fatty acid synthesis and normalization of plasma free fatty acid and triglyceride levels. Overall weight gain in addition to adiposity was reduced in the doubly deficient mice. The former was largely attributable to decreased food intake and may result from decreased hypothalamic expression of the appetite-stimulating neuropeptide agouti-related protein, mRNA expression and activity of gluconeogenic enzymes also was lower in the doubly deficient mice, contributing to significantly lower blood glucose levels. The results of this study suggest that inactivation of ChREBP expression not only reduces fat synthesis and obesity in ob/ob mice but also results in improved glucose tolerance and appetite control. carbohydrate response element-binding protein doi:10.1152/ajpendo.00027.2006

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682-1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region -904/-883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the -893/-888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the -893/-888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1[beta] (IL-1[beta]) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the -893/-888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1[beta]-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the -904/-883 region augmented IL-1[beta] stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at -893/-888 serves to trans-repress basal expression and IL-1[beta] induction of the iNOS promoter. transcription; glomerulus; inducible nitric oxide synthase; promoter; inflammatory cytokines; transcription factors; upstream stimulatory factor

To better understand the biology of leptin during prenatal life, the developmental and spatial regulation of leptin was studied in ovine fetuses. Fetal plasma leptin increased steadily between days 40 and 143 postcoitus (PC), but it was unrelated to fetal weight or placental weight at day 135 PC. Leptin gene expression was detected in fetal brain and liver during most of gestation and in fetal adipose tissue after day 100 PC. At day 130 PC, expression in fetal perirenal adipose tissue was ~10% of maternal expression. In contrast, leptin gene expression was never detected in the placenta and other uteroplacental tissues. When ewes were fed 55% of requirements between days 122 and 135 PC, fetal plasma leptin remained constant despite acute reduction in maternal concentration. We conclude that fetal plasma leptin originates mostly from nonadipose tissue in early pregnancy and, in addition, from fetal adipose tissue near term. The role of fetal plasma leptin remains uncertain given the lack of nutritional regulation and association with fetal growth. pregnancy; placenta; nutrition

Kcn1 encodes a potassium (K+) channel in a variety of mammalian cells. Molecular analyses of this gene identified a TATA-box-like region instead of a typical TATA box in the 5'-flanking region. The coding region was intronless and it is suggested that this 5'-flanking region is the major transcription initiation site. Kcn1 expression was stimulated by adenosine 1,3-cyclic monophosphate and localized at the kidney, aorta, brain and heart.

Regulation of the human renin (hREN) gene was investigated in a model using a human pulmonary carcinoma cell line (Calu-6) expressing this gene. Transfection analysis ofhREN constructs identified multiple weak regulatory elements in the 5'-flanking region and a classic silencer element in the first intron of the coding region. Hybridizations and nuclear runoff transcription showed that forskolin induces a 100-fold increase in hREN mRNA levels but not in hREN promoter activity. These results suggest that hREN expression in Calu-6 cells is mediated by transcriptional and posttranscriptional mechanisms.

The c-fos gene is suppressed in senescent cells. Its expression is mediated by adenosine 3',5'-cyclic monophosphate (cAMP) which acts as a second messenger in the promoter regions. To study the relation between c-fos gene expression and cAMP in senescence, IMR fibroblasts were analyzed for the presence of cAMP stimulators such as prostaglandin E2 and forskolin, and the abundance of c-fos and junB mRNA. The results revealed a markedly low cAMP response element binding (CREB) protein, a transcription factor which enhances gene expression. They suggest that decline in CREB is associated with cell aging.

Rabbit cortical collecting duct expresses the colonic form of H-K-ATPase, and its expression is higher in alkali-loaded subjects than in acid-loaded subjects. H-K-ATPase may be useful in HCO3 secretion and K+ absorption depending upon the cellular and subcellular positioning. Alteration in acid/base balance could have a bidirectional influence on H-K-ATPase activity.

The transcription rate of the sodium-phosphate (Na+Pi) transporter gene is decreased in hypophosphatemic (Hyp) mice indicating that the transcriptional regulation of the gene is abnormal in Hyp mouse. Western blot analysis of the brush-border membrane proteins from mouse kidneys indicate a 50% decrease in Na+Pi phosphate uptake in Hyp mouse. The Hyp mouse is a good specimen as the disorders are found to be inherited as X-linked traits in both mouse and humans.

Proenkephalin gene regulation in the neuroendocrine hypothalamus is reviewed using in situ hybridization, use of transgenic mice, and the adaptation of electrophoretic mobility shift assays to complex tissues. The proenkephalin gene has served as a model to study the transcription regulation in cell lines, and in central and peripheral neuroendocrine tissues. The possible future developments in this field of study are presented.

Analysis of the transcription factor C/EBPalpha and surfactant protein in lung cells reveled that C/EBPalpha is involved in the development and maintenance of the surfactant system in lung type II cells. An increase in lipidmetabolizing epithelial cells was observed where the C/EBP alpha mRNA levels were substantially enriched in the type II cells alveolar epithelial cells.

Analysis of the effects of hypoxia on chloramphenicol acetyltransferase (CAT) reporter gene and on mitochondrial manganese-containing SOD (MnSOD) gene in mouse lung reveled that the lung MnSOD in male mice has specific decrease after hypoxia exposure. The CAT protein expression also decreases with the in males while in females it remains in control. For both the native MnSOD and the MnSOD-CAT reporter genes, the mRNA were constant after hypoxia.

Hormones regulate Na+/K+-ATPase in various tissues and control hormonal activity in both the short- and the long-term. The thyroid hormone and aldosterone are responsible for the long-term changes and how the genes express themselves mediates what these hormones do. The catecholamines are responsible for short-term regulation and the reversible phosphorylation of the pump catalytic subunit mediates what their activity.

Radioimmuno assays and Northern blot analysis indicate the stimulation of growth hormone (GH) expression in the pituitary gland of fetal rats by thyroid hormone at 17 days of gestation. The presence of glucocorticoid is crucial for this GH gene expression. Simultaneous enhancement of glucocorticoid and thyroid hormone levels causes the GH cells to mature after 19 days.

A study of the protein and gene expression of transforming growth factor-beta1 (TGF-beta1) in the kidneys of the nonobese diabetic (NOD) and biobreeding (BB) rodent models with spontaneous insulin-dependent diabetes mellitus reveals that the BB rat kidney exhibits a threefold increase in TGF-beta1 mRNA levels after 3 days of diabetes, while in the NOD kidney the levels are increased threefold after 7 to 9 days of diabetes, both accompanied by renal hypertrophy. Immunohistochemical studies reveal that the cortical tubular cells of both models exhibit increased levels of the TGF-beta1 protein. The absence of TGF-beta1 transcript level enhancement indicates the tissue specificity of the response.

Nitric oxide synthase-III (NOS III) protein and mRNA increase in quantity during late gestation, attaining peak level near term, and decrease in the post natal period. NOS-III modulates the potential for pulmonary endothelial NO production at the time of birth. NOS-III gene expression mediates vasomotor tone in the mesenteric and the cerebral circulation and its regulation relates to variations in the transcription or mRNA stability while NOS-I expression is mediated by post-transcriptional mechanisms.

Human surfactant protein (SP) encoding genes SP-A1 and SP-P2 in fetal lung tissues are differentially affected by glucocorticoids and 3',5'-cyclic monophosphate (cAMP). SP-P2 responds to a higher degree than SP-A1 to the inhibitory function of glucocorticoids and inducive effects of cAMP. SP-A2 mRNA transcripts occur more than the SP-A1 transcripts in the lung tissues that are tested with dibutyryl (DB) cAMP.

Spatial, temporal and stimulus-responsive gene expression regulation and pathophysiological features of mice lungs are investigated using a transgenic model. Transgenic mice composed of DNA fragments from artificial chromosomes of yeast are produced and used for gene targeting and transgene expression formation. The transgenic model is used also to study cellular defense systems and functions, gene therapy, immunology response, pathogenesis and tumorigenesis.

Blunted adenosine 3',5'-cyclic monophosphate (cAMP) responses related to aging in obese Sprague-Dawley rats resulted in modified regulation of fatty acid synthase (FAS) and lipoprotein lipase (LPL) gene. Isoproterenol activated cAMP deposit in isolated adipocytes quickly decreased the expression of the mRNAs for LPL and FAS. The expression of FAS and LPL mRNAs in cells from older rats is not restricted by isoproterenol.

The analysis of changes in intracellular calcium concentrations in Madin-Darby canine kidney (MDCK) reveals that phosphate or calcium deficiency in cells is responsible for regulating sodium-dependent phosphate cotransport (Na-Pi) through different cellular pathways. Intracellular Ca concentration is diminished due to phosphate deficiency, and behaves as a stimulant in the sodium-coupled alanine transport. The velocity of phosphate uptake is a function of gene transcription and is accelerated by stimulation from 8-(N, N'-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB8).

The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ~1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II-III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway. spermatogenesis; urea flux; Sertoli cell; residual bodies

Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep. Am J Physiol Lung Cell Mol Physiol 280: L279-L285, 2001.--Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at I day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs. lipopolysaccharide; surfactant protein B; gene regulation

Vasopressin-dependent upregulation of aquaporin-2 gene expression in glucocorticoid-deficient rats. Am J Physiol Renal Physiol 279: F502-F508, 2000.--We determined alterations in renal aquaporin-2 (AQP2) gene expression in association with impaired water excretion in glucocorticoid-deficient rats. After adrenalectomy, Sprague-Dawley rats were administered aldosterone alone by osmotic pumps (glucocorticoid-deficient rats). As a control, both aldosterone and dexamethasone were administered. These animals were subjected to the studies on days 7-14. The expressions of AQP2 mRNA and protein in kidney of the glucocorticoid-deficient rats were increased by 1.6- and 1.4-fold compared with the control rats, respectively. An acute oral water load test verified the marked impairment in water excretion in the glucocorticoid-deficient rats. One hour after the water load, the expressions of AQP2 mRNA and protein were significantly reduced in the control rats, but they remained unchanged in the glucocorticoid-deficient rats. However, there was no alteration in [[sup.3]H]arginine vasopressin (AVP) receptor binding and AVP [V.sub.2] receptor mRNA expression in the glucocorticoid-deficient rats. A [V.sub.2]-receptor antagonist abolished the increased expressions of AQP2 mRNA and protein in the glucocorticoid-deficient rats. These results indicate that augmented expression of AQP2 participates in impaired water excretion, dependent on AVP, in glucocorticoid deficiency. arginine vasopressin; water retention

Identification of a novel kidney-specific gene downregulated in acute ischemic renal failure. Am J Physiol Renal Physiol 279: F426-F439, 2000.--To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at ~5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression. molecular cloning; medullary rays; proximal tubules; polymerase chain reaction-select

Margana, Ramgopal, Kiflu Berhane, M. Nurul Alam, and Vijayakumar Boggaram. Identification of functional TTF-1 and Sp 1/Sp3 sites in the upstream promoter region of rabbit SP-B gene. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L477-L484, 2000.--Surfactant protein B (SP-B) is essential for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B mRNA is expressed in a cell type-restricted manner in alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. In NCI-H441 cells, a lung cell line with characteristics of Clara cells, a minimal promoter region comprising -236 to +39 nucleotides supports high-level expression of chloramphenicol acetyltransferase reporter activity. In the present investigation, we characterized the upstream promoter region, -236 to -140 nucleotides, that is essential for promoter activity. Deletion mapping identified two segments, -236 to -170 and -170 to -140 nucleotides, that are important for promoter activity. Mutational analysis and gel mobility shift experiments identified thyroid transcription factor-1, Sp1, and Sp3 as important trans-acting factors that bind to sequences in the upstream promoter region. Our data suggest that SP-B promoter activity is dependent on interactions between factors bound to upstream and downstream regions of the promoter. lung; gene regulation; respiratory distress syndrome; transcription factors; surfactant protein B; thyroid transcription factor-1

Yang, G., A. Madan, and P. A. Dennery. Maturational differences in hyperoxic AP-1 activation in rat lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L393-L398, 2000.--Immature organisms (neonates; [is less than] 12 h old) have vastly differing responses to hyperoxic injury than adults. A common feature of hyperoxic gene regulation is involvement of activator protein (AP)-1. We evaluated lung AP-1 binding as well as that of the AP-1 subunit proteins c-Fos, c-Jun, phosphorylated c-Jun, Jun B, and Jun D after exposure to [is greater than] 95% [O.sub.2] for 3 days. Unlike adults, neonates showed no increased AP-] binding in hyperoxia despite a high affinity of the AP-1 binding complexes for phosphorylated c-Jun and Jun D as demonstrated by supershift of these antibodies with the AP-1 complexes. Moreover, neonatal lungs exhibited two distinguishable AP-1 binding complexes, whereas adult lungs had one. In neonates, sequential immunoprecipitation revealed that the lower AP-1 complex was composed of proteins from both the Fos and Jun families, whereas the upper complex consisted of Jun family proteins, with predominance of Jun D. In adults, the single AP-1 complex appeared to involve other Fos or non-Fos or non-Jun family proteins as well. Neonatal lungs showed a higher level of Jun B and Jun D immunoreactire proteins in both air and hyperoxia compared with those in adult lungs. These results suggest that significant maturational differences in lung AP-1 complexes exist and that these may explain transcriptional differences in hyperoxic gene regulation. antioxidant; oxidant stress; sequential immunoprecipitation heine oxygenase

Coon, Steven L., Luis A. Zarazaga, Benoit Malpaux, Jean-Paul Ravault, Loys Bodin, Pierre Voisin, Joan L. Weller, David C. Klein, and Philippe Chemineau. Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities. Am. J. Physiol. 277 (Endocrinol. Metab. 40): E792-E797, 1999.--This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 [+ or -] 24.4 vs. H: 344.1 [+ or -] 33.0 pg/ml, P [is less than] 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 [+ or -] 6.7 vs. H: 119.1 [+ or -] 9.2 mg, P [is less than] 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes. pineal gland; arylalkylamine N-acetyltransferase; hydroxyindole O-methyltransferase