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E Gao

Publications and source records attributed to E Gao.

At least 19 recordsLinked to original sources
PubMed1998

Cyclic AMP-responsive expression of the surfactant protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor-1.

Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.

Animals↗
PubMed1998

Principles and philosophy of modeling in biomedical research.

Despite widespread applications in biomedical research, the role of models and modeling is often controversial and ill understood. It is usual to find that fundamental definitions, axioms, and postulates used in the modeling process have become tacit assumptions. What is essential, however, is a clear vision of the fundamental principles of modeling. This is even more compelling for new and emerging interdisciplinary fields that use techniques from previously separate scientific disciplines. This article outlines and reviews the central nature and philosophy of modeling, the rules that govern it, and its underlying key integral relationship to the 'scientific method'. A comprehensive understanding of these issues is indispensable to successful research and meaningful progress in all facets of biomedicine.

Communication↗
PubMed1998

Mathematical considerations for modeling cerebral blood flow autoregulation to systemic arterial pressure.

The shape of the autoregulation curve for cerebral blood flow (CBF) vs. pressure is depicted in a variety of ways to fit experimentally derived data. However, there is no general empirical description to reproduce CBF changes resulting from systemic arterial pressure variations that is consistent with the reported data. We analyzed previously reported experimental data used to construct autoregulation curves. To improve on existing portrayals of the fitting of the observed data, a compartmental model was developed for synthesis of the autoregulation curve. The resistive arterial and arteriolar network was simplified as an autoregulation device (ARD), which consists of four compartments in series controlling CBF. Each compartment consists of a group of identical vessels in parallel. The response of each vessel category to changes in perfusion pressure was simulated using reported experimental data. The CBF-pressure curve was calculated from the resistance of the ARD. The predicted autoregulation curve was consistent with reported experimental data. The lower and upper limits of autoregulation (LLA and ULA) were predicted as 69 and 153 mmHg, respectively. The average value of the slope of the CBF-pressure curve below LLA and beyond ULA was predicted as 1.3 and 3.3% change in CBF per mmHg, respectively. Our four-compartment ARD model, which simulated small arteries and arterioles, predicted an autoregulation function similar to experimental data with respect to the LLA, ULA, and average slopes of the autoregulation curve below LLA and above ULA.

Animals↗
PubMed1998

Age-related decline in beta adrenergic and adenosine A1 receptor function in the heart are attenuated by dietary restriction.

Previously published reports from this laboratory have shown that the antiadrenergic effect of adenosine A1 agonists declines with age in the rat heart [ J Mol Cell Cardiol 29:593-602] and that this decline may be caused by a decrease in coupling between adenosine A1 receptors (AdoA1R) and guanine nucleotide-binding proteins [ Circ Res 81:1065-1071]. Dietary restriction (DR; 60% calories of ad libitum) has been shown to attenuate age-related changes in cellular signal transduction pathways. Therefore, the present study investigated whether DR altered the age-related changes in AdoA1R-mediated function in senescent rat hearts. Ventricular membranes were isolated from the hearts of ad libitum (AL) fed and DR male F344 rats that were 6, 12 and 24 months of age. In AL rats, there was an age-related decline in isoproterenol (ISO)-stimulated adenylyl cyclase when compared with the 6-month-old rats. The decline in ISO-stimulated cyclase was attenuated in DR animals. In AL rats, inhibition of ISO-stimulated adenylyl cyclase by the AdoA1R agonist, N6-p-sulfophenyladenosine (SPA) decreased with age. In DR rats, the age-related decline in inhibition was attenuated. Previous results from this laboratory indicated that in AL fed rats, there was an age-related decrease in the percentage of high-affinity binding sites for SPA, from 55% at 6 months to 23% at 24 months. Diet restriction attenuated this age-related shift in high-affinity binding sites so that the percentage of high-affinity sites at 24 months was 42%. Our results suggest that DR maintains AdoA1R function by preventing a loss of high-affinity AdoA1R sites.

Adenylyl Cyclases↗
PubMed1997

The basic helix-loop-helix-zipper transcription factor USF1 regulates expression of the surfactant protein-A gene.

Expression of the rabbit pulmonary surfactant protein A (SP-A) gene is lung-specific, occurs primarily in type II cells, and is developmentally regulated. We previously identified two E-box-like enhancers, termed the distal binding element (DBE) and proximal binding element (PBE), in the 5'-flanking region of the rabbit SP-A gene. In the present study, the PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert was isolated which is highly similar in sequence to human upstream stimulatory factor 1 (hUSF1). By use of reverse transcription polymerase chain reaction, two isoforms of rabbit USF1 (rUSF1) mRNAs were identified in fetal rabbit lung and other tissues. The levels of rUSF1 mRNAs reach a peak in fetal rabbit lung at 23 days gestation, in concert with the time of initiation of SP-A gene transcription. Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and isolated type II cells with the DBE and PBE were supershifted by the addition of anti-rUSF1 IgG. Binding activity was enriched in type II cells compared with lung fibroblasts. Overexpression of rUSF1s in A549 adenocarcinoma cells positively regulated SP-A promoter activity of cotransfected reporter gene constructs. It is suggested that rUSF1s, which bind to two E-box elements in the SP-A gene 5'-flanking region, may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.

Animals↗
PubMed1997

The effect of age on adenosine A1 receptor function in the rat heart.

Adenosine is an important regulatory metabolite in the heart where it has a cardioprotective function. In the ventricle, the cardioprotective action of adenosine is mediated through the adenosine A1 receptor and inhibition of adenylyl cyclase. In order to investigate the effect of age on adenosine signal transduction in the heart, the effect of specific adenosine A receptor agonists on adenylyl cyclase activity was measured in crude cardiac ventricular membranes isolated from 1-, 6- and 24-month-old Fisher 344 rats. There were no differences in basal cyclase activity with age. Consistent with observations from other laboratories, isoproterenol- and forskolin-stimulated cyclase activity decreased with age. In addition, there was an age-related decline in the capacity of adenosine to inhibit stimulated adenylyl cyclase. The specific A1 adenosine receptor agonists, N6-cyclopentyladenosine (CPA) and N6-p-sulfophenuladenosine (SPA) inhibited isoproterenol- and forskolin-stimulated adenylyl cyclase activity in cardiac membranes from 1-month and 6-month-old rats; however, CPA and SPA did not inhibit adenylyl cyclase in membranes from 24-month-old rats. These data indicate that in addition to the age-related decline in beta-adrenergic receptor function with age, there is also a decrease in adenosine A; receptor-mediated responses. In contrast, carbachol acting through muscarinic receptors, caused the same inhibition of adenylyl cyclase at all ages. Therefore, the age-related decline in inhibitory signal transduction is specific to the adenosine A1 receptor. The age-related defect is probably at the level of the adenosine/receptor interaction and/or the receptor/guanine nucleotide binding protein interaction.

Adenosine↗
PubMed1997

A theoretical model of cerebral hemodynamics: application to the study of arteriovenous malformations.

A comprehensive computer model of the cerebral circulation, based on both hydrodynamics and electrical network analysis, was used to investigate the influences of arteriovenous malformations (AVM) on regional cerebral hemodynamics. The basic model contained 114 normal compartments: 55 arteries, 37 veins, 20 microvessel groups (MVG), one compartment representing systemic and extracranial vascular resistance, and one representing the heart. Each microvessel group, which represented the arteriolar bed, consisted of 5000 microvessels. Cerebral blood flow autoregulation was simulated by a formula that determined the resistance and therefore the flow rate of the microvessel groups (arterioles) as a function of perfusion pressure. Elasticity was introduced to describe the compliance of each vessel. Flow rate was made a controlling factor for the positive regulation of the diameters of conductance vessels by calculation of shear stress on the vessel wall (vessel dilation). Models containing an AVM were constructed by adding an AVM compartment and its feeding arteries and draining veins. In addition to the basic model, AVM models were simulated with and without autoregulation and flow-induced conductance vessel dilation to evaluate the contributions of these factors on cerebral hemodynamics. Results for the model with vessel dilation were more similar to clinical observations than those without vessel dilation. Even in the presence of total vasoparalysis of the arteriolar bed equivalent, obliteration of a large (1000 mL/min) shunt flow AVM resulted in a near-field CBF increase from a baseline of 21 to a post-occlusion value of no more than 74 mL/100 g/min, casting doubt on a purely hemodynamic basis for severe hyperemia after treatment. The results of the simulations suggest that our model may be a useful tool to study hemodynamic problems of the cerebral circulation.

Animals↗
PubMed1997

Cerebral arteriovenous malformation feeding artery aneurysms: a theoretical model of intravascular pressure changes after treatment.

OBJECTIVE: A quantitative model may be used to estimate the magnitude of expected pressure changes along the vascular tree with shunt ablation and may provide information to assess the hemodynamic risk of arteriovenous malformation (AVM) treatment. METHODS: A computer model of the cerebral circulation was applied to estimate the changes in intravascular pressure, velocity, biomechanical stress, and shear stress that might be expected from either endovascular or surgical ablation of AVMs. Two AVM sizes and two feeding artery constellations were simulated. The effect of different shunt flows on vascular pressure was modeled. In each simulation, AVMs were occluded in a stepwise fashion. The effects of systemic hypertension and hypotension in various vascular zones were also simulated. RESULTS: As large (1000 ml/min) AVMs were occluded, the mean feeding arterial pressure increased from 18 to 68 mm Hg; the percent-occlusion at half-maximal pressure increase was 92%. For medium (500 ml/min) AVMs, feeding arterial pressure increased from 37 to 66 mm Hg; the percent-occlusion at half-maximal pressure increase was 71%. During manipulation of systemic pressure, hemodynamic changes in the circulation close to the nidus were proportionally less than changes in systemic pressure; the degree of proportionality depended on the magnitude of AVM shunt flow. CONCLUSION: In this simulation, shunt obliteration increased pressure in the nidus and feeding arteries with little effect on the proximal circulation. The shunt provided a "buffering" effect, i.e., higher flow fistulas were exposed to smaller variations in intravascular pressure in feeding artery and nidal pressures during manipulation of systemic pressure.

Adult↗
PubMed1997

Reduced adenosine A1 receptor and G alpha protein coupling in rat ventricular myocardium during aging.

Adenosine A1 receptor (A1-AdoR) function in rat ventricles has previously been shown to decrease with age. In the present study, using the ligand [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and coimmunoprecipitation of A1-AdoRs with their associated G proteins, we determined the specific binding of A1-AdoR and A1-AdoR/G protein coupling in ventricular myocardium of 6- to 24-month-old Fischer 344 rats. The densities (Bmax) of A1-AdoRs were 5.8 +/- 0.8 fmol/mg protein in 6-month-old rats and 6.1 +/- 1.4 fmol/mg protein in 24-month-old rats, and the dissociation constants (Kd) were 0.32 +/- 0.04 nmol/L in 6-month-old rats and 0.34 +/- 0.05 nmol/L in 24-month-old rats (P > .05). Analysis of the dose-dependent displacement of [3H]DPCPX binding by the selective A1-receptor agonist, N6-p-sulfophenyladenosine (SPA), yielded two affinity binding sites in both 6- and 24-month-old rats. However, the proportion of high-affinity A1-AdoRs was significantly lower in 24-month-old rats (23.5%) compared with 6-month-old rats (54.9%) (P < .05). In solubilized ventricular membranes, specific [3H]DPCPX binding sites were detected in immunoprecipitates of G alpha i3 and G alpha o antisera but not with antibodies for other G alpha proteins. The basal coimmunoprecipitation of A1-AdoR with G alpha i3 and G alpha o proteins decreased by 22% and 21%, respectively, in ventricular membranes of 24-month-old rats compared with that in 6-month-old animals. A1-AdoR stimulation with SPA increased the coprecipitation of A1-AdoR with G alpha i3 and G alpha o proteins by 287% and 245%, respectively, in 6-month-old rats but only by 129% and 140%, respectively, in 24-month-old rats (P < .01). In the absence of changes in A1-AdoR density and G alpha protein levels, an age-related decline in high-affinity A1-AdoR binding sites and a reduction in the association of A1-AdoR with G alpha proteins suggest that the age-related decrease in ventricular A1-AdoR-mediated response is related to a reduction in the coupling between A1-AdoR and their G proteins.

Adenylyl Cyclase Inhibitors↗
PubMed1997

Preliminary study of the effects of metoprolol and propafenone on ventricular arrhythmia with positive ventricular late potential.

OBJECTIVE: To compare the effects of metoprolol and propafenone in patients with ventricular arrhythmia (VA) of positive ventricular late potential (VLP) and to discuss the effect of medicine on high risk VA. METHODS: 30 A total of 128 patients (78 males and 50 females) with VA of positive VLP were randomly divided into 3 groups. Groups A and B were given metoprolol and propafenone respectively, group C was given vitamin C as placebo. Twenty-four hours dynamic electrocardiogram and VLP were examined before treatment and 4 weeks after treatment. RESULTS: Propafenone could effectively control VA but could not reverse positive VLP to negative meanwhile it had proarrhythmia effect (2/40), metoprolol could effectively control VA (38/46) and reverse positive VLP to negative (39/46) with no obvious side effects. The dosage from 50 to 100 mg/day made no difference. VA and VLP had no significant changes in group C. CONCLUSIONS: Metoprolol is superior to other medicine for VA of positive VLP when the patients have no contraindication of preceptor blocking agents.

Adult↗
PubMed1996

Heat shock proteins and experimental autoimmune encephalomyelitis (EAE): I. Immunization with a peptide of the myelin protein 2',3' cyclic nucleotide 3' phosphodiesterase that is cross-reactive with a heat shock protein alters the course of EAE.

We describe sequence similarity and immunologic cross-reactivity between a peptide of the mycobacterial hsp, HSP65, and the myelin protein 2',3' cyclic nucleotide 3' phosphodiesterase (CNP). We demonstrate that immunization with the homologous cross-reactive CNP peptide (hsp-CNP peptide) has significant biological consequences. Rats immunized with hsp-CNP peptide in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) produce large amounts of peptide-specific antibody. Isotypes of antibodies in animals immunized with peptide in CFA are IgG1 and IgG2a. Isotypes of antibodies in rats immunized with peptide in IFA are predominantly IgG1, with low titers of IgG2a. T cell proliferative responses to HSP65 are present in rats immunized with peptide in CFA. T cell responses to HSP65 initially are absent in rats immunized with peptide in IFA but develop over time. T cell proliferative responses to hsp-CNP peptide were not detected. None of the groups of rats developed clinical or histologic evidence of experimental autoimmune encephalomyelitis (EAE). To induce EAE, rats preimmunized with hsp-CNP peptide were challenged with guinea pig spinal cord (GPSC) emulsified in CFA. Rats preimmunized with peptide in CFA developed severe EAE. Rats preimmunized with hsp-CNP peptide in IFA were protected from EAE, with both a lower incidence and severity of disease. Injecting the murine monoclonal antibody recognizing the shared HSP65 and CNP epitope did not protect against EAE. Our data suggest that a Th2 pattern of immune response to a CNP peptide that itself is non-encephalitogenic protects against EAE. Immune responses to either hsp or myelin proteins cross-reactive with hsp may play an important role in the development of EAE.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1996

Characterization of two baboon surfactant protein A genes.

The gene encoding surfactant protein A (SP-A) is expressed in type II pneumonocytes and is developmentally and hormonally regulated in fetal lung tissue. SP-A is encoded by a single-copy gene in rabbits, dogs, rats, and mice. By contrast, the human genome contains two similar genes, hSP-A1 and hSP-A2, which are differentially regulated during development and differentially regulated by adenosine 3',5'-cyclic monophosphate (cAMP) and glucocorticoid treatment of human fetal lung in culture. In the present study, we have isolated and characterized baboon genomic clones containing two highly similar SP-A genes. Restriction mapping of these clones, together with Southern analysis of genomic DNA, indicates that these comprise two distinct baboon SP-A genes. Sequence comparison of DNA upstream of the transcription initiation sites and within the 3'-untranslated regions encoded by exon VI indicates that one of the baboon SP-A genes (bSP-A1) is more similar to hSP-A1, whereas the other (bSP-A2) is more similar to hSP-A2. Primer extension analysis of baboon lung mRNA indicates that both baboon SP-A genes utilize conserved transcription initiation sites. Reverse transcriptase-polymerase chain reaction analysis of RNA isolated from lung tissues of fetal baboons of 160 days gestational age indicates that both bSP-A1 and bSP-A2 are expressed in baboon fetal lung and that mRNA transcripts of bSP-A1 and bSP-A2 genes are primarily comprised of sequences encoded by exons I and III-VI. However, minor transcripts of the bSP-A1 gene containing exon II and exon II plus an extension also were detected. The presence of two SP-A genes in the baboon suggests that duplication of the SP-A gene occurred > 26.5 million years ago, before divergence of the baboon lineage from the man-gorilla-chimpanzee clade.

Amino Acid Sequence↗
PubMed1996

Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members.

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5'-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CREsp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CREpal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CREsp-a to CREpal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CREsp-a and CREpal. By UV cross-linking analysis, an approximately 43-kilodalton protein complex was found to interact both with CREsp-a and CREpal; however, purified CREB was ineffective in binding CREsp-a but did bind CREpal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CREsp-a; whereas, a supershift was evident using CREpal as a probe. Moreover, in competition EMSA using radiolabeled CREsp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CREpal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CREsp-a. Additionally, expression of a CREsp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CREpal:HIS3 yeast strain. By competition EMSA using mutagenized CREsp-a oligonucleotides, the critical protein-binding nucleotides in CREsp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.

Activating Transcription Factor 2↗
PubMed1993

Identification of enhancers in the 5'-flanking region of the rabbit surfactant protein A (SP-A) gene and characterization of their binding proteins.

The gene encoding surfactant protein A (SP-A) is expressed in type II pneumonocytes and is developmentally and hormonally regulated in fetal lung. In the present study, rabbit lung nuclear proteins were found to bind to two genomic elements within the 5'-flanking region of the rabbit SP-A gene, termed distal binding element (DBE; -986 to -977 base pairs) and proximal binding element (PBE; -87 to -70 base pairs). Binding activity was enriched in type II pneumonocytes as compared with whole lung tissue. Although binding activity was undetectable in nuclear proteins from rabbit liver and kidney, low levels of binding activity were detected in nuclear proteins from cardiac and skeletal muscle. DNase I footprinting indicated that lung nuclear proteins protected the palindromic sequence CCCACGTGGG in the DBE. The underlined core sequence is an E box motif; a similar sequence (CCCTCGTG) is present within the PBE. Both elements competed for binding to the same size species of nuclear proteins of M(r) approximately 69,000, 45,000, and 22,000. In type II cells transfected with fusion genes containing SP-A 5'-flanking DNA linked to the human growth hormone structural gene, mutagenesis of the DBE or PBE resulted in a marked reduction of basal and cyclic AMP-stimulated fusion gene expression. These findings suggest that the DBE and PBE act as enhancers that interact with the same or related trans-acting proteins and serve an important role in type II cell-specific, cyclic AMP-mediated regulation of SP-A gene expression.

Animals↗
PubMed1993

Multiple mechanisms by which protein kinase A potentiates inositol 1,4,5-trisphosphate-induced Ca2+ mobilization in permeabilized hepatocytes.

The mobilization of Ca2+ from intracellular stores by Ins(1,4,5)P3 in suspensions of permeabilized rat hepatocytes was potentiated by preincubating intact cells with adenosine 3':5'-cyclic phosphorothioate (cpt-cAMP), or by addition of the catalytic subunit of cyclic-AMP-dependent protein kinase (PKA) after cell permeabilization. This action of PKA involved both an enhancement in Ins(1,4,5)P3 sensitivity and an increase in the size of the Ins(1,4,5)P3-releasable Ca2+ pool. Inclusion of the protein phosphatase inhibitor okadaic acid in the permeabilization medium augmented the effects of PKA. Treatment with PKA catalytic subunit also increased the rate of ATP-dependent Ca2+ sequestration. To determine whether the effects of PKA on the Ca(2+)-release mechanism were secondary to alterations in the Ca2+ load of the Ins(1,4,5)P3-sensitive stores, a method was developed using Mn2+ as a Ca2+ surrogate to examine the permeability properties of the Ins(1,4,5)P3-gated channels independent of Ca2+ fluxes. This approach utilized the ability of Mn2+ to quench the fluorescence of fura-2 compartmentalized within intracellular Ca2+ stores in an Ins(1,4,5)P3-dependent manner, with thapsigargin added to block the ATP-activated Ca2+ pump and to ensure that the Ca2+ stores were fully depleted of Ca2+. The initial rate and extent of Mn2+ quenching of compartmentalized fura-2 was increased in a dose-dependent manner by Ins(1,4,5)P3. PKA activation increased both the initial rate and the extent of Mn2+ quenching at sub-maximal Ins(1,4,5)P3 doses, but there was no effect on the quench rate in the presence of saturating Ins(1,4,5)P3. However, the amount of compartmentalized fura-2 that could be quenched by Mn2+ in the presence of maximal Ins(1,4,5)P3 was increased by PKA. These data suggest two distinct actions of PKA on the Ins(1,4,5)P3-sensitive Ca2+ stores. (1) Modification of the ion-permeability properties of the Ins(1,4,5)P3 receptor/channel through an increase in the sensitivity to Ins(1,4,5)P3 for channel opening. (2) A recruitment of Ca2+ stores from the Ins(1,4,5)P3-insensitive pool. Both actions were independent of the Ca(2+)-loading state of the stores. Imaging studies of single permeabilized hepatocytes showed that the Ins(1,4,5)P3-sensitive stores were distributed throughout the cell and PKA enhanced the rate of Ins(1,4,5)P3-stimulated Mn2+ quench in individual cells, without modifying the subcellular distribution of Ins(1,4,5)P3-sensitive stores.

Animals↗
PubMed1993

Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene.

Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, -378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A-1766:hGH and SP-A-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)2AMP (Bt2cAMP; 1 mM). When type II cells were transfected with the SP-A-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt2cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt2cAMP was apparent. Cyclic AMP stimulation of expression of the SP-A-1766:hGH, SP-A-991:hGH, and SP-A-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCl-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10(-7) M) antagonized the stimulatory effect of Bt2cAMP on expression of SP-A:hGH fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A-991+670:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10(-10) M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗