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K D Bloch

Publications and source records attributed to K D Bloch.

At least 55 records · Page 3Linked to original sources
PubMed1995

Hypertension in mice lacking the gene for endothelial nitric oxide synthase.

Nitric oxide (NO), a potent vasodilator produced by endothelial cells, is thought to be the endothelium-dependent relaxing factor (EDRF) which mediates vascular relaxation in response to acetylcholine, bradykinin and substance P in many vascular beds. NO has been implicated in the regulation of blood pressure and regional blood flow, and also affects vascular smooth-muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Abnormalities in endothelial production of NO occur in atherosclerosis, diabetes and hypertension. Pharmacological blockade of NO production with arginine analogues such as L-nitroarginine (L-NA) or L-N-arginine methyl ester affects multiple isoforms of nitric oxide synthase (NOS), and so cannot distinguish their physiological roles. To study the role of endothelial NOS (eNOS) in vascular function, we disrupted the gene encoding eNOS in mice. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure.

Amino Acid Oxidoreductases↗
PubMed1995

Three members of the nitric oxide synthase II gene family (NOS2A, NOS2B, and NOS2C) colocalize to human chromosome 17.

Nitric oxide synthases (NOSs) are a family of enzymes responsible for the synthesis of nitric oxide from L-arginine and molecular oxygen. Three human NOS enzymes (I, II, and III) with differing cellular distribution and regulatory mechanisms have been identified. To determine whether additional NOSs are encoded in the human genome, a bovine NOS II-related cDNA was used to screen two human genomic libraries. Clones containing three independent genes were isolated. One clone encoded the previously identified NOS II gene (NOS2A). The two other genes specified amino acids homologous, but not identical, to human NOS II (NOS2B and NOS2C). Southern blot hybridization demonstrated that all three genes are present in the human genome. DNA from human-mouse somatic cell hybrids were used to determine the chromosomal location of the NOS II-related genes. All three NOS II-related genes colocalized to human chromosome 17 between bands p13.1 and q25. These observations suggest that there is more than one NOS II-related gene in the human genome. This finding may have important implications for the design of NOS isoform-specific inhibitors.

Amino Acid Sequence↗
PubMed1995

Constitutive endothelial nitric oxide synthase gene expression is regulated during lung development.

Nitric oxide (NO), a potent vasodilator, is a free-radical gas synthesized from L-arginine by nitric oxide synthases (NOS). NO appears to have an important role in perinatal changes in pulmonary vascular resistance. We previously identified mRNA encoding the constitutive endothelial NOS (ceNOS) isoform in human pulmonary tissue. To begin investigating functions of this enzyme in perinatal pulmonary development, we measured ceNOS mRNA and immunoreactivity in the developing rat lung. With the use of RNA blot hybridization, abundant pulmonary ceNOS mRNA was detected during the late fetal and postnatal period. The highest levels were detected within 24 h after birth, and elevated mRNA levels persisted for 16 days. In contrast, much lower levels of ceNOS mRNA were found in adult rat lung. With the use of immunoblot techniques, ceNOS protein levels were found to be correlated with mRNA levels. To identify the pulmonary cell types expressing the ceNOS gene, in situ hybridization with a digoxigenin-labeled cRNA probe was performed on sections from lungs of 1-day-old and adult rats. In lungs from 1-day-old rats, ceNOS mRNA was detected in alveolar and serosal epithelial cells as well as in endothelial cells lining small and medium-sized blood vessels. In contrast, in adult lungs, ceNOS gene transcripts were detected in rare endothelial cells. These observations suggest that ceNOS gene expression is regulated during lung development and that ceNOS is available to participate in the postnatal reduction of pulmonary vascular resistance. ceNOS gene expression in nonendothelial cells in the neonatal rat lung suggests that NO may also contribute to nonvascular functions in the developing lung.

Amino Acid Oxidoreductases↗
PubMed1995

In vivo lipopolysaccharide pretreatment inhibits cGMP release from the isolated-perfused rat lung.

Administration of bacterial lipopolysaccharide (LPS) to rats stimulates synthesis of nitric oxide (NO), a free radical molecule that activates soluble guanylate cyclase, thereby increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration and inducing systemic vasodilatation. To investigate the effect of endotoxemia on the pulmonary NO/cGMP signal transduction system, we measured the release of cGMP by isolated-perfused lungs of rats that received an intraperitoneal injection of LPS (1 mg/kg) or saline 2 days earlier. Over 90 min, 1.4 +/- 0.78 and 0.079 +/- 0.016 nmol cGMP accumulated in pulmonary perfusates of saline- and LPS-treated rats, respectively (P < 0.05). Despite addition to the perfusate of Zaprinast, superoxide dismutase, or A23187, markedly less cGMP was released from the lungs of rats exposed to LPS than from the lungs of control rats. In contrast, after ventilation with 100 parts per million NO gas, cGMP accumulating in the perfusate of the lungs of both groups of rats was markedly increased, and the quantity of cGMP released from the lungs of LPS-treated rats was similar to that released by control rat lungs (2.8 +/- 0.57 vs. 3.3 +/- 0.88 nmol, P = NS). With the use of immunoblot techniques, equal concentrations of constitutive endothelial NO synthase were detected in the lungs of rats treated with saline or LPS. These results demonstrate that the NO/cGMP signal transduction system is abnormal in the lungs of rats exposed to LPS, at least in part, at the level of endothelial NO synthase activation.

Animals↗
PubMed1995

Nitric oxide responses of air-breathing and water-breathing fish.

Nitric oxide (NO), exogenously administered or endogenously produced by NO synthase (NOS), is an important regulator of lung ventilation and perfusion in mammals. This study attempts to investigate the evolutionary history of this system in fish and its possible relationship to air breathing. The gas bladder of Hoplerythrinus unitaeniatus (air-breathing teleost) and Oncorhynchus mykiss (non-air-breathing teleost) and the lung of Lepidosiren paradoxa (air-breathing dipnoan) all exhibited elevated guanosine 3',5'-cyclic monophosphate (cGMP) levels in response to 1 microM sodium nitroprusside. Only the H. unitaeniatus gas bladder responded to 10 microM acetylcholine chloride (ACh) with increased cGMP levels. The ACh response was inhibited by N omega-nitro-L-arginine methyl ester, which inhibits NOS. These data suggest that although tissues from each species may respond to exogenous NO, only the gas bladder of H. unitaeniatus appears to synthesize NO through NOS. This is the first report of constitutive NOS outside of the central nervous system in a teleost. These results also imply that NOS did not necessarily coevolve with air breathing in fish.

Acetylcholine↗
PubMed1995

Inhaled nitric oxide does not alter endotoxin-induced nitric oxide synthase activity during rat lung perfusion.

Nitric oxide (NO) has been demonstrated to decrease its own synthesis in tissue preparations. We tested the hypothesis that endogenous NO synthesis induced by lipopolysaccharides (LPS) would be decreased by exogenous NO during isolated lung perfusion. Rats were pretreated with either saline or LPS 48 h before lung harvest. Endogenous NO synthase activity was measured as conversion of L-[14C]-arginine to L-[14C]citrulline during 90 min of perfusion. NO (100 ppm) was added to the ventilating gas during perfusion of lungs from one group of control or LPS-treated rats. A second group of control or LPS-treated rats was exposed chronically to 100 ppm NO for the 48 h before lung harvest, in addition to receiving 100 ppm NO added to the ventilating gas during lung perfusion. We conclude that conversion of L-[14C]arginine to L-[14C]citrulline was minimal in control lungs and increased in response to LPS pretreatment. NO added to the ventilating gas for the 90 min of ex vivo perfusion did not alter the rate of L-[14C]citrulline production. In vivo exposure to 100 ppm NO for 48 h did not alter the induction of inducible NO synthase activity as measured during ex vivo lung perfusion. This indicates that inhaled NO does not exert negative-feedback inhibition on inducible NO synthase in the ex vivo rat lung.

Administration, Inhalation↗
PubMed1995

Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hypertrophy, and growth retardation in the hypoxic newborn rat.

Breathing low oxygen levels for several weeks produces progressive pulmonary artery hypertension and smooth muscle hypertrophy and hyperplasia in many species. Because nitric oxide (NO) is an important regulator of pulmonary vascular tone, we examined whether the continuous inhalation of low levels of NO gas would attenuate pulmonary arterial structural changes in hypoxic rat pups. Nine-day-old rat pups and their mothers continuously breathed at FIO2 0.21 or 0.10 with or without adding 20 ppm (by volume) NO for 2 weeks. Lung tissue was obtained for vascular morphometric analysis, and the hearts were dissected to measure right ventricular weight and levels of mRNA encoding rat atrial natriuretic factor (rANF). In addition, femur and skull length were radiographically determined. Breathing at FIO2 0.10 for 14 days increased pulmonary arterial wall thickness and the proportion of muscular arteries in the lung periphery. Right ventricular weight and right ventricular rANF gene expression increased, whereas body weight and skeletal growth were reduced (all P < .05). Continuous inhalation of 20 ppm NO at FIO2 0.10 for 2 weeks decreased hypoxic pulmonary vascular structural changes and somatic growth retardation and prevented the increase of right ventricular weight and right ventricular rANF mRNA levels. These observations suggest that chronically breathing NO attenuates pulmonary vascular smooth muscle hypertrophy and/or hyperplasia and extension into distal arterial walls, right ventricular hypertrophy, and growth retardation of newborns breathing at a low oxygen level.

Administration, Inhalation↗
PubMed1995

Regional and maturation-associated expression of endothelin 2 in rat gastrointestinal tract.

Endothelin 2 (ET2), also referred to as vasoactive intestinal contractor peptide, is a member of a family of vasoactive peptides. ET2 is a potent constrictor of intestinal smooth muscle, and the mRNA that encodes it has been detected in murine intestinal extracts. To further investigate the potential physiological roles of ET2, we characterized the cellular distribution of ET2 gene expression in adult rat gastrointestinal tract. Using an RNAse protection assay, an overall proximal to distal gradient of increasing ET2 gene expression was observed from stomach to colon. In situ hybridization studies confirmed this finding and demonstrated ET2 mRNA localized in lamina propria stromal cells. Moreover, ET2 gene expression in stromal cells increased from crypt to villous tip. The results demonstrate that ET2 is produced by stromal cells in villi throughout the intestine. Increased ET2 gene expression at the villous tip is associated with more mature overlying epithelial cells, suggesting a possible role for this vasoactive peptide in intestinal epithelial differentiation or secretory activity.

Animals↗
PubMed1995

The cell proliferation-associated protein Ki-67 is a target of autoantibodies in the serum of MRL mice.

BACKGROUND: Ab in the serum of patients with autoimmune diseases have been used to identify, characterize, and purify many autoantigens. EXPERIMENTAL DESIGN: Serum from a patient (Ge) with Sjögren's syndrome was used to identify cDNA clones encoding novel autoantigens. This patient's serum was chosen for study because it contained antinuclear Ab that were different from those frequently detected in patients with autoimmune diseases. RESULTS: Ge serum identified a cDNA clone encoding part of protein Ki-67, a cell proliferation-associated protein. The Ki-67 protein (pKi-67) was not previously known to be a target of autoantibodies. To investigate the association between Ab directed against pKi-67 and autoimmune diseases, sera from autoimmune mice were tested for reactivity with a recombinant fragment of pKi-67. Ab were detected in serum from MRL/MpJ(-)+/+ and MRL/MpJ-lpr/lpr mice but not in serum from other autoimmune mice or control animals. CONCLUSIONS: Protein Ki-67 joins the proliferating cell nuclear Ag (PCNA) as an example of a cell proliferation-associated protein that is a target of autoantibodies. The presence of anti-pKi-67 Ab in MRL mice, but not other autoimmune mice, suggests that anti-pKi-67 Ab may be specific markers for the systemic lupus erythematosus-like illness that develops in these animals. Further characterization of the immune response directed against this Ag may provide clues to the etiology and pathogenesis of autoimmune disease in these animals.

Animals↗
PubMed1994

Expression of the endothelin-1 and oxytocin genes in the hypothalamus of the pregnant rat.

Endothelin (ET)-1, a neuropeptide and possible neuromodulator, has been found in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) of the rat in the distribution of oxytocin (OT) neurons. Within the hypothalamus of the pregnant rat, we investigated the developmental expression of the ET-1 gene and the possibility of coordinate expression of the ET-1 and OT genes. Blots containing hypothalamic mRNAs from 4-, 14-, 18-, and 21-day-old pregnant rats were hybridized to a 32P-labeled probe specific to the rat ET-1 gene. Hypothalamic tissue contained an ET-1 transcript of approximately 2.3 kb size. ET-1 mRNA abundance increased significantly in the SON and PVN from early to late gestation (P = 0.005 and 0.05, respectively). Blots containing hypothalamic mRNA were rehybridized to a 32P-labeled probe specific to exon C of the rat OT gene. OT gene expression increased significantly within both the hypothalamic SON (p = 0.0009) and PVN (P = 0.003) as gestation advanced. The sizes of the hypothalamic ET-1 and OT transcript sizes remained unchanged throughout gestation. Hypothalamic ET-1 and OT transcripts display stage-specific increases during gestation. ET-1 may be a neuroendocrine regulator of pregnancy-related events in the rat, and may act alone or in concert with OT.

Animals↗
PubMed1994

The immunoreactive region in a novel autoantigen contains a nuclear localization sequence.

Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens. Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides. In the present study, serum from a patient with Sjögren's syndrome was used to identify a novel autoantigen which was designated Ge-1. Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified. Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins. Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein. This immunoreactive region contained the protein's putative nuclear localization sequence (NLS). To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase. After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus. The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies.

Amino Acid Sequence↗
PubMed1994

Effects of inhaled nitric oxide on pulmonary hemodynamics and gas exchange in an ovine model of ARDS.

Inhaling low concentrations of nitric oxide (NO) gas causes selective pulmonary vasodilation of ventilated lung regions. NO activates soluble guanylate cyclase, increasing guanosine 3',5'-cyclic monophosphate (cGMP). Inhibition of NO synthesis enhances hypoxic pulmonary vasoconstriction. Therefore we examined independent and combined effects of NO inhalation and infusion of NG-nitro-L-arginine methyl ester (L-NAME), an NO synthesis inhibitor, on pulmonary vascular pressure-flow relationships, gas exchange, and plasma cGMP levels in anesthetized and mechanically ventilated sheep with acute lung injury induced by bilateral lavage. After lavage, inhaling 60 ppm by volume of NO decreased pulmonary arterial pressure (PAP) and resistance without any systemic hemodynamic effects, increased arterial PO2, and decreased venous admixture (Qva/QT; all P < 0.05) without altering cardiac output (QT), mixed venous PO2, or O2 uptake, major determinants of intrapulmonary shunt. During NO inhalation, PAP-left atrial pressure gradient (PAP-LAP) and Qva/QT were reduced (both P < 0.05) independently of QT, which was varied mechanically. L-NAME infusion produced systemic and pulmonary vasoconstriction and increased PAP-LAP gradient across the entire range of QT, whereas Qva/QT, was not changed. NO inhalation after L-NAME infusion produced pulmonary vasodilation and decreased Qva/QT to the same degree as NO inhalation alone. Five to 10 min after inhalation of 60 ppm NO, before and after L-NAME infusion, arterial plasma cGMP levels were increased by 80% (both P < 0.05). With NO breathing after L-NAME, we measured a consistent transpulmonary cGMP arteriovenous gradient [31 +/- 8 and 33 +/- 7 (SE) pmol/ml at 5 and 10 min, respectively; both P < 0.05]. NO inhalation before or after L-NAME administration in this acute lung injury model reduced Qva/QT, most likely by increasing cGMP concentration in ventilated lung regions and causing selective pulmonary vasodilation.

Administration, Inhalation↗
PubMed1994

Antenatal glucocorticoid corrects pulmonary immaturity in experimentally induced congenital diaphragmatic hernia in rats.

Congenital diaphragmatic hernia, a highly lethal condition, displays at term the pulmonary biochemical and morphologic immaturity characteristic of premature delivery. We hypothesized that antenatal glucocorticoid, now the standard treatment to prevent hyaline membrane disease in premature human beings, might correct the parameters of the pulmonary biochemical and morphologic immaturity in severe congenital diaphragmatic hernia. A total of 112 fetal rats with or without nitrofen-induced congenital diaphragmatic hernias from 34 pregnancies were treated antenatally with either saline or dexamethasone. Antenatal dexamethasone increased the lung disaturated phosphatidylcholine content, reduced the lung glycogen concentration, reduced the saccular septal thickness, and increased the mean saccular size and volume fraction of saccules in the lungs of rats with large congenital diaphragmatic hernia in comparison with similar rats not so treated. All differences were statistically significant. Antenatal glucocorticoid therapy was efficacious in treating rats with nitrofen-induced congenital diaphragmatic hernia. This encouraging finding warrants further investigation in a large animal model with surgically created congenital diaphragmatic hernia.

Amino Acid Oxidoreductases↗
PubMed1994

Cosegregation of the endothelin-3 locus with blood pressure and relative heart weight in inbred Dahl rats.

OBJECTIVE: To determine whether the endothelin-1 or endothelin-3 genes are genetically linked with blood pressure and relative heart weight in segregating rat populations, in the context of an elevated dietary sodium chloride intake. METHODS: Endothelin-1 and endothelin-3 genotypes of rats in segregating populations, derived from crosses of Dahl salt-sensitive (SS/Jr) rats with contrasting inbred strains, including Lewis rats, spontaneously hypertensive rats and Dahl salt-resistant (SR/Jr) rats, were determined using restriction fragment length polymorphisms. Segregating populations were fed a high (8%)-sodium chloride diet. Linkage of genotype with blood pressure or relative heart weight was determined by analysis of variance. Chromosomal location of the rat endothelin-3 gene was determined by genotyping a panel of recombinant inbred strains. RESULTS: Two alleles for the endothelin-1 gene and three alleles for the endothelin-3 gene were identified. The endothelin-1 locus did not cosegregate with blood pressure or relative heart weight. The endothelin-3 locus cosegregated with blood pressure and relative heart weight in an SS/Jr x F1 (SS/Jr x SR/Jr) population, but not in populations containing a higher percentage of genes from the SR/Jr strain. The endothelin-3 and seminal vesicle protein-1 loci were linked and located on rat chromosome 3. CONCLUSION: The endothelin-3 gene is, or is linked to, a locus on chromosome 3 that regulates blood pressure and relative heart weight in inbred Dahl rats, and these effects were strongly dependent on the genetic background.

Animals↗
PubMed1993

cAMP regulates soluble guanylate cyclase beta 1-subunit gene expression in RFL-6 rat fetal lung fibroblasts.

Endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) activates soluble guanylate cyclase, thereby stimulating the synthesis of guanosine 3',5'-cyclic monophosphate (cGMP). To investigate the regulation of this important EDRF/NO receptor, we studied soluble guanylate cyclase gene expression in a rat fetal lung fibroblast cell line (RFL-6). 3-Isobutyl-1-methylxanthine, forskolin, and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), agents which increase intracellular cAMP, decreased the concentration of mRNA encoding the beta 1-subunit of soluble guanylate cyclase in RFL-6 cells. To investigate whether a decrease in beta 1-subunit mRNA concentration was reflected in diminished capacity to produce cGMP, forskolin-treated RFL-6 cells were exposed to the NO-donor compound sodium nitroprusside. Exposure to forskolin reversibly reduced the ability of RFL-6 cells to increase cGMP in response to NO. These observations suggest that cAMP can modulate the cellular response to EDRF/NO by decreasing the expression of one of the subunits of soluble guanylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase.

Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.

3T3 Cells↗
PubMed1991

cDNA cloning and chromosomal assignment of the endothelin 2 gene: vasoactive intestinal contractor peptide is rat endothelin 2.

Four members of the endothelin family of vasoactive and mitogenic peptides have been identified: human endothelins 1, 2, and 3 (ET1, ET2, and ET3, respectively) and mouse vasoactive intestinal contractor (VIC). To characterize the mRNA encoding ET2, a 192-bp fragment of the ET2 gene, amplified by the polymerase chain reaction from human genomic DNA, was used to screen cell lines and tissues for ET2 gene expression. ET2 mRNA was detected in a cell line (HTB119) derived from a human lung small cell carcinoma, and an ET2 cDNA was cloned from a cDNA library prepared from HTB119 mRNA. DNA prepared from human-mouse somatic hybrid cell lines was used to assign the gene encoding ET2 (EDN2) to the 1p21----1pter region of chromosome 1, demonstrating that EDN2 is not linked to genes encoding ET1 (EDN1; chromosome 6) and ET3 (EDN3; chromosome 20). Southern blot hybridization revealed a single gene in human and rat genomes that hybridized with the ET2 gene fragment, and the rat gene was cloned. The endothelin peptide encoded by the rat gene differed from ET2 at 1 of 21 residues and was identical to mouse VIC. We conclude that VIC is the mouse and rat analogue of the human ET2 gene.

Amino Acid Sequence↗