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J S Pollock

Publications and source records attributed to J S Pollock.

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

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells.

OBJECTIVE: Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. METHODS: Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. RESULTS: Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. CONCLUSIONS: These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.

Animals↗
PubMed1994

Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease.

The cerebral microcirculation undergoes specific biochemical changes in Alzheimer's disease. In this study, we have compared the nitric oxide synthase (NOS) activity of brain microvessels isolated from Alzheimer and control brains. L-[3H]-citrulline, the stable co-product generated with nitric oxide (NO) from L-[3H]-arginine, was measured as an indicator of NOS activity. The results indicated a significant increase in NOS activity in microvessels isolated from Alzheimer brains. In addition, using antibodies to both the endothelial and inducible NOS isoforms, we demonstrated a significant increase in enzyme level in Alzheimer-derived vessels. Elevated vascular production of NO, a potentially neurotoxic mediator in the CNS, may contribute to the susceptibility of neurons to injury and cell death in Alzheimer's disease.

Aged↗
PubMed1994

Upregulation of neuronal nitric oxide synthase and mRNA, and selective sparing of nitric oxide synthase-containing neurons after focal cerebral ischemia in rat.

Nitric oxide synthase-containing neurons are presumed to be resistant to neurodegeneration and neurotoxicity, however this resistance has not been demonstrated after focal cerebral ischemia. We therefore measured the temporal profile of neuronal nitric oxide synthase (NOS-I) mRNA and immunoreactivity and NADPH-diaphorase reactivity over a one week period after permanent middle cerebral artery (MCA) occlusion in 48 male Wistar rats and compared these data to ischemic cell damage as evaluated on hematoxylin and eosin (H & E) stained sections by light microscopy. NOS-I mRNA increased as early as 15 min after MCA occlusion in the ipsilateral striatum and maximal expression of NOS-I was found in the ipsilateral cortex and striatum 1 h after MCA occlusion. The numbers of NOS-I-containing neurons in the ipsilateral cortex and striatum were significantly greater (P < 0.05) than NOS-I-containing neurons in the contralateral hemisphere at 2-48 h after the onset of ischemia. The number of NOS-I-containing neurons peaked at 4 h after MCA occlusion. Neurons exhibited shrinkage or were swollen at 1 to 4 h after MCA occlusion. At 24-48 h after ischemia, neurons in the ischemic lesion appeared to be eosinophilic or ghost like on H & E stained sections. However, some of these neurons retained morphological integrity on the NOS-I immunohistochemical sections. At 168 h after ischemia, all neurons within the lesion appeared necrotic on H & E stained sections; however, scatterred neurons expressed NOS-I and NADPH-diaphorase. The rapid upregulation of NOS-I and mRNA in the ischemic lesion suggests that NOS-I is involved in focal cerebral ischemic injury; the expression of NOS-I by neurons that retain their morphological structure in the area of the infarct suggests that NOS-I-containing neurons are more resistant to the ischemic insult. Our data also indicate a close association of NOS-I immunoreactivity and NADPH-diaphorase reactivity in ischemic brain.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.

Amino Acid Oxidoreductases↗
PubMed1994

Immunochemical detection of inducible NO synthase in human lung.

Type II (inducible) nitric oxide synthase (NOS) may play an important role in pulmonary pathophysiology, yet it remains controversial whether human tissues are capable of expressing this protein. Therefore, a polyclonal antibody (8196) was raised against type II NOS from induced RAW 264.7 macrophages and used to investigate the expression of this enzyme in human lung tissue. Anti-type II NOS antibody did not cross-react with either neuronal (type I) or endothelial (type III) constitutive NOS, whereas a 130-kDa protein was detected in cytosol from induced macrophages or liver removed from lipopolysaccharide (25 mg/kg)-treated rats. Cells or tissues that lacked NOS activity did not express immunoreactive proteins. Similarly, in grossly normal human lung tissue, no immunoreactivity was detected with the anti-type II NOS antibody. In contrast, strong immunoreactivity was detected in alveolar macrophages present in lung tissue from a patient with bronchiectasis and acute bronchopneumonia. These data demonstrate that human alveolar macrophages are able to express type II NOS and support a role for this enzyme in pulmonary inflammatory pathophysiology.

Acute Disease↗
PubMed1994

Maturation-related changes in endothelial nitric oxide synthase immunolocalization in developing ovine lung.

It is unknown whether high fetal pulmonary vascular tone is due in part to absent or decreased endothelial nitric oxide synthase (eNOS), the enzyme that produces nitric oxide in the vascular endothelium. To determine the timing of appearance and maturational changes of eNOS in the developing pulmonary circulation, we performed immunohistochemistry in lungs from fetal, neonatal, and adult sheep. Using a mouse monoclonal antibody against bovine aortic eNOS, we found immunoreactive eNOS selectively in the endothelium and it was present at all fetal ages. Immunoreactivity was seen as early as 29% gestation in the developing capillaries coursing through fetal mesenchyme. By 6 days after birth, immunoreactivity was decreased in most vessels and nearly absent in the distal pulmonary arteries of adult animals. We conclude that immunoreactive eNOS is present very early in fetal life and appears to decrease postnatally. We speculate that the early presence of eNOS in the fetal lung supports a possible role for endogenous nitric oxide activity in the regulation of vascular tone or angiogenesis in the developing pulmonary circulation.

Aging↗
PubMed1994

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Amino Acid Oxidoreductases↗
PubMed1993

Nitric oxide synthases in neuronal cells, macrophages and endothelium are NADPH diaphorases, but represent only a fraction of total cellular NADPH diaphorase activity.

NADPH diaphorase activity is used as a histochemical marker for neuronal nitric oxide (NO) synthase; however, it remains unclear whether these activities are directly correlated in all tissues. In N1E-115 neuroblastoma cells, NADPH diaphorase activity was found primarily in the particulate fraction, whereas NO synthase activity was mostly soluble. Non-induced macrophages expressed significant NADPH diaphorase activity (which was mostly particulate) but virtually no NO synthase activity. Induction of macrophages produced marked increases in both NO synthase and NADPH diaphorase activities in the soluble and particulate fractions. In endothelial cells, both NO synthase and NADPH diaphorase activities were found mostly in the particulate fraction. Purified NO synthases from brain (type I), macrophages (type II), and endothelium (type III) all showed NADPH diaphorase activity; relative activities were: macrophage > endothelium > brain. These data indicate that all known NO synthases are NADPH diaphorases; however, NO synthases represent only a fraction of total cellular NADPH diaphorase activity and these activities are not always co-localized.

Amino Acid Oxidoreductases↗
PubMed1993

Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain.

In the presence of added flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both cytosolic and particulate nitric oxide synthase (NOS) activities can be detected in rat brain. Developmental changes of the cytosolic and particulate NOS in rat cerebellum and cerebrum were determined biochemically and immunochemically. Particulate NOS activity in the cerebrum increased during the first week of development, but then decreased and became almost undetectable in adult rats. In contrast, the cytosolic NOS in cerebellum showed low activity in newborns, but then constantly increased reaching an 8-fold higher level in adult rats. The activities of cerebellar particulate and cerebral cytosolic NOS also increased slightly during maturation. Western blot analysis using a polyclonal antibody raised against rat cerebellar cytosolic NOS revealed that the particulate and cytosolic fractions of 1-week-old and adult rat brains contained the same 160 kDa NOS protein. The relative content of the NOS protein correlated well with the relative amount of NOS activity in all brain fractions. These results indicate that distribution of NOS in the rat brain changes during maturation, but the same NOS protein is likely to be responsible for activities in immature and mature brains, cytosolic and particulate fractions. We suggest that nitric oxide might play a role in functional differentiation of the brain.

Amino Acid Oxidoreductases↗
PubMed1993

A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative.

In histochemical studies using fixed brain tissue, NADPH-diaphorase has been found to be colocalized with soluble nitric oxide synthase. In the present study, using fresh tissues from eight different regions of rat brain, NADPH-diaphorase activity was found mostly in the particulate fraction, whereas most of the nitric oxide synthase activity was located to the cytosolic fraction. Also, the distribution of NADPH-diaphorase activity among brain regions was different from that of nitric oxide synthase. Pretreatment of the fractions with paraformaldehyde virtually abolished the NADPH-diaphorase activity in the particulate fraction, whereas 40-60% of the NADPH-diaphorase activity remained intact in the cytosolic fraction. These results suggest that during fixation most NADPH-diaphorase activity is inactivated and only some of the NADPH-diaphorase activity associated with soluble nitric oxide synthase remains intact.

Amino Acid Oxidoreductases↗
PubMed1993

Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

Adolescent↗
PubMed1993

Effects of lorazepam on oxygen saturation before cardiac surgery.

We have studied patients presenting for coronary artery bypass surgery on two nights before surgery. Oxygen saturation during a night when the patient received no night sedation was compared with the night before surgery, when lorazepam 3-4 mg was given. There was no difference between the two nights when the total time at oxygen saturations less than 90% were compared, but the minimum oxygen saturation value when the patient received lorazepam was significantly smaller. No patient had significant sleep apnoea.

Coronary Artery Bypass↗
PubMed1993

Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies.

We have produced specific monoclonal antibodies (MAb) against particulate bovine aortic endothelial nitric oxide synthase. In Western blots, native and cultured bovine aortic endothelial cells as well as cultured bovine microvascular endothelial cells possess immunoreactive NO synthase. In dot blots, MAb H210 and H32 detect 1 ng and 100 pg of purified endothelial NO synthase, respectively. Both antibodies are specific to the endothelial NO synthase and do not cross-react with other known isoforms of NO synthase, namely from the brain, from cytokine/endotoxin-induced macrophages, or from cytokine/endotoxin-induced vascular smooth muscle cells. Immunohistochemical studies demonstrated the specificity of endothelial NO synthase for endothelial cells in various bovine and human tissues. Many types of endothelial cells, macrovascular, microvascular, arterial, and venous were found to possess this specific isoform of NO synthase. Electron microscopy showed the enzyme to be associated with the plasma membrane, membranes of cytoplasmic vesicles, and in the cytoplasm in human umbilical vein endothelial cells. The results demonstrate that particulate endothelial NO synthase is present in a site to act rapidly to produce NO for release into the blood or toward the smooth muscle in many vascular beds.

Amino Acid Oxidoreductases↗
PubMed1993

Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase.

Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Cerebral endothelial nitric oxide synthase expression after focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: The purpose of this study was to measure the temporal profile of expression of the endothelial nitric oxide synthase (NOS) in cerebral microvessels after middle cerebral artery occlusion in the rat. METHODS: Middle cerebral artery occlusion was performed on 24 male Wistar rats by extracranial insertion of a 4-0 nylon monofilament into the internal artery. Three additional rats were used as controls. Animals were killed at 1, 2, 4, 6, 24, 48, 72, and 168 hours after middle cerebral artery occlusion (n = 3 per time point). Rat brains were perfused with buffer, frozen, sectioned, and stained with a monoclonal antibody against endothelial NOS. Adjacent sections were stained with hematoxylin and eosin for evaluation of neuronal damage. RESULTS: The endothelial NOS in the cerebral vessels was upregulated at 1 hour after induction of ischemia throughout the ischemic region. The induction of the endothelial NOS progressively increased up to 24 hours of ischemia. In the periphery of the area of necrosis in the cortex, a delayed (24-hour) upregulation of the endothelial NOS remained constant throughout the duration of ischemia. CONCLUSIONS: The rapid and intense differential expression of the endothelial NOS in the core and peripheral areas of the lesion indicates a role for endothelial NOS in ischemic cell damage and suggests that the increased expression of NOS may mediate changes in the cerebral blood flow.

Amino Acid Oxidoreductases↗
PubMed1993

Isoforms of nitric oxide synthase: functions in the cardiovascular system.

Various cell types, including endothelial cells, can synthesize nitric oxide (NO). Three different isoforms of NO synthase have been characterized, purified and cloned. Isozyme I is present in neuronal cells of the brain (where NO may mediate synaptic plasticity), in peripheral non-adrenergic non-cholinergic (NANC) neurons (where NO acts as an atypical neurotransmitter relaxing vascular and non-vascular smooth muscle), and in various specialized epithelial cells. Macrophages can be induced with bacterial endotoxin and/or cytokines to express isozyme II. The high concentrations of NO produced by this isoform have cytostatic effects on parasitic microorganisms and tumour cells. A similar isozyme can be induced in the vascular wall (presumably in smooth muscle cells) in sepsis and during cytokine therapy. The large amounts of NO produced by this enzyme contribute to the symptoms of septic shock, such as vasodilatation and microvascular endothelial damage. Endothelial cells contain isoform III of NO synthase which seems to be unique for this cell type. Endothelium-derived NO is a physiologically significant vasodilator and inhibitor of platelet aggregation and adhesion. In addition, vascular NO can prevent leukocyte adhesion to the endothelium by interfering with the adhesion molecule CD11/CD18, and NO has also been shown to inhibit the proliferation of vascular smooth muscle cells. Hence, NO represents a protective factor against vascular damage and probably atherogenesis.

Amino Acid Oxidoreductases↗
PubMed1992

General anaesthesia in the dental surgery: a comparison of propofol and methohexitone.

General anaesthesia was assessed in 100 dental patients in general dental practice using either propofol or methohexitone. Mean induction doses were 1.7 mg/kg and 1.5 mg/kg for propofol and methohexitone, respectively. Anaesthesia was maintained with nitrous oxide and oxygen, supplemented in the majority of cases with halothane. Both groups were comparable in relation to satisfactory surgical conditions and recovery. The maximum heart rate following induction of anaesthesia was significantly greater in the group given methohexitone. Despite the satisfactory results obtained in this study the need for full resuscitation facilities will encourage the trend towards most dental general anaesthesia taking place in a hospital environment.

Adult↗