Search PubMed⌕ Search

Biomedical subjects

R G Knowles

Publications and source records attributed to R G Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1994

Inhibition of hepatic gluconeogenesis by nitric oxide: a comparison with endotoxic shock.

Isolated hepatocytes incubated in the presence of the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholino-sydnonimine (SIN-1) displayed a time- and dose-dependent inhibition of glucose synthesis from lactate plus pyruvate as the substrate which correlated with NO production, but not nitrite production. Neither the parent compound of SNAP, N-acetyl-DL-penicillamine (NAP), nor nitrite or nitrate had any significant effect on glucose output, indicating that the inhibition was due to the generation of NO within the incubation medium. The concentrations of NO required for this effect (< 800 nM) are within the range reported to occur in intact tissues and in vivo. The magnitude of the inhibitory effect of SNAP (approximately 50%) was comparable with that of endotoxin treatment of the rat with lactate plus pyruvate as the substrate. When the effect of SNAP on glucose synthesis and lactate plus pyruvate synthesis from a number of different substrates was examined, this showed a pattern comparable with that observed after endotoxin treatment of the rat, suggesting that NO may be the inhibitory mediator of the effects of bacterial endotoxin on hepatic gluconeogenesis. The NO donor had no effect on the flux through 6-phosphofructo-1-kinase, supporting the concept that the primary site of inhibition of gluconeogenesis by both NO and endotoxin resides at the level of phosphoenolpyruvate formation.

Animals↗
PubMed1994

Nitric oxide synthase activity in human gynecological cancer.

Nitric oxide is generated by the NO synthases, a family of isoenzymes expressed in a wide range of mammalian cells. In the vascular and nervous systems distinct isoforms generate NO to act as a signal transduction mechanism. The isoform induced by cytokines, on the other hand, provides a sustained release of NO which mediates some cytotoxic and cytostatic effects of the immune system. Solid tumors are a heterogeneous population of cell types, including tumor, vascular, and infiltrating immune cells. Studies in vitro show that NO synthase can be present in many of these cells. However, its presence in situ in solid human tumors has not been reported. In this study, we have investigated NO synthase activity and its cellular localization in malignant and nonmalignant human gynecological tissue. Nitric oxide synthase activity was observed in malignant tissue, was highest (> or = 250 pmol/min/g tissue) in poorly differentiated tumors, and was below detectable levels in normal gynecological tissue. Furthermore, investigations with a polyclonal NO synthase antibody revealed immunoreactivity only in malignant tissue. This was associated with NO synthase activity and localized to tumor cells. Thus NO synthase is present in human gynecological tumors, and its presence seems to correlate inversely with the differentiation of the tumor.

Amino Acid Oxidoreductases↗
PubMed1994

Induction of nitric oxide synthases early in pregnancy.

OBJECTIVE: We have previously demonstrated that calcium-dependent nitric oxide synthase is induced by estrogen and that by the end of pregnancy nitric oxide synthase of both endothelial and neuronal origin is increased in various maternal tissues. This rise in activity may be crucial for the alterations in muscle activity necessary for a successful pregnancy. If so, the increase in nitric oxide synthase activity must occur early in gestation. STUDY DESIGN: We tested the hypothesis that pregnancy increases nitric oxide synthase activity early in gestation by measuring in heart, kidney, skeletal muscle, and esophagus of time-mated guinea pigs the conversion by nitric oxide synthase of carbon 14-labeled L-arginine to carbon 14-labeled citrulline and the concentration of cyclic guanosine monophosphate, the second messenger of nitric oxide. RESULTS: Calcium-dependent nitric oxide synthase activity was increased twofold to fourfold by pregnancy in each tissue examined. The rise began by 0.14 gestation (9 of 63 +/- 2 days) and reached a plateau by 0.30 gestation (19 days), which was then maintained until term. The treatment of pregnant animals with tamoxifen decreased nitric oxide synthase activity to nonpregnant values in the heart, where tamoxifen is an estrogen receptor antagonist, but not in kidney, skeletal muscle, and esophagus. Cyclic guanosine monophosphate also rose progressively in each tissue studied until about 0.70 gestation before declining in skeletal muscle, kidney, and heart. It remained elevated in the esophagus. CONCLUSION: These studies demonstrate that nitric oxide synthase activity in maternal tissues rises early in pregnancy and is associated with an increase in cyclic guanosine monophosphate, the second messenger of nitric oxide. These findings are consistent with the hypothesis that an increase in nitric oxide synthase plays a role in smooth muscle adaptations of pregnancy.

Amino Acid Oxidoreductases↗
PubMed1994

Pregnancy increases guanosine 3',5'-monophosphate in the myometrium independent of nitric oxide synthesis.

The mechanism for myometrial quiescence during pregnancy is unknown. cGMP plays an integral role in the relaxation of smooth muscle, and nitric oxide (NO) is the most important endogenous activator of soluble guanylate cyclase. The purpose of this study was to determine the effect of gestational age on myometrial cGMP and NO synthase (NOS) activity in the guinea pig. Myometrial cGMP content (measured by RIA) rose slowly until 0.49 (fraction of pregnancy completed) gestation before abruptly increasing to 200 times the non-pregnant control value. It then declined precipitously after 0.87 gestation. Of the known isoenzymes of NOS, the messenger RNAs coding for both endothelial and neuronal NOS could be amplified from the myometrium of pregnant and nonpregnant animals using reverse transcriptase-polymerase chain reaction, but inducible NOS messenger RNA was not found. Myometrial calcium-dependent NOS activity (measured by the conversion of L-[U-14C]arginine to [U-14C]citrulline) declined slowly with advancing gestation (r2 = 0.096; slope = -0.34; P = 0.01), but never differed significantly from the activity in nonpregnant animals [31.1 +/- 11 (term pregnancy) vs. 56.9 +/- 16 (nonpregnant) pmol/min.g; P = NS]. Calcium-independent activity declined shortly after conception, and then rose toward the nonpregnant level (r2 = 0.19; slope = 0.45; P = 0.0009). However, at no time was it significantly different from that in the nonpregnant animal. Pregnancy had no effect on myometrial L-arginine and L-citrulline content. The administration of L-nitro-arginine methyl ester (200 mg/kg) to inhibit NOS dramatically increased blood pressure and reduced fetal renal NOS activity, but had no effect on the myometrial cGMP content. Estradiol (500 micrograms/kg for 5 days) modestly increased cGMP, but in contrast to many tissues in which estradiol increases NOS, it had no effect on myometrial NOS activity. We conclude that pregnancy dramatically increases cGMP by a mechanism independent of NOS. The stimulus remains to be identified. The temporal change in cGMP concentration is consistent with the hypothesis that cGMP mediates myometrial quiescence during pregnancy.

Animals↗
PubMed1993

A quantitative analysis of the control of glutamine catabolism in rat liver cells. Use of selective inhibitors.

1. At a physiological concentration of glutamine (0.5 mM), 87% of the total transport across the plasma membrane of liver cells isolated from fed rats involved the Na(+)-dependent system N; this was substantially inhibited by L-histidine. The residual Na(+)-independent component was attributed to system L on the basis of inhibition by 2-amino-2-norbornanecarboxylate and L-tryptophan. 2. Catabolism of glutamine by intact liver cells or by isolated mitochondria was inhibited by glutamate gamma-hydrazide with IC50 values of 13.7 +/- 3.5 microM and 22.6 +/- 3.8 microM respectively and a maximal inhibition of approx. 75%. The site of inhibition was identified as glutaminase; glutamate gamma-hydrazide inhibited this enzyme in cell-free extracts (IC50 37.8 +/- 7.7 microM) but had no activity against glutamate dehydrogenase or transport of glutamine, whether across mitochondrial or plasma membranes. 3. The major control site in cells from fed animals incubated with 0.5 mM L-glutamine was glutaminase (flux control coefficient 0.96). Appreciable control also resided in both plasma membrane transport systems, with coefficients of 0.51 for system N and -0.46 for system L, such that both interacted to provide a fine control of the intracellular concentration of the amino acid. Similar values were obtained by computer simulation based on theoretical determination of elasticities. 4. Previous controversy about the locus of regulation of hepatic glutamine metabolism is resolved by this distribution of control.

Animals↗
PubMed1993

Uptake of acetaminophen (paracetamol) by isolated rat liver cells.

The characteristics of the uptake of acetaminophen (N-acetyl-p-aminophenol or paracetamol, APAP) in incubations of isolated rat liver cells were consistent with diffusion of the drug being the predominant mechanism of APAP influx in these cells at concentrations above 0.5 mM. At lower substrate concentrations (below 0.5 mM) a saturable component was apparent. Both uptake processes could have a role in the control of the metabolism of APAP, because, at low concentrations, there was no intracellular accumulation of unconjugated drug, all the APAP entering the cell being converted to sulphate and glucuronide. After addition of drug, there was a lag phase of approximately 5 min before APAP-glucuronide and APAP-sulphate appeared in the incubation medium; during this time both conjugates accumulated inside the cells. These results have implications for our understanding of the mechanisms of APAP transport, and indicate how these processes may affect the drug's overall metabolism.

Acetaminophen↗
PubMed1992

Effect of treatment in vivo of rats with bacterial endotoxin on fructose 2,6-bisphosphate metabolism and L-pyruvate kinase activity and flux in isolated liver cells.

The effect of treatment of rats with bacterial endotoxin on fructose 2,6-bisphosphate (Fru-2,6-P2) metabolism was investigated in isolated liver cells prepared from 18 h-starved animals. The results obtained support the hypothesis that a stimulation of 6-phosphofructo-1-kinase (PFK-1) activity and an inhibition of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) may be one mechanism underlying the inhibition of gluconeogenesis from lactate and pyruvate by endotoxin. We suggest that the stimulation of PFK-1 and inhibition of Fru-1,6-P2ase activity is the result of a 2-3-fold increase in Fru-2,6-P2. The latter is not due to changes in the total activity or phosphorylation state of the bifunctional 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, but appears to be the result of a decrease in the cytosolic concentration of phosphoenolpyruvate (PEP), an inhibitor of PFK-2 activity. The effect of endotoxin is resistant to the presence of glucagon, which has comparable effects in cells prepared from both control and endotoxin-treated animals. The mechanism by which endotoxin treatment of the rat decreases PEP and gluconeogenesis remains to be established. However, it does not involve alterations in either the total activity or the phosphorylation state of pyruvate kinase, nor does it involve increased flux through this enzyme in the intact cell, which is in fact decreased in this model of septic shock. It is suggested that the decreased flux may result from a lower rate of formation of PEP, suggesting that the prime lesion in sepsis is an inhibition of one or more of the steps leading to PEP formation.

Animals↗
PubMed1992

Measurement of glucuronidation by isolated rat liver cells using [14C]fructose.

We have developed a simple and sensitive method for the study of the relative rates of glucuronidation of compounds, in isolated liver cells, based on the incorporation of 14C from fructose into glucuronide conjugates. Liver cells from fasted rats are used to minimize any reduction of the specific activity by glycogenolysis. Although rates of glucuronidation are lower in isolated liver cells from fasted rats than in those from fed rats, because of a reduction in the concentration of UDP-glucuronic acid, it is possible to compare the rates of glucuronidation of different compounds. Radiolabelled glucuronides are separated from [14C]fructose and [14C]glucose, produced by the liver cells, by normal-phase HPLC on a polar amino-cyano column. The specific activity of the glucuronide was found to be approximately 50% of that of the [14C]fructose. Absolute amounts of glucuronide can be determined by measuring the specific activity of the [14C]glucose, also produced by liver cells from fructose, which reflects that of the glucose-6-phosphate and hence the UDP-glucuronic acid used for glucuronidation, although for the measurement of relative rates this would not be necessary. We have used this method to examine the kinetics of the glucuronidation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The method should be applicable to the study of the rates of glucuronidation of a range of aglycones and, unlike other methods, does not require glucuronide standards or radiolabelled aglycone.

Acetaminophen↗
PubMed1992

Enhancers of nonspecific immunity induce nitric oxide synthase: induction does not correlate with toxicity or adjuvancy.

A range of bacterial products and related synthetic compounds, either alone or in combination, enhance nonspecific resistance to infection and tumors. These compounds, which vary in their other properties such as pyrogenicity, toxicity and adjuvancy, were used to assess the hypothesis that nonspecific resistance is mediated by induction of the L-arginine: nitric oxide (NO) pathway. The results obtained show that agents which stimulate nonspecific immunity, such as endotoxin, muramyl dipeptide (MDP) and combinations of monophosphoryl lipid A (MPL) with either trehalose dimycolate (TDM) or MDP cause a substantial induction of Ca(2+)-independent NO synthase in murine lung. In contrast, agents which do not stimulate nonspecific resistance, such as either MPL or TDM alone or threonyl MDP (ThrMDP), do not induce NO synthase. This difference in the ability of MDP and ThrMDP to induce NO synthase in the lung in vivo was also manifest in peritoneal macrophages in vivo as well as being evident in the greater than 100-fold greater potency of MDP in inducing the enzyme in vitro in lung slices. In contrast to the good correlation between induction of NO synthase and induction of nonspecific resistance, no correlation was observed with either the toxic effects of these agents or their adjuvancy.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1992

Nitric oxide as a signal in blood vessels.

In the five years since the discovery that nitric oxide is produced as a signal in blood vessels, a great deal has been discovered about the processes involved. This article reviews current knowledge about the vascular cell synthesis, effects and subsequent destruction of this messenger molecule.

Amino Acid Oxidoreductases↗
PubMed1991

Constitutive and inducible nitric oxide synthases incorporate molecular oxygen into both nitric oxide and citrulline.

Nitric oxide (NO) is synthesized by a number of cells from a guanidino nitrogen atom of L-arginine by the action of either constitutive or inducible NO synthases, both of which form citrulline as a co-product. We have determined the source of the oxygen in both NO and in citrulline formed by the constitutive NO synthase from the vascular endothelium and brain and by the inducible NO synthase from the murine macrophage cell line J774. All these enzymes incorporate molecular oxygen both into NO and into citrulline. Furthermore, activated J774 cells form NO from omega-hydroxyl-L-arginine, confirming the proposal that this compound is an intermediate in the biosynthesis of NO.

Amino Acid Oxidoreductases↗
PubMed1991

Antitumor activity of gamma-interferon in ascitic and solid tumor models of human ovarian cancer.

We have investigated the action of recombinant human gamma-interferon (rHuIFN-gamma) against human ovarian cancer xenografts growing as ascites or as bulky solid i.p. tumors in nude mice. Both forms of the disease responded to i.p. rHuIFN-gamma with significant increases in mouse survival time, and in 2 of 3 ascitic models the mice were cured of peritoneal disease. The activity of rHuIFN-gamma was dose and schedule dependent, and xenografts derived from 3 different patients showed a heterogeneity of response. Peak i.p. levels of rHuIFN-gamma in nude mice bearing multiple i.p. solid tumors were similar to those found in ovarian cancer patients receiving i.p. rHuIFN-gamma, but clearance was more rapid in the mice. Rat gamma-interferon had no antitumor activity at the same doses and schedules although it had some biological activity in the nude mice. Histological examination of treated tumors revealed increased necrosis and loss of cellular organization with large areas of hypocellular epithelial mucin. These changes were preceded by a fall in tumor tryptophan and a rise in tumor kynurenine. We conclude that rHuIFN-gamma has a direct dose related antitumor effect on ovarian cancer xenografts that is preceded by increased metabolism of tryptophan.

Animals↗
PubMed1991

Synthesis of nitric oxide in the bovine retina.

In the absence of light, high concentrations of cGMP open ion channels in the plasma membranes of rod outer segments. The source of stimulation of retinal guanylate cyclase is not known. Nitric oxide is a potent stimulator of guanylate cyclase in other cell systems. The present data demonstrate that nitric oxide synthase, an enzyme responsible for the production of nitric oxide, is present in retina, and specifically in the rod outer segments. This enzyme uses L-arginine as a substrate and is NADPH- and calcium- dependent. L-arginine-derived nitric oxide may be a source of activation of guanylate cyclase in the retina.

Amino Acid Oxidoreductases↗
PubMed1991

Widespread tissue distribution, species distribution and changes in activity of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthases.

The distribution of Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase (NOS) was studied in rabbits and in control and endotoxin-treated rats and guinea-pigs. There was a widespread localization of NOS which differed for the two forms of the enzyme and which showed marked differences between species. Endotoxin induced the activity of the Ca(2+)-independent NOS in many tissues and also increased the activity of Ca(2+)-dependent NOS in the rat ileum and caecum. These results demonstrate the differential distribution of NOSs in control and endotoxin-treated animals and emphasize the widespread biological role of nitric oxide (NO).

Amino Acid Oxidoreductases↗
PubMed1991

Quantitative studies of sulphate conjugation by isolated rat liver cells using [35S]sulphate.

We have developed a simple, rapid and sensitive method for the study of sulphate conjugation in isolated liver cells based on the incorporation of 35S from [35S]sulphate. Excess [35S]sulphate is removed by a barium precipitation procedure, leaving [35S]sulphate conjugates in solution. We have used this method to examine the kinetics of sulphation of N-acetyl-p-aminophenol (acetaminophen), 4-nitrophenol and 1-naphthol in isolated rat liver cells. The efficiency of recovery of the sulphate conjugates was greater than 86%. The method is applicable to the quantitative study of sulphate conjugation of any substrate which forms a sulphate conjugate that is soluble in the presence of barium, without the need for standards or radiolabelled sulphate acceptors.

Acetaminophen↗