22 results on '"Kudolo, G. B."'
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2. Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium
- Author
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Kudolo, G. B., primary, Yang, Y-Q., additional, Chen, D-B., additional, Jones, M. A., additional, and Harper, M. J. K., additional
- Published
- 1995
- Full Text
- View/download PDF
3. Platelet-activating factor antagonists and implantation in rabbits
- Author
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Norris, C. J., primary, Peairs, W. A., additional, Kudolo, G. B., additional, Newton, E. R., additional, and Harper, M. J. K., additional
- Published
- 1994
- Full Text
- View/download PDF
4. Rabbit blastocysts accumulate platelet-activating factor (PAF) and lyso-PAF in vitro
- Author
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Jones, M. A., primary, Kudolo, G. B., additional, and Harper, M. J. K., additional
- Published
- 1992
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5. Further characterization of the second oestrogen-binding species of the rat granulosa cell
- Author
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Kudolo, G. B., Elder, M. G., and Myatt, L.
- Abstract
Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A–Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4°C (1·72 ± 0·27(s.e.m.) × 108mol/h) and 25°C (4·50 ± 0·36 × 108mol/h) was faster than with the oestrogen receptor (7·20 ± 0·15 × 107mol/h and 1·23 ± 0·15 × 108mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25°C (rate constants k−1= 0·30±0·07/min and k−2= 3·73±0·57 × 10−3/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10·3S, Stokes radius >5·2 nm) in low ionic strength buffers and as a small species (4·4S, Stokes radius 3·5 nm) in buffers containing 0·4 m-KCl. However, the SOB fractionated as 2–3S, Stokes radius 3·7–4·0 nm at low ionic strength and as 5·8S, Stokes radius 3·5 nm in 0·4 m-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4·6S, mol. wt 69–72 000 on SDG.J. Endocr.(1984) 102, 93–102
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- 1984
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6. Oestrogen receptor in the granulosa cell during postnatal development of the rat ovary
- Author
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Kudolo, G. B., Elder, M. G., and Myatt, L.
- Abstract
Numbers of granulosa cells obtained from follicles of immature rats increased from 1·6 × 105cells/ovary on day 8 to 7·1 × 106cells/ovary on day 40 of age, the day of vaginal opening and first pro-oestrus. Very high levels of cytosol oestrogen receptor were found on day 8 (175 000 sites/cell) but by day 19 20 000 sites/cell were found. Nuclear receptor concentrations were highest on day 12(5400 ± 1470 (s.d.) sites/cell) and again on day 21 (5400 ± 2300 sites/cell). After day 21 both cytosol and nuclear oestrogen receptor concentrations fell and remained low until nuclear concentrations rose at day 40. Two consecutive daily injections of FSH/LH (5 i.u.) increased cell number over control in animals killed on day 22, gave no significant alteration in animals killed on day 26 or 28 but decreased numbers in animals aged 32 and 35 days. Only on day 22 was the increase in cell number associated with an increase in nuclear oestrogen receptor concentrations. Indeed on days 32 and 35 increased nuclear receptor concentrations were associated with a decreased cell number.J. Endocr.(1987) 112,333–338
- Published
- 1987
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7. A novel oestrogen-binding species in rat granulosa cells
- Author
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Kudolo, G. B., Elder, M. G., and Myatt, L.
- Abstract
The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd0·35 ±0.09 nmol/l) and cytosol (Kd0·39 ± 0·03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75–96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd45·8± 6·9 nmol/l (capacity 16·7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd0·58 ± nmol/l (capacity 2·8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured.The second oestrogen-binding species displayed affinity for oestradiol-17β, oestriol, oestrone, testosterone, 5α-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors. It can be further distinguished from the oestrogen receptor by differential ammonium sulphate precipitation and the stability of its ligand binding at temperatures above 55 °C where the oestrogen receptor–ligand interaction is rapidly lost.Concentrations of nuclear oestrogen receptor in granulosa cells (2200 sites/cell) were similar to those found in other target tissues but a high proportion of this receptor (70%) was 'unoccupied' or available for binding at 4 °C and the majority (75%) was resistant to extraction with 0·4 m-KCl. As the second oestrogen-binding species could not be detected in granulosa cell nuclei it is unlikely to be involved directly in eliciting genomic responses to hormonal stimulation. It is more probable that it regulates the level of the free intracellular steroid to which the oestrogen receptor of the granulosa cell (the predominant site of oestrogen biosynthesis) is exposed.J. Endocr.(1984) 102, 83–91
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- 1984
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8. Reproduction in the vervet monkey (Cercopithecus aethiops): endometrial oestrogen and progestin receptor dynamics during normal and prolonged menstrual cycles
- Author
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Kudolo, G. B., Mbai, F. N., and Eley, R. M.
- Abstract
Sixteen individually caged adult female vervet monkeys (Cercopithecus aethiops), whose reproductive parameters had been studied for 5 years, were hysterectomized/ovariectomized during three reproductive states; i.e. the late follicular (15·4 ± 4·7 (s.d.) days) and luteal (27·8 ± 4·7 days) phases of the normal cycle (20–50 days), and during prolonged intermenstrual intervals (PII; 99·0 ± 2·5 days since the previous menses). These latter animals showed characteristics of both follicular and luteal phases; i.e. their ovaries contained both corpora lutea and large antral follicles and systemic oestradiol and progesterone concentrations were raised. Analysis of cytoplasmic oestrogen and progestin receptors (CER and CPR) revealed that endometrium during PII had CER levels of 0·58 ± 0·07 pmol/mg protein, similar to those of the follicular phase (0·60 ± 0·12); CPR levels (1·20 ± 0·87) were not different from those of the luteal phase (1·05 ± 0·45). The ratio of CPR to CER during the luteal phase was about tenfold higher than that of the follicular phase. Levels during PII were intermediate between the two phases. Under receptoractivating conditions, the DNA-binding components of the PII cytoplasmic fraction underwent over 40% loss while those present during both phases of the normal cycle doubled. The hormone-binding sites at all times remained intact indicating that the DNA and hormone-binding sites are distinct on both CER and CPR.Less than 50% interaction of CER/CPR with DNA-cellulose was obtained, indicating the presence of only limited quantities of cytoplasmic activating factors which may be a prerequisite for receptor binding to the genome. During PII, factors which deactivate DNA-binding sites may also have been induced. Extensive accumulation of nuclear oestrogen receptor was evident in PII endometrium with 80% being salt-resistant. This level is higher than that in the follicular and luteal phases (37 and 52% respectively). These data, suggesting a possible aberration of receptor activation in vitroand receptor processing in vivo, may be indicative of endometrial dysfunction during PII. This could lead to a delay in menstruation.J. Endocr.(1986) 110,429–439
- Published
- 1986
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9. The effect of 3-month ingestion of Ginkgo biloba extract (EGb 761) on pancreatic beta-cell function in response to glucose loading in individuals with non-insulin-dependent diabetes mellitus.
- Author
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Kudolo GB
- Subjects
- Blood Glucose metabolism, C-Peptide blood, Diabetes Mellitus, Type 2 drug therapy, Diet, Female, Glucose Tolerance Test, Humans, Hypoglycemic Agents administration & dosage, Insulin blood, Insulin metabolism, Islets of Langerhans drug effects, Male, Middle Aged, Plant Extracts administration & dosage, Plant Extracts adverse effects, Plant Extracts pharmacology, Vasodilator Agents administration & dosage, Vasodilator Agents adverse effects, Vasodilator Agents pharmacology, Vasodilator Agents therapeutic use, Diabetes Mellitus, Type 2 metabolism, Ginkgo biloba, Hypoglycemic Agents therapeutic use, Islets of Langerhans metabolism, Phytotherapy, Plant Extracts therapeutic use
- Abstract
In the first report (Journal of Clinical Pharmacology 2000; 40:647-654), it was shown that ingestion of 120 mg of Ginkgo biloba extract (EGb 761) daily for 3 months by normal glucose-tolerant individuals caused a significant increase in pancreatic beta-cell insulin and C-peptide response, measured as the area under the curve (AUC0-->120) during a 2-hour standard (75 g) oral glucose tolerance test (OGTT). This follow-up study was designed to determine the effect of the same Ginkgo biloba treatment on glucose-stimulated pancreatic beta-cell function in non-insulin-dependent diabetes mellitus (NIDDM) subjects. In diet-controlled subjects (fasting plasma glucose [FPG], 117 +/- 16 mg/dl; fasting plasma insulin [FPI], 29 +/- 8 microU/ml; n = 6), ingestion of Ginkgo biloba produced no significant effect on the insulin AUC0-->120 (193 +/- 53 vs. 182 +/- 58 microU/ml/h, before and after ingesting Ginkgo biloba, respectively). In hyperinsulinemic NIDDM subjects taking oral hypoglycemic medications (n = 6) (FPG 143 +/- 48 mg/dl; FPI 46 +/- 13 microU/ml), ingestion of Ginkgo biloba caused blunted plasma insulin levels from 30 to 120 minutes during the OGTT, leading to a reduction of the insulin AUC0-->120 (199 +/- 33 vs. 147 +/- 58 microU/ml/h, before and after Ginkgo biloba, respectively). The C-peptide levels increased, and so the AUC0-->120 did not parallel the insulin AUC0-->120, creating a dissimilar insulin/C-peptide ratio indicative of an enhanced hepatic extraction of insulin relative to C-peptide. Thus, in pancreatic beta-cells that are already maximally stimulated, ingestion of Ginkgo biloba may cause a reduction in plasma insulin levels. Only in NIDDM subjects with pancreatic exhaustion (FPG 152 +/- 46 mg/dl; FPI 16 +/- 8 microU/ml; n = 8), who also took oral hypoglycemic agents, did Ginkgo biloba ingestion significantly increase pancreatic beta-cell function in response to glucose loading (insulin AUC0-->120 increased from 51 +/- 29 to 98 +/- 20 microU/ml/h, p < 0.0001), paralleled by a C-peptide AUC0-->120 increase from 7.2 +/- 2.8 to 13.7 +/- 6.8 (p < 0.0001). Whether this increase is due to "resuscitation" of previously exhausted islets or increased activity of only the remaining functional islets is unclear. However, not even in this group did increased pancreatic beta-cell activity cause a reduction of blood glucose during the OGTT. It is concluded that ingestion of Ginkgo biloba extract by an NIDDM subject may increase the hepatic metabolic clearance rate of not only insulin but also the hypoglycemic agents. The result is reduced insulin-mediated glucose metabolism and elevated blood glucose.
- Published
- 2001
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10. The effect of 3-month ingestion of Ginkgo biloba extract on pancreatic beta-cell function in response to glucose loading in normal glucose tolerant individuals.
- Author
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Kudolo GB
- Subjects
- Adult, Alzheimer Disease drug therapy, Alzheimer Disease psychology, C-Peptide blood, Female, Glucose Tolerance Test, Humans, Insulin blood, Islets of Langerhans physiology, Male, Middle Aged, Plant Extracts pharmacology, Ginkgo biloba, Glucose pharmacology, Islets of Langerhans drug effects, Plants, Medicinal
- Abstract
Ginkgo biloba extract is ingested on a chronic basis for enhancing mental focus and improving cognitive function in the elderly. This study was undertaken to determine the effect of Ginkgo biloba extract on glucose-stimulated pancreatic beta-cell function in normal glucose-tolerant individuals. Twenty individuals (14 females and 6 males, ages 21-57 years) underwent a 2-hour 75 g oral glucose tolerance test (OGTT) before and after ingestion of Ginkgo biloba extract (120 mg/day at bedtime) for 3 months. Ginkgo biloba extract ingestion caused a decrease in systolic blood pressure from 125 +/- 15 to 118 +/- 12 mmHg (p < 0.05) and in diastolic blood pressure from 86 +/- 10 to 68 +/- 10 (p < 0.01). Fasting plasma insulin and C-peptide were increased, and the insulin and C-peptide areas under the curve during the OGTT changed from 136 +/- 55 to 162 +/- 94 microU/ml/h (p = 0.1232) and 9.67 +/- 5.34 to 16.88 +/- 5.20 ng/ml/h (p < 0.001), respectively. The observed dissimilar insulin/C-peptide response curves may be due to Ginkgo biloba-induced increase of the rate of insulin metabolic clearance.
- Published
- 2000
11. Urinary platelet-activating factor excretion is elevated in non-insulin dependent diabetes mellitus.
- Author
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Kudolo GB and DeFronzo RA
- Subjects
- Adult, Blood Glucose metabolism, Body Mass Index, Diabetes Mellitus, Type 2 urine, Humans, Insulin Resistance, Kidney Function Tests, Mexican Americans, Middle Aged, Proteinuria urine, Diabetes Mellitus, Type 2 metabolism, Platelet Activating Factor urine
- Abstract
Proteinuria is currently considered a very sensitive predictor of diabetic nephropathy, but 20-25% of all diabetic patients with negative Albustix reaction excrete higher than normal (< 20 mg/24 h) amounts of albumin in their urine. It is our hypothesis that platelet-activating factor (PAF), a potent glycerophospholipid that acts as a chemical mediator for a wide spectrum of biological activities, including increased vascular permeability, may be produced in significant amounts during periods preceding microalbuminuria. In this study, we compared urinary PAF excretion in Mexican-American subjects who were diagnosed with non-insulin dependent diabetes mellitus (NIDDM) with their healthy control counterparts. The age of the NIDDM subjects (45.9 +/- 2.1 years) was not significantly different from the healthy control group, which was 39.4 +/- 2.7 years (P < 0.0672). The NIDDM subjects (body mass index, 29.9 +/- 1.1 compared to 26.1 +/- 0.9 kg/m2 in healthy controls) were characterized by significantly increased (P < 0.05) fasting plasma glucose (192 +/- 11 vs. 97 +/- 4 mg/dl in healthy controls), fasting insulin (20.9 +/- 2.4 vs. 12.3 +/- 1.6 microU/ml), fasting C-peptide (2.93 +/- 1.26 vs. 1.48 +/- 0.51 ng/ml), and hemoglobin A1c (10.3 +/- 0.7 vs. 5.6 +/- 0.3%), respectively. The urine output for the NIDDM and control subjects were 1942 +/- 191 ml/24 h and 1032 +/- 94 ml/24 h, respectively, and urinary albumin excretion (UAE) rates were estimated to be 38 +/- 7 micrograms/min and 11 +/- 1 micrograms/min, respectively. The NIDDM subjects produced significantly increased levels of urinary PAF (2606.3 +/- 513.1 ng/24 h compared with 77.9 +/- 14.1 ng/24 h in controls (or 1706.3 +/- 420.8 ng/ml compared with 85.4 +/- 17.8 pg/ml of urine, in NIDDM and control subjects, respectively). We found that urinary PAF excretion was significantly correlated with microalbumin excretion (r = 0.7) especially at UAE rates greater than 30 mg/day and more importantly, some NIDDM patients with negative Albustix reaction (i.e. normal UAE) produced significantly more PAF, suggesting that PAF excretion may precede microalbuminuria and that subtle injury to the kidneys are present in NIDDM long before overt albuminuria ensues, urinary PAF measurements could potentially therefore serve as a sensitive indicator of renal injury in diabetes mellitus. These results lend further credence to our hypothesis that PAF may be the biochemical compound linking the various members of the insulin resistance syndrome.
- Published
- 1999
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12. Plasma PAF acetylhydrolase in non-insulin dependent diabetes mellitus and obesity: effect of hyperinsulinemia and lovastatin treatment.
- Author
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Kudolo GB, Bressler P, and DeFronzo RA
- Subjects
- 1-Alkyl-2-acetylglycerophosphocholine Esterase, Adult, Cholesterol blood, Cholesterol, LDL blood, Female, Glucose Tolerance Test, Humans, Insulin blood, Male, Middle Aged, Anticholesteremic Agents therapeutic use, Diabetes Mellitus, Type 2 enzymology, Hyperinsulinism enzymology, Lovastatin therapeutic use, Obesity enzymology, Phospholipases A blood
- Abstract
Insulin resistance is characterized principally by impaired insulin-mediated glucose uptake which provokes a compensatory increase in pancreatic beta-cell secretory activity. For a time this may produce well-controlled plasma glucose levels but as the insulin resistance worsens the augmented insulin production becomes inadequate to keep plasma glucose at euglycemia leading to the development of non-insulin dependent diabetes mellitus (NIDDM), accompanied by hyperinsulinemia and hyperglycemia. A number of metabolic defects are associated with NIDDM including obesity, hypercoagulability, cardiovascular disease risk factors such as hypertension and dyslipidemia and these constitute the insulin resistance syndrome. The identity of the biochemical factor that might link all these defects is not yet known. We have hypothesized that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) may be such a link. In this study, we measured plasma acetylhydrolase (EC.1.1.48), which degrades PAF to the inactive metabolise lyso-PAF, as a surrogate for PAF activity in three groups of hypercholesterolemic subjects: lean controls (n = 9), non-diabetic obese (n = 6) and NIDDM subjects (n = 6). The ages and body mass indices of the subjects were 46 +/- 3.1 and 24.2 +/- 2.2 for the lean controls, 52 +/- 2.5 and 28.7 +/- 0.9 for the NIDDM subjects and 60 +/- 2 and 27.6 +/- 2.1 for the obese, non-diabetic subjects (mean +/- S.E.M.). The measurements were made before and after therapy with the cholesterol-lowering drug lovastatin, a 3-hydroxy 3 methylglutaryl (HMG) coenzyme. A reductase inhibitor (40 mg/day) for 3 months. Fasting plasma glucose (FPG) levels were 91 +/- 11, 96 +/- 3 and 146 +/- 11 mg/dl, for the lean, obese and NIDDM subjects, respectively, before therapy began. Lovastatin did not affect FPG in any of the three subject groups. Before treatment, the fasting plasma insulin (FPI) levels were 6.1 +/- 0.92, 10.83 +/- 2.03 and 14.68 +/- 3.64 mU/l for the lean, non-diabetic obese and NIDDM subjects, respectively. After lovastatin therapy only the obese group exhibited a significant change in FPI (15.35 +/- 2.47 mU/l) (P < 0.05). Total cholesterol levels were similar in all three groups both before and after lovastatin therapy but within each group lovastatin therapy significantly reduced the total cholesterol by 32, 29 and 34% in the lean, obese and NIDDM subject groups respectively (P < 0.0001). Lovastatin therapy reduced LDL-cholesterol levels by 40, 32 and 46% in the lean, obese and NIDDM subjects, respectively, but produced no significant effect on HDL or triglyceride levels. Before therapy, the plasma acetylyhydrolase activities were 104 +/- 7, 164 +/- 7 and 179 +/- 7 nmol/ml per min in the lean, obese and NIDDM subjects, respectively. Lovastatin therapy reduced plasma acetylhydrolase levels to 70 +/- 7, 87 +/- 6 and 86 +/- 7 nmol/ml per min in the lean, obese and NIDDM subjects, respectively. Plasma acetylhydrolase activity was predominantly (> 80%) associated with LDL cholesterol both before and after lovastatin treatment. Also, plasma acetylhydrolase activity significantly correlated with fasting plasma insulin levels before lovastatin therapy but not after. Taken together, this study clearly implicates PAF metabolism in three defects associated with the insulin resistance syndrome: hypercholesterolemia, obesity and NIDDM. Additionally, we conclude that chronic hyperinsulinemia may play a significant role in the production of plasma acetylhydrolase.
- Published
- 1997
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13. Chronic hyperinsulinemia inhibits platelet-activating factor (PAF) biosynthesis in the rat kidney.
- Author
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Kudolo GB, Koopmans SJ, Haywood JR, and DeFronzo RA
- Subjects
- 1-Alkyl-2-acetylglycerophosphocholine Esterase, Acetyltransferases metabolism, Animals, Blood Pressure, Chronic Disease, Insulin blood, Insulin Resistance, Male, Phospholipases A blood, Rats, Rats, Sprague-Dawley, Hyperinsulinism metabolism, Kidney metabolism, Platelet Activating Factor biosynthesis
- Abstract
A number of risk factors for cardiovascular disease, including hypertension, are associated with the insulin resistance syndrome. The hallmark of this syndrome is an impairment in insulin action which provokes a compensatory increase in pancreatic beta-cell insulin secretion leading to chronic hyperinsulinemia. Indirect studies show that platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, PAF), a potent antihypertensive lipid produced by the kidney, may be decreased by hyperinsulinemia. The present study was designed to evaluate the effect of chronic hyperinsulinemia on renal PAF metabolism, arterial blood pressure and whole body insulin sensitivity. Chronic catheterized, unstressed rats were infused with saline or insulin plus glucose to create a chronic condition of sustained euglycemic (approximately 130 mg/dl) hyperinsulinemia (approximately 90 mU 1. or 3-fold over basal levels). PAF is a metabolically unstable compound being susceptible to rapid degradation to the biologically inactive lyso-PAF, a metabolite which also serves as a precursor for PAF synthesis. PAF synthesis and counter-regulatory prostaglandins may be derived from the same arachidonate precursor. The enzymes which catalyze these reactions were measured in plasma and in the subcellular fractions of the kidneys. Compared to saline-treated rats, sustained physiologic hyperinsulinemia for 7 days: (i) decreased insulin-mediated glucose disposal by 30%; (ii) caused an increased plasma PAF:acetylhydrolase, which degrades PAF to lyso-PAF, without any change in cytosolic PAF:acetylhydrolase activity; and (iii) completely inhibited microsomal lyso-PAF:acetyl CoA acetyltransferase activity which catalyzes the conversion of lyso-PAF back to bioactive PAF. The increased catabolism of PAF in plasma, combined with decreased renal PAF biosynthesis, would be expected to decrease circulating PAF levels leading to a rise in blood pressure. However, blood pressure remained unchanged. The sustained hyperinsulinemia stimulated plasma membrane CoA-independent transacylase activity, which is responsible for the mobilization of arachidonates into lyso-PAF, to form l-alkylarchidonoyl-glycerophosphocholine. The latter is the stored precursor for the synthesis of PAF and vasodilatory prostaglandins, which may have offset the effect of decreased PAF. We hypothesize that hyperinsulinemia may alter the blood pressure only if the balance between the synthesis/catabolism of PAF and vasodilatory prostaglandins is disrupted.
- Published
- 1997
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14. Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium.
- Author
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Kudolo GB, Yang YQ, Chen DB, Jones MA, and Harper MJ
- Subjects
- Acetyltransferases metabolism, Animals, Cell Separation, Cells, Cultured, Chromatography, Gas, Chromatography, Thin Layer, Embryonic Development, Epithelium metabolism, Female, Flame Ionization, Phospholipid Ethers metabolism, Phospholipid Ethers pharmacology, Platelet Activating Factor analogs & derivatives, Platelet Activating Factor metabolism, Pregnancy, Rabbits, Type C Phospholipases metabolism, Endometrium metabolism, Platelet Activating Factor pharmacokinetics, Pregnancy, Animal metabolism
- Abstract
Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.
- Published
- 1995
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15. Lyso-PAF:acetyl-CoA acetyltransferase and CDP-choline cholinephosphotransferase activities in the rabbit endometrium.
- Author
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Kudolo GB and Harper MJ
- Subjects
- Animals, Dithiothreitol pharmacology, Female, Kinetics, Microsomes enzymology, Phosphatidylcholines metabolism, Platelet Activating Factor biosynthesis, Rabbits, Acetyltransferases metabolism, Diacylglycerol Cholinephosphotransferase metabolism, Endometrium enzymology
- Abstract
Endometrial platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) levels change significantly during the pre-implantation period in the rabbit uterus, but under in vitro culture conditions, constitutive PAF biosynthesis by isolated endometrial tissues is not easily demonstrable. Rapid metabolism of PAF relative to its synthesis may account for this disparity because we have recently shown that in stromal cells there is a significant build-up of lyso-PAF suggesting that lyso-PAF-acetyl-CoA acetyltransferase might be a limiting factor. In the glandular epithelial cells however, the lyso-PAF build-up was replaced by a significant accumulation of a neutral lipid which was tentatively identified as 1-O-hexadecyl-2-acetylglycerol. It was hypothesized that, during endometrial growth and development, this lipid might serve as the substrate for the alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme for PAF synthesis via the de novo pathway. We have therefore examined the activities of lyso-PAF:acetyl-CoA acetyltransferase and the CDP-cholinephosphotransferase enzymes. Microsomal preparations containing lyso-PAF:acetyl-CoA acetyltransferase activity catalyzed the incorporation of [3H]acetyl-CoA lyso-PAF into two distinct lipid products. One co-migrated with authentic PAF and the other with 1-O-hexadecyl-2-acetylglycerol, the latter being formed subsequent to PAF formation. The alkylacetylglycerol CDP-choline cholinephosphotransferase enzyme, which would potentially utilize the alkylacetylglycerol synthesized via the remodeling pathway, was also demonstrable. Unlike the species present in other tissues however, it was found to be sensitive to the presence of 10 mM DTT. The diacylglycerol CDP-choline cholinephosphotransferase species was also demonstrable and supported the synthesis of both PAF and phosphatidylcholine, in the absence of DTT, when only the synthesis of phosphatidylcholine was expected. It is hypothesized that the rabbit endometrium possesses active enzymes which may catalyze PAF synthesis via both the de novo and 'remodeling' pathways.
- Published
- 1995
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16. Platelet-activating factor antagonists and implantation in rabbits.
- Author
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Norris CJ, Peairs WA, Kudolo GB, Newton ER, and Harper MJ
- Subjects
- Animals, Drug Administration Schedule, Female, Pregnancy, Rabbits, Embryo Implantation drug effects, Phospholipid Ethers pharmacology, Platelet Activating Factor antagonists & inhibitors
- Abstract
In an initial experiment, rabbits were injected i.v. with a platelet-activating factor (PAF) antagonist CV-3988 twice a day on days 5 and 6 of pregnancy. Some inhibition of implantation was observed. This effect could not be reproduced in subsequent experiments at the same or at larger or smaller doses. The non-metabolized analogue of PAF, N-carbamyl-PAF (C-PAF) had an inhibitory effect on implantation only when given at toxic concentrations. When CV-3988 and C-PAF were given together on days 5 and 6, there was no effect on implantation. None of the other PAF antagonists tested--BN52021, SRI63,441, WEB2086 or TCV-309--at various doses could inhibit implantation when given on the same days of pregnancy. TCV-309, at 0.1 mg kg-1 i.v. given on days 2-4 of pregnancy, was also ineffective. These results provide no clear support for a role of PAF in implantation in rabbits.
- Published
- 1994
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17. Pregnancy-associated remodeling of rabbit endometrial platelet-activating factor receptors.
- Author
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Kudolo GB and Harper MJ
- Subjects
- Animals, Female, Guanosine Triphosphate metabolism, Kinetics, Membranes metabolism, Pregnancy, Rabbits, Embryonic Development physiology, Endometrium metabolism, Platelet Activating Factor, Platelet Membrane Glycoproteins, Pregnancy, Animal metabolism, Receptors, Cell Surface metabolism, Receptors, G-Protein-Coupled
- Abstract
[3H]PAF binding parameters (affinity constants and binding capacities) were estimated for endometrial membranes obtained on days 3 (when the morulae first enter the gravid uterus), 4, 5 and on day 6, about 22 h before the blastocyst becomes irremovably attached to the endometrium. The two-site receptor system which characterized the binding on a large proportion of purified membranes from days 3 (60%) and 6 (75%) had the following parameters: day 3, Kd1 (nM) 0.15 +/- 0.04, Bmax1 (pmol/mg protein) 0.04 +/- 0.01; Kd2 (nM) 17.60 +/- 6.40, Bmax2 (pmol/mg protein) 2.92 +/- 0.91 (n = 6), and day 6, Kd1 (nM) 0.33 +/- 0.06, Bmax1 (pmol/mg protein) 0.11 +/- 0.02; Kd2 (nM) 7.42 +/- 1.02, Bmax2 (pmol/mg protein) 1.75 +/- 0.31 (n = 3). The remaining membranes, including all preparations of days 4 and 5, exhibited only one class of binding sites which were unlike the putative type 1 PAF receptor. The most significant observations were the apparent pregnancy-associated changes in the parameters of the PAF binding sites. However, regardless of the type of PAF binding exhibited, all the binding sites bound GTP. [3H]PAF dissociation from bound complexes was biphasic at 25 degrees C. The rate constants were k-1, 0.27 +/- 0.09 min-1 and k-2, 3.45 +/- 1.19 x 10(-3) min-1, for the rapid and slow dissociating components, respectively. In view of the inability to resolve the experimental data from days 4 and 5 into multiple sites, and of the occurrence of receptor interconversions on days 3 and 6, it is postulated that there may be cell membrane macromolecular reorganization/remodeling of proteins and cytoskeleton as a result of the arrival of blastocyst in the uterus and/or the dynamic interplay of local uterine PAF and the hormonal milieux during the peri-implantation period of pregnancy.
- Published
- 1992
18. Binding of platelet-activating factor to oviductal membranes during early pregnancy in the rabbit.
- Author
-
Yang YQ, Kudolo GB, and Harper MJ
- Subjects
- Animals, Binding, Competitive, Female, In Vitro Techniques, Kinetics, Membranes metabolism, Platelet Activating Factor analogs & derivatives, Platelet Activating Factor antagonists & inhibitors, Pregnancy, Rabbits, Radioligand Assay, Statistics as Topic, Fallopian Tubes metabolism, Platelet Activating Factor metabolism, Pregnancy, Animal metabolism
- Abstract
The present study explores the ability of rabbit oviductal membranes to bind tritiated platelet-activating factor [3H]PAF on days 3 and 6 of pregnancy. Under optimal conditions (25 degrees C, 120 min) equilibrium saturation analysis revealed only one class of binding sites, characterized by Kd s(nM), 80.03 +/- 11.60 and 11.17 +/- 7.09 and Bmaxs, (pmol/mg protein), 5.25 +/- 2.23 and 1.08 +/- 0.22 (N = 3, mean +/- SEM) for ampullar membranes on days 3 and 6, respectively. The corresponding values for isthmic membranes were Kds, 86.56 +/- 12.01 and 52.43 +/- 30.49 and Bmaxs, 9.41 +/- 0.67 and 2.88 +/- 1.96 for days 3 and 6, respectively. Significant differences between days 3 and 6 were observed only in the binding affinities for the ampullar membranes and the binding capacities for the isthmic binding sites. [3H]PAF binding was inhibited in the following order of decreasing potency: lyso-PAF greater than PAF C18:0 greater than U66985 greater than PAF C16:0 for day 3 ampullar membranes; and lyso-PAF C16:0 greater than PAF C18:0 greater than U66985 greater than PAF C16:0 for day 6 ampullar membranes. These studies show the existence of specific oviductal membrane PAF binding sites, the binding parameters of which may be related to the stage of pregnancy, rather than to the spatial location along the oviduct. The relative proportion of endosalpinx to myosalpinx between the ampulla and isthmus may have masked inherent differences and account for the relatively low affinity binding. The physiological significance of oviductal membrane PAF binding is yet to be established.
- Published
- 1992
19. Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period.
- Author
-
Kudolo GB, Kasamo M, and Harper MJ
- Subjects
- Animals, Autoradiography, Binding Sites, Blastocyst metabolism, Female, Phospholipid Ethers metabolism, Pregnancy, Rabbits, Receptors, Cell Surface metabolism, Embryo Implantation physiology, Endometrium metabolism, Platelet Activating Factor metabolism, Platelet Membrane Glycoproteins, Receptors, G-Protein-Coupled
- Abstract
This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [3H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [3H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [3H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [3H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [3H]PAF C18:0. Intraluminally administered [3H]PAF C18:0 and intravenously injected [3H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.
- Published
- 1991
- Full Text
- View/download PDF
20. Estimation of platelet-activating factor receptors in the endometrium of the pregnant rabbit: regulation of ligand availability and catabolism by bovine serum albumin.
- Author
-
Kudolo GB and Harper MJ
- Subjects
- Animals, Binding, Competitive, Carrier Proteins, Chromatography, Thin Layer, Female, Freezing, Ligands, Pregnancy, Rabbits, Receptors, Cell Surface drug effects, gamma-Globulins pharmacology, Endometrium metabolism, Platelet Membrane Glycoproteins, Pregnancy, Animal metabolism, Receptors, Cell Surface metabolism, Receptors, G-Protein-Coupled, Serum Albumin, Bovine pharmacology
- Abstract
High affinity receptors have been demonstrated for the potent phospholipid autacoid, platelet-activating factor (PAF C18:0; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) in a variety of tissues, including the endometrium. Because of the relative instability of PAF and our previous demonstration that lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphorylcholine), the major metabolite of PAF, displaced [3H]PAF from endometrial PAF receptor sites, we have examined the ability of bovine serum albumin (BSA) to prevent degradation of PAF and have characterized PAF and lyso-PAF binding sites in purified rabbit endometrial membranes isolated on Day 6 of pregnancy. In buffer containing the phospholipase A2 inhibitors, quinacrine (10 microM) and dibromoacetophenone (2 microM), and 0.25% BSA, 87.4 +/- 3.2% of added [3H]PAF C18:0 remained intact after incubation at 25 degrees C for 150 min. The metabolic products, lyso-PAF and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (alkylacyl-GPC), only amounted to 5.2 +/- 3.2 and 3.3 +/- 1.1, respectively. At the same concentration, rabbit serum albumin (RSA) also significantly protected [3H]PAF C18:0 from metabolism, but bovine gamma globulin (BGG) was ineffective. The presence of 0.25% BSA, however, did not protect [3H]lyso-PAF C18:0 from extensive catabolism: the major product formed was [3H]alkylacyl-GPC. Insignificant amounts of [3H]PAF were formed. Under the same conditions (25 degrees C, 150 min) in the presence of 0.25% BSA, saturation analysis revealed the presence of two types of PAF C18:0 receptors in the endometrial membranes. Type 1 sites had a Kd of 0.42 +/- 0.03 nM (mean +/- SD; n = 3) and binding capacity of 0.11 +/- 0.01 pmol/mg protein. Type 2 receptor sites had a Kd of 5.96 +/- 0.35 nM and a binding capacity of 1.59 +/- 0.22 pmol/mg protein. Thus, in the presence of BSA, the binding capacities of the two classes of receptors were markedly reduced compared to values generated previously in its absence. The Kd of the Type 1 sites was not significantly changed by the presence of BSA. A single class of saturable high-affinity binding sites was demonstrable for lyso-PAF C18:0: Kds ranged from 0.76 +/- 0.58 to 11.1 +/- 0.62 nM, depending on which method of analysis was used (Eadie-Hofstee, Scatchard-Rosenthal, or the Lundon nonlinear method). The binding capacities were equally varied, ranging from 0.15 +/- 0.08 to 15.17 +/- 4.95 pmol/mg protein.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1990
- Full Text
- View/download PDF
21. Characterization of platelet-activating factor binding sites on uterine membranes from pregnant rabbits.
- Author
-
Kudolo GB and Harper MJ
- Subjects
- Animals, Cations, Cell Membrane metabolism, Chromatography, Thin Layer, Female, Hydrogen-Ion Concentration, Kinetics, Ligands, Platelet Activating Factor analogs & derivatives, Platelet Activating Factor antagonists & inhibitors, Pregnancy, Rabbits, Platelet Activating Factor metabolism, Platelet Membrane Glycoproteins, Pregnancy, Animal metabolism, Receptors, Cell Surface metabolism, Receptors, G-Protein-Coupled, Uterus metabolism
- Abstract
One of the earliest signs of endometrial preparation for blastocyst implantation is a localized increase in capillary permeability, an event that is essentially inflammatory in character and thought to be a prerequisite for subsequent decidual tissue formation. Platelet-activating factor (PAF), chemically identified as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, is a very potent vasoactive compound that recently has been implicated in the implantation process. In the present study, PAF binding sites are characterized in the rabbit uterus. A specific, reversible, saturable, and thermally labile binding of [3H]PAF to uterine membranes has been demonstrated, exhibiting multiple binding sites. The equilibrium dissociation constant (Kd) of the higher affinity binding site (type 1) was 3.6 +/- 0.4 nM (mean +/- SD) with a binding capacity (Bmax) of 3.4 +/- 1.6 pmol/mg protein. The second (lower affinity) binding site (type 2) had an apparent Kd of 114.6 +/- 13.5 nM and a Bmax of 164.3 +/- 17.6 pmol/mg membrane protein, under the conditions of maximal [3H]PAF binding, 25 degrees C, 150 min. Incubations at 4 degrees C for up to 3 h yielded only 30% of the Bmax observed at 25 degrees C. In crude and purified endometrial membrane preparations in which the PAF binding was predominantly located, the affinity of the binding for PAF was significantly higher than for the whole uterus, giving Kds of 1.5 +/- 0.8 and 0.8 +/- 0.5 nM; these latter values were not significantly different. However, the Bmax values of 3.9 +/- 0.9 pmol/mg protein and 376.8 +/- 163.3 fmol/mg protein for the two endometrial preparations, respectively, did differ significantly. Kinetic analysis at 25 degrees C resulted in a calculated Kd of 3.28 +/- 1.14 nM, which did not differ from the value for for the whole uterus at the same temperature, but was greater than for the endometrial preparations. Using 4 nM [3H]PAF to selectively label only the type 1 binding sites, the relative potencies of PAF and its antagonists in displacing [3H]PAF were lyso-PAF greater than CV3988 greater than PAF greater than U66985 greater than A02405 greater than BN52021 greater than U66982. The antagonists SRI 63,441 and L652,731 were ineffective in displacing [3H]PAF at up to 5000-fold molar excess of [3H]PAF. [3H]Lyso-PAF binding at 4 nM was displaceable by PAF. All cations tested, i.e. Ca2+, Mg2+, K+, Na+, and Li+, inhibited [3H]PAF binding. Serine hydrolase inhibitors, diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), inhibited binding, but bacitracin, leupeptin, and antipain stabilized it.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1989
- Full Text
- View/download PDF
22. Platelet-activating factor (PAF) and blastocyst-endometrial interactions.
- Author
-
Harper MJ, Kudolo GB, Alecozay AA, and Jones MA
- Subjects
- Animals, Female, Pregnancy, Rabbits, Receptors, Cell Surface physiology, Uterus physiology, Blastocyst physiology, Embryo Implantation, Endometrium physiology, Platelet Activating Factor physiology, Platelet Membrane Glycoproteins, Receptors, G-Protein-Coupled
- Published
- 1989
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