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Biomedical subjects

G M Hawksworth

Publications and source records attributed to G M Hawksworth.

At least 19 recordsLinked to original sources
PubMed2001

Differential expression and synthesis of natriuretic peptides determines natriuretic peptide receptor expression in primary cultures of human proximal tubular cells.

INTRODUCTION: The natriuretic peptides and natriuretic peptide receptors may play a beneficial role in hypertension and heart failure and possibly in opposing associated detrimental cellular changes in the heart, vasculature and kidney. These responses may be, in part, modulated by the natriuretic peptide clearance receptor rather than the natriuretic peptide receptors (NPR-A or NPR-B). OBJECTIVE: To investigate the expression of the natriuretic peptide receptors (NPR-A,-B,-C) and the natriuretic peptides (ANP, BNP, CNP) in primary cultures of human proximal tubular cells and the role played by endogenously released natriuretic peptides in natriuretic peptide receptor expression. RESULTS: Northern analysis demonstrated that freshly isolated human proximal tubular cells express the NPR-C only. However, at confluence mRNA transcripts for both the NPR-A and -B were expressed, accompanied by a significant cyclic guanosine monophosphate (cGMP) response to ANP and CNP, indicating the development of functionally active receptors. A significant increase in immunoreactive ANP, BNP and CNP in the cell supernatant accompanied the appearance of these receptors. Incubation of freshly isolated cells with exogenous ANP, BNP, CNP or with the NPR-C specific ligand C(4.23)ANF induced similar changes in receptor expression, suggesting that these changes were mediated via the NPR-C rather than the NPR-A or -B. CONCLUSIONS: Significant changes in peptide and receptor expression occur during cell culture and may be integrally linked, with functionally active NPR-A and -B occurring in response to an increase in the expression of the natriuretic peptides possibly acting at the NPR-C.

Atrial Natriuretic Factor↗
PubMed2000

The expression and secretion of atrial natriuretic factor and brain natriuretic peptide by rat proximal tubular cells.

We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NPR) in primary cultures of rat proximal tubular (RPT) cells using Northern blot assay for peptides and receptors and radioimmunoassay and immunohistochemical analysis for atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide. Freshly isolated cells expressed mRNA coding for ANF, BNP, and the NPR-C. The presence of ANF and BNP in freshly isolated cells was confirmed by immunocytochemical staining. As cells approached confluence, there was a marked increase in mRNA expression for ANF and BNP. Immunocytochemical analysis and radioimmunoassay confirmed that both these peptides were co-localised in RPT cells and present in the cell supernatant. These changes in peptide expression were associated with a concurrent decrease in the expression of the NPR-C and the appearance of the NPR-A and -B. These results confirm that freshly isolated RPT cells possess the components of an autocrine natriuretic peptide system and that growth in primary culture is associated with changes in both peptide system and that growth in primary culture is associated with changes in both peptide and receptor subtype expression, raising the possibility that the endogenous production of ANF and BNP may be involved in the control of control cell growth.

Animals↗
PubMed2000

Characterization of the inspiratory manoeuvre when asthmatics inhale through a Turbohaler pre- and post-counselling in a community pharmacy.

Dose emission from a Turbohaler has been shown to be dependent on the rate of inhalation, with an optimal flow of 60 l min(-1) recommended. Some patients may need counselling to achieve this fast inhalation. Inhalation rate profiles of 24 asthmatics were measured when they inhaled through a placebo Turbohaler. The setting was a community pharmacy when the asthmatics came to collect their next supply of medication. Profiles were measured before and after counselling on how to use the Turbohaler. The mean (SD) peak inhalation rate through the Turbohaler pre- and post-counselling was 48.0 (16.8) and 54.7 (17.6) l min(-1), and their inspiratory volume was 1.75 (0.68) and 1.94 (0.62) l, respectively. Their mean (SD) percent predicted FEV1 was 57.0 (18.9)%. After counselling, 12 patients achieved an inhalation rate of > 60 l min(-1) and a further four obtained > 55 l min(-1). Emphasis should be placed on counselling patients prescribed all types of inhaled devices rather than concentrating on metered dose inhalers.

Administration, Inhalation↗
PubMed2000

Induction, regulation, degradation, and biological significance of mammalian metallothioneins.

MTs are small cysteine-rich metal-binding proteins found in many species and, although there are differences between them, it is of note that they have a great deal of sequence and structural homology. Mammalian MTs are 61 or 62 amino acid polypeptides containing 20 conserved cysteine residues that underpin the binding of metals. The existence of MT across species is indicative of its biological demand, while the conservation of cysteines indicates that these are undoubtedly central to the function of this protein. Four MT isoforms have been found so far, MT-1, MT-2, MT-3, and MT-4, but these also have subtypes with 17 MT genes identified in man, of which 10 are known to be functional. Different cells express different MT isoforms with varying levels of expression perhaps as a result of the different function of each isoform. Even different metals induce and bind to MTs to different extents. Over 40 years of research into MT have yielded much information on this protein, but have failed to assign to it a definitive biological role. The fact that multiple MT isoforms exist, and the great variety of substances and agents that act as inducers, further complicates the search for the biological role of MTs. This article reviews the current knowledge on the biochemistry, induction, regulation, and degradation of this protein in mammals, with a particular emphasis on human MTs. It also considers the possible biological roles of this protein, which include participation in cell proliferation and apoptosis, homeostasis of essential metals, cellular free radical scavenging, and metal detoxification.

Animals↗
PubMed2000

Lymphocyte metallothionein mRNA responds to marginal zinc intake in human volunteers.

Marginal Zn deficiency is thought to be prevalent in both developed and developing countries. However, the extent of Zn deficiency is not known, due to the lack of a reliable diagnostic indicator. Blood plasma and erythrocyte concentrations of metallothionein (MT) reflect Zn status, but measurement of MT is dependent on the availability of sensitive immunoassays. Our aim was to show whether measurement of T lymphocyte MT-2A mRNA, using a competitive reverse transcriptase (RT)--polymerase chain reaction (PCR) assay, could indicate Zn status in human subjects in a residential Zn-depletion study. In the study, the Zn intake of seven volunteers was maintained at 13.7 mg/d for 5 weeks (baseline) followed by 4.6 mg/d for 10 weeks (marginal intake) and then 13.7 mg/d (repletion) for 5 weeks. The quantitative assay was developed using standard techniques and concentrations of MT-2A mRNA were normalized by reference to beta-actin mRNA which was also measured by competitive RT--PCR assay. An alternative method of measuring the PCR product using capillary electrophoresis with laser-induced fluorescence detection was also evaluated. There was considerable inter-individual variation in MT-2A mRNA concentration and the mean level at the end of the baseline period was 10.3 (SE 3.7) fg MT-2A mRNA/pg beta-actin mRNA, which then decreased by 64 % during the low Zn intake period. After repletion, MT-2A mRNA returned to baseline concentrations. In contrast, plasma Zn was unchanged by marginal Zn intake or repletion. The effect of low Zn in all individuals was consistent. We conclude that this assay is a sensitive method of evaluating marginal changes in dietary Zn intake.

Electrophoresis, Capillary↗
PubMed1999

Clinical pharmacy interventions by community pharmacists during the dispensing process.

AIMS: To evaluate the professional contact between the community pharmacist and general practitioner during the dispensing process on issues other than the legality or simple clarification of the prescription. METHODS: Fourteen community pharmacists from five adjacent localities completed details of each clinical pharmacy intervention during 1 week of each month for a period of 1 year. Each week of the month was randomly selected. When a community pharmacist had to contact the prescriber, during the dispensing of a prescription, the following data were recorded: brief patient details, the prescribed drug therapy, the reason for intervention, the outcome and the time taken. The main outcome measures were the type and nature of each intervention, the BNF category of the drug involved and the time taken. A multidisciplinary clinical panel assessed the potential of each intervention to alter the outcome of the patient's clinical management and to prevent a drug related hospital admission. These assessments were ranked between 0 and 10 (100% confident). RESULTS: During a period covering 1 week per month over 1 year, 1503 clinical pharmacy interventions were made out of 201 000 items dispensed. When normalized for the dispensing volume of each community pharmacy the lower the number of items dispensed then the greater was the percentage of interventions (P=0.013). The clinical panel decided that between 19 (0.01% of the total items dispensed) and 242 (0.12%) interventions may have prevented a drug-related hospital admission, 71 (0.04%) to 483 (0.24%) could have prevented harm whilst 103 (0.05%) to 364 (0.18%) had the potential to improve the efficacy of the intended therapeutic plan. The panel also decided that 748 (0.37%) interventions improved the clinical outcome and could have saved a visit to or by the general practitioner. Conclusion Clinical pharmacy provided by a community pharmacist during the dispensing process has the potential to provide a valuable contribution to health care.

Community Pharmacy Services↗
PubMed1998

Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction.

The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

Bismuth↗
PubMed1998

Autoregulation of 5-fluorouracil metabolism.

5-Fluorouracil (5-FU) is a commonly used anticancer agent for the treatment of gastrointestinal, head and neck, and breast tumours. This study determined the influence of 5-FU on dihydropyrimidine dehydrogenase (DPD) activity, the enzyme responsible for its in vivo degradation. DPD activity was measured in mononuclear cells obtained prior to and after the administration of 5-FU in 20 patients with colorectal cancer. Following the results from the human studies, DPD activity was measured in Sprague-Dawley rat liver up to 72 h after administration of 5-FU 200 mg/kg as a single injection. Total liver P450 content and the production of testosterone metabolites (indicative of CYP3A activity) were also analysed to determine the specificity of 5-FU-associated alteration in rat liver metabolism. Human mononuclear cell DPD activity decreased by a median of 38.7% following the administration of 5-FU (P = 0.001). 5-FU-induced alterations in rat liver DPD were also observed, with the lowest activity occurring 48 h after injection (50% of control activity; P = 0.009). Rat liver DPD activity returned to near control values by 72 h postinjection. Rat liver total P450 content and CYP3A activity were not significantly different in 5-FU treated or control tissues. Thus, 5-FU demonstrates autoregulation of its metabolism through inhibition of DPD activity. Although this inhibition appears to be specific for DPD, the mechanism for enzyme inhibition is not clear. These findings may aid in the design of 5-FU treatment regimens and provide the basis for further studies into the regulation of DPD.

Animals↗
PubMed1998

Glutathione S-transferases in human renal cortex and neoplastic tissue: enzymatic activity, isoenzyme profile and immunohistochemical localization.

1. Glutathione S-transferase (GST) activity in the cytosol of renal cortex and tumours from eight men and eight women was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST activities ranged from 685 to 2192 nmol/min/mg protein in cortex (median 1213) and from non-detectable (minimum 45) to 2424 nmol/min/mg protein in tumours (median 469). The activities in the tumours were lower than those in the normal cortices (p < 0.05). 2. In men, the activity in the cortical cytosol was in all cases higher than that measured in the corresponding tumours (p < 0.05). In women, the difference in activity between cortices and tumours was not significantly different (p > 0.05). 3. The age of the patients ranged from 42 to 81 years (median 62) and was not found to play a role in the levels of GST activity observed in cortex or in renal tumours from either sex. 4. Immunoblotting and immunohistochemical studies confirmed that GST-alpha was the predominant form expressed both in normal cortex and tumour and probably accounted for most of the GST activity present in these samples. GST-mu and GST-phi were expressed in both tumours and normal cortex and, while in some cases the level of expression in the cortices was higher than that found in the tumours, the reverse was also observed. Within the GST-mu class, GST M1/M2 was only detected in one sample (tumour), which showed the highest overall expression of GST-mu. GSTM3 was the predominant isoenzyme of the mu class in normal and tumour tissue, whereas GTM4 and GSTM5 were not detected. 5. These differences could have functional significance where xenobiotics or cytotoxic drugs are specific substrates for the different classes of GSTs.

Adenocarcinoma, Clear Cell↗
PubMed1998

Investigation of the potential interaction between terfenadine and tedisamil in human liver microsomes.

1. The potential drug-drug interaction of terfenadine and tedisamil has been investigated. Terfenadine is a widely used antihistamine drug with the potential for QTC prolongation. Tedisamil is a potassium channel blocking agent known to produce bradycardia and prolong the effective refractory period in man. 2. Tedisamil and terfenadine were incubated with human liver microsomes for 30 min at 37 degrees C. No significant inhibition of terfenadine biotransformation was seen with 0.1 or 10 microM tedisamil as the formation of the terfenadine alcohol and acid metabolites were unaffected. 3. Based on the in vitro results it is suggested that tedisamil will not interact pharmacokinetically with terfenadine as it does not impair metabolism of terfenadine.

Adult↗
PubMed1997

Structure-activity relationship for two lipoxygenase inhibitors and their potential for inducing nephrotic syndrome.

In a study of structure-activity relationship with drug-induced nephropathy two lipoxygenase inhibitors, the N-hydroxyurea derivative 70C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxyurea) and the N-hydroxamic acid analogue 360C ((E)-N-{3-[3-(4-fluorophenoxy) phenyl]-1-(R, S)-methylprop-2-enyl}-N-hydroxamic acid), were administered to rats. 70C and 360C were dosed to female Wistar rats at 100 mg/kg po daily for 7 days. Another group of rats was given a single intravenous bolus dose of puromycin aminonucleoside (PAN) at 100 mg/kg. Urine samples were collected from all groups during the study and plasma samples were collected after 7 days. Kidneys were excised and fixed for examination by electron microscopy. 70C- and PAN-treated groups both showed early changes in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. This foot process loss was associated with decreases in total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, creatinine, and urea were recorded. Marked proteinuria was observed in both the 70C and PAN groups. The foot process loss together with increased proteinuria, hypoalbuminemia, hypercholesterolemia, and lipemia are all characteristic of the human condition, Minimal Change Nephrotic Syndrome. All the biochemical and morphological investigations showed that 360C-treated rats were similar to the control group, suggesting that the hydroxyurea moiety of 70C is responsible, either directly or indirectly, for the induction of the nephrotic syndrome seen in rats.

Administration, Oral↗
PubMed1997

Selective antagonism of the AT1 receptor inhibits angiotensin II stimulated DNA and protein synthesis in primary cultures of human proximal tubular cells.

The hypertrophy of renal proximal tubular cells occurs as an adaptive response to a variety of stimuli and may be involved with the progression of renal disease. Angiotensin II acting alone or in combination with other growth factors has been implicated in this process. The aims of this study were to identify the role of both angiotensin II and the angiotensin receptor subtypes in DNA synthesis and protein synthesis in human renal proximal tubular cells. Primary cultures of human renal proximal tubular cells were incubated with angiotensin II (10(-10) M, 10(-8) M, 10(-6) M) for 24 to 120 hours either alone or in combination with losartan, PD123319 or 8-bromo-cAMP. Incubation of human proximal tubular cells with angiotensin II (10(-10) M, 10(-8) M) induced a significant early increase in [3H]thymidine uptake by 19% and 56% (P < 0.01), respectively, and a later increase in total protein content by 30% (P < 0.01). The effect of angiotensin II upon DNA and protein synthesis was inhibited by 8-bromo-cAMP and losartan but not by PD 123319, indicating that the responses are mediated via the AT1 receptor and dependent upon the inhibition of adenylate cyclase.

8-Bromo Cyclic Adenosine Monophosphate↗