Biomedical subjects

R E Olver

Publications and source records attributed to R E Olver.

At least 37 records · Page 2Linked to original sources

PubMed1999

P2Y2 receptor-mediated inhibition of ion transport in distal lung epithelial cells.

1 Rat foetal distal lung epithelial cells were plated onto permeable supports where they became integrated into epithelial sheets that spontaneously generated short circuit current (ISC). 2 Apical ATP (100 microM) evoked a transient fall in ISC that was followed by a rise to a clear peak which, in turn, was succeeded by a slowly developing decline to a value below control. Apical UTP evoked an essentially identical response. 3 UDP and ADP were ineffective whilst ATP had no effect when added to the basolateral solution. These effects thus appear to be mediated by apical P2Y2 receptors. 4 The rising phase of the responses to ATP/UTP was selectively inhibited by anion transport inhibitors but persisted in the presence of amiloride, which abolished the inhibitory effects of both nucleotides. Thus, apical nucleotides appear to evoke a transient stimulation of anion secretion and sustained inhibition of Na+ absorption. 5 Basolateral isoprenaline (10 microM) elicited a rise in ISC but subsequent addition of apical ATP reversed this effect. Conversely, isoprenaline restored ISC to its basal level following stimulation with ATP. Apical P2Y2 receptors and basolateral beta-adrenoceptors thus allow their respective agonists to exert mutually opposing effects on ISC.

Adenosine Triphosphate↗

PubMed1999

Systematic review of antistaphylococcal antibiotic therapy in cystic fibrosis.

BACKGROUND: The respiratory tract in patients with cystic fibrosis is frequently colonised with Staphylococcus aureus. There is great diversity of clinical practice in this area of cystic fibrosis. A systematic review was conducted to study the evidence relating antistaphylococcal therapy to clinical outcome in patients with cystic fibrosis. METHODS: A search strategy already evaluated for the study of the epidemiology of cystic fibrosis clinical trials was used. This yielded 3188 references from which 13 clinical trials of antistaphylococcal therapy were identified. RESULTS: Substantial heterogeneity was observed between trials. In the 13 clinical trials a total of 19 antibiotics were used to assess a wide variety of outcome measures (11 clinical, six laboratory). Both intermittent and continuous treatment strategies were used. Sputum clearance of S aureus was more frequently achieved than any other beneficial outcome. A beneficial effect on pulmonary function was rarely measured or observed. Although five randomised clinical trials were identified, the extent of heterogeneity precluded the use of meta-analysis for further synthesis of information. CONCLUSIONS: Antistaphylococcal treatment achieves sputum clearance of S aureus in patients with cystic fibrosis. Prophylactic antistaphylococcal treatment in young children with cystic fibrosis is likely to be of clinical benefit. It remains to be determined whether the use of "prophylactic" versus "intermittent" antistaphylococcal therapy in cystic fibrosis is associated with improved lung function and/or chest radiographic scores, an increase in bacterial resistance, or earlier acquisition of Pseudomonas aeruginosa. A large randomised clinical trial lasting approximately two years is urgently required to address this problem.

Anti-Bacterial Agents↗

PubMed1998

Differential regulation of Na+ and Cl- conductances by PTX-sensitive G proteins in fetal lung apical membrane vesicles.

In apical membrane vesicles (AMV) prepared from late gestation fetal guinea pig lung we show that conductive 22Na+ uptake is modulated by at least two pathways involving pertussis toxin (PTX)-sensitive G proteins. Intravesicular incorporation of 100 microM GTPgammaS into vesicles resuspended in NaCl caused a significant stimulation (P<0. 05) of conductive Na+ uptake in AMV to 150+/-10% (n=10) of control, whereas GDPbetaS reduced uptake to 65+/-9% (n=4) of control. This contrasting response to GTPgammaS and GDPbetaS is characteristic of a G protein mediated pathway. GTPgammaS induced a significantly smaller stimulation, 125+/-8% (n=5) of control, in the presence of the relatively impermeant anion isethionate (Ise-). Taken together, these data indicate modulation of both Na+ and Cl- channels in the apical membrane by co-localised G protein(s). Treatment with PTX stimulated conductive 22Na+ uptake to 171+/-20% (n=13) of control in AMV resuspended in NaCl, but did not have a significant effect, 94+/-19% of control, in the presence of NaIse indicating the existence of tonic activation of Cl- channels in these AMV under resting conditions. As the combined effects of PTX and GTPgammaS diminished uptake, we propose that the G protein(s) responsible for Na+ channel activation in response to GTPgammaS is PTX-sensitive and that additional PTX-insensitive G proteins might also modulate 22Na+ uptake in these AMV. The presence of Gialpha1, Gialpha2, Gialpha3 and Goalpha in this apical membrane preparation was confirmed by PTX catalysed [32P]ADP-dependent ribosylation and Western blotting. Incubation of AMV with 200 microM DTT caused an inhibition of conductive Na+ uptake in AMV resuspended in NaCl or NaIse to 66+/-8% (n=11) and 64+/-8% (n=6) of control respectively. Pre-treatment with DTT did not affect the ability of GTPgammaS to stimulate conductive Na+ uptake suggesting that the regulation of 22Na+ uptake in late gestation guinea pig fetal lung AMV is unlikely to involve an associated regulatory protein.

Adenosine Diphosphate Ribose↗

PubMed1998

Culture substrate-specific expression of P2Y2 receptors in distal lung epithelial cells isolated from foetal rats.

ATP and UTP did not evoke [Ca2+]i signals in rat foetal lung epithelial cells grown on glass but elicited clear responses in cells grown into functionally polarised epithelia on permeable supports. Moreover, P2Y2 receptor mRNA could not be detected in cells on glass by the polymerase chain reaction but this mRNA species was clearly expressed by polarised cells. P2Y2 receptor expression thus appears to be a feature of the polarised phenotype.

Adenosine Triphosphate↗

PubMed1998

G protein-coupled prostaglandin receptor modulates conductive Na+ uptake in lung apical membrane vesicles.

Because G protein-regulated cation channels in type II pneumocytes constitute the most likely pathway for alveolar Na+ entry, we explored the hypothesis that a G protein-coupled prostaglandin (PG) E2 receptor controls perinatal lung alveolar Na+ transport. [3H]PGE2 binding to the alveolar apical membrane was trypsin sensitive and showed a rank order of competitive inhibition: PGE2 = PGE1 > PGD2 > PGF2 alpha. Kinetic analysis demonstrated both high-affinity [dissociation constant (KD) = 2.1 +/- 0.7 nM; maximal binding (Bmax) = 27 +/- 7 fmol/mg protein] and low-affinity (KD = 28 +/- 2 nM; Bmax = 265 +/- 29 fmol/mg protein) binding sites. Modulation of high-affinity GTPase activity identified a similar potency order (IC50 = 11 mM for PGF2 alpha vs. 10-50 microM for other PGs), suggesting that the receptors are G protein coupled. Finally, 1 microM PGE2 (approximately IC25) increased conductive 22Na+ uptake into membrane vesicles only in the presence of 100 microM intravesicular GTP. The KD value for the high-affinity binding site together with the rank order of PG effect on ligand binding and G protein function places this PG receptor in the EP3 subtype, whereas Na+ uptake studies suggest that it helps maintain perinatal lung Na+ homeostasis.

Animals↗

PubMed1997

Direct modulation of G-proteins by polyunsaturated fatty acids: a novel eicosanoid-independent regulatory mechanism in the developing lung.

Basal and fatty-acid-modulated G-protein function was studied in 1-3-day-pre-term, fetal guinea-pig, type II (fATII) pneumocyte apical membrane. Unstimulated (tonic) high-affinity GTPase activity (measured as [gamma-32P]GTP hydrolysis rate) was high and 77% pertussis toxin-insensitive. Alteration of this activity was used as a marker of G-protein regulation. Arachidonic acid (AA) showed a dose-dependent (IC50 = 48+/-8 microM) inhibition of activity at concentrations significantly below critical micellar concentrations; this effect was mimicked by other polyunsaturated fatty acids (IC50 for linoleic acid = 47 +/- 2 microM; IC50 for oleic acid = 106 +/- 11 microM). Saturated fatty acids showed no effect. The effect of AA on ouabain-insensitive ATPases in the same preparation was significantly lower, suggesting a specificity of the GTPase modulation effect. AA modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5'-lipoxygenase, cyclo-oxygenase and P-450. In order to explore further the mechanism of AA-G-protein interaction, the effect of AA on the time course and equilibrium binding of [35S]GTP[S] to apical membrane was studied. Consistent with our GTPase assay data, AA inhibited binding with an IC50 value of 71+/-1 microM; stearic acid did not mimic this effect. This is the first report of unsaturated-fatty-acid-specific modulation of lung G-protein function: since AA also up-regulates perinatal lung alveolar Na+ transport, we suggest this lipid/G-protein switch helps maintain pulmonary fluid homoeostasis around birth.

Animals↗

PubMed1997

Conductive Na+ transport in fetal lung alveolar apical membrane vesicles is regulated by fatty acids and G proteins.

We have characterised G protein and fatty acid regulation of the Na+ conductance in purified apical membrane vesicles prepared from late gestation fetal guinea-pig lung. Addition of 100 microM GTP gamma S or beta gamma-methylene-GTP, irreversible G protein activators, stimulated conductive 22Na+ uptake (ratio of experimental to control 1.35 +/- 0.02 and 1.34 +/- 0.05, respectively). Conversely, the addition of GDP beta S, an irreversible G protein inhibitor, reduced conductive 22Na+ uptake from 1.00 (control) to 0.79 +/- 0.04. A range of saturated (myristic, palmitic, stearic), monounsaturated (elaidic, oleic) and polyunsaturated (linoleic, arachidonic) fatty acids all stimulated conductive 22Na+ uptake, by between 1.18 +/- 0.05 to 1.56 +/- 0.13 over the control. Both arachidonic acid and GTP gamma S-dependent stimulation were abolished in the presence of 10 microM amiloride. The non-metabolisable analogue of arachidonic acid, eicosa-5,8,11,14-tetraynoic acid also stimulated conductive 22Na+ uptake. Furthermore, addition of indomethacin and nordihydroguairetic acid, inhibitors of cyclooxygenase and lipoxygenase pathways of arachidonate metabolism respectively, did not affect the arachidonic acid stimulation suggesting a direct effect of fatty acid upon the Na+ channel Since mepacrine (50 microM), a phospholipase A2 inhibitor, did not affect the GTP gamma S-stimulated conductive 22Na+ uptake, and inhibition of G protein turnover by GDP beta S did not attenuate the arachidonic acid response we conclude that these two regulatory pathways modulate alveolar Na+ transport directly and independently of each other.

5,8,11,14-Eicosatetraynoic Acid↗

PubMed1997

Inwardly rectifying K+ currents of alveolar type II cells isolated from fetal guinea-pig lung: regulation by G protein- and Mg2+-dependent pathways.

K+ currents in alveolar type II cells, isolated from fetal guinea-pig lung, were studied using the whole-cell patch-clamp technique. Inwardly rectifying (IR) K+ currents were observed when cells were bathed in symmetrical KCl-rich solutions. When extracellular K+ was replaced by Na+, inward currents were greatly decreased and the zero-current potential moved from 0 mV to -69 mV, indicating high K+ selectivity. In recordings with an intracellular KCl-rich solution, containing 1.12 mM Mg2+ and 10(-8 )M free Ca2+, IR K+ currents slowly diminished with time. Addition of the irreversible G protein activator, guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]), to the intracellular solution accelerated the rate of current run-down. In experiments where the intracellular solution was Mg2+ free, current run-down was abolished. The rate of current run-down was found to increase with increasing free intracellular [Mg2+]. Raising the intracellular free [Ca2+] to 10(-6 )M under Mg2+-free conditions had no effect on the K+ currents. Extracellular Ba2+ blocked the IR K+ currents in a concentration- and voltage-dependent manner. Tolbutamide, a blocker of ATP-sensitive K+ (KATP) channels, had no effect on the currents. The single channel underlying the whole-cell IR K+ currents displayed inward rectification and had a conductance of 31 pS in symmetrical KCl-rich solutions. We demonstate that mRNA coding for IRK1 is expressed in this cell preparation. Possible functions for this channel are discussed.

Animals↗

PubMed1997

What is core? Guidelines for the core curriculum in paediatrics.

A key point that the UK General Medical Council addressed in its recommendations on the undergraduate medical education was the concept of 'core curriculum' (General Medical Council 1993). Although enthusiastic for the idea of reducing factual overload, many medical teachers found themselves facing the task of how to define what a core curriculum is, what should be included and why. Predictably, our initial response is to include common and important topics, but how common is common, and how does one determine the relative importance of topics? We do not claim to have unravelled all the ambiguities surrounding the subject nor to have resolved all the controversies that are inevitably encountered. We hope, however, to describe some of the principles that governed our approach and put forward some guidelines, that may contribute to the debate.

Child↗

PubMed1996

G protein regulation of alveolar ion channels: implications for lung fluid transport.

Vectorial ion flow across the alveolar epithelium provides the driving force for lung fluid secretion in the prenatal lung and for fluid reabsorption at birth and thereafter into adult life. Fluid secretion is dependent upon 'active' accumulation of Cl- into the lumen of the developing lung and any factor which interupts the production of this liquid template results in life-threatening abnormalities in lung growth. The direction of fluid flow is reversed at birth to bring about the reabsorption of the lung fluid so that gaseous exchange can be initated successfully in the neonate. This functional switch at birth involves active Na+ reabsorption; failure of this mechanism to activate adequately contributes to respiratory distress in the newborn. Although beta 2-adrenoceptor-cAMP-protein kinase activation provides a basic model for the switch, it is clear that local regulation of many of the components in the switching cascade are important to the overall efficiency with which it is achieved. Co-localized with apical Cl- and Na+ channels are pertussis toxin-sensitive G proteins, which exert their regulatory effects principally by direct protein-channel interactions. The modulation of channels by local G proteins is finely tuned by negative feedback mechanisms, which may include a novel double-bond specific fatty acid regulation of G protein turnover and the paracrine effects of locally produced eicosanoids. An understanding of how these overlapping pathways integrate to produce the smooth and ordered transition from Cl- secretion to Na+ absorption will allow the design of rational treatments for conditions that are characterized by disfunctional lung fluid homeostasis.

Age Factors↗

PubMed1996

Nebulised antipseudomonal antibiotic therapy in cystic fibrosis: a meta-analysis of benefits and risks.

BACKGROUND: To establish the benefits and risks of nebulised antipseudomonal therapy in cystic fibrosis the results of relevant randomised controlled trials were combined. METHODS: The therapeutic end points compared were (a) number of pulmonary exacerbations requiring treatment with systemic antibiotics, (b) measurable alteration in respiratory tract pseudomonal load, (c) alteration in lung function on spirometric assessment, (d) development of resistance in respiratory tract Pseudomonas strains to the nebulised antipseudomonal used in each randomised controlled trial, and (e) renal and auditory impairment. RESULTS: Five studies were suitable for meta-analysis, eight others could not be included because of inadequate outcome description or the lack of appropriate randomisation. Meta-analysis shows benefit for nebulised antipseudomonal antibiotic therapy with no demonstrable adverse effect other than a possible increase in in vitro antibiotic resistance of Pseudomonas aeruginosa of the respiratory tract. CONCLUSIONS: Although inferences drawn from individual randomised controlled trials concerning the benefits and risks of this form of therapy are conflicting, pooled effect size establishes benefit with nebulised antipseudomonal antibiotic therapy and emphasises its relevance to the integration of information in other areas of controversy relating to the treatment of this disease.

Administration, Inhalation↗

PubMed1994

Conductive cation transport in apical membrane vesicles prepared from fetal lung.

In order to characterise the apically-located conductive cation pathway of the type II pneumocyte, apical plasma membranes were prepared from mature fetal guinea pig lung. The protocol yielded purified apical membranes that enriched 19-fold with the brush border enzyme marker alkaline phosphatase; there was no significant contamination with other cellular membranes. A technique for imposing an outwardly-directed electrochemical Na+ gradient was used to amplify conductive 22Na+ uptake into vesicles. Uptake of 22Na+ was time-dependent, proportional to the magnitude of the Na+ gradient, specific and sensitive to the amiloride analogues phenamil and EIPA (apparent minimum Ki values of 50 nM and 10 microM, respectively, with maximum uptake inhibition of 42% and 39% at 100 microM). Uptake experiments in which the outwardly-directed Na+ gradient was replaced by outwardly-directed gradients of small monovalent cations and molecular cations were performed. The Na+/K+ permeability ratio was 1.2:1, and over the extended range of small monovalent cations, a permeability sequence of Na+ > K+ > Li+ > Rb+ > Cs+ was observed, indicating the presence of fixed negative charge in or spatially close to the pore. The molecular cation permeability sequence of NH4+ > methylamine+ > dimethylamine+ > choline+ > N-methyl-D-glucamine+ > tetraethylammonium+ > tetramethylammonium+, after transformation, gives an estimate of 8 A for the conducting pore diameter. These data are consistent with the presence in the apical membrane of fetal type II pneumocytes of a cation specific channel with low Na+ selectivity and amiloride sensitivity.

Animals↗

PubMed1994

Amiloride-sensitive Na+ channels in fetal type II pneumocytes are regulated by G proteins.

We have used the patch-clamp technique to record single channels in excised membrane patches from type II pneumocytes freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. The 10/56 patches exhibited spontaneous channel activity with a mean open-state probability (NPo) of 0.5 +/- 0.1. In symmetrical Na(+)-rich solutions, the channels had a unitary conductance of 11.1 +/- 0.5 pS and showed current reversal at approximately 0 mV. Superfusing the inner membrane leaflet of the patch with a K(+)-rich solution resulted in single-channel current activity with a conductance of 5.6 +/- 0.2 pS being resolved. Current reversed at +22.1 +/- 1.9 mV, which is compatible with a PNa+/PK+ of 1.8 +/- 0.1. The addition of 0.1 mM guanosine 5'-O-(3-thiotriphosphate) to the cytoplasmic face of the patch elicited channel activity in 12/31 previously quiescent patches, whereas, in spontaneously active patches, channel NPo was increased. Amiloride, in the concentration range 0.4-4 microM, reduced the frequency of observed spontaneous (or activatable) channel activity, reduced NPo, and induced flickery channel behavior. No activity was seen in the presence of 10 microM amiloride in the pipette. This is the first direct observation of a G protein regulated Na(+)-conductive pathway in alveolar epithelium, and it may represent one route by which the alveolar epithelium of the fetus can regulate the Na(+)-driven fluid reabsorption necessary for the adaptation of the newborn lung to air breathing at birth.

Amiloride↗

PubMed1993

G protein-regulated large-conductance chloride channels in freshly isolated fetal type II alveolar epithelial cells.

Using the patch-clamp technique, we have recorded single channels in cell-attached and inside-out excised patches from the plasma membrane of type II alveolar epithelial cells freshly isolated from fetal guinea pig lung by elastase digestion and differential filtration. In cell-free patches the channels were highly selective for Cl- (PCl:Pcat = 9:1), had a large unitary conductance (375 pS +/- 23 pS), and current reversal of 0 mV in either symmetrical Na(+)-rich solutions or when the inner membrane leaflet was bathed in a K(+)-rich solution. The large-conductance Cl- channel exhibited little or no voltage inactivation at positive potentials, remained open for a significant amount of time at potentials negative to -40 mV, and was blocked at all potentials by 0.1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Channel activity was independent of intracellular calcium concentration. Bath addition of the nonmetabolizable analogue of GTP, GTP gamma S (0.1 mM), caused a voltage-dependent inhibition of channel activity [open probability (Po) plot was shifted by at least +25 mV]. Smaller channels (25 +/- 3 pS) were recorded in the cell-attached configuration with a current-voltage (I-V) relationship which was compatible with a Cl- conductance. On excision, the patches previously containing small-conductance channels exhibited only large-conductance Cl- channel behavior. These large-conductance, G protein-regulatable Cl- channels may provide a route for alveolar cell Cl- exit and as such may be an integral part of the mechanism responsible for secretion of fetal lung fluid.

Animals↗

PubMed1991

Effect of benzamil on sheep tracheal epithelium.

Apical membrane potential (Va), voltage divider ratio (f), transepithelial potential (Vt) and transepithelial resistance (Rt) were measured in isolated sheep tracheal epithelium. Individual membrane resistances and electromotive forces were calculated by a method described in the text. Whereas Va hyperpolarized and fa increased in tissues acutely exposed to amiloride or benzamil, the values for these parameters in tissues bathed for 1 h with mucosal benzamil (0.38 microM) were not significantly different from those found in control tissues. Circuit analysis revealed a higher value for both apical and basolateral membrane resistances in the benzamil-treated tissues. These results suggest a decrease in the basolateral potassium conductance secondary to the initial decrease in the apical sodium conductance, thereby ensuring that the electrical driving force for other ions across the apical membrane is restored to normal in the face of a hyperpolarizing perturbation to the apical membrane.

Amiloride↗

PubMed1990

Sodium-proton exchange across the apical membrane of the alveolar type II cell of the fetal sheep.

In order to detect and characterise Na(+)-H+ countertransport in the fetal lung epithelium we have studied under a variety of conditions the effect of an outward facing H+ gradient on Na+ uptake into purified apical membrane vesicles prepared from alveolar type II cells. Kinetic analysis of the data reveals both a diffusional and a saturable component of total Na+ uptake. Evidence for the presence of a Na(+)-H+ exchanger is demonstrated by (1) stimulation of Na+ uptake by proton loading of vesicles both in the presence and absence of chemical voltage clamping; (2) saturation kinetics with respect to external Na+ with a Km of 16 mM and a Vmax of 2.1 nmol/mg protein per min; (3) amiloride inhibition of Na+ uptake driven by pH gradient. We conclude that although diffusion may be the major component of total Na+ uptake at physiological external Na+ concentration, Na(+)-H+ countertransport provides a possible mechanism for the acidification of fetal lung liquid in-vivo in addition to its established role in intracellular pH and volume regulation.

Amiloride↗

PubMed1990

Ionic permeabilities of the cell membranes of sheep tracheal epithelium.

1. The ionic permeabilities of the apical and basolateral membranes of sheep tracheal epithelial cells have been evaluated by studying the effect of ion replacements on the apical (Va) and basolateral (Vb) membrane potentials and apical voltage divider ratio (fa). 2. The results provide evidence for the existence of conductances for Cl-, Na+ and K+ at the apical membrane, and of an important K+ conductance at the basolateral membrane. These characteristics agree with the model for Cl- secretion and Na+ absorption proposed for tracheal epithelium of other species. 3. The short-circuit current (Isc) across this tissue was found to increase significantly when HCO3- and CO2 were added to the bathing solutions. The addition of 0.5 mM-SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid) to the serosal bathing solution decreased Isc only if HCO3- and CO2 were present in the bathing solutions. These results suggest the existence of a HCO3- secretion in sheep tracheal epithelium under short-circuit conditions. 4. The results obtained in HCO3- replacement studies are compatible with an electrically silent HCO3- entry across the basolateral membrane and an electrogenic exit at the apical membrane of the cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1990

Dibutyryl cAMP induces a gestation-dependent absorption of fetal lung liquid.

The maturation of the adenosine 3',5'-cyclic monophosphate-(cAMP) dependent pathway controlling fetal lung liquid secretion was examined in experiments in which the lungs of chronically catheterized fetal lambs (123-141 days gestational age) were exposed to dibutyryl cAMP (DBcAMP, 10(-4) M). The effect of DBcAMP was markedly gestation dependent, with the greatest effect observed in the most mature fetuses. In immature fetuses (less than 130 days, mean age 125 days) DBcAMP caused slowing of secretion, with maximal effect at 5 h. With increasing maturity the effect of DBcAMP was more pronounced and occurred earlier so that in mature fetuses (mean age 140 days) lung liquid absorption took place, with maximal effect at 2 h. Changes in lung liquid volume flow induced by DBcAMP could be blocked by addition of 10(-4) M amiloride to lung liquid. It is concluded that 1) DBcAMP induces a change in lung liquid secretion that, like epinephrine, is mediated via an increase in Na+ permeability of the apical membrane of the lung epithelium and 2) the rate-limiting step in the maturation of this process must lie beyond the generation of intracellular cAMP.

Absorption↗