Mechanisms underlying K+-induced hyperpolarizations in the presence and absence of phenylephrine were investigated in endothelium-denuded rat mesenteric arteries (for all mean values, n=4).Myocyte resting membrane potential (m.p.) was −58.8±0.8 mV. Application of 5 mM KCl produced similar hyperpolarizations in the absence (17.6±0.7 mV) or presence (15.8±1.0 mV) of 500 nM ouabain. In the presence of ouabain +30 μM barium, hyperpolarization to 5 mM KCl was essentially abolished.In the presence of 10 μM phenylephrine (m.p. −33.7±3 mV), repolarization to 5 mM KCl did not occur in the presence or absence of 4-aminopyridine but was restored (−26.9±1.8 mV) on addition of iberiotoxin (100 nM). Under these conditions the K+-induced repolarization was insensitive to barium (30 μM) but abolished by 500 nM ouabain alone.In the presence of phenylephrine + iberiotoxin the hyperpolarization to 5 mM K+ was inhibited in the additional presence of 300 nM levcromakalim, an action which was reversed by 10 μM glibenclamide.RT–PCR, Western blotting and immunohistochemical techniques collectively showed the presence of α1-, α2- and α3-subunits of Na+/K+-ATPase in the myocytes.In K+-free solution, re-introduction of K+ (to 4.6 mM) hyperpolarized myocytes by 20.9±0.5 mV, an effect unchanged by 500 nM ouabain but abolished by 500 μM ouabain.We conclude that under basal conditions, Na+/K+-ATPases containing α2- and/or α3-subunits are partially responsible for the observed K+-induced effects. The opening of myocyte K+ channels (by levcromakalim or phenylephrine) creates a ‘K+ cloud' around the cells which fully activates Na+/K+-ATPase and thereby abolishes further responses to [K+]o elevation.
EDHF; Na+/K+-ATPase isoforms; ouabain; potassium; hyperpolarization; immuno-histochemistry; phenylephrine; iberiotoxin; 4-aminopyridine
The apamin-sensitive small-conductance Ca2+-activated K+ channel (SKCa) was characterized in porcine coronary arteries.In intact arteries, 100 nM substance P and 600 μM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8±0.8 mV and 24.1±1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8±0.3 mV).In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K+ conductance of 6.8±0.04 pS. The open-probability was increased by Ca2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7±0.03 pS inhibited by d-tubocurarine was observed infrequently.Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SKCa was detected by RT – PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions.Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells.In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SKCa containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.
EDHF; porcine coronary artery; endothelium; apamin; 1-EBIO; SK1; SK2; SK3
In intact mesenteric arteries, increasing [K+]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 μM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K+]o. In endothelium-denuded vessels, increasing [K+]o by 5 mM hyperpolarized the smooth muscle but K+ had no effect after depolarization by 10 μM phenylephrine. On subsequent exposure to iberiotoxin plus 4-aminopyridine, the repolarizing action of 5 mM K+ was restored. In endothelium-intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K+-mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine-induced efflux of K+ via smooth muscle K+ channels produces a local increase in [K+]o which impairs repolarization to added K+. Thus, studies involving vessels precontracted with agonists which increase [K+]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium-derived K+.
EDHF; K+; smooth muscle; endothelium; hyperpolarization; repolarizarion; K+ channels
In porcine coronary arteries, smooth muscle hyperpolarizations produced by the nitric oxide donor, NOR-1, and the prostacyclin analogue, iloprost, were compared with those induced by substance P and bradykinin and attributed to the endothelium-derived hyperpolarizing factor (EDHF).In the presence of 300 μM L-nitroarginine and 10 μM indomethacin, iloprost-induced hyperpolarizations were partially inhibited by 10 μM glibenclamide whereas those to NOR-1, substance P and bradykinin were unaffected.Hyperpolarizations produced by maximally-effective concentrations of NOR-1 and NS1619 were identical (to −65 mV). They were significantly less than those generated by either substance P or bradykinin (to approximately −80 mV) and were abolished by iberiotoxin 100 nM, a concentration which had essentially no effect on responses to substance P or bradykinin.Incubation of segments of intact arteries for 16–22 h in bicarbonate-buffered Krebs solution had little effect on EDHF responses to substance P or bradykinin. In contrast, after incubation for this period of time in HEPES-buffered Tyrode solution or Krebs containing 10 mM HEPES the EDHF response to substance P was abolished and that to bradykinin was markedly reduced. The residual bradykinin-induced hyperpolarization following incubation in Tyrode solution was inhibited by iberiotoxin and by 10 μM 17-octadecynoic acid.We conclude that substance P activates only the EDHF pathway in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors. Incubation in HEPES-buffered Tyrode solution abolishes the EDHF responses to substance P and bradykinin to reveal an additional hyperpolarizing mechanism, associated with the opening of K+ channels, activated only by bradykinin.
EDHF; iberiotoxin; porcine coronary artery; gap junctions; HEPES; bradykinin; prostacyclin; substance P; nitric oxide donor; hyperpolarization
effects of long term treatment of asthma with the short acting β agonist fenoterol have been established in both epidemiological and
clinical studies. A study was undertaken to investigate the efficacy
and safety of long term treatment with salbutamol and salmeterol in
patients with mild to moderate bronchial asthma.
METHODS—In a two
centre double dummy crossover study 165 patients were randomly assigned
to receive salbutamol 400 µg qid, salmeterol 50 µg bid, or
placebo via a Diskhaler. All patients used salbutamol as required for
symptom relief. The study comprised a four week run in and three
treatment periods of 24 weeks, each of which was followed by a four
week washout. Asthma control was assessed by measuring mean morning and
evening peak expiratory flow rate (PEFR), a composite daily asthma
score, and minor and major exacerbation rates. Washout assessments
included methacholine challenge and bronchodilator dose response tests.
Analysis was by intention to treat.
RESULTS—Data from 157 patients were analysed. Relative to placebo, the mean morning PEFR
increased by 30 l/min (95% CI 26 to 35) for salmeterol but did not
change for salbutamol. Evening PEFR increased by 25 l/min (95% CI 21 to 30) and 21 l/min (95% CI 17 to 26), respectively (p<0.001).
Salmeterol improved the asthma score compared to placebo (p<0.001),
but there was no overall difference with salbutamol. Only daytime
symptoms were improved with salbutamol. The minor exacerbation rates
were 0.29, 0.88, and 0.97 exacerbations/patient/year for salmeterol,
salbutamol and placebo, respectively (p<0.0001 for salmeterol).
The corresponding major exacerbation rates were 0.22, 0.51 and 0.40, respectively (p<0.03 for salmeterol). For salbutamol the asthma score
deteriorated over time (p<0.01), and the time spent in major
exacerbation was significantly longer compared with placebo (12.3 days
(95% CI 4.2 to 20.4)) versus 8.4 days (95% CI 5.2 to 11.6), p = 0.02). There was no evidence of rebound deterioration in asthma
control, lung function, or bronchial hyperresponsiveness following
cessation of either active treatment, and no evidence of tolerance
to salbutamol or salmeterol.
treatment with salmeterol is effective in controlling asthma symptoms
and reduces minor more than major exacerbation rates. Salbutamol was
associated with improved daytime symptoms but subtle deterioration in
asthma control occurred over time. Salbutamol should therefore be used
only as required.
AIMS: To study the expression of cell adhesion molecules in the renal biopsy specimens of patients with systemic vasculitis and Henoch-Schönlein purpura (HSP); to correlate this with the severity of glomerular inflammation. METHODS: Renal biopsy specimens obtained from eight patients with untreated systemic vasculitis (four with Wegener's granulomatosis and four with microscopic polyarteritis), eight with HSP and nine controls (four with normal histopathology and five with thin glomerular basement membrane disease) were stained using the alkaline phosphatase anti-alkaline phosphatase method with monoclonal antibodies directed against intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. RESULTS: Biopsy specimens of normal kidneys expressed ICAM-1 in glomerular endocapillary cells, Bowman's capsule epithelium, interstitial cells and interstitial vascular endothelium, and VCAM-1 in Bowman's capsule epithelium, proximal tubular epithelium and interstitial vascular endothelium. No staining with antibody directed against E-selectin was seen in any of the biopsy specimens. Biopsy specimens of patients with a vasculitic glomerulonephritis (segmental necrotising glomerulonephritis) expressed VCAM-1 in glomerular endocapillary cells (four of eight patients with systemic vasculitis; two of eight patients with HSP). In patients with a systemic vasculitis glomerular VCAM-1 expression was associated with a more severe renal lesoin (44, 50, 60, and 65% of glomeruli involved) than in those not showing glomerular VCAM-1 expression (3, 3, 11, and 39% of glomeruli involved). CONCLUSION: Expression of VCAM-1 by glomerular endocapillary cells in renal biopsy specimens raises the possibility that recruitment of VLA-4 bearing leucocytes may contribute to glomerular injury in Wegener's granulomatosis and microscopic polyarteritis.
Increasing financial barriers to primary health care against a background of social and economic decline are likely to have contributed to asthma morbidity and mortality in New Zealand. Although there would not have been a sufficient increase in asthma prevalence to have accounted for the threefold increase in mortality rates, whether or not there was an increase in asthma severity in the late 1970s remains open to debate. Misuse or poor use of newly available and potent bronchodilator medications by those with the most severe asthma may simply have contributed to further delays in obtaining appropriate care and therefore to an increase in frequency of severe attacks in the community. Despite substantial increases in the use of bronchodilator therapy in New Zealand, there was no immediate improvement in indices of either asthma morbidity or mortality. The initial reduction in mortality rates in the 1980s happened at a time when first admissions for asthma were still increasing and seems to be best explained by an improvement in utilisation of hospital services (which were free until 1992) rather than a reduction in asthma severity. However, the recent reductions in all measures of asthma morbidity and further reduction in asthma mortality since 1989 does now suggest a reduction in asthma severity and would be best explained by the substantial increase in medium and high dose inhaled corticosteroid use, and to the endorsement of the current management strategies for asthma which are being promoted internationally and which were given considerable publicity in New Zealand in 1989 and 1990. Whilst sales of inhaled beta agonists were higher in 1991 than 1989, this may not reflect their pattern of use by individual patients since the need for an increase in inhaled beta agonist treatment has been accepted as indicating a lack of control and the need for either starting or increasing the dose of inhaled steroid treatment.
BACKGROUND--Studies of mortality from asthma have suggested that a very severe asthma attack identifies a group at greatly increased risk of subsequent death from the disease. This study compares the demographic characteristics of asthmatic patients who required management in an intensive care unit for a severe life threatening attack between 1981 and 1987 with a group who died of asthma between 1980 and 1986. The outcome of the group admitted to an intensive care unit is described. METHODS--The groups comprised all cases aged between 15 and 49 years arising from the Auckland Area Health Board (AAHB) population who required admission to an intensive care unit for asthma between 1981 and 1987 (n = 413) and all deaths from asthma in those aged 15 to 49 years arising from the New Zealand population between 1980 and 1986 (n = 466). Details of age, sex, and information on the day and month of the attack were collected. For the group requiring admission to an intensive care unit, outcome in terms of mortality and readmission to intensive care was determined. RESULTS--The age distributions of the two groups were dissimilar, with the severe life threatening attack group having an excess of asthmatic patients under 30 years old. The distribution of events by calendar month was uniform in both groups, but there was an unexpected increase in frequency of attacks on Sundays in both groups. Over the study period, mortality fell from 5.3 per 100,000 to 3.5 per 100,000 but the admission rate to intensive care increased from 10.8 per 100,000 to 17.9 per 100,000. At least 24% of asthma deaths occurring in the AAHB region during the study period had previously experienced a severe life threatening attack. CONCLUSIONS--The similarities between the groups suggest that asthmatic patients who experience severe life threatening attacks are likely to come from the same subgroup of the asthma population as those who die. The group who experience severe life threatening attacks are at high risk of subsequent morbidity and mortality and further studies may produce information relevant to reducing mortality from asthma.
OBJECTIVES--To determine the relative effects of cigarette smoking and mineral dust exposure on numbers and activity of circulating phagocytes, plasma nutritional antioxidant state, and pulmonary function in South African gold miners. METHODS--Pulmonary function was assessed spirometrically, whereas reactive oxidant generation by circulating phagocytes, and plasma concentrations of the nutritional antioxidative nutrients vitamin C and vitamin E and beta carotene were measured with chemiluminescence, spectrophotometry, or high performance liquid chromatography respectively. RESULTS--Cigarette smoking, but not mineral dust exposure, was associated with increased numbers and pro-oxidative activity of circulating neutrophils and monocytes, decreased plasma concentrations of vitamin C, and pulmonary dysfunction. DISCUSSION--In this study group occupational exposure to mineral dust has not been found to promote increases in the numbers or reactivity of circulating phagocytes or to be a significant cause of pulmonary dysfunction, the changes found being due primarily to cigarette smoking.
The transcription of the Drosophila melanogaster 68C salivary gland glue gene Sgs-3 involves the interaction of a distal and a proximal regulatory region. These are marked in vivo by a specific chromatin structure which is established sequentially during development, starting early in embryogenesis. The distal region is characterized by a stage- and tissue-specific DNase I hypersensitive site. A stage- and tissue-specific factor, GEBF-I, binds in this region and is missing in 2B5 mutant larvae which lack Sgs-3 transcripts. This binding involves the simultaneous interaction with two distinct DNA sequences which induces conformational changes in the protein. Salivary glands acquire competence to respond to ecdysone in the mid-third larval instar, whereafter the hormone rapidly induces both the GEBF-I protein and Sgs-3 transcription.
Pentamidine isethionate was bacteriostatic against Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus sanguis, Micrococcus sp., and Candida albicans. S. aureus was inhibited by concentrations of 16 to 64 micrograms/ml and killed by 64 to greater than or equal to 128 micrograms/ml. Staphylococcal killing was consistently greater in the presence of cations and was unaffected by methicillin resistance.
Factors that could affect adsorption of monodispersed poliovirus to cell culture monolayers were evaluated. These included varying the virus adsorption period under static and nonstatic (rocked) conditions and altering the rocking rate. The effects of several soluble proteins on plaque formation, enumeration, and size were also evaluated. Rocking involved the mechanical spread of viruses over cell culture monolayers for 1 to 4 h. Rocked cultures exhibited significantly higher (P less than 0.05) plaque counts than corresponding static cultures. Optimal plaque counts were obtained after a 2-h adsorption period with rocking; increasing the period to 4 h did not significantly increase PFU. Optimal counts were not obtained until greater than or equal to 4 h with static adsorption. Plaque counts were not affected by increasing the rocking rate above one oscillation per minute, but a slower rocking rate resulted in a significant decrease in plaques. Adsorption of poliovirus in the presence of 3% solutions of beef and meat extracts, acid-precipitated oyster protein, two brands of skim milk, and 3 and 10% fetal bovine serum was compared with adsorption in protein-free controls. Significant reductions (P less than 0.05) in plaque counts occurred with one brand of skim milk, whereas 3% beef extract yielded highly significant reductions (P less than 0.01) in plaque counts and appreciable decreases in plaque sizes. Salinities of protein-containing virus inocula were high for beef and meat extracts but somewhat below physiological levels for the remaining inocula. Beef extract-associated reductions in PFU were eliminated after the extracts were dialyzed. Plaque reductions were associated with dialyzable components of the beef extract but not with the inoculum salinity.
Misclassification of the oestrogen status of a human breast tumour cytosol, arising from different sources and magnitudes of error in the dextran-coated charcoal (DCC) method, have been investigated using both practical and computer simulated data analysed by Scatchard and Mass Action models. The minimum detectable receptor site concentration, relative or absolute numerical bias and imprecision which are complex and integral functions of misclassification, have been calculated from practical data and for a range of experimental conditions likely to be encountered in practice. The Mass Action model was found to be superior and the computer program, designed to investigate the effects of methodological errors on quantitative aspects of the assay, may be a useful aid for analytical design and internal quality control of the receptor assay.
A double-blind trial of perioperative intravenous metronidazole treatment to prevent infections at the operative site and unexplained fever after abdominal hysterectomy was conducted in 106 patients. Metronidazole prophylaxis reduced the rate of recovery of anaerobes from vaginal swabs for several days and prolonged the high rate of vaginal carriage of enterococci and aerobic gram-negative bacilli following hysterectomy. Although the fever index, calculated from the duration of a temperature above 37.3 degrees C, was significantly lower in the metronidazole-treated group than in the placebo-treated group, the frequency of postoperative infections, the proportion of patients requiring antibiotic treatment and the average duration of hospital stay were similar in the two groups. These results do not support the reported value of perioperative metronidazole prophylaxis in patients undergoing abdominal hysterectomy.