Proliferation of the human urothelium is induced by atypical ß1 -adrenoceptors.

We wanted to assess whether ß-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively. Urothelial cells were cultured for 24 h in the presence of the ß-adrenoceptor agonists isoprenaline (ß1/2/3 ), dobutamine (ß1 ), salbutamol (ß2 ), BRL 37344 (ß3 ), CGP 12177 (a partial ß-agonist) or ß-adrenoceptor antagonists (metoprolol; ß1 , propranolol; ß1/2 ). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the ß1 -adrenoceptor. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of ß1 -adrenoceptors in both urothelial cell lines. The present data show that the urothelium expresses atypical ß1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation.

Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder.

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction.

The potential of non-adrenergic, non-cholinergic targets in the treatment of interstitial cystitis/painful bladder syndrome.

Regulation of bladder function involves both divisions of the autonomic nervous system. However, in addition to the classical autonomic transmitters, noradrenaline and acetylcholine, other autonomic transmitters and other signalling components play important roles in physiology and pathophysiology of the lower urinary tract. Several substances of neuronal non-adrenergic, non-cholinergic (NANC) systems have already proven to considerably influence functional responses in the inflamed urinary bladder. Interstitial cystitis (IC) or painful bladder syndrome (PBS) is a chronic inflammatory bladder disease, characterized by urinary frequency, urgency and pelvic pain. IC/PBS is difficult to diagnose, especially because the etiology of the condition is largely unknown. Despite the unclear nature of the cause and manifestation of IC/PBS, it has been shown that the disease involves a significant NANC component. Here, we review the possible roles of ATP, adenosine, nitric oxide, vasoactive intestinal polypeptide, substance P, and pituitary adenylate cyclase-activating peptide in the contribution to IC/PBS development and manifestation of IC/PBS symptoms.

Coupled nitric oxide and autonomic receptor functional responses in the normal and inflamed urinary bladder of the rat.

Both divisions of the autonomic nervous system are involved in regulation of urinary bladder function. Several substances, other than noradrenaline and acetylcholine, seem to play important roles in physiology and pathophysiology of lower urinary tract. In the current study, we aimed to examine if there exist interplays between nitric oxide (NO) and autonomic transmitters and if such interactions vary in different parts of the urinary bladder in healthy and cyclophosphamide (CYP)-induced cystitic rats; when administered to the animals (100 mg/kg; i.p.), the cytotoxic CYP metabolite acrolein induces bladder inflammation. In the current study a series of in vitro functional studies were performed on detrusor muscle strip preparations. Stimulation with electrical field stimulation (EFS), methacholine, adenosine 5´-triphosphate (ATP), and adrenaline evoked contractile responses in isolated bladder preparations that were significantly reduced in cyclophosphamide (CYP)-treated rats. While the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 10(-4) M) did not affect contractile responses in normal, healthy strip preparations, it significantly increased the contractile responses to EFS, methacholine and adrenaline, but not to ATP, in the bladders from the CYP-treated rats. In the CYP-treated rats, the ATP-evoked relaxatory part of its dual response (an initial contraction followed by a relaxation) was 6-fold increased in comparison with that of normal preparations, whereas the isoprenaline relaxation was halved in the CYP-treated. While L-NNA (10(-4) M) had no effect on the isoprenaline-evoked relaxations, it reduced the ATP-evoked relaxations in strip preparations from the bladder body of CYP-treated rats. Stimulation of beta(2)- and beta(3)-adrenoceptors evoked relaxations and both responses were reduced in cystitis, the latter to a larger extent. In the trigone, the reduced ATP-evoked contractile response in the inflamed strips was increased by L-NNA, while L-NNA had no effect on the ATP-evoked relaxations, neither on the relaxations in healthy nor on the larger relaxations in the inflamed trigone. The study shows that both contractile and relaxatory functions are altered in the state of inflammation. The parasympathetic nerve-mediated contractions of the body of the bladder, evoked by the release of ATP and acetylcholine, were substantially reduced in cystitis. The relaxations to beta-adrenoceptor and purinoceptor stimulation were also reduced but only the ATP-evoked relaxation involved NO.

Pharmacological modulation of the micturition pattern in normal and cyclophosphamide pre-treated conscious rats.

In the current study, we wanted to assess the influence of muscarinic receptors, nitric oxide and purinoceptors on the micturition pattern of conscious normal and cyclophosphamide (CYP) pre-treated rats. The micturition parameters were assessed using a metabolic cage. Rats were pre-treated with either saline or CYP, to induce cystitis, followed by treatment with either the muscarinic M1/M3/M5 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), the nitric oxide synthase blocker N(ω)-nitro-L-arginine methyl (L-NAME), the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) or a combination of 4-DAMP with PPADS or L-NAME. Voiding volumes per micturition event were significantly lower in CYP pre-treated than in saline pre-treated rats. Neither 4-DAMP nor L-NAME had any effect in the normal rats, whereas PPADS reduced the micturition volume per event. In CYP pre-treated rats, 4-DAMP and L-NAME significantly increased voiding volumes per event and micturition frequency, respectively. 4-DAMP dose-dependently reduced the differences in micturition activity between saline and CYP pre-treated rats. We show that cystitis changes the urodynamics in conscious rats and that this change seems to depend on the production of NO and on altered muscarinic receptor effects. The altered muscarinic receptor responses are likely to per se involve NO-mediated mechanisms.

Landfill leachate treatment with willows and poplars--efficiency and plant response.

Irrigation of willow and poplar short-rotation coppice with landfill leachate is an increasingly interesting treatment option. Minimal leaching to groundwater and disturbance to plant growth must be ensured, but in such systems, where various site-specific factors interact, a case-specific approach is needed to determine potential hazards. This paper compares the effect of leachate irrigation on willow grown in clay lysimeters and poplar grown in sand lysimeters. Leachate irrigation increased willow biomass production, but not that of poplar. Near-zero nitrate-N concentrations were found in drainage water for both species after 2 years of irrigation. Ability to retain total N and P, and TOC was relatively high for willow, taking into account the large amounts supplied, and better than for poplar. To reduce environmental risks the irrigation load should be reduced, but if leachate concentrations are reduced, the irrigation load can be as high as 6mm/day.

Changes in muscarinic receptors in the toad urothelial cell line TBM-54 following acrolein treatment.

1. In cyclophosphamide-induced cystitis in the rat, cholinergic function of the bladder and muscarinic receptor expression are altered. In the present study, we investigated whether the toad urothelial cell line TBM-54 expresses functional muscarinic receptors and whether changes in muscarinic receptors can be induced in vitro by treating cells with acrolein, a metabolite of cyclophosphamide causing cystitis. 2. The occurrence of muscarinic receptors on cells was assessed by microphysiometry, a method analysing receptor function by measuring changes in the extracellular acidity rate (ECAR) in response to receptor stimulation. 3. Challenging untreated cells with the muscarinic receptor agonist carbachol gave rise to a concentration-dependent increase in changes in ECAR, with a maximal response at 1 mmol/L carbachol of 51 +/- 6%. Pre-incubating cells with different muscarinic receptor antagonists (i.e. pirenzepine (M(1) receptor selective), methoctramine (M(2)/M(4) receptor selective) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M(3)/M(1)/M(5) receptor selective)), gave rise to a concentration-dependent decrease in the effects of carbachol (0.5 mmol/L) on changes in ECAR. 4. Western blot analysis was used to determine the expression of all muscarinic receptor subtypes (M(1)-M(5)) by the cell line. Following acrolein treatment, cells were markedly less sensitive to carbachol and the expression of muscarinic M(2) receptors was decreased, whereas the expression of muscarinic M(3) receptors was increased. 5. In conclusion, the urothelial cell line TBM-54 expresses functional muscarinic receptors and exposure to acrolein leads to a modulation in the expression of muscarinic receptors. Consequently, acrolein may have direct effects on muscarinic receptor function and expression that contribute to the pathogenesis of cyclophosphamide-induced cystitis.

Fate of heavy metals after application of sewage sludge and wood-ash mixtures to short-rotation willow coppice.

Short-rotation willow coppice (SRWC), grown on farmland in Sweden for energy-biomass production, was fertilised with sewage sludge and wood-ash mixtures on the basis of the permitted annual phosphorus supply. Two identical experiments were conducted in central Sweden, on two newly harvested commercial SRWC fields. The maximum legally permitted amount of the sludge-ash mixture, sludge only, ash only, and twice the maximum permitted sludge-ash amount, were applied. The aim was to assess the effect of pH changes following treatment, on the ability of SRWC to take up large amounts of Cd and other metals. The remediation effect of SRWC was also studied. Under the experimental conditions applied, uptake by SRWC was unaffected by pH changes. The differences between the amounts of metals experimentally applied, less the uptake by SRWC after a potential harvest, were broadly within the permitted limits. For Cd, a reduction of total amounts in soil was observed.

Effects of non-synaptic neuronal interaction in cortex on synchronization and learning.

During neural communication by means of action potentials, small electromagnetic (EM) fields are also generated. We use a three layered cortical neural network model to study the effects of EM fields and gap junctions on spatio-temporal network activity. We investigate the possible role of these effects in synchronizing activity, a phenomenon which has been observed in the olfactory cortex and the hippocampus. The simulation results support the notion that fast synchronization of activity in distant parts of the neural network are made possible by means of EM fields and/or gap junctions. The results also indicate that these effects, to a certain extent, are beneficial to system performance.

Evolution of T4-related phages.

Much progress has been made in understanding T-even phage biology in the last 50 years. We now know the entire sequence of T4, encoding nearly 300 genes, only 69 of which have been shown to be essential under standard laboratory conditions; no specific function is yet known for about 140 of them. The origin of most phage genes is unclear, and only 42 genes in T4 have significant similarity to anything currently included in GenBank. Comparative analysis of related phages is now being used to gain insight into both the evolutionary origins and interrelationships of these phage genes, and the functions of their protein products. The genomes of phages isolated from Tbilisi hospitals, Long Island sewage plants, the Denver zoo, and Khabarovsk show basic similarity. However, these phages show substantial insertions and deletions in a number of regions relative to each other, and closer investigation of specific sequences often reveals much more complex relationships. There are only a few cases in T4-related phages in which there is evidence for evolution through DNA duplication. These include the fibrous products of genes 12, 34, and 37; head proteins gp23 and gp24; and the Alt enzyme and its downstream neighbors. T4 also contains 13 apparent relatives of group I and group II intron homing endonucleases. Distal portions of the tail fibers of various T-even phages contain segments closely related to tail-fiber regions of other DNA coliphages, such as Mu, P1, P2, and lambda. Horizontal gene transfer clearly emerges as a major factor in the evolution of at least the tail-fiber regions, where site-specific recombination probably is involved in the exchange of host-range determinants.

The forced oscillation technique in children with respiratory disease.

The single-frequency forced oscillation technique was used to estimate the total respiratory impedance, resistance, and reactance at 2, 4, and 12 Hz of children who had cricoid stenosis (CS; n = 11), provoked bronchoconstriction (PB; n = 6), or cystic fibrosis (CF; n = 13). The selection criteria for patients who had CS and CF were abnormal forced expiratory volume in 1 sec (FEV!) and/or maximal expiratory flow at 50% vital capacity (Vmax 50%). Sixteen of the 17 patients who had CS or PB were found to have resistance (Rrs) values outside the normal range at 4 Hz; at 2 Hz, 14 were found to have abnormal values and, at 12 Hz, only 9. The reactance in the CS and PB groups was somewhat less discriminative than Rrs at all frequencies. Of the 13 patients who had CF, only 4 had abnormal Rrs values; in this clinical condition, the reactance at 2 Hz was the most discriminative variable, being abnormal in 6 of the 13 patients. Irrespective of the clinical group, the Rrs was, in absolute terms, highest at 2 Hz, and it decreased with increasing frequency. This pattern of negative frequency dependence was, however, not obviously specific for any of the tested clinical conditions and offered no information in addition to a single low-frequency determination.

Respiratory resistance and impedance magnitude in healthy children aged 2-18 years.

Two hundred eighteen healthy children aged 2 to 18 years were studied using a modification of the forced oscillation technique. Reference values with height as predictor were determined for total respiratory resistance and impedance during inspiration, expiration, and throughout the whole respiratory cycle at an oscillation frequency of 4 Hz and, in a subpopulation of 61 children, at frequencies of 2 and 12 Hz. Mean total inspiratory resistance, determined at 4 Hz, decreased with growth from 1.3 kPa X 1(-1) X s at 2 years of age to 0.3 kPa X 1(-1) X s at 18 years. Variability in the results between individuals, expressed in terms of coefficient of variation, was found to be +27% and -21%, respectively, and within individuals, 9%. Resistance during expiration was on average 16% higher than during inspiration and the variability within individuals was 11%. A marked decrease in resistance was found in small children when the frequency was increased from 2 to 12 Hz. The frequency dependence of respiratory resistance observed in small children changes gradually with growth, in parallel with the reduction of total respiratory resistance, to an adult pattern in which no significant change in resistance can be noted between frequencies of 2 and 12 Hz.

Forced oscillation technique and maximum expiratory flows in bronchial provocation tests in children.

Variables obtained with the forced oscillation technique (total respiratory resistance and impedance at 2, 4 and 12 Hz) and with the forced vital capacity manoeuvre (FVC, FEV1, MMEF, MEF75, MEF50, MEF25) were used in the evaluation of the bronchial allergen challenge test in 2 groups of asthmatic children. Each method was applied in 15 provocation tests and the changes of the different variables, allowing for their respective variations, were compared with the clinical assessment. Agreement with the clinical evaluation was found in 15 of 15 occasions with the forced oscillation technique and in 12 of 15 occasions with the forced vital capacity manoeuvre. With the forced oscillation technique a bronchial reaction was detected in 6 of 10 patients at 1/10 of the allergen concentration that gave positive clinical signs and the single most sensitive variable was resistance determination at 4 Hz, with significant change in 5 of 10 cases. The forced vital capacity manoeuvre allowed detection of a bronchial reaction preceding clinical signs at a tenfold lower allergen concentration in 2 of 9 cases and the most sensitive variables were FEV1 and MEF25.

Nitrogen single breath test, flow-volume curves and spirometry in healthy children, 7-18 years of age.

One hundred and forty school children, 75 boys and 65 girls, aged 7-18 years, underwent the following lung function tests: closing volume, slope of the alveolar plateau, maximal expiratory and inspiratory flows at 75, 50 and 25% of vital capacity, static and dynamic lung volumes. Lung volumes and maximal flows increase with height in an exponential way, the exponents varying between 2.6 and 3.1, while closing volume in per cent of vital capacity and the slope of the alveolar plateau decrease linearily with increasing height. Regression equations are given for each sex with height as determinant.