Protein kinase A modulates A-type potassium currents of larval zebrafish (Danio rerio) white muscle fibres.

AIMS: Potassium (K(+)) channels are involved in regulating cell excitability and action potential shape. To our knowledge, very little is known about the modulation of A-type K(+) currents in skeletal muscle fibres. Therefore, we sought to determine whether K(+) currents of zebrafish white skeletal muscle were modulated by protein kinase A (PKA). METHODS: Pharmacology and whole-cell patch clamp were used to examine A-type K(+) currents and action potentials associated with zebrafish white skeletal muscle fibres. RESULTS: Activation of PKA by a combination of forskolin + 3-isobutyl-1-methylxanthine (Fsk + IBMX) decreased the peak current density by approximately 60% and altered the inactivation kinetics of A-type K(+) currents. The specific PKA inhibitor H-89 partially blocked the Fsk + IBMX-induced reduction in peak current density, but had no effect on the change in decay kinetics. Fsk + IBMX treatment did not shift the activation curve, but it significantly reduced the slope factor of activation. Activation of PKA by Fsk + IBMX resulted in a negative shift in the V(50) of inactivation. H-89 prevented all Fsk + IBMX-induced changes in the steady-state properties of K(+) currents. Application of Fsk + IBMX increased action potential amplitude, but had no significant effect on action potential threshold, half width or recovery rate, when fibres were depolarized with single pulses, paired pulses or with high-frequency stimuli. CONCLUSION: PKA modulates the A-type K(+) current in zebrafish skeletal muscle and affects action potential properties. Our results provide new insights into the role of A-type K(+) channels in muscle physiology.

Calpain activation and not oxidative damage mediates L-2-chloropropionic acid-induced cerebellar granule cell necrosis.

Possible biochemical events involved in L-2-chloropropionic acid (L-CPA)-induced delayed cerebellar granule cell necrosis following N-methyl-D-aspartate activation were studied in vivo. We examined whether the calcium-sensitive proteolytic enzymes, the calpains, may be activated by L-CPA or whether the generation of excess quantities of cytotoxic free radicals may play a role in the neurotoxicity produced by oral administration of L-CPA (750 mg/kg, pH 7.0). Evidence for free radical-induced cellular damage was examined using biochemical approaches such as examining brains from L-CPA-treated rats for increased lipid peroxidation, DNA damage, or protein oxidation. Second, the ability of antioxidants to provide neuroprotective activity against L-CPA-induced neurotoxicity was examined in vivo. Western blotting using antibodies against spectrin (alpha-fodrin) demonstrated evidence for calpain (EC 3.4.22.17) activation in the cerebellum, but not in the cerebral cortex of L-CPA-treated rats at 36 and 48 hr after L-CPA dosing. In contrast, there was no evidence for oxidative damage to cerebellar proteins or lipids in L-CPA-treated rat brains compared to controls. We also could not find evidence for DNA damage using the TUNEL method for the detection of single- and/ or double-strand breakage in situ in L-CPA-treated brains. We examined whether a number of reported antioxidants may be effective against L-CPA-induced neurotoxicity. The aminosteroids U74389G and U83836E, the free radical scavengers 3-methyl-1-phenylpyrazolin-5-one and N-tert-butylphenylnitrone, and the iron chelator N-ethoxy-2-ethyl-3-hydroxypyridin-4-one were all ineffective in attenuating L-CPA neurotoxicity. We suggest that L-CPA-induced cerebellar necrosis is the result of calpain activation which results in the degradation of cytoskeletal proteins and other proteins necessary for cellular biochemistry. We could find no evidence of oxidative damage to cerebellar proteins, lipids, or DNA as a result of excess amounts of free radicals, and selective antioxidants were unable to provide neuroprotection against L-CPA neurotoxicity, suggesting that oxidative stress does not play a role in the granule cell necrosis.

Methylene chloride-induced DNA damage: an interspecies comparison.

DNA single-strand (ss) breaks were detected in the livers of B6C3F1 mice immediately following exposure to 4000-8000 p.p.m. methylene chloride (MC) for 6 h. This damage was undetectable 2 h after exposure, suggesting an active DNA repair process. Similarly, DNA ss breaks were detected in whole lung homogenates taken from mice exposed to 2000-6000 p.p.m. MC. The DNA of mouse Clara cells incubated in vitro with MC was also damaged at concentrations of 5 mM MC and above. Pre-treatment of mice with the glutathione depletor buthionine sulphoximine (BSO) caused a decrease in the amount of DNA damage detected, suggesting a GST-mediated mechanism. DNA damage was also reduced in Clara cells when incubated in vitro with MC in the presence of BSO. In CHO cells induction of DNA damage was dependent upon exogenous MC metabolism by mouse liver S100 fraction (but not microsomes) in the presence of GSH. DNA ss breaks were not induced by MC in hamster hepatocytes in vitro at concentrations from 5 to 90 mM MC, nor in eight individual samples of normal human hepatocytes exposed to MC at similar concentrations. The ability of MC to induce DNA ss breaks in the four species studied is entirely compatible with the known carcinogenicity of this chemical in animals and offers experimental evidence to suggest that humans would not be susceptible to MC-induced liver cancer. The DNA ss breaks correlate with the metabolism of MC by the GST pathway and provide an explanation for the lack of sensitivity of hamsters and rats to MC-induced liver cancer.

Trichloroacetic acid: investigation into the mechanism of chromosomal damage in the in vitro human lymphocyte cytogenetic assay and the mouse bone marrow micronucleus test.

Trichloroacetic acid (TCA) was tested for its ability to induce chromosomal damage in cultured human peripheral blood lymphocytes and in bone marrow cells of male and female C57BL/6JfBL10/Alpk mice. Two in vitro cytogenetic assays were conducted with TCA. In the first TCA, as free acid, was added to whole blood cultures at final concentrations of 500, 2000 and 3500 micrograms/ml in the presence and absence of an auxiliary metabolic activation system (rat liver S9-mix). Statistically significant increases in the percentage of aberrant cells compared with solvent control values were observed in cultures treated with TCA at 2000 and 5000 mu/ml. Investigation into the effects of TCA on the pH of the culture medium revealed significant reductions in pH at both these TCA concentrations. Neutralized TCA was then tested at concentrations of 500, 2,000 and 5000 micrograms/ml, also in the presence and absence of S9-mix. No statistically or biologically significant increases in the percentage of aberrant cells were observed in any of these cultures. In the mouse micronucleus test, neutralized TCA was administered in two equal intraperitoneal doses 24 h apart to C57BL/6JfBL10/Alpk mice (337, 675 and 1080 mg/kg in males; 405, 810 and 1300mg/kg in females). These dose levels represent 25%, 50% and 80% of the median lethal dose (MLD) in this strain of mouse. Bone marrow samples were taken 6 and 24 h after the second dose and the chromosomal damage assessed by analysis of the bone marrow for micronuclei. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the solvent control dosed animals were observed in either sex at the 6 h sampling time or in the females at the 24 h sampling time. A small but statistically significant increase in micronucleated polychromatic erythrocytes was observed in male mice 24 h after a dose of 675 mg/kg (50% MLD). Since no increases were noted at the 25 or 80% MLD, and the levels recorded are within the range of the concurrent solvent control values, the small increase observed in the males at the 50% MLD is considered not to be biologically significant. Flow cytometric studies on suspensions of isolated liver cell nuclei revealed that changes in FITC binding (indicating altered chromatin conformation) were induced by pH changes alone and were not caused by neutralized TCA.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of implantation of osmotic pumps on rat thyroid hormone and testosterone levels in the plasma, an implication for tissue 'S' phase studies.

In order to monitor the effect of the procedures required to s.c. implant osmotic pumps into rats on plasma thyroid and testosterone hormone levels, male Fischer 344 rats (8-10 weeks old) were divided into six groups of 10 rats and the groups treated in the following manner: (1) controls housed 5 per cage; (2) controls housed individually; (3) animals anaesthetised for surgery and individually housed; (4) anaesthetised, sham operated and individually housed; (5) anaesthetised, s.c. implanted with osmotic pumps containing saline and individually housed; (6) anaesthetised, s.c. implanted with osmotic pumps containing 5-bromo 2-deoxyuridine (BRDU) and individually housed. Four days after performing the surgery the study was terminated and the level of hormones in the plasma determined by radio immunoassay (RIA). Tri-iodothyronine (T3) and thyroxine (T4) plasma levels (free and total) were significantly decreased with each additional step in the procedure used for the s.c. implantation of an osmotic pump containing BRDU, when compared with the individually housed controls. Similarly, testosterone plasma levels were significantly decreased by the s.c. implantation of osmotic pumps, implying a 'stress' response might occur following implantation. These observations might need to be considered by investigators when performing toxicological research which, as part of the study, uses osmotic pumps for the delivery of the nucleotide precursor required for monitoring cells in 'S' phase.

Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.

The effect of chlorinated paraffins on hepatic enzymes and thyroid hormones.

Male rats and mice were administered chlorinated paraffins (CPs) by daily gavage in corn oil for 14 days. Chlorowax 500C (short chain CP with 58% chlorination), Cereclor 56L (short chain CP with 56% chlorination) and Chlorparaffin 40G (medium chain CP with 40% chlorination) were the CPs studied at dose levels of 0, 10, 50, 100, 250, 500 and 1000 mg/kg for both rats and mice. The no effect levels for hepatic peroxisome proliferation for the above chemicals, as determined by the CN- insensitive palmitoyl co-enzyme A beta-oxidation (PCO) assay, were calculated as 184, 600 and 473 mg/kg and 180, 120 and 252 mg/kg for rats and mice, respectively, whilst those for percent liver weight/body weight were calculated as 74, 51 and 31 mg/kg and 215, 70 and 426 mg/kg for rats and mice, respectively. The short chain CPs were more potent peroxisome proliferators than the medium chain CP, with the mouse proving to be more responsive than the rat. Rats administered the highest dose of CPs showed a depressed plasma thyroxine (T4) level, with a concomitant increase in the plasma concentrations of thyroid stimulating hormone (TSH). The decreased plasma T4 levels appeared to be the result of increased T4 glucuronidation.

Trichloroacetic acid: studies on uptake and effects on hepatic DNA and liver growth in mouse.

Trichloroacetic acid (TCA) was tested in mice for its ability to cause single-strand breaks (SSBs) in hepatic DNA in the presence and absence of liver growth induction. Male B6C3F1 mice were given 1, 2 or 3 daily doses of TCA (500 mg/kg p.o.) as a neutralized solution (sodium salt) and killed 1 h after the final dose. Some mice were given a single dose of TCA (500 mg/kg p.o.) as the acid or as a neutralized solution and killed 24 h after. Liver nuclei were prepared and the induction of DNA SSBs assayed. TCA gave no significant response. Absorption and distribution studies were conducted with radiolabelled trichloro[2-14C]acetic acid, which was administered by gavage (500 mg/kg) as aqueous free acid, neutral aqueous solution (sodium salt) or free acid in corn oil. The absorption and distribution of TCA was similar in all cases: the chemical was absorbed rapidly after dosing, maximum plasma and liver concentrations of free radiolabel being achieved in less than 1 h. Within the first 4 h following dosing there was no evidence of binding to DNA or other macromolecules in plasma and very little 'covalent' binding was detected in liver, indicating that at times when maximum DNA single-strand breakage has been reported there was no significant binding to liver cells. Studies on liver growth parameters (hyperplasia and peroxisome proliferation) with TCA revealed that the chemical induced small but significant increases in both parameters. No SSB induction was detected in association with either liver growth phenomenon elicited by TCA. We have thus found no evidence that TCA causes SSBs in the hepatic DNA of treated mice, in contrast to previous observations by other investigators.

Lung rejection and bronchial hyperresponsiveness to methacholine and ultrasonically nebulized distilled water in heart-lung transplantation patients.

Acute denervation of the lungs occurs after heart-lung transplantation (HLT), affecting both afferent and efferent nerves below the tracheal anastomosis. After surgery, the carina and main bronchi are perfused by mediastinal collaterals derived from the coronary arteries, and the intrapulmonary airways by retrograde blood flow from pulmonary artery collaterals. During acute rejection, the lungs are subjected to inflammation, particularly perivascular lymphocytic infiltrates. Rejection can be diagnosed by transbronchial biopsy (TBB). We report the bronchial responses to inhaled methacholine and ultrasonically nebulized distilled water (USNDW) in 16 HLT patients 2 wk to 43 months after surgery, relating them to the lung histopathology from concurrent TBB. Methacholine bronchial hyperresponsiveness was common, but it was not associated with airway epithelial or submucosal inflammation or perivascular lymphocytic infiltration. Six patients had a modest response to USNDW (fall in FEV1 greater than 10%). The responsiveness to USNDW was not associated with enhanced methacholine responsiveness or epithelial and mucosal inflammation. However, it was more commonly seen in patients with lung rejection and perivascular infiltrates. Methacholine hyperresponsiveness in HLT patients could therefore reflect denervation hypersensitivity of airway smooth muscle muscarinic receptors. The modest response to USNDW in some patients cannot be a result of a vagal reflex but could reflect a pathologic vascular response associated with lung rejection. These observations offer insight into the possible mechanisms of bronchial hyperresponsiveness in disease.

Sites of deposition of aqueous aerosols: a study of efficiency of delivery systems for lung ventilation imaging in man.

A study was made of the deposition of 99Tcm-DTPA aerosol in the components of a jet nebulizer-based aerosol production system. Three impaction devices were compared: a ball-bearing separator, a virtual impactor and a step separator. In addition a comparison was made of two types of tubing which carried aerosol from nebulizer to mouthpiece: corrugated and smooth-walled tubing. The retention of aerosol following inhalation was measured in five normal volunteers using different patterns of breathing. Using an aerosol production system which included a ball-bearing separator only a mean of 11% of the radioactivity loaded into the nebulizer was emitted as an aerosol. Some 18% remained in the ball-bearing separator. The ball-bearing and step separators produced similar total outputs (7% and 6% minimum), the step separator producing marginally higher mean output/min. The virtual impactor produced a lower output than the other two impactors studied, only 1%. A larger proportion of the aerosol output was deposited on corrugated tubing (7%) compared with smooth-walled tubing (1%). The retained fraction of the aerosol inhaled by subjects ranged from 16% to 43%. A higher fraction was retained when subjects inhaled deeply and held their breath for 10 s between each breath. The efficiency of radionuclide deposition from aerosol generator to patient ranged from 1.1% to 6% and was determined more by the retention in the subject than by choice of separator or tubing.

Clinical experience in the management of pulmonary opportunist infection and rejection in recipients of heart-lung transplants.

The main clinical problems that follow heart-lung transplantation are opportunist infections of the lungs and pulmonary rejection. Of 23 patients undergoing heart-lung transplantation, eight had opportunist infections and 12 had at least one episode of pulmonary rejection. Cardiac rejection occurred in only one patient, who did not need treatment. Of the 12 patients who had pulmonary rejection, nine recovered fully after augmented immunosuppression with high dose corticosteroids, although one patient required additional low dose corticosteroids for eight months before making a full recovery. Fatal opportunist lung infection followed treatment for rejection in two patients. One patient developed obliterative bronchiolitis. Of the eight patients with opportunist infections, five had primary cytomegalovirus pneumonitis, acquired from the donor. All three patients treated with acyclovir died, whereas the two treated with hyperimmune globulin and dihydroxy proxymethylguanine recovered fully. Two patients developed Pneumocystis carinii pneumonia, which was treated successfully in one patient with intravenous sulphadimidine and trimethoprim. The other patient died after a further episode of rejection and aspergillus bronchitis. One patient developed a tuberculous empyema. The calculated actuarial survival at one year was 78% and at two years 67.2%. Although it is still in its innovative stage heart-lung transplantation appears to have complications and results similar to those of transplantation of other organs.