Günter Scholtysik

Bovine dilated cardiomyopathy : proteomic analysis of an animal model of human dilated cardiomyopathy

Bovine hereditary dilated cardiomyopathy (bCMP) is endemic in Switzerland and hearts from diseased animals display important clinical and biochemical similarities to human DCM. Recent research has identified at least one protein (myoglobin) to be significantly reduced in bovine DCM. Using a proteomic approach, we have separated over 1125 protein species from bovine ventricular tissue. Gel analysis and protein characterisation have identified a number of proteins whose abundance is significantly altered in bovine DCM. Twenty‐four proteins are of decreased abundance in diseased tissue, whilst 11 proteins are of increased abundance in the diseased state. A combination of amino acid compositional analysis, peptide mass profiling, N‐terminal microsequencing and MultiIdent (http://www.expasy.ch/sprot/multiident.html) has been employed in order to elucidate the identities of the differentially expressed proteins. Using these techniques we have currently determined the identity of 12 of the 35 altered proteins. We have also detected three proteins that are differentially expressed in genotypically diseased but phenotypically normal animals, identifying a possible mechanism for the onset of the disease. The possibility that inappropriate ubiquination of proteins plays an important role in the disease is discussed. A database of bovine proteins is currently being established. The identity of the proteins affected, together with a comparison of the human and bovine expression patterns, is displayed.

Topical application of synthetic pyrethroids to cattle as a source of persistent environmental contamination

Following the application of permethrin or cyhalothrin to cattle for the control of ectoparasites, the occurrence and persistence of these chemicals was assessed on the animals and in their environment. The release of permethrin from ear tags containing 1 g of the drug on cattle was followed for 65 days and lead to concentrations of 5 to 35 micrograms of permethrin per gram of hair on the shoulders. On the flanks of the animals, the corresponding values were 10 times lower. Across the 1.5 acre pasture, high concentrations of permethrin were measured at various locations and long after treatment: 6 micrograms/g on bark of a birch after one week, 5 micrograms/g on a pole of the fence after two weeks, 1 microgram/g in grass from a resting site of the animal after six weeks, and 0.5 microgram/g in bark of a pine tree after three month and two weeks after the animals had left the pasture. In similar assays, cyhalothrin applied to milk cows as a pour-on preparation was monitored. One week following treatment with 0.2 g/animal, hair cut from the shoulders contained 5 micrograms/g of the insecticide, which disappeared with a half-life of 12 days. Dust collected two weeks after the pour-on treatment from the milk barn where the cows were milked twice daily contained 47 micrograms/g of cyhalothrin, which disappeared with a half-life of 44 days. These results show that synthetic pyrethroids used on farm animals can be the source of widespread and persistent contamination.

Bovine hereditary cardiomyopathy: an animal model of human dilated cardiomyopathy

UNLABELLED Bovine hereditary cardiomyopathy (bCMP) displays clinical characteristics of human idiopathic dilated cardiomyopathy (DCM). We studied isometric force of contraction in right ventricular trabeculae, plasma and tissue catecholamines, beta- and alpha 1-adrenoceptor density, Gi proteins and adenylyl cyclase activity in eight hearts with bCMP and eight control hearts (right and left atria and ventricles each). RESULTS Compared to control, the potency of isoprenaline in bCMP was eight-fold decreased, whereas the maximal positive inotropic effect of isoprenaline as well as the efficacy and potency of calcium were unchanged. Plasma noradrenaline was increased by 240%. Tissue noradrenaline and adrenaline were decreased by 36-63% and 58-69%, whereas dopamine was increased by 105-218%. beta-adrenoceptor density was drastically reduced by 90%, but binding affinity was unchanged. alpha-Adrenoceptor density and binding affinity were unchanged. Total PTX-substrates were increased in bCMP by 28-99%. Basal adenylyl cyclase activity was decreased by 36-47%. Similarly, stimulation by GTP, GMPPNP, isoprenaline, sodium fluoride, manganese or forskolin was attenuated by 26-62% (atria) and 45-66% (ventricles). In conclusion, we found marked activation of the sympatho-adrenergic system, downregulation of beta-adrenoceptors, upregulation of Gi proteins, global desensitization of adenylyl cyclase and selective subsensitivity to beta-adrenergic inotropic stimulation. These results closely resemble the characteristic alterations in the beta-adrenoceptor-G protein-adenylyl cyclase pathway in human heart failure, indicating that they are general features of heart failure. The similarity to human DCM, the inheritance and the availability of large tissue samples make bCMP a suitable model for human DCM.

Effects of ketanserin and DOI on spontaneous and 5‐HT‐evoked peristalsis of the pig ureter in vivo

The influence of 5‐hydroxytryptamine (5‐HT) receptor agonists and antagonists on the ureter motility was investigated in vivo on intact ureters of anaesthetized pigs. Drugs were administered intravenously or topically. 5‐HT induced a dose‐dependent increase in the frequency of ureter contractions in anaesthetized pigs when given intravenously (0.0001 – 1 mg kg−1; ED50 0.066 mg kg−1) or topically (0.001 – 1 mg ml−1; EC50 0.043 mg ml−1). Significant increases in heart rate and blood pressure were observed when the drug was given intravenously but not topically. The 5‐HT2A agonist, DOI (1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane) increased the frequency of ureteral contractions in a dose‐dependent manner (1 – 300 μg kg−1 i.v.). Calculation of ED50 indicated this compound to be about 1.5 times more potent with an efficacy of 23% compared to 5‐HT. The 5‐HT2A/2C antagonist, ketanserin (0.5 mg kg−1) and the 5‐HT2C antagonist, methysergide (1 mg kg−1) antagonized the 5‐HT‐induced ureter peristalsis when given intravenously. Contraction amplitude, blood pressure and heart rate were not affected by the antagonists. Intravenous (0.0001 – 1 mg kg−1) and topical (0.0001 – 1 μg ml−1) ketanserin significantly decreased the frequency of spontaneous ureteral contractions to about 30% of controls, which could be partly reversed by 5‐HT (0.3 mg kg−1 i.v.). The contraction amplitude, contractions of the contralateral, saline perfused ureter, heart rate and mean arterial blood pressure were not affected. Thus, contractility of porcine ureter is mediated by 5‐HT2 receptors. Their antagonists ketanserin and methysergide seem to be promising drugs for treatment of acute ureteric colic or in preparing the ureter for ureteroscopy.

Cell culture in nephrotoxicity testing

The need to develop alternative methods has become obvious thanks to organizations concerned with animal welfare who ask for more humane and ethically acceptable methods, but also thanks to scientists who call for finer and more differentiated methods. In industrial toxicology where the numbers of animals used are particularly large there is increasing concern as to reducing the number of animals, replacing animal experiments by other methods or at least refining the tests performed on animals in order to diminish pain and discomfort. Being responsible for the elimination of many xenobiotics, the kidneys are exposed to much higher quantities of toxicants than other organs. The high blood flow compared to the organ weight and the ability of the tubular cells to concentrate toxins within them are two further factors which account for the vulnerability of the kidney to a large number of toxic substances. Considering that damage to the kidney can be a threat to the whole organism, it is clear that nephrotoxicity testing is an essential step in the development of new drugs. Cell culture is one of many in vitro methods in renal toxicology. Its advantages in comparison to in vivo tests are: (1) the opportunity to investigate the mechanisms of toxicity at the cellular level, (2) the possibility of long-term storage and repeated experimentation under similar conditions (especially when using cell lines), and (3) saving of time and test substance. However, since the cells are taken out of their normal environment and since some important factors of nephrotoxicity are omitted such as the metabolism of the drugs in the liver or the dynamics of blood flow in the kidney, cells in culture may not be considered as a replacement for animal experiments.