L. P. Jager

The effect of catecholamines and ATP on the smooth muscle cell membrane of the guinea-pig taenia coli

Abstract A comparative study was made of the effects of some catecholamines and purine-like compounds on the smooth muscle cell membrane of the guinea-pig taenia coli, using the double sucrose-gap method. The inhibitory junction potential (IJP) might be mediated by purinergic or adrenergic nerves. Therefore, the effects of these compounds on the membrane resistance and the membrane potential and also on the IJP were measured in the presence of guanethidine and atropine. The potency of the catecholamines tested to mimic the IJP with regard to membrane hyperpolarization and decrease of membrane resistance suggested an action on α-adrenoceptors: adrenaline > noradrenaline ⪢ isoprenaline. Equipotent concentrations of adrenaline and adenosine triphosphate (ATP) were found to exert the same hyperpolarization of the smooth muscle membrane in both the presence and absence of chloride. From the effects observed with phentolamine and tolazoline it is concluded that the IJP is not mediated by adrenergic nerves, for the adrenaline response was abolished, whereas the IJP was unaffected. Furthermore, the ATP-blocking potencies of phentolamine and imidazole could not be demonstrated. On the other hand, the palallelism observed between the responses obtained with ATP and with transmural stimulation is in favor of the hypothesis that the IJP is mediated by purinergic nerves.

Some pharmacokinetic observations of carbadox medication in pigs.

Concentrations of carbadox and a first metabolite, desoxycarbadox, were measured in contents of the porcine gastrointestinal tract after in-feed administration of carbadox in therapeutic dosages (100-150 ppm). The levels of carbadox in the relevant parts of the gastrointestinal tract were found to be lower than the MIC-values reported for enteropathogenic microorganisms at their sites of action. The presented observations do not provide a pharmacological rationale for the therapeutic use of carbadox in the treatment of dysentery and diarrhoea in swine. The carbadox levels encountered in the proximal part of the gut (stomach, duodenum) however, seem to indicate that in-feed administration of 50 ppm carbadox can provide an effective prophylaxis against Treponema hyodysenteriae, a causative agent in swine dysentery. The timecourse of the blood levels of carbadox and desoxycarbadox after in-feed administration of carbadox (50 ppm) and the concentration profiles in the gastrointestinal tract are discussed with regard to the disposition of this drug in pigs.

Hypoaldosteronism in piglets induced by carbadox

An exploratory study was made of the mechanisms underlying the toxic action of carbadox in young pigs: dehydration, loss of appetite and at autopsy seemingly specific and selective structural alterations of the glomerular zone of the adrenal cortex. Administration of carbadox in the feed, in dosages of 150 ppm (approximately 6 mg·kg−1 b.wt·day−1) caused a rapid decline in the plasma aldosterone levels (to 10% of control) followed by significant changes in the sodium and potassium levels in blood. Characteristic for the toxic action of carbadox are the rapid and seemingly selective and specific alterations in the aldosterone-releasing zona glomerulosa of the adrenals. Our results indicate that with carbadox a functional and possibly reversible extirpation of the adrenal zona glomerulosa can be achieved in pigs.

Carbadox-induced inhibition of aldosterone production in porcine adrenals in vitro.

Carbadox, a growth promoter used in pig husbandry, inhibited aldosterone production in sliced porcine adrenals in vitro. The adrenal slices, obtained from 3-5-wk-old piglets, were maintained in a Krebs solution and exposed to 1-40 mug carbadox/ml for 1 hr. Aldosterone was estimated (using a solid-phase radioimmunoassay kit) in samples taken hourly for up to 5 hr after carbadox exposure. The results showed a dose-dependent inhibition of aldosterone production by carbadox, with 25-35% inhibition by a concentration of 40 mug/ml. The findings support the hypothesis, based on clinical and histopathological evidence from in vivo experiments in pigs, that carbadox interferes with aldosterone production in the adrenal zona glomerulosa.

Screening for drug-induced alterations in the production and release of steroid hormones by porcine adrenocortical cells in vitro

To screen drugs rapidly and at minimal expense for their potential to alter steroidogenesis, an in vitro model using porcine adrenocortical cells was developed. Pregnenolone, progesterone, deoxycorticosterone or corticosterone (all at 1 muM) were used as substrates. Drug-induced changes in the production/release of aldosterone were measured after 1-hr incubation. With pregnenolone, drug-induced effects on the release of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, HO-progesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. For assessment of cell viability and the amount of steroids produced/released, a cheap, simple modified Krebs solution was at least comparable to an elaborate cell culture medium. Within the conditions adopted, the cell suspension reacted to varying potassium concentrations as expected. ACTH stimulated steroid production/release only without added substrate. 11 agents known to interfere with steroid biogenesis were tested at 0.1-100 muM. Although all known points of action of the test compounds were located, several showed additional activity. Spironolactone shifted steroid biogenesis from aldosterone and cortisol towards androgenic steroids. Aminoglutethimide inhibited the release of aldosterone with corticosterone as substrate, but not with deoxycorticosterone or progesterone as substrate, revealing an alternative pathway in the biogenesis of aldosterone by-passing corticosterone. Trilostane (0.1-1 muM) completely blocked conversion of pregnenolone to progesterone and OH-progesterone; the release of androstenedione was at most only halved, whereas the release of dehydroepiandrosterone and testosterone was greatly enhanced. This implies isoenzymes of 3beta-hydroxysteroid dehydrogenase/isomerase with different sensitivities towards trilostane. Mitotane, metyrapone, ketoconazole and etomidate all inhibited the mitochondrial P450 (11beta 18 ) enzymes. In addition, mitotane and ketoconazole also inhibited (albeit to a lesser extent) endoplasmic enzymes involved in transformations at C21 and at C17, respectively. Cyproheptadine blocks all transformations with progesterone or HO-progesterone as starting point. Finasteride reduced the release of most steroids, except the androgens, presumably by inhibition of transformations at C3 and at C11. Carbadox and related quinoxalines inhibited not only C18 oxidation but also C21 hydroxylation. Steroidogenesis in these porcine adrenocortical cells in vitro could be described as similar to that in other mammals. A notable feature was that inhibition of the release/production of a steroid hormone was usually accompanied by an increased release of other steroid hormones. This screening model also yields information about the point of action of drugs interfering with steroidogenesis.

Differential effects of nitrofurans on the production/release of steroid hormones by porcine adrenocortical cells in vitro

Changes in the biogenesis of corticosteroids caused by nitrofurans were studied. The three nitrofurans used: furazolidone, furaltadone and nitrofurantoin, altered the steroid production/release by porcine adrenocortical cells in vitro during 1 h incubations. With pregnenolone as a substrate the nitrofurans inhibited aldosterone production/release. Although the nitrofurans differed in potency (nitrofurantoin > furazolidone > furaltadone) maximum inhibition occurred at 100 microM. In this concentration the nitrofurans changed also the release/production of other corticosteroids. The output of corticosterone and cortisol decreased by 50%. The production/release of deoxycortisol stayed the same. In contrast the output of progesterone and 17alpha-hydroxyprogesterone increased to more than 200% of control. The nitrofurans slightly reduced the output of androstenedione. No significant increases of the production/release of other steroids (testosterone, dehydroepiandrosterone, estradiol-17beta and estrone) by the cell suspension could be observed. The profile of the nitrofuran-induced changes lead to the conclusion that nitrofurans interfere with mitochondrial enzymes. These enzymes, presumably cytochrome P450(11,18) mediate the hydroxylation and the oxidation at C11 and C18, the final steps in the biogenesis of aldosterone, corticosterone and cortisol. The rapid and reversible fall in the output of these steroids occurs in vitro at concentrations which are below therapeutic blood concentrations seen in vivo. At higher concentrations the nitrofurans hinder the biogenesis of androgens. Thus nitrofurans can also affect steps in the steroid biogenesis located in the endoplasmatic reticulum.

Effects of atipamezole, detomidine and medetomidine on release of steroid hormones by porcine adrenocortical cells in vitro

The 4-substituted imidazole type alpha2-adrenoceptor ligands atipamezole, detomidine, and medetomidine were screened for actions on the release of aldosterone by a suspension of porcine adrenocortical cells with deoxycorticosterone (1 microM) as substrate. Progesterone, pregnenolone or corticosterone (all at 1 microM) were also used as substrates. With pregnenolone as substrate, drug-induced effects on the output of nine steroids (aldosterone, corticosterone, cortisol, deoxycortisol, testosterone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone) were monitored simultaneously. The alpha2-adrenoceptor antagonist atipamezole was a potent inhibitor of aldosterone release (range 10-1000 nM). The sedative alpha2-adrenoceptor agonists medetomidine and detomidine also inhibited aldosterone release (range 10-1000 nM). With pregnenolone as substrate, the inhibition induced by 4-substituted imidazoles of the release of corticosterone and cortisol was more pronounced than that of aldosterone. Androstenedione and deoxycortisol release was enhanced. The 4-substituted imidazoles atipamezole, detomidine, and medetomidine inhibited mitochondrial cytochrome P450(11beta/18) in vitro. This inhibition was unrelated to their alpha2-adrenoceptor actions. The 4-substituted imidazole type alpha2-adrenoceptor ligands used to control sedation/anaesthesia can alter the steroid-based defence mechanisms of the body.

Effects of α1-antagonists on production and release of aldosterone and other steroid hormones by porcine adrenocortical cells in vitro

Quinazoline type alpha1-adrenoceptor antagonists (range 10-100 microM) inhibited aldosterone release of a cell suspension of porcine adrenocortical cells, potency order: doxazosin > prazosin > trimazosin. Phenoxybenzamine also inhibited the aldosterone release at a concentration of 100 microM. Alpha1-adrenoceptor antagonists from other chemical classes had no measurable effect on the aldosterone output from adrenocortical cells in vitro. Agonists selective for either alpha1- or beta-adrenoceptors did not affect the aldosterone release. The inhibition of the aldosterone release induced by quinazolines was similar with different substrates. The small differences between the drug-induced inhibitions could be ranked as corticosterone = progesterone > pregnenolone = deoxycorticosterone. The doxazosin (10 microM)-induced changes in the release of nine steroids indicated that quinazoline-type alpha1-antagonists interfere with enzymes of the aldosterone biogenesis pathway involved in C18-oxidation and C21beta-hydroxylation, reducing the release of both aldosterone and corticosterone. At higher concentrations (100 microM), the C21beta-hydroxylation in the cortisol biogenesis pathway is also affected, decreasing the output of cortisol and deoxycortisol, but increasing the output of progesterone and OH-progesterone. Simultaneously, the C17-oxidation and side-chain cleavage is also inhibited, decreasing the output of androstenedione. The rank order of phenoxybenzamine (100 microM)-induced inhibition of the aldosterone release with different substrates is pregnenolone > corticosterone = progesterone > deoxycorticosterone. With pregnenolone as substrate, the output of aldosterone, corticosterone, and cortisol was reduced to the same extent. The dehydroepiandrosterone, androstenedione, and progesterone release was enhanced. It seems that phenoxybenzamine is a rather selective inhibitor of the mitochondrial P450(11beta/18) enzymes.

Comparative Toxicity of Three Quinoxaline-di-N-dioxide Feed Additives in Young Pigs

The quinoxaline-di-N-dioxide derived feed additives carbadox, olaquindox, and cyadox are widely used as growth promoters in modern pig husbandry (Aumaitre and Raynaud 1978; Bronsch et al. 1976; Herzig et al. 1984). Carbadox and olaquindox have also been demonstrated to protect against several enteric pathogens such as Treponema hyodysenteriae, an anaerobic spirochete involved in the pathogenesis of swine dysentery (Blobel and Schliesser 1979). In earlier studies from this institute it was demonstrated that doses of carbadox in feed of less than twice the recommended one of 50 ppm induced toxic side-effects, causing specific adrenal lesions (Van der Molen et al. 1985), and it was suggested that carbadox interacts with the biosynthesis of aldosterone (Van der Molen et al. 1986a, b).

Residues of Carbadox Metabolites in Edible Pork Products

Carbadox (methyl-3-[2-quinoxalinylmethylene]carbazate-N1, N4-dioxide; CBX) is a widely used feed additive for young pigs (Trasher et al 1969), but because of its adrenal toxicity there is some risk in using it (Van der Molen et al 1985, 1986). CBX is rapidly metabolized; known biotransformation products involve the three N-O-reduced compounds (N1-, N4-, and N1,N4-desoxycarbadox; DCBX), and quinoxaline-2-carboxylic acid (QCA). QCA has been identified as the major residual metabolite in pigs (Federal Register 1972; Ferrando et al 1975). Since CBX and DCBX are suspected carcinogens, CBX may only be administered to swine under the age of 4 months, and a withdrawal period of 4 weeks has been imposed.