Reversibility of the acute toxic effect of Cyclosporin A on pancreatic B cells of Wistar rats

Summary. This study investigated if and to what extent the acute toxic effect of Cyclosporin A on pancreatic Wistar rat B cells is reversible. After 2 weeks of treatment rats developed marked glucose intolerance accompanied by reduced pancreatic insulin content due to a loss of B cells, diminished islet DNA synthesis and decreased B-cell insulin content. Cyclosporin A had accumulated in the pancreas. Three weeks after withdrawal of Cyclosporin A, pancreatic tissue concentrations of Cyclosporin A were still 100 times larger than in serum. Glucose tolerance, however, had already improved, associated with an increase of B-cell insulin content and apparent islet replication, and the insulin response of isolated islets was reduced. Five weeks after the withdrawal of Cyclosporin A, glucose tolerance was normal, but pancreatic insulin content and relative B-cell volume were still diminished in comparison to vehicle-treated controls. Eight weeks after withdrawal, the morphometric parameters had also been normalized. The results suggest that the loss of pancreatic B cells is caused by a toxic destruction, possibly combined with an apparent decrease of replicatory activity. The acute toxic effects of Cyclosporin A in pancreatic B cells are stepwise reversible.     

Insulin receptors in lizard brain and liver: structural and functional studies of α and β subunits demonstrate evolutionary conservation

Summary Specific insulin receptors are present in the liver and brain of the lizard Anolis carolinesis. In this study, the specific binding of ¹²⁵I-insulin to the receptors showed time, temperature and pH dependency. Specific binding to crude membranes prepared from brain was 1–2% of the total radioactivity added compared to 4–5% in the crude membranes prepared from liver. Solubilization and wheat germ agglutinin purification of the membranes resulted in an increase in the specific binding (per mg of protein) between 6 and 32 times for liver membranes and 13–186 for brain membranes. Binding inhibition of tracer insulin by unlabeled porcine insulin was characteristic for insulin receptors with 50% inhibition for liver crude membranes at 60 ng/ml of porcine insulin and 0.7 ng/ml for purified brain insulin receptors. Chicken insulin was 2- to 3-fold more potent and proinsulin about 100 times less potent than porcine insulin. The -subunits of liver and brain had apparent molecular weights on sodium dodecyl sulfate polyacrylamide gel electrophoresis of 135 kDa and 120 kDa respectively. Apparent molecular weights of subunits were 92 kDa for both tissues. Insulin stimulated phosphorylation of the subunit of both brain and liver receptors. Both tissues demonstrated tyrosine-specific phosphorylation, which was stimulated by insulin, of exogenously added artificial substrates. In addition, purified brain insulin receptor preparations contained an endogenous protein with apparent molecular weight of 105 kDa, whose phosphorylation was stimulated by insulin (10^–7 mol/l). This phosphoprotein was not immunoprecipitated by anti-insulin receptor antibodies. These studies suggest that the structural differences between brain and liver receptors previously demonstrated in the rat are also present in the lizard, which is about 300,000,000 years older than the mammalian species. Thus, there is strong evolutionary conservation of the brain insulin receptor.     

Modifying effects of disulfiram on DNA adduct formation and persistence of benzaldehyde in N-nitroso-N-methyl-benzylamine-induced carcinogenesis in rats

Summary Whereas disulfiram (DSF) is known to inhibit tumor formation resulting from a number of chemical carcinogens, such inhibition does not apply to nitrosamines. In the present study, biochemical and morphological findings were examined to elucidate the effect of DSF on long-term application of N-nitroso-N-methylbenzylamine (NMBA). HPLC and fluorescence detection were used to determine O⁶-methylguanine (O⁶-MG) in DNA obtained from the respiratory tract of rats subjected to long-term simultaneous application of DSF and NMBA. After 2 days of treatment, more O⁶-MG was detected in the proximal portion of the respiratory tract, including the trachea and main bronchi, than in the distal portion. The findings were reversed after 10 and 30 days, at which time formation of the DNA adduct was substantially higher in the distal portion of the respiratory tract, despite increases in both portions. The biochemical results corresponded to morphological findings. Initially, mereased numbers of metabolizing goblet cells appeared in mucous cell hyperplasia in the proximal respiratory tract. Subsequently, the hyperplasia migrated to distal regions of the respiratory tract; at this stage, the goblet cells disappeared from the proximal portion, which now revealed toxic degeneration, atrophy and subsequent squamous metaplasia of the mucous lining and squamous papillomas.At various times during a 40-day period, 2 to 7 times more O⁶-MG in pulmonary DNA was detected in rats treated with DSF and NMBA, than with NMBA alone, whereby distinct amounts of O⁶-MG were found in the latter animals. In contrast to the above-mentioned morphological findings, no morphological alterations occurred in the respiratory tract of the animals treated with NMBA alone. It is therefore conceivable that the above pathological lesions resulted not merely from the presence of DNA adducts, but also from an additional, previously unspecified effect.As benzaldehyde (BA) is formed in equimolar amounts in NMBA metabolism and DSF has been demonstrated to inhibit aldehyde metabolism, this aldehyde is a possible candidate for such an effect. In the present study, rats were therefore treated with BA, DSF, or NMBA, or combinations thereof. Histomorphological evaluation of these experiments revealed that long-term application of BA alone led to the following alterations in the respiratory tract: goblet cell hyperplasia, hyperplasia of the peribronchial lymphatic system, mucous epithelial atrophy and accompanying peerivasculitis — the same alterations seen under long-term application of NMBA and DSF. Furthermore, these changes were most pronounced in the group with concomitant application of NMBA, DSF, and BA. It is therefore conceivable that BA plays a role in pathological changes observed under the influence of NMBA.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986Supported by Breuninger Stiftung, D-7000 Stuttgart; AWG-Analysen-Geräte GmbH, D-7970 Leutkirch, FRG; Asta-Werke AG, D-6000 Frankfurt am Main. FRG; Cyanamid GmbH, D-8190 Wolfratshausen, FRG, Farmitalia Carlo Erba GmbH, D-7800 Freiburg i Br., FRG     

Callosal projections from area SII to SI in monkeys: anatomical organization and comparison with association projections

The present research was aimed at ascertaining in the macaque monkey the reciprocity of the heterotopical callosal connections between SI and SII, with particular regard to the connectivity of the hand representation, and at comparing the topographical and laminar pattern of these callosal connections with those of association connections entertained by these areas. Horseradish peroxidase (HRP) was unilaterally injected into area SI in five monkeys. The sites of HRP delivery included the trunk and the hand zones preliminarily identified by recording multi-unit responses to peripheral stimulation by means of microelectrodes. Anterograde and retrograde labelling was studied in SII of both sides. The results showed the complete reciprocity of the heterotopical callosal connections between SI and SII. In the latter area both callosal axon terminals and neurones were found, which were labelled from either the trunk or the hand zone of contralateral SI. Labelling of callosal axon terminals occurred mainly in layer IV and in the lowermost part of layer III. Labelled callosal neurones were mainly in the lower half of layer III, whereas few occurred in infragranular layers. Topographically, the distribution of callosal terminals and cell bodies duplicated the distribution of association terminals and cell bodies labelled in SII on the side ipsilateral to HRP injection. The laminar pattern of termination of association fibres from SI was similar to that of callosal fibres. However, the distribution of association-projecting neurones in SII showed a striking difference from that of callosal-projecting neurones. Unlike the latter neurones, which were mainly located in supragranular layers, association cell bodies overwhelmingly dwelt in layers V and VI and were less numerous in layers II and III. This laminar pattern of association SII-SI cells corresponds to the "feed-backward" model and fits the laminar pattern of their axon terminations (Friedman: Brain Res. 273: 147-151, '83). The association and callosal inputs and outputs of area SII are discussed in relation to the function of the forward and backward type of reciprocal connections entertained with SI in the ipsilateral hemisphere and to the function of SII in the interhemispheric exchange of somatosensory information.     

Body weight set point studies in weanling rats with dorsomedial hypothalamic lesions (DMNL rats)

In order to further characterize a previously postulated "organismic" set point, weanling DMNL and control (CON) rats were maintained on lab chow ad lib (AL) for 55 post-operative days. Subsequently, some DMNL and CON rats were food-restricted (REST) to 80% of the food intake of their AL-fed counterparts for 24 days. At this point, representative rats from each group were killed by decapitation and the remaining animals were re-fed AL and killed 7 and 22 days thereafter. At the end of REST, both DMNL and CON showed significant weight loss, which was greater in CON than in DMNL rats. After 7 days of refeeding, DMNL rats normalized their body weights but re-fed CON still weighed less than AL-fed CON 22 days after refeeding. Food intake in formerly REST groups overshot on refeeding for 7 days, but this was significant only in DMNL rats. Notably, during this time formerly REST-DMNL ate as much as AL-fed CON. Efficiency of food utilization was normal in DMNL during AL feeding and became reduced on REST as it did in REST-CON. Notably, on refeeding formerly REST-DMNL rats overshot that of AL-fed DMNL rats by the same magnitude as previously REST-CON overshot the values of AL-fed CON. After 22 days of refeeding, this overshoot was still evident in DMNL but not in CON. At the end of the REST period, plasma insulin and glucose were similar in AL-fed DMNL and AL-fed CON. They were significantly and comparably reduced in both REST-DMNL and REST-CON compared to the AL-fed DMNL and AL-fed CON. On refeeding these changes normalized within seven days. At the end of REST, plasma free fatty acid concentrations were higher in REST-DMNL and REST-CON than in AL-fed DMNL and AL-fed CON. After seven days of refeeding they normalized only in formerly REST-CON. Plasma glycerol and total protein were normal throughout all groups, as was carcass protein. Carcass fat was equivalently reduced in both DMNL and CON at the end of REST and normalized 7 days after refeeding. AL-DMNL had the same carcass fat as AL-CON and REST-DMNL had the same carcass fat as REST-CON. In conjunction with previously reported normal anabolic hormone levels the data suggest that DMNL rats are not growth-retarded but are merely scaled down in size without compromise of their homeostatic competence. We take this as strong evidence for the existence of an "organismic" set point.     

Ouabain enhances release of acetylcholine in the heart evoked by unilateral vagal stimulation

Summary The aim of the study was to elucidate peripheral effects of ouabain on the parasympathetic innervation of the heart, effects that could contribute to the experimentally and clinically well established vagal effect of cardiac glycosides. The experiments were carried out with ouabain concentrations of 3×10^–7 and 10^–6 mol/l, which were considered therapeutic, as they increased force of contraction and did not elicit arrhythmias in incubated chicken atria.In atrial preparations of chickens and guinea-pigs the negative chronotropic and inotropic effects of acetylcholine (ACh) were not altered by 3×10^–7 mol/l ouabain. Resting efflux of ACh from perfused chicken hearts was increased by ouabain from 10 to a maximum of 30 pmol/g min, whereas release of ACh evoked by bilateral vagal stimulation at 3 or 20 Hz for 1 min was unchanged (resting release subtracted). In contrast, release of ACh caused by unilateral vagal stimulation was augmented by ouabain up to 200% of the control. Release by unilateral stimulation (80 pmol/g; 20 Hz) was calculated for each experiment by averaging the releases evoked by consecutive stimulation of the right and left nerves. Ouabain infused for 90 min did not alter the tissue content of ACh (5.5 nmol/g).Within 2 days after unilateral (left) vagal transsection (denervation of cardiac ganglia) the release of ACh evoked by stimulation of the intact nerve (20 Hz) increased from about 80 to 200 pmol/g, whereas the release from the lesioned nerve markedly declined. One day after denervation, ouabain had lost the ability to facilitate the release of ACh evoked by stimulation of the intact nerve, whereas the release by stimulation of the lesioned nerve was still increased.It is concluded that ouabain at therapeutic concentrations increased resting release of ACh but did not influence the mechanism of action potential-evoked release of ACh. The effect of exogenous ACh on sinus node activity was not enhanced by ouabain. The observation that ouabain increased release of ACh caused by unilateral, but not by bilateral vagal stimulation was explained by an increase in the number of activated postganglionic neurons arising from those (contralateral) ganglia that received a subthreshold input from the stimulated vagus nerve.Supported by the Deutsche ForschungsgemeinschaftSome of the results are part of the M.D. theses of M. Feinauer and B. Ullrich     

Spinal facilitation in cholinergic-sympathetic efferents by desipramine

Summary Electrodermal potentials (EDPs) evoked by electrical stimulation of the cholinergic-sympathetic system at different levels were recorded in the forepaws of anaesthetized cats and used as a measure of sudomotor activity. After pretreatment with yohimbine (0.25 mg/kg i.v.) to block ₂-adrenoceptors, unilateral electrical stimulation of the hypothalamus (square wave pulses 1 ms duration, 16 Hz, 2 s train length at intervals of 1 min) induced EDPs in both forepaws. Injection of the inhibitor of neuronal catecholamine reuptake, desipramine (1 mg/kg i.v.), facilitated the EDPs in both forepaws, even though access of the drug to the sweat glands was prevented by application of a tourniquet to one paw. The facilitation was abolished by injection of the specific ₁-adrenoceptor antagonist, prazosin (0.5 mg/kg i.v.). An equal enhancement of this effect by desipramine (1 mg/kg i.v.) and its abolition by prazosin (0.5 mg/kg i.v.) was obtained in cats with the brain stem transected at the level of the medulla oblongata and electrical stimulation of the spinal cord at C1. EDPs evoked in the right forepaw by preganglionic electrical stimulation of the right stellate ganglion were inhibited by desipramine (1 mg/kg i.v.).From these and previous results it is concluded that inhibition of neuronal reuptake of catecholamines results in facilitation of activity in sudomotor efferents. This effect is mediated by spinal ₁-adrenoceptors and provides an explanation of the occasional occurrence of excessive sweating in psychiatric patients treated with tricyclic antidepressants.     

n-Hexane urine elimination and weighted exposure concentration

Summary The concentration of n-hexane in urine was determined in 30 subjects occupationally exposed to n-hexane (median value 59.6 mg/m³) in a shoe factory. The measurement of the substance was performed by means of a Hewlett-Packard 5880 gas chromatograph supplied with a Hewlett-Packard 5970 Mass Selective Detector. The analyses were performed by the head space method (constant volume method, after determination of the urine partition coefficient by the multiple phase equilibration method). The authors found a significant correlation between the n-hexane urine concentrations (g/1, Cu) and the n-hexane environmental concentrations (mg/m³, Ci) (r = 0.84; Cu = 0.0669 x Ci + 0.8396).This work was supported by the research funds given to Fondazione Clinica del Lavoro, Pavia by the Health Council     

Brainstem afferents to the omnipause region in the cat: a horseradish peroxidase study

"Omnipause" neurons (OPNs), located in the nucleus raphe pontis and the reticular formation, actively suppress saccadic eye movements during intersaccadic intervals. To determine the brainstem afferents that may inhibit the OPNs and thereby allow a saccade to occur, we injected horseradish peroxidase into the raphe pontis of four cats at the site of physiologically identified OPNs. Labeled neurons were found in a number of brainstem nuclei. The greatest concentrations, composed of small to medium-sized neurons, were located in a group of nuclei around the habenulopeduncular tract, in the rostral mesencephalic reticular formation, in the deep layers of the superior colliculus, and in parts of the subjacent cuneiform and subcuneiform reticular nuclei. Smaller numbers were found in the nucleus reticularis pontis oralis. Caudal to the injection site, labeled neurons were scattered in parts of the nuclei reticularis gigantocellularis, paragigantocellularis dorsalis, and paragigantocellularis lateralis. A few neurons were labeled in a restricted region of the causal part of the nucleus prepositus hypoglossi and in the nucleus reticularis medullaris ventralis. Larger numbers of neurons were labeled in the dorsal column nuclei and in parts of the cochlear nuclei. Smaller numbers were found in the spinal trigeminal nucleus, the lateral nucleus of the superior olive, and the fastigial nucleus of the cerebellum. The nonreticular brainstem projections may contribute sensory information in a number of modalities since OPNs respond to visual, somesthetic, and auditory stimuli. Our findings indicate a number of regions that may contain neural elements impinging on the OPNs. The best prospects for a saccade initiation signal from one of the labeled populations appear to be the meso-diencephalic reticular formation and/or the superior colliculus.     

Preoptic area unit activity during sleep and wakefulness in the cat

The spontaneous discharge of 86 preoptic area (POA) neurons was recorded extracellularly in chronically prepared cats during wakefulness (W), slow-wave sleep (SWS), and REM sleep. Of these, the percentage of units exhibiting maximal discharge rates in SWS and REM sleep (84%) was significantly greater than that of those exhibiting a maximal discharge rate in W (16%). Furthermore, those neurons that discharged rapidly in sleep (fast units) generally had a reduced discharge rate in W. Sixteen of the 86 units showed a strong tendency to discharge in bursts during SWS but not during W or REM sleep. The mean coefficient of variation and the mean discharge rate for these bursting cells in SWS were significantly greater than the corresponding values for the same cells in W and REM sleep, and for the nonbursting cells in SWS. Because POA stimulation is known to initiate behavioral and electrocortical signs of sleep, it is suggested that "fast units" in SWS with reduced discharge rates in W, may be "hypnogenic" cells.