, which resulted in a K i value for MB of 0.58+0.02 mmol l 71. 6 In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant anity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L-arginine-NO-pathway, in particular when muscarinic receptor stimulation is involved.

1 This study was designed to investigate the mechanism(s) of the negative inotropic eects of a 1 -adrenoceptor agonists observed in rat isolated left atria after exposure to free radicals. 2 Ouabain and calphostin C were used in contraction experiments to block the sodium pump and protein kinase C. Methoxamine-induced phospholipase C and Na + /K + ATPase activities were measured. 3 Methoxamine (300 mM) increased contractile force by 1.6+0.2 mN in control atria but decreased contractile force in electrolysis-treated atria by 2.0+0.1 mN (P50.05), as determined 10 min after methoxamine addition. In contrast, the positive inotropic eects of endothelin-1 (30 nM) and isoprenaline (10 mM) were reduced from 2.6+0.3 to 1.3+0.1 mN and from 2.6+0.3 to 1.7+0.2 mN, respectively, by electrolysis treatment (P50.05), but not converted into a negative inotropic action. 4 In an inositol phosphate assay we observed that the stimulation of phospholipase C by methoxamine was attenuated by electrolysis when the (electrolyzed) medium from the organ bath was used, but the phospholipase C responses were restored by the use of fresh medium. However, fresh medium did not counteract the negative inotropic eect of methoxamine. Accordingly, the negative inotropic eect of methoxamine is not directly related to the impaired phospholipase C responses seen in atria subjected to electrolysis. 5 Ouabain (10 mM) and the protein kinase C inhibitor calphostin C (50 nM), completely prevented the negative inotropic eect of 300 mM methoxamine in electrolysis-treated atria. 6 Measurement of the Na + /K + ATPase activity, revealed that in control atria, a 1 -adrenoceptor stimulation with 300 mM methoxamine, decreased the Na + /K + ATPase activity by 14.4+7.7%. In contrast, methoxamine increased the Na + /K + ATPase activity by 48.8+8.9% (P50.05) in electrolysistreated atria. Interestingly, this increase in Na + /K + ATPase activity was completely counteracted by calphostin C (1.4+0.1% over basal). 7 These results indicate that the negative inotropic eects of a 1 -adrenoceptor agonists, observed in rat isolated left atria exposed to free radicals, are likely to be caused by protein kinase C-mediated phosphorylation and subsequent activation of the Na + /K + ATPase.

We investigated the influence of hyper- and hypothyroidism on basal parameters of isolated perfused hearts of rats. In addition the effects of different extracellular calcium concentrations ([Ca2+]o), the calcium entry promoter Bay K8644 and the alpha 1-adrenoceptor agonist methoxamine were investigated. Since alterations in alpha-adrenoceptor density could explain the increased sensitivity to methoxamine in hearts from hypothyroid rats, alpha 1-adrenoceptor density in the left ventricle was also established. Different time-schedules of exposure to hyper- and hypothyroidism were used to investigate whether the influence of chronic dysthyroid states on alpha 1-adrenoceptor density is transient and time-dependent. Simultaneously myocardial noradrenaline and adrenaline tissue concentrations were determined, since they might correlate with the observed changes. Hyperthyroidism was induced by feeding rats for 1, 4 and 8 weeks with 5 mg/kg L-thyroxine (T4)-containing rat chow. Hypothyroid rats were obtained by adding 0.05% propylthiouracil (PTU) to the drinking water during 1, 4 and 8 weeks. For the functional experiments animals were treated during 4 weeks, to mimic the clinical situation of a chronic endocrine disease. Langendorff hearts from hyperthyroid hearts showed an increased maximally developed relaxation velocity, whereas Langendorff hearts from hypothyroid rats showed an increased left ventricular pressure (LVP). We observed an increased maximal inotropic response to [Ca2+]o in hearts from both hyperthyroid and hypothyroid rats, indicating that both dysthyroid states interfere with the handling of calcium ions by the contractile apparatus. Unchanged responses to Bay K8644 in hearts from hyperthyroid and depressed responses in hearts from hypothyroid rats suggest that the involvement of L-type calcium channels is rather unlikely. Furthermore, the reflex increase in coronary flow in response to enhanced contractile force appeared to fail in hearts from hypothyroid rats. Sensitivity of the response to methoxamine was increased in hearts from hypothyroid rats, which was accompanied by a decrease in the number of myocardial alpha 1-adrenoceptors. Both T4 and PTU treatment resulted in a non-transient decrease of alpha 1-adrenoceptor density in left ventricular tissue. Furthermore, hypothyroidism increased the percentage of alpha 1A-binding sites, whereas in hyperthyroidism the distribution of the alpha 1-adrenoceptor subtypes was not affected. Myocardial tissue concentrations of noradrenaline and adrenaline were unchanged in hyperthyroid rats and decreased in hypothyroid rats. The present study indicates that thyroid hormones have a direct rather than a sympathetically mediated effect on alpha 1-adrenoceptor mediated myocardial functions.