Preventing effects of a novel anti-parkinsonian agent zonisamide on dopamine quinone formation.

The neurotoxicity of dopamine (DA) quinones as dopaminergic neuron-specific oxidative stress is considered to play a role in the pathogenesis and/or progression of Parkinson's disease (PD), since DA quinones conjugate with several key PD pathogenic molecules (e.g., tyrosine hydroxylase, alpha-synuclein and parkin) to form protein-bound quinone (quinoprotein) and consequently inhibit their functions. Zonisamide (ZNS) is used as an anti-epileptic agent but also improved the cardinal symptoms of PD in recent clinical trials in Japan. To evaluate the effects of ZNS on excess cytosolic free DA-induced quinone toxicity, we examined changes in DA quinone-related indices after ZNS treatment both in in vitro cell-free system and in cultured cells. Co-incubation of DA and ZNS in a cell-free system caused conversion of DA to stable melanin via formation of DA-semiquinone radicals and DA chrome. Long-term (5 days) treatment with ZNS decreased quinoprotein and increased DA/DOPA chromes in dopaminergic CATH.a cells. ZNS significantly inhibited quinoprotein formation induced by treatment with tetrahydrobiopterin and ketanserin that elevate cytosolic free DA in the cells. Our results suggest that the novel anti-parkinsonian agent ZNS possesses preventing effects against DA quinone formation induced by excess amount of cytosolic DA outside the synaptic vesicles.

Identification of a 5-hydroxytryptamine (5-HT2) receptor on guinea pig small intestinal crypt cells.

Radioligand labeling of [3H]ketanserin was examined in suspensions of dispersed guinea pig small intestinal mucosal cells prepared by modification of the EDTA-chelation method described by M. M. Weiser (J. Biol. Chem. 248: 2536-2541, 1973). Preferential incorporation of [3H]thymidine was used to confirm that suspensions were enriched in crypt cells. At 25 degrees C, binding of [3H]ketanserin to dispersed enterocytes was rapid, maximal by 5 min, saturable (dissociation constant = 1.5 nM), 65 +/- 5% specific, stable, and reversible. The maximal number of binding sites per cell was 92,000 (range 86,000-105,500). Binding was temperature dependent, with maximal binding at 37 degrees C, and was inhibited by 5-hydroxytryptamine (5-HT) (half-maximal inhibition of [3H]ketanserin binding observed in response to 1 microM 5-HT) and ketanserin (half-maximal inhibition of [3H]ketanserin binding observed in response to 1 nM ketanserin) but not by the 5-HT1P antagonist N-acetyl-5-hydroxytryptophyl 5-hydroxytryptophan amide (5-HTP-DP) or the 5-HT3 antagonist 3-tropanyl-indole-3-carboxylate methiodide (ICS-205-930). The second messenger system coupled to the putative mucosal 5-HT2 receptor was examined. 5-HT stimulated a concentration-dependent production of inositol 1,4,5-trisphosphate (IP3) in the dispersed enterocytes. This was maximal at 1 min and was inhibited in a concentration-dependent manner by ketanserin. 5-HTP-DP and ICS-205-930 had no effect on 5-HT-stimulated production of IP3. These data provide evidence for the existence of a mucosal 5-HT2 receptor located on guinea pig small intestinal crypt cells.

Effects of the serotonin-antagonist ketanserin on the function of ischaemic and normally perfused myocardium and modification by beta-1-blockade in anaesthetized normotensive dogs.

The 5-HT-2 antagonist ketanserin (KAS) has been successfully used to treat acute hypertension in coronary bypass surgery. The present study was performed to investigate the effect of KAS on ischaemic myocardium. In 11 anaesthetized (piritramide) dogs, systolic contraction (sdL) and end-diastolic length (edL) of myocardium supplied by the left descending coronary artery (LAD) and the left circumflex coronary artery (LCX) were measured by sonomicrometry simultaneously with aortic pressure (AoP), left ventricular dP/dtmax and end-diastolic pressure (LVedP), heart rate (HR), stroke volume, and LAD flow (QLAD). Regional ischaemia to decrease sdLLAD (-48%) was achieved by LAD stenosis (QLAD -47%). Concomitantly, edLLAD increased by 8%. However, the other variables did not change. Then KAS was given i.v. (0.15 + 0.15 + 0.30 + 0.6 mg/kg) at 15-min intervals. Following KAS, prestenotic sdLLAD recovered in a dose-dependent manner. LVedP and edLLAD decreased, sdLLCX increased, and the other variables were not affected. This functional recovery of ischaemic myocardium was attenuated by pretreatment with metoprolol (MET, 1 mg/kg) prior to LAD stenosis. The ischaemic area was not irreversibly damaged, however, as proven by the recovery of prestenotic sdLLAD values after release of the stenosis. The improved systolic shortening of ischaemic myocardium following KAS did not result from restored QLAD due to post-stenotic vasodilation or break up of platelet aggregates (QLAD did not increase) or from reduced afterload (AoP did not decrease). Obviously, it was mediated by beta-1-receptors, as shown by the attenuation of the beneficial effect of KAS by pretreatment with MET.

Inhibition by intraventricular microinfusion of ketanserin of the cardiovascular responses to peripheral administration of methoxamine in rats.

The cardiovascular responses induced by intracerebroventricular or intravenous administration of ketanserin in normotensive rats were evaluated. Ketanserin, which is an antagonist at 5-HT2 receptors, when microinfused into the third cerebral ventricle, did not induce significant cardiovascular effects, except for a slight and transitory hypotensive response after the microinfusion of the highest dose (200 micrograms). However, at doses which were unable to affect directly blood pressure or heart rate, ketanserin, microinfused into the same site, antagonized the pressor response induced by peripheral administration of methoxamine, an alpha 1-adrenoceptor agonist. Furthermore, peripheral administration of ketanserin produced dose-dependent hypotension and bradycardia and antagonized the pressor effect of an intravenous bolus injection of methoxamine. In conclusion, the present experiments confirmed the ability of ketanserin to produce cardiovascular effects when administered peripherally and provides evidence for an involvement of ketanserin-sensitive receptors in the brain in the regulation of phasic responses during experimentally induced hypertension.

Comparison of two different arterial tissues suggests possible 5-hydroxytryptamine2 receptor heterogeneity.

A comparison of activities and affinities of several known and novel compounds in the isolated rat thoracic aorta (RA) and the rabbit femoral artery (RFA) was undertaken to evaluate these two tissues for use in screening for functional 5-hydroxytryptamine2 (5-HT2) receptor activity. Affinities for 5-HT and for ketanserin against 5-HT-elicited contractions in both vascular tissues suggested the presence of homogeneous 5-HT2 receptors with values consistent with other reported 5-HT2 receptor preparations. However, further studies showed compounds which exhibited either partial agonism in the RFA and competitive antagonism of 5-HT in the RA, or antagonism of 5-HT in both arteries with different affinities. Affinity constants calculated from the isolated vascular tissue studies were compared with affinity constants calculated for inhibition of [3H]ketanserin binding in the frontal cortices of both the rat and the rabbit. There were no significant differences between the pKi values in the rat and the rabbit cortical membranes or between these pKi's in either species and the pA2 values in the RA. Several of the affinities for both the partial agonists and the antagonists in the RFA were significantly different from the binding pKi and the pA2 values in the RA. These findings suggest identity between the [3H]ketanserin binding site in the cortices of both species and the 5-HT2 receptor in the RA; however, the contractile 5-HT receptor in the RFA, although showing some characteristics of a 5-HT2 receptor, is significantly different. We suggest that there may be functional subtypes of the vascular 5-HT receptor.

Ethanol does not affect serotonin receptor binding in rodent brain.

The effects of ethanol on serotonin (5-hydroxytryptamine, 5-HT) receptor binding in rat and mouse brain were determined under in vitro conditions and in mouse brain following seven days of ethanol ingestion. 5-HT1A receptor characteristics were measured utilizing the agonist [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([ 3H]DPAT), and 5HT2 receptor-binding studies utilized the antagonist [3H]ketanserin. At the highest concentration of ethanol tested in vitro (680 mM), there was only 25% inhibition of [3H]DPAT binding in rat and mouse brain and 14% inhibition of [3H]ketanserin binding in rat brain. Effects of an anesthetic concentration of ethanol (100 mM) on agonist binding in the presence and absence of the guanine nucleotide GTP were also evaluated in vitro in mouse brain. In no case did ethanol (100 mM) significantly affect 5-HT1A or 5-HT2 receptor-binding characteristics. When 5-HT receptor characteristics were measured after mice consumed ethanol for seven days, there was no change in either 5-HT1A or 5-HT2 receptor-binding properties in any of the brain areas examined.

The alpha 2-adrenoceptor antagonist SK&F 104078 has high affinity for 5-HT1A and 5-HT2 receptors.

The affinity of SK&F 104078, a putative selective postjunctional alpha 2-adrenoceptor antagonist, was determined at 5-HT1A and 5-HT2 receptors in rat brain. SK&F 104078 had moderate affinity towards alpha 2-adrenoceptors (pKi 6.7) but displayed higher affinity at 5-HT1A (pKi 8.1) and 5-HT2 (pKi 7.6) receptors. If SK&F 104078 is used in functional studies to define heterogeneity within of alpha 2-adrenoceptors, care must be taken to define the role of 5-HT1A and 5-HT2 receptors in the response being measured.