Dopamine inhibits prostaglandin F2α-induced depolarization of rabbit jejunal arteries via activation of DA1-receptors

Summary In rabbit jejunal arteries, the membrane potential of single smooth muscle cells decreased on the application of noradrenaline 3 mol/1. LY 171555 1 mol/1 did not change, whereas SKF 38393 10 mol/1 reversed the effect of noradrenaline. When prostaglandin F₂ (PGF₂) was used to evoke depolarization in the presence of prazosin 0.1 mol/1, rauwolscine 1 mol/1 and propranolol 1 mol/1, both SKF 38393 10 mol/1 and dopamine 10 mol/1 repolarized the membrane. SCH 23390 1 mol/1 antagonized the effects of SKF 38393 10 mol/1 and dopamine 10 mol/1. Thus, the change in membrane potential is mediated by a DA₁-recep-tor.     

Presynaptic dopamine DA2-receptors in rabbit jejunal arteries

Summary Excitatory junction potentials (e.j.ps) evoked by nerve stimulation with 15 pulses at 1 Hz were recorded from muscle cells of rabbit isolated jejunal arteries. LY 171555 1 mol/l, SKF 38393 10 mol/l, dopamine 10 ol/l and clonidine 0.1 mol/l depressed all e j.ps in the train. The percentage inhibition was inversely related to the number of pulses. S- and R-sulpiride, 10 mol/l, domperidone 1 mol/l, SCH 23390 1 mol/l and rauwolscine 1 mol/l did not change, or even depressed the first e j.ps. Of these compounds only S- and R-sulpiride, 10 mol/l and rauwolscine 1 mol/l facilitated the late e.j.ps. The percentage facilitation increased with the number of pulses until a maximum was reached; rauwolscine 1 ol/l had the largest effect. S- and R-sulpiride, 10 mol/l, as well as domperidone 1 ol/l antagonized the action of LY 171555 1 mol/l. S-Sulpiride was more potent than its R-isomer. SCH 23390 1 mol/l and rauwolscine 1 mol/l blunted the effect of SKF 38393 10 mol/l. Rauwolscine 1 mol/l slightly reduced the inhibition by dopamine 10 mol/l; S-sulpiride 10 mol/l was antagonistic only in the presence of rauwolscine 1 mol/l. When rauwolscine 1 mol/l, prazosin 0.1 mol/l, propranolol 1 mol/l and cocaine 10 mol/l was added to the medium, dopamine 10 mol/l continued to produce the same depression of e j.ps, as in the absence of these compounds. Under such conditions S-sulpiride 10 mol/l also counteracted dopamine 10 gmol/l. Rauwolscine 1 mol/l prevented the effect of clonidine 0.1 mol/l. The antagonists were not absolutely selective against only one type of agonist. We suggest that both presynaptic DA₂- and postsynaptic DA₁-receptors are present in rabbit jejunal arteries. The activation of either receptor-type may depress the e j.ps. Dopamine interferes with neuroeffector transmission due to ₂-adrenoceptor agonist properties; its DA₂-effect is unmasked only after ₂-adrenoceptor blockade. There was no evidence for a co-transmitter function of dopamine. Send offprint requests to P. Illes at the above address     

Effect of phorbol esters and polymyxin B on modulation of noradrenaline release in mouse atria

Summary Phorbol 12-myristate 13-acetate (PMA; 0.03, 0.1 and 1.0 mol/l), a protein kinase C activating phorbol ester, significantly enhanced the stimulation-induced (S-I) outflow of radioactivity at 5 Hz stimulation in mouse atria preincubated with [³H]-noradrenaline, whereas a phorbol ester which does not activate protein kinase C, phorbol 13-acetate (0.1 mol/l), had no effect. This suggests that protein kinase C may have a role in modulating sympathetic neurotransmission.Polymyxin B (7 and 21 mol/l), an inhibitor of protein kinase C, had no effect on the S-I outflow of radioactivity. However, it had a significant inhibitory effect in a concentration of 70 mol/l. Polymyxin B (21 mol/l) reduced the facilitation of the S-I outflow of radioactivity produced by PMA (0.03 mol/l), 8-bromo-cyclic AMP (90 mol/l), tetraethylammonium chloride (300 mol/l), and idazoxan (0.1 mol/l). Furthermore, when a higher frequency of stimulation was applied (10 Hz rather than 5 Hz), polymyxin B (21 pmol/1) by itself inhibited the S-I outflow of radioactivity.In the presence of a concentration of PMA (0.1 mol/l) that was maximally effective in enhancing the S-I outflow of radioactivity, both idazoxan (0.1 mol/l) and 8-bromocyclic AMP (90 mol/l) still enhanced the S-I outflow. This suggests that these agents are not operating through protein kinase C and further suggests that the inhibitory effect of polymyxin B on these agents cannot be due to inhibition of protein kinase C. The effects of clonidine on the S-I outflow were not affected by a maximally effective concentration of PMA (0.1 mol/l). These results suggest that protein kinase C is not involved in a ₂-adrenoceptor mediated modulation of noradrenaline release. Send offprint requests to I. F. Musgrave at the above address     

Effects of the novel dopamine DA2-receptor agonist carmoxirole (EMD 45609) on noradrenergic and purinergic neurotransmission in rat isolated kidney

Summary The effects of the classical dopamine DA₂-receptor agonist quinpirole (LY 171555) and the recently characterized DA₂-receptor agonist, carmoxirole (EMD 45609), on neurotransmission in rat isolated kidney were investigated. After preincubation with ³H-noradrenaline, the renal nerves were electrically stimulated. The stimulation induced (S-I) outflow of radioactivity was taken as an index of noradrenaline release. Quinpirole (0.3 mol/l) inhibited S-I outflow of radioactivity and pressor responses to renal nerve stimulation (RNS) at 1 Hz. Both effects of quinpirole were blocked by the DA₂-receptor antagonist S(–)-sulpiride (10 mol/l). The ₁, ₂-adrenoceptor antagonist phentolamine (1 mol/l) did not block the inhibitory effect of quinpirole. Carmoxirole (0.003 and 0.03 mol/l) did not alter and carmoxirole (0.3 mol/l) even enhanced S-I outflow of radioactivity, however, pressor responses to RNS were markedly reduced by carmoxirole (0.003–0.3 mol/l). Pressor responses to RNS were also markedly reduced by the ₁-adrenoceptor antagonist prazosin (0.1 mol/l). Carmoxirole (0.3 mol/l), prazosin (0.1 mol/l) and phentolamine (1 mol/l) totally abolished pressor responses to exogenous noradrenaline (0.05 mol/l). In contrast, quinpirole (0.3 mol/l) did not alter pressor responses to exogenous noradrenaline (0.05 mol/l). Furthermore, carmoxirole (0.003–0.3 mol/l) markedly reduced pressor responses induced by the ₁-adrenoceptor agonist methoxamine (1 mol/l) but even the highest concentration of carmoxirole (0.3 mol/l) had no effect on pressor responses induced by bolus injections of either neuropeptide Y (1.5 ng) or angiotensin II (1 ng). Phentolamine (1 mol/l) by itself markedly enhanced S-1 outflow of radioactivity and pressor responses to RNS were virtually unchanged. In the presence of phentolamine carmoxirole (0.03 and 0.3 mol/l) and quinpirole inhibited S-I outflow of radioactivity and pressor responses to RNS. Phentolamine resistant pressor responses to RNS were also inhibited by the P_2X-receptor desensitizing agent , -methylene adenosine triphosphate (mATP, 1 mol/l), which by itself in the presence of phentolamine did not alter S-I outflow of radioactivity. The inhibitory effects of carmoxirole (0.3 mol/l) in the presence of phentolame (1mol/l) were antagonized by S(–)-sulpiride (10 mol/l). The data suggest that activation of prejunctional DA₂-receptors by quinpirole inhibits noradrenaline release and thereby reduces pressor response to RNS at 1 Hz in rat isolated kidney. Carmoxirole activates prejunctional inhibitory DA₂-receptors, but this effect is masked by simultaneous blockade of inhibitory prejunctional -adrenoceptors. Pressor responses to RNS at 1 Hz in rat isolated kidney are largely due to neuronally released noradrenaline whereas phentolamine resistant pressor responses to RNS at 1 Hz are most likely due to ATP, which is co-released with noradrenaline. Carmoxirole inhibits pressor responses to RNS at 1 Hz as well as pressor responses induced by either exogenous noradrenaline or methoxamine by blocking postjunctional ₁-adrenoceptors. In addition carmoxirole and quinpirole seem to block phentolamine resistant pressor responses by inhibiting ATP release through activation of prejunctional DA₂-receptors. Send offprint requests to L. C. Rump at the above address     

Response of rat small intestinal active aldohexose transport to elevation of mucosal cyclic AMP by forskolin and 3-isobutyl-1-methylxanthine in vitro

Summary The phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX) was able to elevate rat small intestinal cyclic AMP levels to 300% of basal values. Active jejunal d-glucose transport was enhanced parallel to the rise of intracellular cyclic AMP levels to 140% of control values at 100 mol/l IBMX. Transport parameters, as determined in a three compartment model in vitro using a dual label method, indicate increased uphill glucose transport at the site of the brush border membrane, higher intracellular accumulation of the sugar, with unchanged passive permeabilities. Phlorizin-inhibited d-glucose transport and l-glucose transfer in the rat were not affected by the persisting cyclic AMP elevation produced by IBMX. Stimulating effects could also be demonstrated with d-galactose as a substrate. IBMX 100 mol/l also increased active d-glucose as well as 3-O-methylglucose transport in mouse jejunum.Stimulatory effects on intestinal hexose transport and mucosal cyclic AMP levels were also found with the adenylate-cyclase activator forskolin. In the present study, forskolin effects on jejunal mucosal cyclic AMP levels were enhanced in the presence of 100 mol/l IBMX, resulting in a 20-fold increase compared to controls at 20 mol/l forskolin. The concentration response for the effect of forskolin in the presence of 100 mol/l IBMX on d-glucose transport did not produce a significant increase compared to transport stimulation with IBMX alone. At higher concentrations of forskolin however, glucose transport decreased to levels well below the IBMX controls.The elevation of cellular cyclic AMP levels had no effects on passive permeability.Both IBMX 100 mol/l as well as forskolin 20 mol/l inhibited rat jejunal net fluid transport by 40%, combination of both agents resulted in a 55% reduction of net fluid absorption in everted sacs of rat jejunum.These results indicate a functional relationship between jejunal mucosal cyclic AMP levels and active hexose absorption different from the inhibitory role of cyclic AMP in intestinal fluid transport.A preliminary account of this work has been given at the 26th Spring Meeting (March 1985) of the Deutsche Pharmakologische Gesellschaft in Mainz, FRG     

Mechanisms of the contractile effects of flosequinoxan

In guinea-pig papillary muscles the positive inotropic effect of flosequinoxan (BTS) starting at 100 mol/1 amounted to 287.6 ± 34.2% at 300 mol/l without any effects on time to peak tension (103.9 ± 2%) and relaxation time (107.1 ± 6.7% of predrug value, respectively). 10 mol/l carbachol attenuated the positive inotropic effect of 300 mol/l to 166.5 ± 11.6% (n = 10). The phosphorylation state of the inhibitory subunit of troponin (TnI) and phospholamban(PLB) in [³²P]-labeled guinea-pig ventricular myocytes was increased starting at 100 mol/l amounting to 142.5 ± 12.6% and 130.9 ± 2.2% at 300 mol/l, respectively (n = 5). Furthermore, BTS (300 mol/l) decreased phosphorylase phosphatase activity by 23.1%. It is concluded that the contractile effects of BTS are accompanied by enhanced phosphorylation of regulatory proteins which could in part be due to inhibition of phosphorylase phosphatase activity.     

Phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex

Summary Possible antagonist effects of phentolamine at presynaptic serotonin autoreceptors were studied in slices of the occipito-parietal cortices of the rabbit and the rat. The slices were preincubated with ³H-serotonin and then superfused and stimulated electrically with single pulses or pulse trains. Nitroquipazine 1 mol/l, a compound that inhibits the high affinity neuronal uptake of serotonin, was present in the superfusion medium in all one pulse-experiments as well as in experiments in which the effect of unlabelled serotonin was examined.In rabbit cortical slices, unlabelled serotonin reduced the single pulse-evoked overflow of tritium. Its concentrationresponse curve was not changed by the selective ₂-adrenoceptor antagonist idazoxan 1 mol/l but was shifted to the right by phentolamine 1 and 10 mol/l. Phentolamine 10 mol/l also shifted to the right the concentration-inhibition curve of the selective 5-HT₁-receptor agonist 5-carboxamidotryptamine. When the slices were stimulated by trains of 30 pulses at 3 Hz, phentolamine 1 and 10 mol/l but not 0.1 mol/l increased the evoked overflow of tritium, the maximal increase amounting to 178%; its effect was enhanced in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l (a drug combination that, when given alone, slightly increased the evoked overflow of tritium). The serotonin receptor antagonist metitepin at concentrations of 0.01–1 mol/l also increased the overflow of tritium elicited by 30 pulses/3 Hz, the maximal increase amounting to 280%; its effect was potentiated in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l but was abolished or almost abolished in the presence of nitroquipazine 1 mol/l plus phentolamine 10 mol/l (a drug combination that, given alone, greatly increased the evoked overflow of tritium). When slices were stimulated by trains of 360 pulses at 3 Hz, there was no apparent antagonism of phentolamine 10 mol/l against the inhibitory effect of unlabelled serotonin. In rat brain cortex slices, unlabelled serotonin reduced the overflow of tritium elicited by 4 pulses delivered at 100 Hz. Again, phentolamine 10 mol/l shifted the concentration-response curve to the right.It is concluded that phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex with pA₂ values of 6.44 and 5.95, respectively. Previous failures to detect the antagonistic effect against exogenous agonists were probably due to stimulation conditions that led to marked endogenous autoinhibition of serotonin release. At least the major part of the increase by phentolamine of the release of serotonin is due to autoreceptor blockade rather than blockade of the presynaptic a2-adrenoceptors at the cortical serotoninergic axons.Send offprint requests to N. Limberger at the above address     

Blockade of α2-adrenoceptors increases opioid μ-receptor-mediated inhibition of the firing rate of rat locus coeruleus neurones

Summary In pontine slices of the rat brain, the frequency of spontaneous action potentials of locus coeruleus (LC) neurones was recorded extracellularly. Noradrenaline 0.1–100 mol/l, UK 14,304 0.01–100 nmol/l, [Met⁵]-enkephalin 1–10,000 nmol/l and [D-Ala², D-Leu⁵]enkephalin 0.1–1,000 nmol/l, all depressed the firing rate. Rauwolscine 1 mol/l antagonized the effects of both noradrenaline and UK 14,304, but potentiated the effects of [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin. Idazoxan 1 mol/l acted in a similar manner. Prazosin 1 mol/l did not change the effects of either noradrenaline or [Met⁵]enkephalin. Naloxone 0.1 mol/l antagonized both [Met']enkephalin and [D-Ala², D-Leu⁵]enkephalin, but failed to alter the effects of either noradrenaline or UK 14,304. Rauwolscine, idazoxan and prazosin, all 1 mol/l, as well as naloxone 0.1 mol/l, did not influence the firing rate when given alone. Desipramine 1 mol/l inhibited the discharge of action potentials in a rauwolscine-antagonizable manner. Noradrenaline 10 mol/l produced the same depression of firing, both in the presence of noradrenaline 1 mol/l and [Met⁵]enkephalin 0.03 mol/l. Likewise, the effect of [Met⁵]enkephalin 0.3 mol/l was the same, irrespective of whether it was added to a medium containing [Met⁵]enkephalin 0.03 mol/l or noradrenaline 1 mol/l. The spontaneous activity of LC neurones is inhibited by somatic ₂-adrenoceptors and opioid -receptors. We suggest that the two receptors interact with each other at a site located between themselves and not in the subsequent common signal transduction system.Send offprint requests to: P. Illes at the above address     

Inhibition of striatal tyrosine hydroxylase by low concentrations of apomorphine

Summary The inhibitory effect of apomorphine on tyrosine hydroxylase (TH) was tested using enzyme preparations from rat striatum, neuroblastoma clone N1E-115 and pheochromocytoma clone PC-12. When the striatal enzyme preparation was incubated at pH 7.2 with (6R,S)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) as cofactor (100–1,000 mol/l), the IC₅₀ for apomorphine was found to be in the 0.1–1 mol/l range depending on the BH₄-concentration used. Changing the incubation medium to pH 6.0 yielded an IC₅₀ of about 2.5 mol/l (BH₄=100 mol/l) Apomorphine was even less effective when 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydroteridine (100 mol/l) was used as cofactor (IC₅₀10 mol/l). Similar results were obtained with the enzyme preparations of the two cell clones.These experiments show that, even in low concentrations, apomorphine inhibits TH directly, provided more physiological test conditions are used. The relevance of these results for the autoreceptor-mediated mechanism of the apomorphine action on catecholamine synthesis is discussed.Abbreviations used BH₄ (6R,S)-l-erythro-5,6,7,8-tetrahydrobiopterin - DMPH₄ 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine - DOPA 3,4-dihydroxyphenylalanine - ELCD electrochemical detection - HPLC high performance liquid chromatography - NEAA non essential amino acid concentrate - NSD 1015 3-hydroxy-benzyl-hydrazine - TH tyrosine hydroxylase     

The depolarizing action of 5-hydroxytryptamine on rabbit isolated preganglionic cervical sympathetic nerves

Summary This study describes a depolarizing action of 5-hydroxytryptamine (5-HT) on rabbit isolated preganglionic cervical sympathetic nerves using an extracellular recording technique. From cumulative concentration-response curves for 5-HT (1 mol/1-1 mmol/1), the mean maximal depolarization was shown to be 277 ± 32 V and EC₅₀ was 9.4 mol/l(6.5–13.6 mol/l, geometric mean, 95% confidence limits, n = 42). The responses to 5-HT displayed marked tachyphylaxis. When cumulative concentration-response curves to 5-HT and 2-methyl-5-HT were determined in the same preparations (n = 4), the mean maximal response to 5-HT was 519 ± 167 V, EC₅₀ 32.2 mol/l (8.8–118 mol/l) and the mean maximal response to 2-methyl-5-HT was 317 ± 63 V, EC₅₀ 35.1 mol/l (12.9–95.5 mol/l, geometric means, 95 % confidence limits). The action of selective 5-HT antagonists was tested on repeated cumulative concentration-response curves to 5-HT. Neither methiothepin (0.1–1 mol/l, _n = 3) nor ketanserin (0.1–1 mol/l, n = 3) had an action on 5-HT responses. The selective 5-HT₃ antagonists MDL 72222, ICS 205-930 and SDZ 206–830 were all potent antagonists of the 5-HT depolarizations. The action of these antagonists was quantified by determining the apparent pA₂ from the dose ratios and a Schild plot. For MDL 72222 (1 nmol/1-0.1 mol/l), the apparent pA₂ was 9.1 ± 0.1 (n = 12), Schild plot: 9.2; for ICS 205–930 (0.1 nmol/l–3 nmol/1), the apparent pA₂ was 10.4 ± 0.1 (n = 11), Schild plot 10.3, and for SDZ 206–830 (0.03 nmol/l-1 nmol/1), the apparent pA₂ was 11.2 ± 0.1 (n = 12), Schild plot 11.2. 5-HT depolarizations were unaffected by hexamethonium (0.5 mmol/1). 5-HT depolarizations were reduced by superfusion with both Na-free (42 ± 8% of controls, n = 4) and Na/Ca-free media (35 ± 7% of controls, n = 4). It is concluded that 5-HT depolarizations of rabbit preganglionic cervical sympathetic nerve are mediated by 5-HT₃ receptors. The data with selective 5-HT₃ receptor antagonists suggest that the receptor profile may be more like that for the 5-HT₃ receptor on the terminals of sympathetic nerves than that for the 5-HT₃ receptor on the soma of superior cervical ganglion cells or on vagal afferent neurones. Send offprint requests to D. I. Wallis at the above address     

Developmental Responses of a Terrestrial Insect Detritivore, <Emphasis Type="BoldItalic">Megaselia scalaris</Emphasis> (Loew) to Four Selenium Species

Megaselia scalaris (Loew) (Diptera: Phoridae) is an important and ubiquitous terrestrial detritivore that consumes both animal and plant material. Because both plants and animals convert selenium pollutants into various forms, the relative toxicities of ecologically relevant concentrations of sodium selenate, sodium selenite, seleno-L-methionine, and Se-(methyl) selenocysteine hydrochloride to larvae were assessed in diet bioassays. In addition, ovipositional preferences of adults and developmental effects on the eggs and larvae were measured. With chronic exposure selenocysteine was the most toxic of the selenium species to the larvae (LC₅₀: 83 g/g wet weight), followed by seleno-L-methionine (LC₅₀: 130 g/g), selenate (LC₅₀: 258 g/g), and selenite (LC₅₀: 392 g/g). Ovipositing females did not discriminate between the highest treatment concentrations of any of the pollutants as compared to the controls, indicating a lack of avoidance behavior. Larval development time was significantly increased with exposure to selenate at 100 g/g wet weight and above, selenite at 300 g/g and above, and at 50 g/g and 25 g/g and above for seleno-L-methionine and selenocysteine respectively. Pupal development was not affected by any of the selenium treatments. Significant differences between male and female adult eclosion times were observed, with females eclosing later than males as selenium concentrations increased. Significant decreases in larval survival relative to controls occurred at the lowest treatment tested (100 g/g) for both selenate and selenite and at 100 g/g for seleno-L-methionine, and 50 g/g for selenocysteine. The population level implications of lack of avoidance of contaminated food, and the effects of increased development times, reduced survivorship, and non-synchronized male and female emergence are discussed.     

Influence of N-allyl-normetazocine on acetylcholine release from brain slices: involvement of muscarinic receptors

Summary The effects of (±)N-allyl-normetazocine on the release of acetylcholine from different areas of guinea-pig and rat brain were investigated. 1. The drug did not modify the electrically (2 Hz) evoked tritium efflux from guinea-pig cerebral cortex, thalamus and caudate nucleus slices, preloaded with ³H-choline 0.1 mol/l and superfused with Krebs solution containing hemicholinium-3 10 mol/l. 2. (±)N-allyl-normetazocine 10 mol/l. enhanced the evoked ³H efflux from guinea-pig brain slices superfused with Krebs solution containing physostigmine 30 mol/l or oxotremorine 0.3 -1 gmol/l; the effect was naloxone-insensitive and was abolished by atropine 0.15 mol/l, but not by pirenzepine 1 mol/l. 3. (±)N-allyl-normetazocine 5 mol/l enhanced the electrically evoked release of endogenous acetylcholine as well, in a naloxone-insensitive way. 4. Both (±) and (+)N-allyl-normetazocine were without effect on ³H efflux from rat caudate nucleus slices electrically stimulated at 0.2 Hz frequency, after preloading with ³H-choline and during superfusion with hemicholinium-3. 5. The results are discussed in view of the antimuscarinic properties of the drug. Send offprint requests to A. Siniscalchi     

Intracerebroventricular injection of a TRH analogue, γ-butyrolactone-γ-carbonyl-l-histidyl-prolinamide induces gastric lesions and gastric acid stimulation in rats

Summary The effects of TRH and its biologically stable analogue, -butyrolactone--carbonyl-l-histidyl-l-prolinamide (DN-1417), on gastric mucosa and acid secretion were examined in rats. Intracerebroventricular (ICV) injection of DN-1417 (0.1–10 g) caused a dose-dependent gastric lesion in the corpus and antrum 6 h after administration. The gastric lesions produced by 1 g of DN-1417 were more severe than those produced by ICV TRH (10 g), intravenous DN-1417 (200 g) and stress. Although the lesion-generating effect of TRH (10 g) tended to be reduced 6 h after the injection, that of DN-1417 (1 g) was sustained during 6 h. Atropine (0.1 and 1 mg/kg s.c.) inhibited DN-1417-induced gastric lesions in a dose-related manner while sulpiride (10 and 30 mg/kg s.c.), haloperidol (1 mg/kg i.p.), phentolamine (1 and 5 mg/kg s.c.) and yohimbine (5 mg/kg s.c.) did not prevent the lesions. ICV DN-1417 also stimulated basal gastric acid secretion and the effect was stronger and longer-lasting than that of TRH. Atropine (0.1 mg/kg s.c.) stopped DN-1417-stimulated gastric acid secretion. In conclusion, the possibility that TRH may be involved in the CNS modulation of gastric mucosal integrity deserves further attention. The enhanced potency of action of DN-1417 over TRH could make ICV injection of this peptide a useful tool for inducing centrally-mediated gastric lesions in rats.     

The site of action of muscle relaxant purine nucleosides

Summary The effects of a series of purine nucleosides, including the novel marine natural product 1-methylisoguanosine, have been examined on muscle relaxation in conscious animals and on spinal reflexes and neuromuscular transmission in mice anaesthetized with sodium pentobarbitone. 1-Methylisoguanosine (5–15 mol kg^–1) and 2-chloroadenosine (1–5 mol kg^–1), both of which cause muscle relaxation in conscious animals, depressed both mono- and polysynaptic spinal reflexes but did not affect neuromuscular transmission. At much higher doses (300 mol kg^–1) both compounds did depress neuromuscular transmission. Adenosine and 1-methyladenosine did not produce muscle relaxation in conscious animals and only slightly depressed polysynaptic reflexes at the highest doses tested (300 mol kg^–1). Theophylline 50 mol kg^–1 enhanced polysynaptic reflexes and antagonized the depression of these reflexes by 1-methylisoguanosine. Neither adenosine nor 1-methylisoguanosine affected the development of tension by isolated diaphragm muscles in vitro. It is concluded that the muscle relaxant purine nucleosides 2-chloroadenosine and 1-methylisoguanosine produce their effects primarily by depressing activity in the central nervous system. Transmission at the neuromuscular junction is not affected at doses in the range of those producing muscle relaxation.     

Role of dopamine receptors in the modulation of acetylcholine release in the rabbit hippocampus

Summary Modulation of acetylcholine release was studied in slices of the rabbit hippocampus preincubated with ³H-choline and then continuously superfused with a medium containing 10 mol/l hemicholinium-3. Electrical field stimulation of the superfused slices elicited an increase in tritium outflow, which was tetrodotoxin-sensitive and largely calcium-dependent. Stimulus-evoked acetylcholine release in the rabbit hippocampal slices was modulated by presynaptic muscarinic autoreceptors, as has been shown previously for the rat hippocampus. Drugs with affinity for - and or -adrenoceptors did not affect the evoked overflow of tritium from rabbit hippocampal slices. In contrast, the dopamine receptor agonist apomorphine (0.1 or 1 mol/l) and exogenous dopamine (1 or 10 mol/l) significantly reduced the evoked outflow by about 10 or 20%, respectively. This effect was antagonized by haloperidol (0.01 mol/l) but not by phentolamine (1 mol/l). Attempts to enhance (using nomifensine 10 mol/l) or reduce (using haloperidol, up to 1 mol/l; or bretylium, 1 mmol/l for 5 min) endogenous dopaminergic transmission in the hippocampal slices did not affect stimulation evoked acetylcholine release. In conclusion, presynaptic dopamine receptors modulating acetylcholine release are present in the rabbit hippocampus, but they seem not to be of physiological significance.     

Dopaminergic modulation of hippocampal noradrenaline release

Summary ³H-Noradrenaline release in the rabbit hippocampus and its possible modulation via presynaptic dopamine receptors was studied. Hippocampal slices were preincubated with ³H-noradrenaline, continuously superfused in the presence of cocaine (30 mol/l) and subjected to electrical field stimulation. The electrically evoked tritium over-flow from the slices was reduced by 0.1 and 1 mol/l dopamine and apomorphine, but significantly enhanced by 10 mol/l apomorphine or by 0.1 and 1 mol/l bromocriptine. If the ₂-adrenoceptor antagonist yohimbine (0.1 mol/l) was present throughout superfusion, the inhibitory effects of dopamine and apomorphine were more pronounced and even 10 mol/l apomorphine and 1 mol/l bromocriptine inhibited noradrenaline release. Qualitatively similar observations were made in the presence of another ₂-antagonist, idazoxane (0.1 mol/l). In the presence of the D2-receptor antagonist domperidone (0.1 mol/l) the inhibitory effects of dopamine were almost abolished, whereas both apomorphine (>1 mol/l) and bromocriptine (>0.01 mol/l) greatly facilitated noradrenaline release. The D2-receptor agonist LY 171555 (0.1 and 1 mol/l) significantly reduced the evoked noradrenaline release whereas the D1-selective agonist SK & F 38393 was ineffective at similar concentrations. The effects of LY 171555 were abolished in the presence of domperidone (0.1 mol/l) but remained unchanged in the presence of yohimbine or idazoxane (0.1 mol/l, each).At 1 mol/l the D2-receptor antagonists domperidone and (-)sulpiride significantly increased the evoked noradrenaline release by about 10%. However, at this concentration, domperidone (but not (-)sulpiride) affected also basal tritium outflow. Bulbocapnine and the preferential D1-receptor antagonists SCH 23390 enhanced the evoked noradrenaline release already at 0.1 mol/l. Their marked facilitatory effects (50 to 60% increase at 1 mol/l) were reduced in the presence of idazoxane (0.1 mol/l) and almost abolished in the presence of 0.1 mol/l yohimbine, whereas the increase due to 1 mol/l (-)sulpiride persisted under these conditions.The evoked tritium efflux from rabbit hippocampal slices preincubated with ³H-serotonin was not affected by dopamine receptor agonists.From our results we conclude that hippocampal noradrenaline, but not serotonin release, is modulated via D2-dopamine receptors. In addition, our results provide evidence for more or less pronounced ₂-adrenoceptor agonistic properties of dopamine and ₂-adrenoceptor antagonistic properties of apomorphine, bromocriptine, SCH 23390 and bulbocapnine in this noradrenaline release model from CNS tissue.     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

5-HT-induced neurogenic relaxations of the guinea-pig proximal colon: investigation into the role of ATP and VIP in addition to nitric oxide

In the guinea-pig proximal colon, 5-hydroxytryptamine (5-HT) relaxes the longitudinal muscle by stimulating neuronal 5-HT receptors, which induces the release of nitric oxide (NO). It was investigated whether the inhibitory neurotransmitters adenosine 5-triphosphate (ATP) and/or vasoactive intestinal polypeptide (VIP) could be involved as well.Antagonists to block the contractile response to 5-HT via 5-HT₂, 5-HT₃ or 5-HT₄ receptors were present throughout the experiments and methacholine was administered to precontract the strips. ATP, VIP and 5-HT induced concentration-dependent relaxations, in the case of 5-HT yielding a non-monophasic concentration-response curve. Tetrodotoxin (TTX; 300 nM), N^G-nitro-l-arginine (l-NNA, 100 M) and their combination did not inhibit the relaxations induced by VIP (up to 0.3 M) or 0.3–3 M ATP but reduced those by 10 M ATP. Suramin (300 M) strongly inhibited the relaxations to ATP and VIP. l-NNA and suramin also inhibited the relaxations to 5-HT. In the presence of l-NNA (100 M), suramin did not significantly inhibit the relaxations to 5-HT. Suramin did not affect the relaxations to isoprenaline, nitroglycerin or exogenous NO (1 M), demonstrating its specificity. Apamin (30 nM) inhibited both the relaxations to ATP (by 70–100%) and to 5-HT; relaxations to isoprenaline were partially inhibited, indicating a non-specific component in the inhibitory action of apamin. However, relaxations to exogenous VIP (up to 0.3 M), NO (1 ,M) and to nitroglycerin were not inhibit ed. In the presence of l-NNA (100 M), apamin inhibited the relaxations to 5-HT only at 30 M. ,\-methylene-ATP (,-Me-ATP; 100 M) did not desensitize the responses to ATP. Reactive blue 2 affected the relaxations to isoprenaline at concentrations necessary to significantly inhibit the relaxations to ATP (i.e. from 10 M onwards). Thus, it was not possible to test either ,-Me-ATP or reactive blue 2 against the relaxations to 5-HT. -Chymotrypsin (0.015 mg·ml^–1) and trypsin (0.005 mg·ml^–1) almost abolished the relaxations to VIP, but did not affect those to isoprenaline and 5-HT. The VIP receptor antagonists [p-Cl-d-Phe⁶, Leu¹⁷]VIP (1 M) and VIP_10–28 (1 and 3 M) did not affect the concentration-response curve to VIP and were hence not tested against 5-HT. Phosphoramidon (1 M) had no effect on the relaxations to VIP or 5-HT.It can be concluded that in the guinea-pig colon longitudinal muscle, VIP and ATP induce relaxation via a direct effect on the smooth muscle, not involving NO. 5-HT-induced relaxations are mediated by NO as well as by a substance which is sensitive to inhibition by suramin but not apamin. It is suggested that this substance is ATP and not a peptide.     

Effects of (+)- and (±)-sotalol on repolarizing outward currents and pacemaker current in sheep cardiac Purkinje fibres

Summary This study was aimed to differentiate the action of (+)- and (±)-sotalol (10–1000 mol/l) on membrane currents which are active during the repolarization of cardiac action potentials Effects where studied in shortened sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique Action potentials were activated at a frequency of 0.25 Hz and membrane currents at 0.03 Hz or 0.05 Hz in most experiments.Out of the currents investigated the transient outward current (i_to) reacted most sensitively to (+)- and (±)-sotalol. I_to-amplitude was decreased on the average to 77% of reference at 10 mol/l and to 53% at 1000 mol/l (+)- or (±)-sotalol. The maximally available ito-current was decreased but the voltage-dependent control of inactivation was left nearly unchanged. The initial inwardly rectifying current (i_Ki), which propels the last repolarization phase of the action potential and controls resting potential to a large extent was reduced on the average to 93% of reference at 10 mol/l and to 62% at 1000 mol/l (+)- or (±)-sotalol. Time-dependent (delayed) outward current (i_K) was on the average not affected by (+)- or (±)-sotalol up to 100 mol/l and was decreased to 84% of reference current under the influence of 1000 mol/l. An initial outward current, which is activated at positive membrane potentials (i_inst) was not clearly affected by (+)- or (±)-sotalol at concentrations up to 1000 mol/l Pacemaker current (i_f) was not influenced by the drugs up to 100 mol/l. Only at 1000 mol/l was the amount of available i_f-current decreased to 79% of reference. (The potential-dependent control of activation was not affected) Time constants of time-dependent currents i_to, i_K and i_f did not change in concentrations up to 1000 mol/l of the drug.Action potential duration increased at (+)- or (±)-sotalol concentrations 10 mol/l and maximal prolongation was achieved at concentrations of 100–300 mol/l Resting potential remained nearly unchanged at these concentrations, but the membranes depolarized at 1000 mol/l. According to our data action potential prolongation in sheep Purkinje fibres under the influence of (+)- and (±)-sotalol correlates to the drug-induced block to i_to-current and inwardly rectifying i_K1-current.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

The involvement of central cholinergic system in the pressor effect of intracerebroventricularly injected U-46619, a thromboxane A2 analog, in conscious normotensive rats

The aim of this study was to determine the involvement of the central cholinergic system in the rise in blood pressure evoked by the thromboxane A2 (TxA2) analog, U-46619, given centrally. Intracerebroventricular (i.c.v.) injections of U-46619 (0.5, 1.0 and 2.0 g) caused dose- and time-related increases in blood pressure and decreased heart rate in awake rats. U-46619 (1 g; i.c.v.) also produced an approximately 65% increase in posterior hypothalamic extracellular acetylcholine and choline levels. Pretreatment with SQ-29548 (8 g; i.c.v.), selective TxA2 receptor antagonist, completely inhibited both the cardiovascular responses and the increase in acetylcholine and choline levels to subsequent injection of U-46619 (1 g; i.c.v.). Atropine (10 g; i.c.v.), nonselective muscarinic receptor antagonist, pretreatment did not affect the cardiovascular responses observed after U-46619 (1 g; i.c.v.). Pretreatment with the nonselective nicotinic receptor antagonist, mecamylamine (50 g; i.c.v.) attenuated the pressor effect of U-46619 (1 g; i.c.v.). Higher doses of mecamylamine (75 and 100 g; i.c.v.) pretreatments did not change the magnitude of the blockade of pressor response to U-46619; however, they abolished the bradycardic effect of U-46619 dose-dependently. Interestingly, pretreatment of rats with methyllycaconitine (10 g; i.c.v.) or -bungarotoxin (10 g; i.c.v.), selective antagonists of 7 subtype of nicotinic acetylcholine receptors (7nAChRs), partially abolished the pressor response to i.c.v. injection of U-46619 (1 g). Similar to the mecamylamine data, the use of higher doses of methyllycaconitine (25 and 50 g; i.c.v.) produced the same magnitude of blockade that was observed after the 10 g methyllycaconitine pretreatment, but it completely abolished the bradycardic effect of U-46619 (1 g; i.c.v.) at the dose of 25 g. The present results show that central administration of U-46619 produces pressor and bradycardic effect and increase in hypothalamic acetylcholine and choline levels by activating central TxA2 receptors. The activation of central nicotinic receptors, predominantly 7nAChRs, partially mediates the cardiovascular responses to i.c.v. injection of U-46619.