Antagonism by γ-aminobutyric acid of the stimulant effect of angiotensin II on cardiac sympathetic ganglia in spinal dogs

Summary The effects of angiotensin II and neuro-aminoacids administered through the right subclavian artery (i. a.) to the cardiac sympathetic ganglia were investigated in spinal dogs. Angiotensin II (1–8 g) elicited a dose-dependent positive chronotropic effect which was reduced after i. a. injection of saralasin (100g). The effect of angiotensin II was not reduced after combined treatment with either hexamethonium (10 mg/kg) plus atropine (0.1 mg/kg) or hemicholinium-3 (5 mg/kg) plus preganglionic stimulation. The dosedependent response to angiotensin II of heart rate was inhibited by GABA (50, 500g), GABOB (500g) and muscimol (50, 100g). The inhibition of the response to angiotensin II by a small dose of GABA (50g), but not by a high one (500g), was antagonized by i. a. injection of picrotoxin (2 mg). The positive chronotropism induced by bethanechol (25, 50g) and a small dose of acetylcholine (25g) were significantly inhibited by a high dose (500g) but not by a low dose (50g) of GABA. These results confirm that angiotensin II stimulates cardiac chronotropism by acting on the angiotensin II receptor located at the cardiac ganglia and show that this stimulant effect is antagonized by GABA.     

In Vitro Cutaneous Disposition of a Topical Diclofenac Lotion in Human Skin: Effect of a Multi-Dose Regimen

Purpose. This study determines comparative bioavailability of diclofenac sodium lotion compared to an aqueous solution after topical application to viable human skin in vitro. In addition, the difference between a single dose and multiple doses (8 times) was also determined. Methods. An in vitro flow-through diffusion cell system was employed, using radiolabelled diclofenac sodium. Results. Multiple doses of lotion (2 l/cm² and 5 l/cm²) delivered a total of 40.1 ± 17.6 g and 85.6 ± 41.4 g diclofenac, respectively, at 48 h, compared to only 9.4 ± 2.9 g and 35.7 ± 19.0 g absorbed after topical application of diclofenac as an aqueous solution (P < 0.05). A single dose study showed no statistical difference between diclofenac delivered in lotion or an aqueous solution. Over 48 h the total absorption from lotion was 10.2 ± 6.7 g and 26.2 ± 17.6 g (2 l/cm² and 5 l/cm², respectively), compared to 8.3 ± 1.5 g and 12.5 ± 5.7 g from an aqueous solution. Both single doses of lotion and aqueous diclofenac showed decreased diclofenac absorption into the receptor fluid between 12 and 24 h. However, when applied multiple times, absorption from lotion was continually increasing up to 48 h. The total dose accountability ranged from 76.8 ± 8.2% to 110.6 ± 15.1% of the applied dose. Conclusions. Diclofenac lotion exhibited enhanced diclofenac percutaneous absorption rate through human skin (mass, flux and partition coefficient) when applied a multiple number of times and this enhanced absorption was maintained over 48 h. This suggests that a constituent of the lotion (DMSO) will enhance human skin absorption of diclofenac when used in a multi-dose regimen, but not after a single dose.     

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.     

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.     

Noradrenaline release from rat sympathetic neurons evoked by P2-purinoceptor activation

The effects of ATP and analogues on the release of previously incorporated ³H-noradrenaline were studied in cultured sympathetic neurons derived from superior cervical ganglia of neonatal rats. Electrical field stimulation (40 mA at 3 Hz) of the neurons for 10 s markedly enhanced the outflow of tritium. ATP applied for 5 s to 2 min at concentrations of 0.01 to 1 mmol/l caused a time- and concentration-dependent overflow with half maximal effects at about 10 s and 100 mol/l, respectively. 2-Methylthio-ATP was equipotent to ATP in inducing ³H-overflow. ADP (100 mol/l), when applied for 2 min, also caused a small ³H-overflow, but , -methylene-ATP (100 mol/l), AMP (100 mol/l), R(–)N⁶-(2-phenylsiopropyl)-adenosine (R(–)-PIA; 10 mol/l) and 5-N-ethylcarboxamidoadenosine (NECA; 1 mol/l) did not. The ³H-overflow induced by 10 s applications of 100 mol/l ATP was abolished by suramin (100 mol/l) and reduced by about 70% by reactive blue 2 (3 mol/l). Electrically evoked overflow, in contrast, was slightly enhanced by suramin, but not modified by reactive blue 2. Xanthine amine congener (10 mol/l) and hexamethonium (10 mol/l) did not alter ATP-evoked release. Removal of extracellular Ca²⁺ from the medium reduced ATP- and electrically induced overflow by about 95%. Tetrodotoxin (1 mol/l) abolished electrically evoked ³H-overflow but inhibited ATP-induced overflow by only 70%. The ₂-adrenoceptor agonist UK 14,304 at a concentration of 1 mol/l diminished both electrically and ATP-evoked tritium overflow by approximately 70%. These results indicate that activation of P₂-purinoceptors stimulates noradrenaline release from rat sympathetic neurons. The release resembles electrically induced transmitter release, but additional mechanisms may contribute. Correspondence to: S. Boehm at the above address     

A comparison of the effects of TMB-8 and nifedipine on pressor responses to α1- and α2-adrenoceptor agonists in pithed rats

TMB-8 has been characterized as an inhibitor of the release of Ca⁺ from intracellular pools. We have studied the modification of the pressor responses to selective _l-adrenoceptor agonists (methoxamine and phenylephrine), and to selective ₂-adrenoceptor agonists (B-HT 920 and B-HT 933) in pithed rats, produced by TMB-8. We have compared this modification with that produced by the calcium antagonist nifedipine. Nifedipine (100 g/kg, 300 g/kg, and 1000 g/kg) inhibited in a dose-dependent manner the pressor responses to the ₁- and ₂-adrenoceptor agonists, the dose-response curves to the ₂-adrenoceptor agonists being shifted further to the right. TMB-8 at a dose of 3000 g/kg did not modify the pressor effects of the _l-adrenoceptor agonists, and neither did it reinforce the inhibition of such responses produced by nifedipine. By contrast, TMB-8 pretreatment (0.03 g/kg, 0.3 g/kg, 3 g/kg, 30 g/kg, 300 g/kg and 3000 g/kg) inhibited the responses to both ₂-adrenoceptor agonists, the inhibition being more pronounced with B-HT 920. A similar effect was obtained with 0.03 g/kg TMB-8 and 0.3 g/kg TMB-8, particularly in the case of B-HT 920. It was stronger with higher doses, but similar for all doses over 3 g/kg. The inhibition of the pressor responses mediated by the stimulation of ₂-adrenoceptors by TMB-8 was less in rats treated with the Ca²⁺ entry promoter BAY K 8644 (300 g/kg), and could also be reduced by the continuous infusion of CaCl₂ (0.25 g/min). These results suggest that in pithed rats TMB-8 may also behave as an inhibitor of the Ca⁺ influx into vascular smooth muscle.     

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.     

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.     

Dose-response study of oxitropium bromide inhaled as a nebulised solution

Twelve patients suffering from partially reversible chronic obstructive pulmonary disease (COPD) took past in a single blind, randomised, 4-way cross-over trial to determine the optimal dose and duration of action of the anticholinergic agent oxitropium bromide (OTB) inhaled as a nebulised solution.Single doses of 500, 1000, 1500 and 2000 g nebulised OTB were compared during a 6 hour-observation period. Lung function test results indicated that 500 and 100 g OTB only induced slight bronchodilatation, whereas 1500 and 2000 g OTB produced a significantly greater increase in mean FEV1 compared to 500 g. There was a trend for 2000 g to be superior to 1000 g, but 2000 g and 1500 g were not significantly different. Significant bronchodilatation (>15% rise in FEV1 from baseline) persisted for 6 h after 1500 g. A significant decrease in airway resistance (Raw) was observed following inhalation of 2000 g. The mean decrease in Raw was 33% after 30 min, 20% after 4 h and 12% after 6 h.In this trial, 2000 g OTB administered by an ultrasonic nebuliser was the optimal dose, but a satisfactory result was also obtained with 1500 g.     

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.     

Differential effects of the selective D2-antagonist raclopride in the nucleus accumbens of the rat on spontaneous and d-amphetamine-induced activity

The effect of the D2-antagonist raclopride was investigated in two test situations, which are presumed to involve dopamine (DA) transmission within the nucleus accumbens of the rat. Local injection of d-amphetamine sulphate (10 g/0.5 l) produced a marked increase in motor activity, measured as motility, locomotion, and rearing, which was dose- and time-dependently antagonised by local injection of raclopride (0.05–5.0 g/0.5 l). After an initial decrease, at low doses (0.05–0.25 g/0.5 l) an apparent enhancement of the d-amphetamine-induced motor activity appeared, which was most clearly seen with rearing. These lower doses, however, did not induce any clear changes in the exploratory activity in a novel environment (i.e., the second test situation). Only the higher doses used (1.0–5.0 g/0.5 l) decreased exploratory activity during the first 5–10 min, also measured as motility, locomotion, and rearing. These data are discussed with respect to the role of D2-receptors within the nucleus accumbens of rats in the motor activity induced by a novel environment and d-amphetamine. Overall, the data underline previous notions that raclopride is a potent antagonist of DA-mediated behaviour.     

The role of brain norepinephrine in clonidine suppression of isolation-induced distress in the domestic chick

Distress vocalizations (DV) induced by social isolation were measured in 1-day-old domestic chicks after intracerebroventricular injections of drugs believed to exert their effects through the noradrenergic system. The -adreno-receptor agonist clonidine reliably suppressed DV rates at doses low as 0.08 g. When given alone, phentolamine, phenoxybenzamine, propranolol, sotalol, and yohimbine (adrenoreceptor antagonists) did not reliably after DV rates at doses that were not toxic. The clonidine DV suppression was reliably reversed by yohimbine (1.75 g), but by none of the other adrenoceptor antagonists or naloxone. 6-Hydroxydopamine at doses as high as 120 g, which essentially eliminated forebrain norepinephrine, failed to suppress DV rates reliably when given alone and, when given in combination with clonidine (0.1 g) or morphine (0.05 g), failed to reverse the severe DV suppression imposed by these drugs. The results suggest that clonidine suppresses DV rates in chicks through interaction with postsynaptic adrenoreceptors or by some means other than prejunctional ₂-adrenoreceptor stimulation.     

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.     

Distinct distribution of opioid receptor types in rat lumbar spinal cord

Summary The distribution of opiate binding sites was studied in sections of rat lumbar spinal cord under conditions selective for , and receptors. While the levels of binding sites were highest in the substantia gelatinosa, elevated levels were also observed in laminae III, IV, V and VIII. In contrast, binding was notable only in lamina I. The levels of typical sites were low, and were concentrated in the substantia gelatinosa. An additional, atypical site was detected using ³H-diprenorphine in the presence of , and receptor blocking agents, and this site was also concentrated in the substantia gelatinosa. Send offprint requests to B. J. Morris at the above address     

Pharmacokinetics and safety of ILX23-7553, a non-calcemic-vitamin D3 analogue, in a phase I study of patients with advanced malignancies

Purpose: Differentiation therapy is an alternative to chemotherapy with potentially less toxicity, improved quality of life, and survival. We conducted a phase I trial of ILX23-7553, a formulation of 1,25-dihydroxy-16-ene-23-yne-vitamin D₃, a 1,25-dihydroxyvitamin D₃ analog with preclinically demonstrated antitumor and differentiating effects and diminished hypercalcemic effects. Patients and methods: The protocol consisted of five daily oral treatments during 14-day cycles at 15 dose levels from 1.3 to 45.0g/m²/day. We treated 42 heavily pretreated patients who had a variety of malignancies with 162 treatment cycles, and obtained pharmacokinetics from three patients at the two highest dose levels. Results: There were no grade 3 or 4 toxicities. Grade 1–2 toxicities included diarrhea, nausea, fatigue, constipation, and one grade 1 hypercalcemia. Average day 6 calcium was 9.26 ± 0.55mg/dl in cycle 1 and 9.30 ± 0.67mg/dl in cycle 2. Pharmacokinetics at dose levels 14 (40g/m²/day) (1 patient) and 15 (45g/m²/day) (2 patients) demonstrated an average C _max of 30.4 ± 7.8pg/ml (0.07nM) and 104 ± 38.2pg/ml (0.25nM), and AUCs of 222.5 ± 225.2pg·h/ml and 855 ± 536pgh/ml, respectively. Eight patients (19%) had stable disease. While in vitro effects have been reported at these concentrations, they were at least 10-fold lower than ED₅₀s, and the study was terminated before an MTD was reached. Conclusion: The drug is safe and has potential benefits at serum concentrations where effects begin to be noted in vitro. Further study is needed with a reformulated higher unit dose compound to determine the safety and efficacy of higher serum concentrations.     

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     

Abolition by calcium antagonists of the autoregulation of renal blood flow

Summary Intra-arterial infusions of Ca antagonistic vasodilator substances such as nifedipine (1–10 g/min) and verapamil (30–100 g/min) inhibited renal autoregulation in the perfused dog kidney. Simultaneous infusion of 30 mg/min of CaCl₂ with nifedipine (3 g/min) or with verapamil (30 g/min) abolished the effects of these Ca antagonists. It is suggested that Ca ions play an important role in establishing renal autoregulation.     

Release of ATP in rat vas deferens: origin and role of calcium

Release of endogenous ATP elicited by electrical (neural) stimulation and exogenous agonists was studied in the rat isolated vas deferens. The aims were to dissect neural and postjunctional contributions to the nerve activity-evoked overflow of ATP and to clarify the role of transmitter receptors and calcium in postjunctional ATP release.In tissues preincubated with [³H]-noradrenaline, electrical stimulation (100 pulses/10 Hz) elicited contraction and an overflow of tritium and ATP. Contractions as well as ATP overflow were reduced by prazosin 0.3 M and even more so by prazosin 0.3 M combined with suramin 300 M. They were also reduced by nifedipine 10 M and even more so by nifedipine 10 M combined with ryanodine 20 M (the additional effect of ryanodine on ATP overflow was not significant). In tissues not pretreated with [³H]-noradrenaline, exogenous noradrenaline 10 M and ,-methylene ATP 10 M elicited contraction and an overflow of ATP. Responses to noradrenaline were blocked by prazosin 0.3 M but not suramin 300 M and were greatly reduced by nifedipine 10 M and in Ca²⁺-free medium. Responses to ,-methylene ATP were blocked by suramin 300 M but not prazosin 0.3 M were reduced by nifedipine 10 M (effect on ATP overflow not significant) and were reduced even more in Ca²⁺-free medium. Neuropeptide Y 0.3 M caused only very small contraction and ATP overflow. The electrically as well as the agonist-evoked ATP overflow correlated well with the contraction responses except in experiments with suramin which retarded the removal, by vas deferens tissue, of ATP from the medium.Itsis concluded that the overflow of ATP from rat vas deferens elicited by electrical (neural) stimulation is at least 90% postjunctional, presumably smooth muscle, in origin. ATP is released from postjunctional cells as a consequence of both ₁-adrenoceptor and P₂-purinoceptor activation. Ca²⁺ is a second messenger in the postjunctional ATP release response; its major part enters through L-type channels. A purely neural overflow of ATP was not isolated under the conditions of the experiments. Correspondence to: R. Bültmann at the above address     

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     

Behavioural and anticonvulsant effects of Ca2+ channel toxins in DBA/2 mice

This study investigated the behavioural and anticonvulsant effects of voltage-sensitive calcium channel blockers in DBA/2 mice.-Conotoxin MVIIC (0.1, 0.3 g ICV/mouse) and-agatoxin IVA (0.1, 0.3, 1 g ICV), which act predominantly at P- and/or Q-type calcium channels, prevented clonic and tonic sound-induced seizures in this animal model of reflex epilepsy (ED₅₀ values with 95% confidence limits for protection against clonic sound-induced seizures were 0.09 (0.04–0.36) g ICV and 0.09 (0.05–0.15) g ICV, respectively and against tonic seizures 0.07 (0.03–0.16) g ICV and 0.08 (0.04–0.13) g ICV, respectively). The N-type calcium channel antagonists-conotoxin GVIA and-conotoxin MVIIA were also tested in this model.-Conotoxin GVIA was anticonvulsant in DBA/2 mice, but only at high doses (3 g ICV prevented tonic seizures in 60% of the animals; 10 g ICV prevented clonic seizures in 60% and tonic seizures in 90% of the animals), whereas-conotoxin MVIIA did not inhibit sound-induced seizures in doses up to 10 g ICV. Both-conotoxin GVIA and-conotoxin MVIIA induced an intense shaking syndrome in doses as low as 0.1 g ICV, whereas-conotoxin MVIIC and-agatoxin IVA did not produce shaking at any of the doses examined. Finally,-conotoxin GI (0.01–1 g ICV) and-conotoxin SI (0.3–30 g ICV), which both act at acetylcholine nicotinic receptors, were not anticonvulsant and did not induce shaking in DBA/2 mice. These results confirm that blockers of N- and P-/Q-type calcium channels produce different behavioural responses in animals. The anticonvulsant effects of-conotoxin MVIIC and-agatoxin IVA in DBA/2 mice are consistent with reports that P- and/or Q-type calcium channel blockers inhibit the release of excitatory amino acids and are worthy of further exploration.