Pharmacokinetics and pharmacodynamics of nitroglycerin and its dinitrate metabolites in conscious dogs: Intravenous infusion studies

Intravenous infusions of nitroglycerin (GTN), 1,2-glyceryl dinitrate (1,2-GDN), and 1,3-glyceryl dinitrate (1,3-GDN) were given to four conscious dogs at 10 g/min, 30 g/min, 50 g/min, and 70 g/min of GTN and 20 g/min and 100 g/min of GDNs. The steady state plasma concentrations (Css)of GTN were reached after about 60 min whereas for 1,2-GDN and 1,3-GDN the Csswere reached at about 150 min after the infusion began. Except for one dog, the Cssof GTN were not proportional to infusion rate, however, all dogs together showed a good linear relationship between Cssof GTN and infusion rates with an average correlation coefficient of 0.917±0.102. Large variability in GTN clearance after various infusion rates was observed in all dogs. The Cssratios of 1,2-GDN/GTN and 1,3-GDN/GTN yield overall averages of 31.5 ±17.2 and 5.47 ±3.19,respectively. Average Cssratios of metabolites 1,2-GDN/1,3-GDN were 5.78±1.23. This ratio is different from those obtained after iv bolus and oral dosing indicating that the biotransformation of GTN to 1,2-GDN and 1,3-GDN differs for each dosing route. The clearances for 1,2-GDN and 1,3-GDN were not changed over the dose range of 20 g/min to 100 g/min. Terminal half-lives of 1,2-GDN and 1,3-GDN postinfusion were similar to those values obtained after a single bolus dose (45 min). It appears that all the GTN dose at steady state can be accounted for by the formation of measurable 1,2-GDN and 1,3-GDN. Large intra- and interdog variations in systolic blood pressure decrease (SPD) following infusions of GTN were observed, however, all dogs showed a clear systolic blood pressure decrease when the highest infusion rate (70 g/min) was given. No significant systolic blood pressure drop was detected following 20 g/min infusions of 1,2-GDN or 1,3-GDN. It was clear that systolic blood pressure in all dogs decreased following 100 g/min infusions of 1,2-GDN or 1,3-GDN. When SPD values were plotted vs. log GTN concentrations following the infusion of 70 g/min of GTN in all four dogs, a counterclockwise hysteresis was observed indicating the significant contribution of the active dinitrate metabolites to GTN pharmacodynamics.This work was supported in part by NIH grant HL32243.     

Tolerance to nitrates and simultaneous upregulation of platelet activity prevented by enhancing antioxidant state

We analysed the induction of tolerance to nitrates both in the vasculature (in vivo) and platelets (ex vivo). Simultaneously, we tested mechanisms underlying the induction of tolerance and interventions to prevent or overcome this phenomenon. For this purpose nitroglycerin (GTN 1.5 g/kg per min i.v.), alone or in combination with ascorbate (55 g/kg per min i.v.) as antioxidant, was infused continuously for a period of 5 days into chronically instrumented dogs. Along with haemodynamic parameters, ex vivo platelet function was continuously monitored. Following the start of GTN infusions there was a maximal coronary dilator response (245±15 m) and, as an index of venodilation, a fall of left ventricular end-diastolic pressure (by 2.3±0.4 mmHg). Both responses declined progressively and disappeared during the infusion period. However, in combination with ascorbate as antioxidant the dilator responses were maintained fully throughout the infusion period. With GTN alone there was a progressive, unexpected upregulation of platelet activity demonstrated by enhanced thrombin-stimulated intracellular Ca²⁺ levels and increases in the microviscosity of platelet membranes (indicating enhanced receptor expression) associated with a progressive impairment in basal, unstimulated cGMP levels. These changes could also be prevented completely by i.v. co-administration of ascorbate. From these results it is concluded that vascular tolerance is closely reflected by simultaneous changes in platelet function and further, that both can be prevented completely by appropriate antioxidants such as ascorbate.     

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     

Mechanisms of action of plant sterols on inhibition of cholesterol absorption

Summary The effects of two different plant sterols on intestinal cholesterol absorption were compared in normal volunteers by an intestinal perfusion study during a control period followed by high dose infusion of sitosterol or sitostanol (3.6 mol/min), to which subjects were allocated in a randomized manner. Cholesterol absorption during the control period was similar in the two groups, averaging 0.88 ± 0.48 mol/min (32 ± 11%) for group I (sitosterol) and 0.68 ± 0.33 mol/min (29 ± 9%) for group II (sitostanol). The infusion of a high dose of sitosterol resulted in a significant reduction of cholesterol absorption to 0.47 mol/min (16%). Following the same dose of sitostanol, cholesterol absorption diminished significantly to 0.15 ± 0.11 mol/min (5.1 ± 2.9%). Overall cholesterol absorption declined during sitosterol infusion by almost 50%, whereas sitostanol infusion caused a reduction of cholesterol absorption by almost 85%. These findings of a more effective inhibition of cholesterol absorption by sitostanol might confirm the observation recorded by others that an increase in hydrophobicity of a plant sterol results in a higher affinity but lower capacity to mixed micells. This may cause an effective displacement of cholesterol from micellar binding and therefore diminished cholesterol absorption.     

Metformin prevents the impairment of endothelium-dependent vascular relaxation induced by high glucose challenge in rabbit isolated perfused kidneys

High glucose concentrations are involved in the development of diabetic-associated vascular complications. We have previously reported that acute high glucose challenge, corresponding to post-prandial glycemia levels observed in patients with type 2 diabetes, blunts ACh-induced endothelium-dependent relaxation of the renal circulation of non-diabetic rabbits. Isolated perfused kidneys from non-diabetic rabbits were acutely exposed (3 h) to normal (5.5 mM—control group) or high (15 mM) D-glucose concentrations in the presence or absence of a continuous infusion of metformin (20 or 100 M). Renal vascular reactivity was evaluated with endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) vasodilating agents. ACh-induced maximal renal vasodilation was reduced by high glucose infusion (15 mM) in comparison to the control group (25±3% and 41±3% respectively; P<0.01), being restored to 41±4% and 43±2% by a simultaneous 3-h infusion of 20 or 100 M of metformin respectively (P>0.05). Perfusion of the kidneys with the angiotensin II-converting enzyme inhibitor captopril (10 M) also significantly prevented the deleterious effects of high glucose challenge in the renal circulation. The use of a continuous infusion of -nitro-L-arginine methyl ester (L-NAME, 100 M) did not affect the protective effect of metformin in the renal circulation (39±4%; P>0.05), while tetraethylammonium (TEA, 10 mM) partially blunted this effect (33±4, P<0.01). Renal vasodilation induced by SNP was not modified by simultaneous infusion of high glucose and/or metformin. It is concluded that the impairment of ACh-induced endothelium-dependent renal vasodilation observed after acute exposure to high glucose concentrations is abolished by metformin administration. These alterations of renal vascular reactivity can be accounted for, at least in part, by the activation of the renal renin-angiotensin system during hyperglycemia. The protective effects of metformin present some EDHF-dependent component and are not related to metabolic pathways dependent on nitric oxide.     

Further studies on the extraneuronal uptake and metabolism of isoprenaline in the perfused rat heart

Summary To simultaneously determine the kinetics of removal, O-methylation and accumulation of ³H-isoprenaline, isolated rat hearts were perfused for 4 min with various concentrations of ³H-isoprenaline. The apparent K _m for the O-methylation of ³H-isoprenaline (3.3±0.5 M) was more than one order of magnitude lower than the corresponding value for the accumulation of unchanged amine (71.3±7.1 M). The apparent K _m for removal was very similar to that for accumulation (63.2±5.9 M). At perfusion concentrations higher than 25 M, i.e. when O-methylation was saturated, removal virtually equalled accumulation. However, at low substrate concentrations removal of ³H-isoprenaline was overwhelmingly followed by O-methylation; this led to a marked difference between rates of removal and those of accumulation.When initial rates of uptake of ³H-isoprenaline were determined after 1.5 min of perfusion of the hearts by the method of Graefe et al. (1978), the uptake of ³H-isoprenaline consisted of two components: a nonsaturable and a saturable (after subtraction of the nonsaturable component from the total uptake).The kinetic constants of the saturable component of uptake were higher than those obtained after 4 min perfusion (see above) (K _m : 110±19 M; V _max: 80±4 nmoles·g^–1·min^–1).Corticosterone competitively inhibited the saturable component of uptake of ³H-isoprenaline (K _m : 1.2 M).During wash out of accumulated ³H-isoprenaline, O-methylation took place predominantly in one of the two extraneuronal compartments. The efflux of 3-O-methyl-³H-isoprenaline (³H-OMI), the O-methylated metabolite of ³H-isoprenaline, was characterized by a half time of about 1.2 min. O-methylation accelerated the loss of radioactivity from the tissue during wash out.The extraneuronal uptake of ³H-isoprenaline was characterized as a pump and leak system by means of steady-state kinetics of accumulation of ³H-isoprenaline. Half saturation of the steady-state accumulation was observed at a concentration of 104.5 ±18.5 M ³H-isoprenaline; the leak component was characterized by a rate constant of 0.0359 min^–1.This study was supported by the Deutsche Forschungsge-meinschaftA preliminary account was presented at the 6th International Congress of Pharmacology (Graefe et al., 1975)     

Celiprolol exerts microvascular dilatation by activation of β2-adrenoceptors

Summary In order to clarify the question whether the ₁-selective adrenoceptor antagonist celiprolol possesses vasodilating properties, isolated vascular networks were perfused with increasing concentrations of celiprolol (in a cumulative manner) ranging from 10^–8 to 10^–4 mol/l. The study was carried out using the isolated mesenteric vascular bed of the guinea pig mesenterium coli. Vascular diameters of four different vascular regions [vessels classified as G1 (585 ± 30 m), G2 (403 ± 25 m), G3 (282 ± 27 m) and G4 (197 ± 13 m)] were assessed by means of microscopic videoangiometry. Perfusion with celiprolol resulted in concentration dependent vasodilation which was more pronounced in G3 and G4 vessels. In addition, cumulative concentration-response curves were determined from responses obtained in the presence of 10^–8, 10^–7, 10^–6 and 10^–4 mol/l ICI 118,551 (a highly selective adrenoceptor antagonist). In the presence of ICI 118,551 at concentrations 10^–6 mol/l, no celiprolol response could be observed. Lower concentrations of ICI 118,551 shifted the celiprolol concentration-response curve to the right in a concentration-dependent manner. Therefore, it is concluded (a) that celiprolol has a vasodilating effect, (b) that this vasodilation is produced by stimulation of ₂-adrenoceptors and (c) that the vasodilating effect is more pronounced in smaller than in larger vessels (G3, G4 vs G1, G2). Send offprint requests to S. Dhein at the above address     

Single-dose toxicokinetics of aluminum in the rat

The toxicokinetics of aluminum (Al) in male Wistar rats was studied after single intragastric (IG) doses of 1000 and 12000 g Al/kg and intravenous (IV) doses of 10, 100, 1000, and 12000 g Al/kg. Serial blood samples, daily samples of urine and feces as well as brain, liver, kidney, spleen, quadriceps muscle, and femur samples were collected. Al was measured by atomic absorption spectrometry. Al blood profiles after IV doses were adequately described by a two-compartment open model. Al toxicokinetics was dose dependent and appeared to plateau at 12000 g/kg. At IV doses between 10 and 1000 g/kg the terminal half-life of elimination from whole blood (t_1/2) increased from 29.9±7.8 to 209.3±32.6 min, and the total body clearance (CL) decreased from 2.45±0.64 to 0.28±0.03 ml min^–1 kg^–1. Following an IV bolus of 10 and 100 g/kg the administered Al was recovered completely from urine (94.4%±9.9% and 98.5%±3.2%). Twenty-nine days after the IV dose of 1000 g/kg daily renal excretion decreased to baseline values while only 55.1%±8.0% of the dose was excreted. Nineteen days after the single IV dose of 1000 g/kg Al accumulated in liver (28.1±7.7 versus 1.7±0.5 g/g of control rats) and spleen (72.5±21.1 versus <0.4 g/g). After the single 1000 g/kg IG dose no absorption of Al was detectable. The IG dose of 12000 g/kg resulted in a maximum blood Al level of 47.9±12.4 g/l after 50 min. The blood concentration time curve fitted a one-compartment open model with a half-life of absorption of 28.2±3.6 min and a t_1/2 of 81.2±20.2 min. Cumulative renal Al excretion was 0.18%±0.10% of the dose and oral bioavailability was 0.02%. Seventeen days after the 12000 g/kg IG dose the Al content in femur samples was increased (2.7±1.3 versus 0.6±0.4 g/g). In no case was fecal elimination of incorporated Al observed.     

Effects of secretogranin II-derived peptides on the release of neurotransmitters monitored in the basal ganglia of the rat with in vivo microdialysis

In vivo microdialysis was used to study the effect of secretogranin II-derived peptides on dynorphin B (Dyn B), dopamine, -aminobutyric acid (GABA), glutamate and aspartate release in the substantia nigra and neostriatum of halothane-anaesthesized rats.In the substantia nigra, local infusion of secretoneurin (secretogranin II 154–186) (1–50 M) increased, in a concentration-dependent manner, extracellular aspartate, glutamate, Dyn B, dopamine and GABA levels. The effect was particularly prominent on aspartate and glutamate levels which, following 50 M of secretoneurin, were increased by >20 and >10 fold, respectively. However, the effect of secretoneurin on Dyn B release appeared to be more specific, since a significant increase (>2 fold) was already observed following 1 M of secretoneurin. In the neostriatum, Dyn B, glutamate, aspartate and GABA levels were also increased by local secretoneurin infusion, but the effect was less prominent than in the substantia nigra. In the substantia nigra, only Dyn B levels were significantly increased following infusion of 10 M of the secretoneurin-C terminal (secretoneurin-15C), whereas Dyn B and GABA levels were increased by the same concentration of the secretogranin II C terminus (YM). Only glutamate and aspartate levels were increased by local infusion of 10 M of secretogranin II 133-151 (LF), a peptide adjacent to secretoneurin in the primary amino acid sequence. In the neostriatum, Dyn B and GABA levels were increased by 10 M of secretoneurin-15C. Dyn B levels were also increased by 10 M of YM, and glutamate and aspartate levels were increased by 10 M of both YM and LF.Thus, secretogranin 11-derived peptides affect extracellular levels of several putative neurotransmitter systems monitored in the basal ganglia of the rat with in vivo microdialysis. The effect of Dyn B appears to be specific and related to a physiological role of secretoneurin, since (i) it occurs in an area where secretoneurin-immunocytochemistry has been observed, (ii) is exerted at comparatively low concentrations, and (iii) is mimicked by secretoneurin-15C. The increases in excitatory amino acid levels produced by high concentrations of secretoneurin and other secretogranin II-derived peptides reflect, perhaps, a potential neurotoxicity produced by abnormal accumulation of these peptides.     

Lead and cadmium in breast milk higher levels in urban vs rural mothers during the first 3 months of lactation

Breast milk from 10 women each from the city of Hamburg and from a rural area was analyzed by atomic absorption spectrometry for contamination with lead and cadmium. Samples were examined at regular intervals for 3 months after birth. On day 5 a diurnal profile was analyzed; on the other days milk was taken before and after the morning feed.Daily permissable intake (DPI) for lead is 5 g/kg/day for children; the DPI for cadmium has as yet been determined only for adults as 400–500 g/week, equivalent to about 1 g/kg/day (WHO 1972). For breast milk as the main source of nutrition in infants, this study shows values of 9.1±2.5 (SD) g/l for lead in the rural population, with a tendency to decrease towards the end of lactation. Urban mothers had 13.3±5.5 (SD) g/l, with a tendency to increase. This difference was significant only on day 45. Mean cadmium content in rural mothers was 17.3±4.9 g/l, with much higher values in the colostrum and a decrease after 15 days. Urban mothers had 24.6±7.3 g/l, again with high colostrum values and a subsequent decrease. These latter values are not significantly different.Calculated daily intake according to these values is presented, based on 840 ml breast milk for a 5.5 kg infant per day. Rural infants ingested 0.9–1.3 g/kg/day of lead, and in the city 1.5–2.3 g/kg/day. Cadmium intake in rural infants amounted from 1.2–1.8 g/kg/day; in Hamburg it was 1.6–2.2 g/kg/day. Thus the daily ingestion of lead was just below the DPI, cadmium ingestion was higher than the DPI for adults. The rural population had lower values in breast milk for both heavy metals than the urban population, although not statistically significant. Compared to earlier reports there was a slight increase in lead concentration and a more significant increase for cadmium. This study shows that the increase in cadmium may have taken place during very recent years, possibly due to the increase in the pH of soil. Therefore, it is suggested that both levels be monitored in a continuous program to prevent any nutritional hazard.     

Phase II trial of 9-aminocamptothecin (NSC 603071) administered as a 120-hour continuous infusion weekly for three weeks in metastatic colorectal carcinoma

9-Aminocamptothecin (9-AC) is a camptothecin derivative with broad antitumor activity in preclinical studies. Prior investigations suggested that prolonged maintenance of 9-AC lactone plasma concentrations above 10 nmol/l and frequent administration of the drug are important determinants of antitumor activity. Our phase II study, therefore, examined a 5-day continuous infusion of 9-AC weekly for 3 weeks in patients with advanced colorectal cancer. Eighteen patients previously untreated for metastatic disease received 480 g/m²/day of 9-AC. No responses were observed in 17 evaluable patients. Severe toxicities included granulocytopenia, nausea, vomiting and diarrhea. The median absolute granulocyte count (AGC) nadir was 2,300/l (range 0–9,000/l) and occurred on day 10. Eight patients received an escalated dose of 600 g/m²/day. The median AGC nadir at the escalated dose was 1,500/l (range: 300–2,700/l) and occurred on day 22. The median number of courses given was 2 (range: 1–8); and the median time to disease progression was 8 weeks (range: 1–40 weeks). 9-AC administered by this schedule lacked antitumor activity in patients with advanced colorectal carcinomas.     

Accumulation of 3H-(±)-noradrenaline by isolated rat liver cells

Summary Using hepatocytes isolated by collagenase perfusion, we studied the accumulation of ³-noradrenaline. Cells incubated during 15 min in the presence of 0.4 mol/l ³H-noradrenaline (without inhibition of noradrenaline metabolism) accumulated 8.32 ± 1.77 pmol/10⁶ cells (n = 3). The accumulation of ³H-noradrenaline in isolated parenchymal liver cells was sensitive to 10 mol/l cocaine (inhibition 36.6 ± 7.9%, n = 3) and 1 mol/l desipramine (inhibition 27.2 ± 6.9, n = 3). Accumulation of ³H-noradrenaline was temperature and sodium dependent (inhibition 33.2 ± 9.4%, n = 9, when Na⁺ was replaced by Tris⁺) and was influenced by the inhibition of the membrane Na⁺-K⁺-adenosine triphosphatase (Na⁺-K⁺-ATPase) by 150 mol/l ouabain (34.7 ± 6.9% inhibition, n = 3). Accumulation of 3H-noradrenaline in the hepatocytes was not affected by the presence of uptake₂ inhibitors, normetanephrine (30 mol/l) and corticosterone (30 mol/l), but was reduced by 30 mol/l isoprenaline (76.3 ± 5.0% inhibition, n = 6). Thus, the system that takes up and accumulates noradrenaline in the isolated rat liver cells possesses some characteristics of both, uptake₁ and uptake₂ systems and appears to be different from other extraneuronal cocaine-sensitive systems, such as the one reported for pulmonary endothelial cells. Send offprint request to M. I. Masana at the above address     

The effects of drugs and denervation on removal and accumulation of noradrenaline in the perfused hind-limb of the dog

Summary The removal of noradrenaline by the autoperfused hind-limb of dogs anaesthetized with pentobarbital, as well as the accumulation of noradrenaline in the saphenous vein were studied. Sensitivity of the perfused vascular area was determined by the response of the perfusion pressure to infusions of noradrenaline.The removal of noradrenaline declined very slowly during infusions lasting for up to 2 h, but edema of the perfused limb occurred after 45 to 60 min; therefore, the duration of infusion was limited to 30 min. During this period, noradrenaline was infused in rates increasing by a factor of 2 and ranging from 0.5 to 16 g/kg per minute. Accumulation capacity was saturated at 1 g/kg · min^–1, but the amount removed increased until a four-to eightfold rate was reached and then levelled off.At a rate of 1 g/kg · min^–1 the influence of drugs and of surgical denervation was investigated in other experimental series. Cocaine, nialamide, phenoxybenzamine and pretreatment with reserpine reduced removal (by 50, 45, 40 and 35%, respectively). Cortexone had no detectable influence on removal with this rate of infusion, but blocked it effectively when 4 g/kg · min^–1 were infused. Accumulation of noradrenaline in the vein was prevented by cocaine or reserpine, slightly reduced by phenoxybenzamine and enhanced by nialamide. The effects of nialamide plus cocaine did not differ significantly from those of cocaine alone, but cortexone plus cocaine completely blocked removal and accumulation. Surgical denervation reduced removal by about 70% and abolished accumulation; reserpine plus nialamide had similar effects. In the case of nialamide, removal progressively diminished during the infusion period and this time dependence of effects was accompanied by a prolongation of noradrenaline washout.Cocaine, reserpine and denervation caused supersensitivity of the perfused vessels to noradrenaline, whereas nialamide and cortexone had no such effect and phenoxybenzamine caused subsensitivity.The pronounced ability of the perfused vessels of the hind-limb to remove noradrenaline from the circulating blood is attributed primarily to neuronal uptake and intraneuronal oxidative deamination; extraneuronal uptake and inactivation seem to play an important role when neuronal mechanisms are saturated (infusion of higher noradrenaline doses) or impaired (after cocaine or denervation).Supported by Instituto de Alta Cultura (Research Project PMC/2). Part of this work was presented at the Fifth International Congress on Pharmacology (S.Francisco, July 23–28, 1972).     

Kinetische Analyse der Calcium-Kompartimente im Meerschweinchenherzen unter Kontrollbedingungen und Strophanthineinwirkung

Summary A quantitative analysis of myocardial Ca-metabolism was carried out on isolated, isovolumetric (10 ml/min) perfused guinea pig hearts by combined determinations of the total Ca-content and kinetics of ⁴⁵Ca-efflux (collecting period 60 min) (Fig. 1). The kinetics of ⁴⁵Ca-uptake was estimated by extrapolating ⁴⁵Ca-efflux curves of heart muscles isotopically loaded for different times (2, 5, 10, 30 60 min) to the end of the loading-period (Fig. 2).From the specific activity it was possible to express the absolute Ca-content of the extrapolated compartments in M Ca/g w.w. The amount of activity in cannules and coronary vessels was estimated by dextranblue (MW about 2 millions).The results indicate the presence of three kinetically defined phases (K₁, K₂, K₃) of calcium movements in guinea pig heart muscle. The content of the Ca compartments under equilibrium conditions (60 min ⁴⁵Ca-loading period) was found to be: K₁=0.29±0.05, K₂=0.56±0.09, K₃=0.30±0.04 moles/g w.w.The half-time of Ca movement in these compartments for ⁴⁵Ca efflux is for K₁ 15.5±1.0 sec, for K₂ 2.2±0.1 min, for K₃ 17.9±0.9 min, and for ⁴⁵Ca uptake K₁<2, K₂3.9± _1.0 ^1.1 , K₃9.2± _1.8 ^2.5 min.Total Ca content has been found to be 1.67±0.06 moles/g w.w. Under control conditions about 65% of total heart muscle Ca was exchangeable. Under the influence of ouabain (1.5·10^–7 M) Ca of the heart muscle exchanged to about 100%. This was caused by an increase in Ca content of compartment K₃ to 0.46±0.06 moles/g w.w. and a significant decrease in total Ca-content to 1.32±0.07 moles/g w.w. The rate of ⁴⁵Ca-exchange was not influenced by ouabain. The value of Ca-turnover for Ca-availability in contraction cycle and the mechanism of ouabain action are discussed.

Ein Teil der Ergebnisse wurde anläßlich der Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft im Mainz mitgeteilt (Krebs et al., 1970).     

Interaction between clonidine and physostigmine in normal rats and in rats after sinoaortic denervation

Summary Clonidine (3–30 g · kg^–1, i.v.) induced a fall in mean arterial pressure in rats after sinoaortic denervation but not in sham-operated animals. Moreover, sinoaortic denervation reduced the bradycardic action of this antihypertensive drug. Pressor and tachycardic response to physostigmine (60 g · kg^–1, i.v.) were greater in denervated than in sham-operated rats. The increase of mean arterial pressure was 26.2 ± 2.2 mm Hg in sham-operated rats (n = 12) and 53.8 ± 2.0 mm Hg in denervated rats (n = 12, P < 0.005).Pretreatment with 3 g · kg^–1 (i. v.) of clonidine did not alter the pressor response to physostigmine (60 g · kg^–1) in either of the two groups; 10 and 30 g · kg^–1 of clonidine reduced the physostigmine-induced increase of mean arterial pressure in sham-operated rats but enhanced the pressor response in denervated animals. Furthermore, an ineffective dose of physostigmine (30 g - kg^–1 i.v.) induced a pressor response after pretreatment with clonidine (10 gg · kg^–1) in denervated rats.Clonidine (10 g · kg^–1) did not affect the pressor effect of 1,1 dimethyl-4-phenylpiperazinium iodide (DMPP: 50 g · kg^–1 i.v.) or phenylephrine (4 g · kg ^–1, i.v.) in either group.The anticholinergic effect of clonidine in sham-operated rats may be explained by an inhibitory action on the release of acetylcholine in several brain structures but the facilitatory effect of clonidine observed in denervated animals is not clear. The results did not suggest a peripheral involvement in this facilitatory effect. Send offprint requests to M. A. Enero at the above address     

Muscarine receptor types mediating autoinhibition of acetylcholine release and sphincter contraction in the guinea-pig iris

Summary The potencies of several muscarine receptor antagonists in blocking either the autoinhibition of acetylcholine release or the muscarinic contraction of the sphincter muscle upon acetylcholine release were investigated in the guinea-pig iris. The agonist at pre- or postjunctional muscarine receptors was acetylcholine released upon field stimulation (5.5 Hz, 2 min) of the irides preloaded with ¹⁴C-choline. The stimulation-evoked ¹⁴C-overflow was doubled in the presence of atropine 0.1 mol/l but unaffected by the agonist (±)-methacholine (50 mol/l). Thus, under the present stimulation conditions, the autoinhibition of acetylcholine release on the guinea-pig iris cholinergic nerves was nearly maximally activated. Isotonic contractions of the irides upon field stimulation consisted of a rapid, atropine (0.1 mol/l). peak phase followed by a sustained contraction which involved a cholinergic and a non-cholinergic stimulation of the sphincter muscle. The M₂-selective antagonists methoctramine (10 mol/l) and gallamine (100 µmol/l). increased both the ¹⁴Goverflow and the peak contractions evoked by field stimulation. In contrast, the M₃-selective antagonist hexahydrosiladifenidol (0.1–10 mol/l) failed to affect the evoked ¹⁴C-release but concentration-dependently (1–10 mol/l) reduced the iris contractions. Pirenzepine (10 mol/l) enhanced the evoked ¹⁴C-overflow and inhibited the peak contractions (0.1–10 mol/l; maximal effect at 10 mol/l). The low potency of the antagonist at both receptor sites indicates that an M₁ muscarine receptor is not involved. The results are consistent with the idea of M₂ muscarine receptors mediating autoinhibition of acetylcholine release in the guinea-pig iris and M₃-like receptors inducing the contraction of the sphincter muscle. Send offprint requests to I. T. Bognar at the above address     

Comparative study of the vasorelaxant activity, superoxide-scavenging ability and cyclic nucleotide phosphodiesterase-inhibitory effects of hesperetin and hesperidin

This study investigated the vasorelaxant activity, superoxide radicals (O₂^–)-scavenging capacity and cyclic nucleotide phosphodiesterase (PDE)-inhibitory effects of hesperidin and hesperetin, two flavonoids mainly isolated from citrus fruits. Hesperetin concentration-dependently relaxed the isometric contractions induced by noradrenaline (NA, 1 M) or by a high extracellular KCl concentration (60 mM) in intact rat isolated thoracic aorta rings. However, hesperetin (10 M–0.3 mM) did not affect the contractile response induced by okadaic acid (OA, 1 M). Mechanical removal of endothelium and/or pretreatment of aorta rings with glibenclamide (GB, 10 M), tetraethylammonium (TEA, 2 mM) or nifedipine (0.1 M) did not significantly modify the vasorelaxant effects of this flavonoid. Hesperetin (10 M–0.1 mM) did not affect the basal uptake of ⁴⁵Ca²⁺ but decreased the influx of ⁴⁵Ca²⁺ induced by NA and KCl in endothelium-containing and endothelium-denuded rat aorta. Hesperetin (10 M–0.1 mM) did not scavenge O₂^– generated by the phenazine methosulfate (PMS)-reduced -nicotinamide adenine dinucleotide (NADH) system. Hesperetin (0.1 mM) significantly reversed the inhibitory effects of NA (1 M) and high KCl (60 mM) on cyclic nucleotide (cAMP and cGMP) production in cultured rat aortic myocytes. Hesperetin preferentially inhibited calmodulin (CaM)-activated PDE1 and PDE4 isolated from bovine aorta with IC₅₀ values of about 74 M and 70 M respectively. In contrast, the 7-rhamnoglucoside of hesperetin, hesperidin (10 M–0.1 mM), was inactive in practically all experiments, although it inhibited basal and cGMP-activated PDE2 isolated from platelets (IC₅₀ values of 32±4 M and 137±34 M respectively). These results suggest that the vasorelaxant effects of hesperetin are basically due to the inhibition of PDE1 and PDE4 activities.     

Food chain transfer and potential renal toxicity of mercury to small mammals at a contaminated terrestrial field site

Mercury concentrations were determined in surface soil and biota at a contaminated terrestrial field site and were used to calculate transfer coefficients of mercury through various compartments of the ecosystem based on trophic relationships. Mercury concentrations in all compartments (soil, vegetation, invertebrates, and small mammals) were higher than mercury concentrations in corresponding samples at local reference sites. Nonetheless, mercury concentrations in biota did not exceed concentrations in the contaminated surface soil, which averaged 269 g g^-1. Plant tissue concentrations of mercury were low (0.01 to 2.0 g g^-1) and yielded soil to plant transfer coefficients ranging from 3.7×10^-5 for seeds to 7.0×10^-3 for grass blades. Mercury concentrations in invertebrates ranged from 0.79 for harvestmen (Phalangida) to 15.5 g g^-1 for undepurated earthworms (Oligochaeta). Mean food chain transfer coefficients for invertebrates were 0.88 for herbivores/omnivores and 2.35 for carnivores. Mean mercury concentrations in target tissue (kidney) were 1.16±1.16 g g^-1 for the white-footed mouse (Peromyscus leucopus), a granivore, and 38.8±24.6 g g^-1 for the shorttail shrew (Blarina brevicauda), an insectivore. Transfer coefficients for diet to kidney were 0.75 and 4.40 for P. leucopus and B. brevicauda, respectively. A comparison of kidney mercury residues measured in this study with values from controlled laboratory feeding studies from the literature indicate that B. brevicauda but not P. leucopus may be ingesting mercury at levels that are nephrotoxic.     

Lack of accumulation of midazolam in plasma and lipoprotein fractions during intravenous lipid infusions in patients on artificial respiration

Summary Severely ill patients often require total parenteral nutrition including intravenous liqid emulsions concurrently administered with lipophilic drugs. Therefore we investigated whether therapeutic application of a mixed medium chain/long chain triglyceride infusion affects the disposition of midazolam necessary for sedation in patients on artificial respiration. The concentrations of midazolam were measured in unfractionated plasma, and in lipoprotein fractions isolated from ex vivo blood samples, including determination of triglycerides and cholesterol; the albumin level was also analysed.Midazolam in the VLDL fraction was only 0.246 g·ml^–1, whereas the total plasma concentration averaged 1.101 g·ml^–1, and the midazolam content of the LDL plus HDL fractions amounted to 1.771 g·ml^–1. Albumin in these lipoprotein fractions was just as unequally distributed. A lipid infusion resulted in a significant elevation of total triglycerides from 157 to 221 mg·dl^–1 and VLDL-triglycerides from 77 to 155 mg·dl^–1. The triglyceride content of the LDL plus HDL fraction rose from 102 to 139 mg·dl^–1. At the same time the midazolam concentration in unfractionated plasma and in the VLDL and the LDL + HDL fractions decreased to 0.899 g·ml^–1, 0.130 g·ml^–1, and 1.265 g·ml^–1, respectively. Cholesterol and albumin concentrations were not affected.The data show for the first time that a significant increase in plasma triglycerides during an intravenous lipid infusion does not result in accumulation of midazolam in lipoproteins, probably because albumin binding of the drug is very strong. The lack of midazolam trapping is important with respect to the safety of concurrent use of lipophilic drugs and intravenous lipid infusions.     

Activation of Cl− channels by avermectin in rat cultured hippocampal neurons

Summary The actions of the insecticide avermectin (AVM) were studied in rat cultured hippocampal neurons with patch-clamp techniques. Application of micromolar concentrations of AVM to voltage-clamped cells gave rise to whole-cell currents, which showed a slow time-course of activation in the order of 10 s, and wash-out periods of typically 20 min. Dose-response curves revealed a half-maximally activating AVM concentration (EC₅₀) of 2.0±0.6 M and a Hill coefficent of 1.5±0.9. The current activated by AVM was carried predominantly by Cl^– ions, as demonstrated by ion-substitution experiments. The Cl^– channel blocker picrotoxinin (100 M) substantially but transiently reduced the AVM response. Outsideout patch recording showed that AVM opened Cl^– channels with a conductance of 40±12 pS. The open-time distribution was characterized by two time constants of 11 ms and 259 ms. It is suggested that AVM directly activates Cl^– channels in mammalian central neurons, which resemble the channels activated by the physiological transmitters GABA and glycine.Correspondence to: J. Bormann at the above address