Tannin inhibition of protein kinase C in airway epithelium

Tannin, a polydisperse polyphenol extracted from cotton bracts (CBE), has been implicated in the pathogenesis of byssinosis, a lung disease of mill workers. CBE tannin inhibits chloride secretion in airway epithelial cells by means of an unknown mechanism(s). Activation of protein kinase C (PKC) by PMA (phorbol 12-myristate 13-acetate) in airway cells increases chloride secretion. The effect of tannin on this PKC pathway was examined, using canine tracheal epithelium mounted in Ussing chambers. PMA addition (10 nM) to the mucosal bath resulted in a 0.36 ± 0.07 Eq/cm² · h (mean ± SEM, n = 20) increase in short-circuit current (I_sc) and a 0.38 ± 0.17 Eq/cm² · h increase in net chloride secretion (J_net). The inactive 4-phorbol had no effect. Tannin addition to the mucosal bath produced a dose-dependent decrease in I_sc and J_net. In tissues pretreated with 2–50 g/ml tannin, and subsequently stimulated with PMA, tannin inhibited PMA stimulation of chloride secretion beginning at a tannin concentration of 10 g/ml (0.09 ± 0.05 Eq/cm² · h [n = 10] increase in I_sc and 0.08 ± 0.03 Eq/cm² · h increase in J_net with PMA after tannin pretreatment). At 50 g/ml tannin, the stimulatory effect of PMA was completely abolished. The known PKC inhibitor, H-7 (20 M), inhibited PMA stimulation, while chelerythrine (2 M) had not effect on PMA-stimulated I_sc and J_net, and calphostin C was toxic to the airway epithelium. In membrane fragments, 2.5 g/ml tannin inhibited the rate of histone III phosphorylation by PMA from 32.1 ± 4.4 nmol/mg protein per min to 20.1 ± 2.7 nmol/mg protein per min (n = 7). In bovine airway cells, tannin pretreatment (2.5 g/ml) decreased the cytosolic activity of PKC but had no effect on PKC translocation to the membrane. We conclude that tannin inhibits chloride secretion in airway epithelial cells in part by inhibiting PKC.Offprint requests to: Michelle M. Cloutier     

Determinants of Lactose Digestion in the Miniature Pig

Although lactose is an important nutrient in thediet of the infant and child, the factors contributingto its digestion have not been clarified adequately. Wesought to determine the degree to which lactase activity and small intestinal transit explainlactose digestion, the average error (SEE) in estimatinglactose digestion using these parameters, and the effectof age. We compared lactose digestion from both a 7% lactose-containing formula and asolution by determining lactose in ileostomy output inpig littermates at 10 days, 4 weeks, and 10 weeks ofage. The entire small intestinal mucosa was assayed for lactase specific activity (mol µmin^-1 µ g protein^-1), totalactivity (mol µ min^-1), andwhole-villus lactase activity. Transit time (min), andtransit rate (cm/min) were measured. Meal type did notaffect lactose digestion. Lactose digestion wasexplained best by lactase specific activity (formula,R² = 0.73, SEE 1.1; solution, R²= 0.69, SEE 1.0; P < 0.001). The next best parameterwas total transit rate (formula, R² = 0.69, SEE = 2.0; solution,R² = 0.46, SEE = 1.3). The relationship withlactase specific activity was age related and thereappeared to be a critical value of lactase specificactivity above which essentially all the lactose was digested.     

Cellular and mitochondrial energy metabolism in the stunned myocardium

Summary The influence of cardiac stunning on the oxidation of fatty acids and the oxidative phosphorylation in mitochondria was investigated. Rat hearts were perfused for 15 min according to the working mode with a Krebs-Henseleit buffer containing glucose (11 mM). The hearts were then maintained in normoxic conditions (C group) or subjected to a 15-min global no-flow normothermic ischemia followed by a 30-min reperfusion (R group). Throughout the perfusion, the aortic and coronary flows, and the heart rate and oxygen consumption were monitored. At the end of the perfusion procedure, a bolus of 1-¹⁴C palmitate was injected in the coronary arterial bed to evaluate the fatty acid oxidation. Two sub-populations of mitochondria were isolated from each heart by either mechanical (ME mitochondria) or enzymic (EE mitochondria) extraction and their respiration properties were evaluated. Furthermore, the mitochondrial energy production (ATP and creatine phosphate) was assessed. During ischemia, the aortic flow was suppressed and recovered only to approximately 50% of the preischemic value during reperfusion. This mechanical stunning was associated with an important reduction of the stroke volume (–37%,p<0.01) and a slight decrease in heart rate (–20%,p<0.001). At the end of reperfusion, the beta-oxidation rate constituted 55±1.7% of the cell palmitate and was similar to that assessed in the C group. The oxygen consumption was decreased to 216±31.0L O₂/min/gww and the venous O₂ concentration increased to 5.1±0.572 L O₂/mL (instead of 2.9±0.342 L O₂/mL in the C group), although due to large SD, only the latter was statistically significant. A decrease in metabolic effeciency (42±14.4 vs 106±16.8 mL/L O₂ in the C group) and an increase in palmitate oxidation to oxygen consumption ratio (77±10.1 vs 47.6±4.25 % beta-oxidized palmitate/L O₂ in the C group) were observed. This increased fatty acid contribution in the oxidation metabolism could be responsible for some oxygen wasting and could contribute to decrease the energy available for the contraction despite the normal cardiac oxygen uptake. Furthermore, the respiration parameters of the mitochondria were similar in the C and R groups when glutamate (20 mM) or palmitoylcarnitine (25 M) were used as substrate. ME mitochondria of R group displayed a reduced rate of ATP production (118±29.5 vs 180±14.5 nmoles/min/mg proteins in the C group) without altered creatine phosphate production. The presence of calcium in the medium (10^–5 M) provoked a decrease in ATP production. These effects were not observed with EE mitochondria. Thus, a decreased energy production resulting from a substrate effect and/or a decreased mitochondrial phosphorylative capacity could be associated with the mechanical stunning.     

Carbachol Prolongs Ventricular Repolarisation through Nitric Oxide Release in an Intact Heart

Aims: The primary aim of the study was to investigate the effect of carbachol on ventricular repolarisation in an intact animal heart. Methods: In five sheep, carbachol was administered to the left circumflex coronary artery (LCX) at 1.0 and 2.5 mol/ml/min respectively for 3 min. Multiple unipolar ECGs were acquired from the epicardium of LCX territory. Activation-recovery interval (ARI) was analysed from these ECGs. Administration of carbachol at 2.5 mol/ml/min was also repeated after pre-treatment with nitro-L-arginine (20 mg/kg), a nitric oxide synthase inhibitor. Results: Carbachol at 1.0 or 2.5 mol/ml/min resulted in a T wave inversion and ARI prolongation in the LCX territory. The increase in ARI at 1.0 and 2.5 mol/ml/min was 38 ± 17 and 58 ± 14 ms respectively (p < 0.05). T wave inversion and ARI prolongation at 2.5 mol/ml/min was diminished by pre-treatment with nitro-L-arginine. Conclusions: Carbachol results in a dose-dependent prolongation in ventricular repolarisation in this open-chest animal model. This effect is partially mediated by endogenous nitric oxide.     

Deleterious effects of digitalis on reperfusion-induced arrhythmias and myocardial injury in ischemic rat hearts: possible involvements of myocardial Na+ and Ca2+ imbalance

Summary Isolated rat hearts were made ischemic for 25 min after an initial recirculating perfusion, followed by 30 min of reperfusion. In some hearts, interventions including administration of ouabain and/or high [K⁺] in the buffer were performed during the first 10 min of reperfusion.During ischemia, intracellular Na⁺ (Na_i) increased from 15 to 64 [mol/g dry weight (dwt). During reperfusion, Na_i declined rapidly (at 10 min of reperfusion: 48 nol/g dwt, at 30 min: 25 mol/g dwt) and regular rhythm was recovered within 10 min in hearts without any intervention during reperfusion.⁴⁵Ca²⁺ uptake increased from 0.8 to 7.5 mol/g dwt after 30 min of reperfusion. Ventricular function recovered by 45 %.A 10-min perfusion with 10 or 50 M of ouabain increased Na_i (17 to 21 or 27 mol/g dwt) with increased left-ventricular (LV) contractile function, but these effects were reversed by combination of high perfusate [K⁺] (20 mM) in non-ischemic hearts.A 10-min reperfusion with ouabain retarded or stopped the decline in Na_i (at 10 min of reperfusion: 54 or 63 mol/g dwt, at 30 min: 32 or 40 mol/g dwt). These amounts of ouabain also increased the incidence of ventricular tachyarrhythmias during reperfusion to 30 % or 50 %, and increased the duration of ventricular fibrillation from 6.5 to 11.5 or 18.0 min.⁴⁵Ca²⁺ uptake reached to 8.8 or 10.0 mol/g dwt, and function recovered only 35 % or 28 %. When high perfusate [K⁺] was combined with ouabain during reperfusion, the retarded decline in Nai, augmented⁴⁵Ca²⁺ uptake, and reduced recovery of function caused by ouabain alone were attenuated. These results suggest that digitalis has toxic effects on reperfused ischemic hearts by inhibition of rapid active outward transport of previously elevated Na_i and potentiation of Ca²⁺ overload.The work was supported in part by grant HL 37936 from the National Heart, Lung and Blood Institute. J. R. Neely was deceased on November 29, 1988     

A study of zinc distribution in erythrocytes of normal humans

Summary The assignment of zinc bound to carbonic anhydrase isoenzymes (CA-I and CA-II) and Cu₂ Zn₂ superoxide dismutase (SOD₁) was investigated in the hemolysates from 21 normal male subjects.Sufficient care was taken to remove leukocytes and platelets. The following values of zinc distribution were obtained:total zinc, 1113.8±22.7 (mean±S.E.) g · 100 ml^–1; CA-I-derived zinc, 866.6±26.2 g · 100 ml^–1; CA-II-derived zinc, 99.9±3.9 g · 100 m^–1; SOD₁-derived zinc, 60.3±1.9 g · 100 ml^–1; the other zinc, 87.0±12.6 g · 100 ml^–1. Namely, 7.6% of the zinc in human erythrocytes is not bound to the carbonic anhydrases and Cu₂Zn₂ superoxide dismutase, but present in available form or attached to other enzymes.     

Granulocyte elastase in assessment of severity of acute pancreatitis

Complexes of granulocyte elastase and ₁-antitrypsin are markers for granulocyte activation. In 75 patients with acute pancreatitis these complexes were immunologically determined daily in plasma during the first week of hospitalization. Patients were classified into three groups: mild pancreatitis (I, 1 complication, N=34), severe pancreatitis (II, 2 complications, N= 29), lethal outcome (III, N=12). Initially, granulocyte elastase (mean±sem) was lower in group I (348±39 g/liter) as compared to groups II (897±183 g/l) and III (799±244 g/liter), P<0.001 for I vs II + III. Initial elastase concentrations >400 g/liter were consistent with a severe or fatal course of the disease but did not distinguish between severe and lethal pancreatitis. In patients with mild or severe disease, mean elastase concentrations decreased continuously during the following days (197±15 g/liter in mild cases, 325±30 g/liter in severe cases at day 7). In patients with lethal disease, however, mean elastase concentrations even increased at day 2 and remained higher than 700 g/liter during the observation period. At days 1 and 2 the predictive value for severe or lethal disease of raised (>400 g/liter) elastase concentrations [positive predictive value (PPV) 82%, negative predictive value (NPV) 81%] was better than that of elevated (>100 mg/liter) C-reactive protein (PPV 73%, NPV 73%), elevated (>4.0 g/liter) ₁-antitrypsin (PPV 59%, NPV 50%), or decreased (<1.5 g/liter) ₂-macroglobulin (PPV 82%, NPV 67%). When the time course of the concentrations of the acute-phase proteins was studied, it was found that rises of granulocyte elastase were followed by elevated C-reactive protein levels after one day, by elevated ₁-antitrypsin levels after two days and by decreased ₂-macroglobulin levels after three to four days. We conclude that granulocyte elastase is a good early marker for the severity of acute pancreatitis. Compared with elevated levels of C-reactive protein and ₁-antitrypsin release of granulocyte elastase reflects an event that precedes acute-phase protein induction.     

Serumeisen-Normalwerte und statistische Verteilung der Einzelwerte bei Mann und Frau

Zusammenfassung Die Bestimmung der Normalwerte des Serumeisen bei 608 Erwachsenen und die Untersuchung des Verteilungstyps der Einzelwerte zeigt folgende Ergebnisse: Bei 503 Männern beträgt der Mittelwert (als arithmetisches Mittel) 109 g Fe/100 ml ±25 und der Normalbereich (als ±2 SD-Bereich) 59 bis 158 g Fe/100 ml, bei 105 Frauen 91 g Fe/100 ml±27 als Mittelwert und 37 bis 145 g Fe/100 ml als Normalbereich. Die Untersuchung der Verteilung mittelsFisher- undKolmogoroff-Test führte zur Annahme, einer näherungsweisen Normalverteilung.

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Summary The determination of normal values of serum iron in 608 adults and the examination of the frequency distribution gives the following results: the arithmetic mean in 503 male persons is 109±25 g Fe/100 ml and the normal range (2-SD-range) 59 to 158 g Fe/100 ml; in 105 female persons 91±27 g Fe/100 ml mean and 37 to 145 g Fe/100 ml normal range. The assumption of approximate normal distribution are controlled by theFisher- andKolmogoroff-test.

     

Oxidant-stimulated chloride secretion in rat jejunum in vitro is mediated by eicosanoids

Specific effects of the cytotoxic secondary lipid oxidation product, 4-hydroxynonenal (10^–8–10^–4 M), on intact sheets of rat jejunum were measured as changes in short circuit current (I_sc) following cumulative addition to either the mucosal or serosal side, using the analogous aldehyde, nonenal, as reference. 4-Hydroxynonenal stimulated I_sc from the serosal side (maximal I_sc = 27.2 ± 3.5 A/cm², mean ± SEM, N = 32) while nonenal stimulated I_sc primarily from the mucosal side (maximal I_sc = 16.2 ± 3.4 A/cm², N = 20). Inhibition by 100 M bumetanide (4-hydroxynonenal: 88.9 ± 3.0%, N = 6, p < 0.05, nonenal: 69.3 ± 2.9%, N = 6, P < 0.05) indicated chloride secretion. Nonenal-induced I_sc was inhibited (72.5 ± 1.2%, N = 8, P < 0.05) by a combination of nordihydroguaiaretic acid (100 M) and piroxicam (10 M), while 4-hydroxynonenal-induced I_sc, was abolished by piroxicam (N = 8, P < 0.001) and inhibited by 1 M tetrodotoxin (69.8 ± 9.7%, N = 6, P < 0.001). These data indicate that 4-hydroxynonenal stimulates chloride secretion mediated by prostaglandins and the enteric nervous system. The site of action (serosal) being opposite to the reference aldehyde.     

Quantification of ischemic stress during repeated coronary artery occlusion in the dog A method for validation of therapeutic effects

Summary In 9 open-chest mongrel dogs 4–6 intermittent 3-min occlusions of the LAD artery were performed with time intervals of about 45 min. Using a -computer, the following variables, were calculated online: energy demand according to the Bretschneider equation (E_t) from digitized hemodynamic data; myocardial oxygen consumption (M O₂) from fiberoptically measured coronary sinus oxygen saturation and coronary sinus blood flow. Coronary occlusion led to a decrease in M O₂ in comparison to E_t. The integral of the difference between M ₂ and E₁ over the entire occlusion time yielded a total O₂-deficiency (DO₂) of 76 (±12%) l O₂/g ischemic tissue and a correlation coefficient with the weights of the intravitally stained ischemic areas of r=0.96. Additional O₂-uptake in relation to E_t during the early perfusion period yielded a correlation to the size of the ischemic area of r=0.95 and an average O₂-repayment (RO₂) of 32 (±14%) l O₂/g ischemic tissue. The determination of total myocardial O₂-deficiency during ischemic stress as well as determination of O₂-repayment during the early reperfusion period could be used to estimate the extent of ischemic stressed myocardium. Subsequently, the evaluation of pharmacological effects on myocardial ischemia should be possible.Supported by the Deutsche Forschungsgemeinschaft. SFB 89-Kardiologie Göttingen     

Stimulation of GTP hydrolysis in guinea pig bronchial membranes by mastoparan

Guanine nucleotide-binding proteins, or G proteins, play an important role in transmitting information from membrane receptors to intracellular effector systems. Activation of G proteins results in the hydrolysis of GTP, and the measurement of GTPase activity represents a means by which the role of G proteins in signal transduction can be investigated. GTPase activity of guinea pig bronchial membranes was measured as the liberation of ³²P_i from [-³²P]GTP. GTPase activity was divided into two components, one possessing a high affinity and the other a low affinity for GTP. The contribution of high- and low-affinity GTPase to total hydrolysis was dependent on Mg²⁺. In the presence of submicromolar Mg²⁺, high-affinity GTPase represented 65–80% of all activity, whereas in the presence of 26 µM Mg²⁺, all detectable hydrolysis was due to the low-affinity GTPase. High-affinity GTPase was stimulated by Mg²⁺ in the 0.15–1.1 M range (2.5-fold maximal stimulation, apparent Km for Mg²⁺ 0.31 M). Mastoparan (1–100 M) caused a concentration-dependent stimulation of high-affinity (but not low-affinity) GTPase (71 ± 13% maximal stimulation, EC₅₀ 0.38 M), suggesting that high-affinity GTPase may be due to a G protein. Carbachol (10 M) and fenoterol (10 M) had no effect on high-affinity GTP hydrolysis, suggesting that under the conditions described, GTPase activity of bronchial membranes is not activated by muscarinic or -adrenergic receptors, respectively. Offprint requests to: K. J. Rhoden     

Quantitative analysis of hyaluronan in the synovial tissues of patients with joint disorders

Quantitative analysis of hyaluronan (hyaluronic acid; HA) in the synovial tissues of patients with joint disorders were performed. HA was found not only in the synovial intimal cells and matrices, but also especially in the alveolar lymphoid follicles and connective tissues surrounding blood vessels in the inflammatory granular synovium which formed the pannus. HA levels in the synovium of patients with rheumatoid arthritis (RA) (459.0±66.2 g/g) where shown to be higher than those in patients with osteoarthritis (246.9±34.8 g/g) and traumatic injury (227.7±35.4 g/g). It follows from the present findings, HA in the synovium might contribute to the high amounts of serum levels of HA in patients with rheumatoid arthritis.     

The circulating molecular weight forms of infused recombinant insulin-like growth factor-I and effects on glucose and fat metabolism in lambs

Summary We have investigated the relationship between the plasma distribution of infused recominant insulin-like growth factor-I across the insulin-like growth factor binding proteins and the resultant effects on glucose and fat metabolism. The studies were performed in 24-h fasted ram lambs which received primed constant infusions of ³H labelled glucose tracer. When isotopic equilibrium had been reached, the animals received 90-min infusions of human insulin-like growth factor-I at various doses (2.5, 20, 40 and 120 g· kg^–1·h^–1, n=3 for each dose). Total plasma insulin-like growth factor-I was significantly elevated by infusion at a rate of 40 g·kg^–1·h^–1 (from 185±14 g/l to 442±41 g/l, p<0.05) and 120g·kg^–1h^–1 (from 181±2 g/l to 953±39 g/1, p<0.005). The plasma concentrations of insulin-like growth factor-I not associated with binding proteins remained undetectable (<15 g/l) at the end of the 2.5 and 20 g·kg^–1·h^–1 doses, but were significantly elevated at the end of the 40 and 120 g·kg^–1·h^–1 infusions (to 71±14 g/l, p<0.05 and 176±55 g/l, p<0.01 respectively). The infused insulin-like growth factor-I associated primarily with 35–60 kilodalton binding proteins. Glucose kinetics were significantly altered only by the highest dose infusion, during which there was a fall in plasma glucose concentration from 3.5±0.2 mmol/l to 1.9±0.2 mmol/l (p<0.05). This was due to a 51% increase in the rate of glucose clearance. There was no significant change in the rate of glucose production. The plasma concentrations of glycerol and non-esterified fatty acid were not changed by any of the doses infused. We conclude that the hypoglycaemic action of infused recombinant insulin-like growth factor-I relates to a marked elevation of free insulin-like growth factor-I in the plasma, but that a threshold concentration of free insulin-like growth factor-I must be exceeded before this action is observed. The hypoglycaemic action of recominant insulin-like growth factor-I results primarily from an increase in glucose clearance while glucose metabolism was more sensitive than fat metabolism to infused recominant insulin-like growth factor-I. Both these actions contrast with those of insulin, and suggest that the acute metabolic effects of recombinant insulin-like growth factor-I are not mediated simply by cross-reaction with insulin receptors.     

Decreased protein catabolism after exercise in subjects with IDDM

Summary We examined whether the increased rates of protein catabolism (proteolysis and leucine oxidation) associated with moderate insulinopenia in subjects with IDDM would be accentuated by prior bicycle exercise (53% VO_2max for 82 min). Insulin infusions maintained plasma glucose concentrations on one study day in tight control (TC: 6 mmol/l) and on a separate day in loose control (LC: 12 mmol/l). Elevations in serum ketone body, plasma NEFA, and whole-blood branched-chain amino acid concentrations on the loose control day during the basal period persisted throughout the post-exercise recovery period. Amino acid kinetics were estimated during a primed, constant infusion of l-[1-¹³C]leucine from plasma dilution of -[1-¹³C]KIC and expired air ¹³CO₂ enrichments. Loose control was associated with increased rates of whole-body leucine oxidation (LC 25±7 vs TC 21±8 mol · kg^–1 · h^–1) and protein degradation (LC 127±12 vs TC 118±18 mol · kg^–1 · h^–1) (both p<0.05). During the 2-h post exercise recovery period, there were significant decreases in rates of leucine oxidation (LC 21±7, TC 16±7) and protein degradation (LC 112±13, TC 107±11), compared to the basal period (both p<0.05, basal vs recovery). Rates of wholebody protein synthesis were unchanged by prior exercise. In conclusion, moderate insulinopenia is associated with significantly higher rates of protein degradation and leucine oxidation in the basal state. Following exercise, net protein catabolism is diminished due to reduced rates of protein degradation in the presence of maintained rates of protein synthesis. The significantly increased concentrations of fat-derived substrates (ketone bodies, NEFA) may have prevented the predicted increases in protein catabolism which we anticipated would follow acute exercise during periods of relative insulin deficiency.Abbreviations IDDM Insulin-dependent diabetes mellitus - KIC -ketoisocaproic acid - ANOVA analysis of variance - VO_2max maximal aerobic capacity     

Dose-dependent effect of continuous subcutaneous verapamil infusion on experimental acute pancreatitis in mice

Calcium antagonists may limit experimental tissue injury by membrane stabilization. We studied the effects of verapamil on pancreatic ultrastructure and zymogen extraction during diet-induced acute pancreatitis. Acute pancreatitis was induced in female Swiss-Webster mice by feeding a choline- and methionine-deficient diet (CD) supplemented with 1% ethionine (CDE). Varying doses of verapamil in normal saline were infused continuously at a rate of 0.5l/hr for 96 hr through subcutaneously implanted osmotic pumps. The pancreata were examined blindly by light microscopy and by electron microscopy. Zymogen extracted from pancreatic tissue was measured and expressed per gram of protein. Mean histological scores, calculated according to a formula that incorporates the extent of necrosis, inflammation, acidophilia, and edema, were 14.1±4, 10.3±2, 9.9±4, 5.9±7, 12.5±4, and 12.7±4 for CDE-fed animals receiving 0, 0.14, 0.28, 0.56, 0.84, and 1.12M verapamil daily, respectively. Animals fed normal diet or CD had scores of 0±0. Histological scores were significantly lower in animals treated with 0.56m verapamil compared to animals who received no verapamil (p<0.05) and was associated with reduced dissolution of the zymogen granule membrane on EM. Mean extracted trypsinogen and chymotrypsinogen content were reduced in the CD- and CDE-fed mice. The reduction in mean trypsinogen content reached statistical significance in CDE-fed mice treated with verapamil 0.56M daily. Mean chymotrypsinogen content was also significantly reduced in the CD-fed mice and in mice treated with 0.56m and 0.84M of verapamil daily. Increasing doses of verapamil protect against diet-induced pancreatitis in mice. This protection is associated with reduction in zymogen granule membrane dissolution.     

Modulation of pacemaker activity in sheep cardiac Purkinje fibers by stimulation of β-adrenoceptor subtypes

The electrophysiological effects mediated by ₁- and ₂- in spontaneously active sheep cardiac Purkinje fibers were investigated using the non-selective agonist (–)-isoproterenol (IPN) and the selective agonists (–)-noradrenaline (₁) and procaterol (₂) in the absence and presence of the selective antagonists bisoprolol (₁) and ICI 118,551 (₂).IPN (0.01 mol/l) increased the spontaneous rate by 54% and the slope of diastolic depolarization by 68% of the respective control values. Further, IPN increased the action potential duration at –20 mV (APD –20 mV) from 96 to 154 ms, reduced the APD –70 mV by 17% and the duration of the diastole by 39% and slightly hyperpolarized the maximum diastolic potential. These effects were partially inhibited by ICI 118,551 (0.03 mol/l), diminished by bisoprolol (0.1 mol/l) and almost completely blocked by the combination of both antagonists. Concentration response curves of IPN were influenced by the selective antagonists as follows: ICI 118,551 (0.03 mol/l) shifted the curves to the right by 0.2–0.4 log units and increased the slope factor. Bisoprolol (0.1 mol/l) induced a greater shift to the right by 1.1–1.5 log units. Combination of bisoprolol with ICI 118,551 shifted the curves to the right by 1.5–1.7 log units.Noradrenaline (0.3 mol/l) elicited similar actions as IPN. Bisoprolol (0.1 mol/l) shifted the concentration response curves of noradrenaline to the right by 1.1–1.9 log units. Actions of procaterol (0.1 mol/l) were weak, attained only 15–35% of the maximal effects of IPN and could be blocked by ICI 118,551 (0.03 mol/l).These results show that the increase of pacemaker activity induced by catecholamines in sheep cardiac Purkinje fibers is predominantly mediated by stimulation of ₁. However, contribution of ₂ mediated effects could be demonstrated.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr, 40008786.     

Gastric secretion and emptying in hypothyroidism

Hypothyroidism is commonly thought to cause decreased gastric emptying and secretion, but these may be related to associated autoimmune disease or chronic changes. Therefore, we measured gastric emptying and secretion in 11 healthy controls and in nine patients (19–54 years old; five females and 4 males) rendered athyreotic by surgery and/or¹³¹I for thyroid cancer. Replacement T₄ was stopped 47–65 days and subsequent replacement T₃ was stopped 33–40 days before the study. All patients were symptomatic with complaints including weight gain, lethargy, and constipation. Deep tendon reflexes had delayed relaxation phase. Serum cholesterol and creatine phosphokinase levels were elevated. Thyroid hormone levels were markedly decreased (means ±se; T₄: 0.7±0.3 g/dl; free T₄: 0.2±0.1 ng/dl; T₃: 28±6 ng/dl) and TSH was markedly increased (88±16 U/ml). Gastric fractional emptying rate (%/min) and hydrogen ion (H⁺) output (meq/hr) were determined before and following two sequential stimulations: a 250-ml water load and an intravenous infusion of pentagastrin (6 g/kg/hr). There were no significant differences between controls and athyreotic patients. Our data demonstrated that short-term, profound, thyroid hormone deficiency does not modify gastric emptying or acid output.The opinions and assertions contained are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of Defense.     

Calcium-channel blockers inhibit human low-density lipoprotein oxidation by oxygen radicals

Summary Previous studies have shown that calcium channel blockers may reduce the development of experimental athero-sclerosis, and that nifedipine may slow the progression of coronary atherosclerosis in humans. The mechanisms responsible for this antiatherogenic effect are still unclear. It has been recently proposed that oxygen free radicals can induce the oxidation of human low-density lipoproteins (LDL) and that oxidized LDL may be an atherogenic stimulus. Previous studies in other systems have shown that calcium channel blockers may effectively inhibit oxygen radical-induced lipid peroxidation in vitro. Thus, the aim of the present study was to investigate whether calcium channel blockers may also reduce LDL modifications induced by oxygen radicals. Isolated human LDL were exposed to oxygen radicals generated by CuSO₄ (10 M for 18 hours) after a 30 minute preincubation with different concentrations (1–100 M) of nifedipine, diltiazem, and verapamil. Lipid peroxidation was measured from malonyldihaldehyde (MDA) production. Oxygen radical-induced damage on apolipoprotein-B₁₀₀ was evaluated by acrylamide and agarose gel electrophoresis. Calcium channel blockers dose-dependently prevented oxidation of both the lipid and protein components of LDL. MDA formation was reduced in LDL pre-incubated with calcium antagonists before exposure to oxygen radicals (% MDA inhibition was 89.8±6.9 with 30 M nifedipine, 68.6±4.9 with 30 M verapamil, and 65.6±7.1 with 30 M diltiazem; p< 0.01 vs. controls). Similarly, apolipoprotein-B₁₀₀ integrity was preserved against oxygen radical attack in the presence of calcium antagonists. Thus, calcium channel blockers reduce the oxidation of human LDL in vitro. These data suggest that reduced formation of atherogenic oxidized LDL may be an additional mechanism for the antiatherosclerotic effects of calcium channel blockers in vivo.     

In vivo assessment of the inotropic and toxic effects of oxidized ouabain

Summary Oxidized ouabain, a product of the oxidative cleavage of the rhamnose ring in ouabain has been found to have a higher inotropic toxic ratio in cultured cardiac myocytes.The purpose of our study was to evaluate the efficacy and toxicity of oxidized ouabain in comparison with ouabain in intact animals. Drugs were infused to healthy cats; the positive inotropic effect, and the time-course of development of arrhythmia were followed and recorded until death. Oxidized ouabain was associated with a higher increase in arterial blood pressure, a mean increase of 41±19% as compared with 21±8% in the ouabain group (p<0.10). There were no significant differences in maximal increases of dP/dt or dP/dt/P (65±29%, 28±10% for oxidized ouabain and 49±16%, 27±11% for ouabain, respectively). The mean doses causing persistent arrhythmia (toxic dose) were 93±23 g/kg of oxidized ouabain vs 39±14 g/kg of ouabain. Lethal arrhythmias were produced by 215±46 g/kg of oxidized ouabain and 62±16 g/kg of ouabain. The radio of toxic to lethal doses was 0.62±0.11 for ouabain vs 0.45±0.09 for oxidized ouabain (p<0.05), but the inotropic to toxic dose ratios were not different.We conclude that oxidized ouabain acts similarly to the known cardiac glycosides in doses which produce inotropic effects in cats, has a lower potency as compared to ouabain, and appears to have a more benign course of intoxication.     

Effects of dilevalol in the anesthetized pig with a partially occluded coronary artery

Summary The beta-adrenoceptor antagonist dilevalol in a total dose of 430 g/kg IV, potently suppressed isoprenaline-induced increases in heart rate and max LVdP/dt (dose ratios of 42±6 and 38±5, respectively, in anesthetized pigs), but a dose of 1430 g/kg did not appreciably modify phenylephrine-induced increases in arterial blood pressure (dose ratio< 4) in both anesthetized and conscious pigs. The actions of dilevalol on ischemic myocardium of anesthetized pigs were investigated following a reduction of left anterior descending artery flow by 85–90%. Dilevalol (300 g/kg), administered after 15 minutes of ischemia, did not affect the ischemia-induced changes in systemic hemodynamics (such as heart rate, max LVdP/dt and cardiac output), myocardial perfusion, and wall-thickening of the ischemic segment during the following 15 minutes of ischemia and 2 hours of reperfusion. The reasons for the lack of antiischemic actions are most likely the absence of negative chronotropy and an absence of afterload reduction by dilevalol.