Relationship between action potential, contraction-relaxation pattern, and intracellular Ca2+ transient in cardiomyocytes of dogs with chronic heart failure

Abnormalities of contractile function have been identified in cardiomyocytes isolated from failed human hearts and from hearts of animals with experimentally induced heart failure (HF). The mechanism(s) responsible for these functional abnormalities are not fully understood. In the present study, we examined the relationship between action potential duration, pattern of contraction and relaxation, and associated intracellular Ca²⁺ transients in single cardiomyocytes isolated from the left ventricle (LV) of dogs (n = 7) with HF produced by multiple sequential intracoronary microembolizations. Comparisons were made with LV cardiomyocytes isolated from normal dogs. Action potentials were measured in isolated LV cardiomyocytes by perforated patch clamp, Ca²⁺ transients by fluo 3 probe fluorescence, and cardiomyocyte contraction and relaxation by edge movement detector. HF cardiomyocytes exhibited an abnormal pattern of contraction and relaxation characterized by an attenuated initial twitch (spike) followed by a sustained contracture ('dome') of 1 to 8 s in duration and subsequent delayed relaxation. This pattern was more prominent at low stimulation rates (58% at 0.2 Hz, n = 211, 21% at 0.5 Hz, n = 185). Measurements of Ca²⁺ transients in HF cardiomyocytes at 0.2 Hz manifested a similar spike and dome configuration. The dome phase of both the contraction/relaxation pattern and Ca²⁺ transients seen in HF cardiomyocytes coincided with a sustained plateau of the action potential. Shortening of the action potential duration by administration of saxitoxin (100 nM) or lidocaine (30 μM) reduced the duration of the dome phase of both the contraction/relaxation profile as well as that of the Ca²⁺ transient profile. An increase of stimulation rate up to 1 Hz caused shortening of the action potential and disappearance of the spike-dome profile in the majority of HF cardiomyocytes. In HF cardiomyocytes, the action potential and Ca²⁺ transient duration were not significantly different from those measured in normal cells. However, the contraction-relaxation cycle was significantly longer in HF cells (314 ± 67 ms, n = 21, vs. 221 ± 38 ms, n = 46, mean ± SD), indicating impaired excitation-contraction uncou pling in HF cardiomyocytes. The results show that, in cardiomyocytes isolated from dogs with HF, contractile abnormalities and abnormalities of intracellular Ca²⁺ transients at low stimulation rates are characterized by a spike-dome configuration. This abnormal pattern appears to result from prolongation of the action potential. Received 22 January 1998; received after revision 16 March 1998; accepted 27 March 1998     

Increased Na+-H+ exchanger activity in the ileal brush-border membrane of spontaneously hypertensive rats

In the present study, we have examined the intestinal Na⁺ transport, through the Na⁺-H⁺ exchanger, in ileal brush-border membrane vesicles (BBMV) isolated from spontaneously hypertensive rats (SHR), and normotensive Wistar Kyoto (WKY) rats as a control group. Na⁺ uptake into ileal BBMV was stimulated in the presence of a proton gradient (pH 5.5 inside/pH 7.5 outside) in SHR and WKY rats, resulting in a transient accumulation (overshoot) in both groups of rats. No overshoot was observed in the absence of a pH gradient. The magnitude of the accumulation was significantly higher in SHR than in WKY rats. Uptake of Na⁺ at equilibrium was identical in the presence and the absence of a proton gradient and was not changed in SHR. The use of amiloride inhibited pH gradient-driven Na⁺ uptake in a dose-dependent manner with a Ki of 90 μM and 100 μM for SHR and WKY rats, respectively. The relationship between proton gradient-driven Na⁺ uptake and external Na⁺ concentration was saturable and conformed to Michaelis-Menten kinetics in both SHR and WKY rats. Lineweaver-Burk analysis of the pH gradient-driven Na⁺ uptake indicated values of V_max that were significantly increased in SHR compared to WKY rats (11.4±0.55 nmol/mg/8 s vs. 4.96±0.78 nmol/mg/8 s for SHR and WKY rats, respectively). In contrast, similar K_m values for Na⁺ were found between SHR and WKY rats (4.0±0.2 mM vs. 4.9±0.6 mM for SHR and WKY rats, respectively). These studies show derangement in ileal BBMV Na⁺ transport of SHR, which is characterized by increased Na⁺-H⁺ exchanger activity. Received 18 December 1996; received after revision 3 February 1997; accepted 7 February 1997     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Dynamic insulin secretion from perifused rat pancreatic islets

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 μM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 μM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K⁺ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED₅₀ = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED₅₀ = 89 nM) and DA (ED₅₀ = 2.2 μM). γ-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances. Received 16 January 1998; received after revision 6 May 1998; accepted 8 May 1998     

Effect of insulin in vivo on the synthesis of free fatty acids (FFA) in chicken heart and skeletal muscle

Summary The effect of insulin on the synthesis of free fatty acids from glucose in the skeletal and heart muscles of chicken is examined. 10 min after glucose-(U-¹⁴C) administration, labeled free fatty acids (FFA) appeared in both skeletal and heart muscles. 0.75 IU of insulin kg^–1 b. wt significantly increased the labeled FFA at the 30, 60 and 120 min intervals, with a maximum at 60 min.     

Production of functional rat liver PSP protein in Escherichia coli

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH₂-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998     

Chemie und Stoffwechsel der Polyenfettsäuren

Summary The chemical structure of the polyenoic fatty acids occuring in organ phosphatides and in fish oils is reviewed. The double bonds of all these polyenoic acids are arranged in divinylmethane pattern. Except some of the C₁₆-polyenoic acids of fish oils, these polyenoic acids belong either to the oleic, linoleic or linolenic acid type and have chain lengths C₁₈, C₂₀, and C₂₂. Polyenoic acids of the oleic acid type are present only in small amounts in phosphatides of mammalian origin. Fish oils are lacking in these but predominantly contain polyenoic acids of the linolenic acid type.Metabolic studies have shown that polyenoic acids of linoleic acid type (e.g. arachidonic acid) originate from linoleic acid and those of the linolenic acid type (e.g. C₂₀-pentaenoic and C₂₂-hexaenoic acid) from linolenic acid-both supplemented exogenous-by extension of the carbon chain by acetate on the side of the carboxylic acid group and introduction of additional double bonds in the divinylmethane pattern directed toward the carboxylic acid group. There is some evidence, however, that a total synthesis of the polyenoic acids of the oleic acid type occurs in the animal body.The transformation of linolenic to C₂₂-hexaenoic acid and some intermediate reactans have been investigated more precisely by means of the tracer method. As far as the biosynthesis of the polyenoic fatty acids is concerned there are no fundamental differences in different vertebrates.

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Nach einem Vortrag auf der gemeinsamen Tagung der deutschen, französischen und schweizerischen Biochemiker in Zürich vom 10.–12. Oktober 1960.     

Primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum

This is the first report on a primary culture of dispersed skin epidermal cells of rainbow trout Oncorhynchus mykiss Walbaum. These primary cells revealed a low seeding efficiency after 3 days (11.6 ± 4.6%), whereas subcultured cells had a higher seeding efficiency at the same time point (75.5 ± 34.0%) and increased in cell number (150 – 200% of seeded cells after 20 to 30 days). The cells were characterized applying histological, immunocytochemical and ultrastructural methods. The culture consisted of undifferentiated keratinocytes. Mucous cells as well as differentiated epithelial cells were absent. To date the cells were cultured for maximally 9 passages and 402 days and therefore provide the possibility for long-term studies. Received 31 March 1998; received after revision 14 July 1998; accepted 14 July 1998     

Nest cell lining of the solitary beeHylaeus bisinuatus (Hymenoptera: Colletidae)

The nest cell lining ofHylaeus bisinuatus (Hymenoptera: Colletidae) was shown by high-resolution solidstate [¹³C]NMR to be composed of lipid polymer and protein. The lipid polymer was shown by reduction and subsequent GC/MS analysis to be comprised of -hydroxy fatty acids (C₂₀, C₂₂, C₂₄ and C₂₆) and fatty alcohols (C₁₆ to C₃₀). The protein portion of the lining had a silk-like amino acid composition.     

Furan fatty acid steryl esters from the marine spongeDictyonella incisa which show inflammatory activity

Summary Three new furan fatty acids, (9Z, 19Z)-3, 6-epoxyhexacosa-3,5,19-tetraenoic acid (1a), (8Z, 11Z, 14Z, 17Z)-3, 6-epoxyeicosa-3,5,8,11,14,17-hexaenoic acid (2a), and (8Z, 11Z, 14Z, 17E)-3, 6-epoxyeicosa-3,5,8,11,14,17-hexaenoic acid (3a), and a new polyunsaturated fatty acid (5Z, 8Z, 11Z, 14Z, 17E)-eicosa-5,8,11,14,17-pentaenoic acid (6a), present in the spongeDictyonella incisa as the respective steryl esters, have been isolated as methyl esters and their structures have been determined by spectral and chemical analysis. The furan fatty acid esters have shown a high inflammatory activity, which suggests their potential role as feeding deterrents.     

Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins

Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish. Received 22 December 1997; received after revision 5 March 1998; accepted 5 March 1998     

Fatty acid metabolism in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid -oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the -oxidation products out of the peroxisome to mitochondria for full oxidation to CO₂ and H₂O.Received 19 February 2003; received after revision 27 March 2003; accepted 27 March 2003     

Über die Fettsäuren aus Spinatchloroplasten

Summary The fatty acids of two samples of chloroplasts fromSpinacia oleracea have been investigated quantitatively. They contain many polyenoic fatty acids (more than 70% of the total fatty acids); amongst these, a relatively high content of C¹⁶-trienoic acid is remarkable. Moreover, the presence of the ³-trans-hexadecenoic acid, recently detected in plants, is noteworthy.     

Sex pheromone conversion and degradation in antennae of the silkworm mothBombyx mori L.

Summary In living antennae of the silkworm mothBombyx mori L. the pheromone compound (E, Z)-10,12-hexadecadienol and hexadecanol are enzymatically converted to their corresponding fatty aldehydes, acids and long-chain fatty acid esters. The pheromone is completely degraded at high rates in the antennae of freshly hatched moths. The polar volatile [³H]metabolites exclusively consist of tritiated water. The half-life of the pheromone is about 2.5 min in males and 0.5 min in females. Drying inactivates the enzymes responsible for pheromone oxidation.Dedicated to Professor Adolf Butenandt on the occasion of his 85th birthday     

Down-regulation of sodium current in chronic heart failure: effect of long-term therapy with carvedilol

Evidence has accumulated recently about the importance of alterations in Na⁺ channel function and slow myocardial conduction for arrhythmias in the infarcted and failing heart. The present study tested a hypothesis that Na⁺ current (I_Na/C) density decreases in chronic heart failure (HF) and that Na⁺ channel (NaCh) functional density can be restored by long-term therapy with carvedilol, a mixed α- and β-adrenergic blocker. Studies were performed using a canine model of chronic HF produced in dogs by sequential intracoronary embolizations with microspheres. HF developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 28 ± 1 %). Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium, and I_Na was measured by whole-cell patch-clamp. The maximum I_NA/C was decreased in failing (n = 19) compared to normal (n = 12) hearts (33.1 ± 1.6 vs 48.5 ± 5.1 pA/pF, mean ± SE, p < 0.001). The steady-state inactivation and activation of I_Na remained unchanged in failing compared to normal hearts. Long-term treatment with carvedilol (1 mg/kg, twice daily for 3 months) normalized I_Na/C in dogs with HF. I_Na/C in HF dogs (n = 6) treated with carvedilol was higher compared to that of non-treated HF dogs (n = 6) (49.4 ± 0.9 vs 29 ± 4.8 pA/pF, p < 0.007). In vitro culture of VCs of failing hearts for 24 h did not restore I_Na/C. However, I_Na/C was partially restored when VCs were incubated for 24 h with BAPTA-AM, an intracellular Ca²⁺ buffer. Thus, we conclude that experimental chronic HF in dogs results in down-regulation of the functional density of NaCh that can be restored by long-term therapy with carvedilol. The mechanism of NaCh down-regulation in HF may be linked to poor Ca²⁺ handling in this stage of disease. Received 4 June 2002; received after revision 1 July 2002; accepted 17 July 2002 RID="*" ID="*"Corresponding author.     

Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the k _cat/K _m values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.     

Gesteigerte Saugfrequenz vonDactynotus jaceae L. (Homoptera,Aphididae) bei Erhöhung des Aminosäurengehaltes in der Wirtspflanze

Summary In isolated shoots ofCentaurea jacea, placed in a solution of 0.5% glutamic acid for 15 h, the concentration of free amino acids in the stems is more than doubled. Compared with the controls, these shoots with an increased amino acid concentration are preferred by aphids in the preference-test.     

α1-Adrenergic stimulation of ketogenesis and fatty acid oxidation is associated with inhibition of lipogenesis in rat hepatocytes

Summary The effect of norepinephrine on fatty acid synthesis (³H₂O incorporation into fatty acids), on fatty acid oxidation to CO₂ and on ketogenesis was studied in isolated hepatocytes of fed rats. After incubation with norepinephrine (50 M), lipogenesis was lower (5.7±1.1 nmoles³H₂O incorporated into fatty acids/mg dry weight/30 min) than in controls (7.5±1.7; n=6, p<0.02). In contrast, (1-¹⁴C) palmitate conversion into total ketone bodies was increased to 10.9±1.8 nmoles/mg/30 min with norepinephrine, vs 8.5±1.6 in controls (p<0.05), and more (1-¹⁴C) palmitate was converted to¹⁴CO₂ with norepinephrine than in controls (1.48±0.10 nmoles/mg/30 min vs 1.06±0.11, p<0.05). The inhibitory effect of norepinephrine on lipogenesis was abolished by addition of the ₁-receptor blocker prazosin, but not by ₂ or -blockers. The results demonstrate that the ketogenic effect of norepinephrine is coupled with an inhibitory effect on lipogenesis which may be explained by diminished activity of acetyl-CoA carboxylase, diminished formation of malonyl-CoA and decreased activity of carnitine palmitoyl transferase I.     

Shortening velocity of single muscle cells isolated from a molluscan smooth muscle

Summary The maximal unloaded shortening velocity (V_max) of smooth muscle cells isolated from the pedal retractor muscle ofMytilus was more than twice as large as that of the whole muscle, suggesting the presence of extracellular components which resist the contraction of the whole muscle. The V_max of the isolated cells was almost constant at cell lengths ranging between 0.5 and 0.831₀ (1₀, optimal length for tension generation) indicating that the intracellular resistance to contraction is negligible within this range of lengths.     

Effect of maternal under-nutrition on pup body weight and hypothalamic endocannabinoid levels

Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB₁ receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB₁ receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development. Received 4 November 2002; received after revision 29 November 2002; accepted 16 December 2002 RID="*" ID="*"Corresponding author.