Calcium overload and cardiac function

The changes in cardiac function caused by calcium overload are reviewed. Intracellular Ca²⁺ may increase in different structures [e.g. sarcoplasmic reticulum (SR), cytoplasm and mitochondria] to an excessive level which induces electrical and mechanical abnormalities in cardiac tissues. The electrical manifestations of Ca²⁺ overload include arrhythmias caused by oscillatory (V_os) and non-oscillatory (V_ex) potentials. The mechanical manifestations include a decrease in force of contraction, contracture and aftercontractions. The underlying mechanisms involve a role of Na⁺ in electrical abnormalities as a charge carrier in the Na⁺-Ca²⁺ exchange and a role of Ca²⁺ in mechanical toxicity. Ca²⁺ overload may be induced by an increase in [Na⁺]_i through the inhibition of the Na⁺-K⁺ pump (e.g. toxic concentrations of digitalis) or by an increase in Ca²⁺ load (e.g. catecholamines). The Ca²⁺ overload is enhanced by fast rates. Purkinje fibers are more susceptible to Ca²⁺ overload than myocardial fibers, possibly because of their greater Na⁺ load. If the SR is predominantly Ca²⁺ overloaded, V_os and fast discharge are induced through an oscillatory release of Ca²⁺ in diastole from the SR; if the cytoplasm is Ca²⁺ overloaded, the non-oscillatory V_ex tail is induced at negative potentials. The decrease in contractile force by Ca²⁺ overload appears to be associated with a decrease in high energy phosphates, since it is enhanced by metabolic inhibitors and reduced by metabolic substrates. The ionic currents I_os and I_ex underlie V_os and V_ex, respectively, both being due to an electrogenic extrusion of Ca²⁺ through the Na⁺-Ca²⁺ exchange. I_os is an oscillatory current due to an oscillatory release of Ca²⁺ in early diastole from the Ca²⁺-overloaded SR, and I_ex is a non-oscillatory current due to the extrusion of Ca²⁺ from the Ca²⁺-overloaded cytoplasm. I_os and I_ex can be present singly or simultaneously. An increase in [Ca²⁺]_i appears to be involved in the short- and long-term compensatory mechanisms that tend to maintain cardiac output in physiological and pathological conditions. Eventually, [Ca²⁺]_i may increase to overload levels and contribute to cardiac failure. Experimental evidence suggests that clinical concentrations of digitalis increase force in Ca²⁺-overloaded cardiac cells by decreasing the inhibition of the Na⁺-K⁺ pump by Ca²⁺, thereby leading to a reduction in Ca²⁺ overload and to an increase in force of contraction.     

(+)-PN200-l 10 and Ouabain Binding Sites in Purified Bovine Adrenomedullary Plasma Membranes and Chromaffin Cells

Bovine adrenal medulla plasma membranes were purified by a differential centrifugation procedure using sucrose and Urografin discontinuous density gradients; the membranes were enriched 10-12-fold in acetylcholinesterase activity and [3H]ouabain binding sites. Specific (+)-[3H]PN200-110 binding to these membranes amounted to 90% of total binding and was saturable and of high affinity (KD = 41 pM; Bmax = 119 fmol/mg of protein) with a Hill coefficient close to 1, a result suggesting the presence of a single, homogeneous population of dihydropyridine receptors. The association and dissociation rate constants were, respectively, 7.5 X 108 M-1 min-1 and 0.023 min-1. Unlabeled (+)-PN200-110 displaced (+)-[3H]PN200-110 binding with a potency 100-fold higher than (-)-PN200-110 (IC50,0.5 and 45nM, respectively). Although the two enantiomers of BAY K 8644 completely displaced (+)-[3H]PN200-110 binding, they exhibited no stereoselectivity (IC50, 69 and 83 nM,respectively). Whereas ( +/- )-nitrendipine very potently displaced (+)-[3H]PN200-110 binding (IC50 = 1.3 nM) verapamil and cinnarizine displaced the binding by only 30 and 40% at 1 microM, and diltiazem increased it by 20% at 10 microM. [3H]Ouabain bound to plasma membranes with a KD of 34 nM and a Bmax of 9.75 pmol/mg of protein, a figure 80-fold higher than the Bmax for (+)-PN200-110. [3H]Ouabain also bound to intact chromaffin cells with a Bmax of 244 fmol/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同因子对毛地黄叶离体培养强心苷含量的影响

对毛地黄幼叶离体培养中,影响毛地黄强心苷含量的因子进行了研究。培养基的组成成分对毛地黄强心苷的合成十分重要,ＭＳ＋２,４－Ｄ１．０ｍｇ／Ｌ＋ＢＡ１．５ｍｇ／Ｌ＋活性炭０．５％有明显促进作用;外源植物激素是合成毛地黄强心苷必需的,在ＭＳ＋２,４－Ｄ１．０ｍｇ／Ｌ基本培养基中附加ＢＡ,ＢＡ的浓度在０．２－１．５ｍｇ／Ｌ范围内,浓度越大,强心苷含量越高;不同继代培养细胞的时期对毛地黄强心苷的合成起重要作用,随着继代的增加,毛地黄强心苷的含量呈渐降趋势;细胞的生长后期对毛地黄强心苷的合成效果最佳;不同光质的光照培养对毛地黄强心苷的积累也有影响,蓝光明显提高强心苷的合成量。     

Increased production of digoxin by digitoxin biotransformation using cyclodextrin polymer inDigitalis lanata cell cultures

Addition of β-cyclodextrin (β-CD) polymer during the biotransformation of digitoxin into digoxin using cell suspension cultures ofDigitalis lanata enhanced the conversion yield. Digitoxin showed better adsorption to CD polymer compared to digoxin, so that the optimization of addition time was found to be necessary. In the case of adding CD polymer 24 hours after the feeding of substrate digitoxin, the highest digoxin production could be achieved. At this period, digitoxin was almost consumed by cells and productivity was proportionally enhanced according as the amount of substrate was increased. Immobilization of CD polymer did not promote the biotransformation. When 3.33 g/L of CD polymer was added, 90% and 50% of digitoxin and digoxin was adsorbed respectively. Thus selective inclusion complex formation could be expected. Adsorption rate was found to be rapid and saturation was obtained within 10 hours of contact.     

Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Δ⁵-3β-Ηydroxysteroid dehydrogenase (Δ⁵-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ⁵-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ⁵-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed. Received: 20 January 1999 / Accepted: 5 May 1999     

Stress-induced expression of cyclophilins in proembryonic masses of Digitalis lanata does not protect against freezing/thawing stress

Using proembryonic masses (PEMs) of Digitalis lanata Erh., it was demonstrated that cold, hormonal or osmotic stress, which increased freezing tolerance during cryopreservation, induced an increasing level of two peptidyl-prolyl-cis/trans-isomerases (PPIases). The difference in pI (9.2 ± 0.2 and 9.5 ± 0.2, ±SD; n = 3) allowed the separation of the two enzymes by free-flow isoelectrophoresis. Both were inhibited by cyclosporin A and thus belong to the cyclophilin family of PPIases. The enzymes differed slightly in their substrate specificity and their relative molecular masses of 18038 ± 4 Da (D. lanataCyp18.0) and 18132 ± 3 Da (D. lanataCyp18.1). Both cyclophilins were blocked N-terminally. Partial internal amino acid sequences from the two cyclophilins, with a length of 34 amino acids, displayed 82% sequence identity to each other. Pretreatment of PEMs with abscisic acid, sorbitol or a combination of both substances led to a 270 ± 30% elevation of the total cytosolic cyclophilin concentration determined with a cyclophylin affinity sensor. During the first 4 d of pretreatment, the total PPIase activity was enhanced up to 230 ± SD% compared with the control culture. The lag phase between maximal PPIase concentration after 4 d of pretreatment and maximal effect of freezing tolerance after 10 d of pretreatment indicated that increasing levels of cytosolic PPIases may be necessary to overcome the stress induced by hormones and osmotica during pretreatment but not to protect against freezing/thawing stress. Received: 17 September 1998 / Accepted: 29 January 1999     

RAPD variation within and among natural populations of outcrossing willow-leaved foxglove (Digitalis obscura L.)

 Random amplified polymorphic DNA (RAPD) markers were used to assess levels and patterns of genetic diversity in Digitalis obscura L. (Scrophulariaceae), an outcrossing cardenolide-producing medicinal plant species. A total of 50 plants from six natural populations on the Iberian Peninsula were analysed by six arbitrarily chosen decamer primers resulting in 96 highly reproducible RAPD bands. To avoid bias in parameter estimation, analyses of population genetic structure were restricted to bands (35 of 96) whose observed frequencies were less than 1–3/n in each population. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of RAPDs (genotypic analysis) showed that most of the variation (84.8%) occurred among individuals within populations, which is expected for an outcrossing organism. Of the remaining variance, 9.7% was attributed to differences between regions, and 5.5% for differences among populations within regions. Estimates of the Wright, Weir and Cockerham and Lynch and Milligan F_ST from null-allele frequencies corroborated AMOVA partitioning and provided significant evidence for population differentiation in D. obscura. A non-parametric test for the homogeneity of molecular variance (HOMOVA) also showed significant differences in the amount of genetic variability present in the six populations. UPGMA cluster analyses, based on Apostol genetic distance, revealed grouping of some geographically proximate populations. Nevertheless, a Mantel test did not give a significant correlation between geographic and genetic distances. This is the first report of the partitioning of genetic variability within and between populations of D. obscura and provides important baseline data for optimising sampling strategies and for conserving the genetic resources of this medicinal species. Received: 7 September 1998 / Accepted: 28 November 1998     

Is there evidence for the post‐invasion evolution of increased size among invasive plant species?

Many plant species grow taller and have higher reproductive capacity where they are nonindigenous invaders than where they are native components of the flora. Traditionally, it has been accepted that this is a plastic response to a benign environment, though recently this assumption has been challenged and a genetic basis for increased plant size has been invoked. We tested the hypothesis that the increased size of certain weed species is genetically, rather than environmentally, based. A common environment growth experiment revealed no significant differences in the size of Carduus nutans , Digitalis purpurea , Echium vulgare or Senecio jacobaea sampled from alien (Australia and New Zealand) or native (Britain and continental Europe) habitats. We conclude that post-invasion genetic changes associated with increased size may be unusual and that the phenomenon, where it occurs, generally reflects a plastic response to a novel environment.