The effects of catecholamines on tension reactivation in cardiac muscle

The effects of adrenaline and the beta-agonist isoprenaline on the time course of tension reactivation were studied in several cardiac tissues. The aim of the study was to assess whether experimental evidence can be found for a role of the sarcoplasmic reticulum in the reactivation of tension. It was assumed that calcium recycles between different parts of the reticulum, and that this recycling may affect tension repriming. Isoprenaline was assumed to enhance such recycling by increasing the uptake of calcium, following its release during a preceding contraction. Isoprenaline (in the range of 40 nM to 4 microM) was found to enhance tension repriming in adult guinea pig atria. However, in adult rat atria, isoprenaline often gave a complex effect, with a smaller degree of repriming at short intervals, and enhanced repriming at longer intervals. This was thought to reflect the balance between the enhancing effect of the drug on calcium recycling and an augmented release from the sarcoplasmic reticulum (SR). In striking contrast, there was no effect of isoprenaline on tension repriming in neonatal guinea pig atria and a retardation in neonatal rat atria. This was interpreted as reflecting the lack of a sarcoplasmic network in the neonatal tissue. The effects of isoprenaline on tension repriming in the frog atrium (which also has a sparse sarcoplasmic reticulum network) were also found to be complex; low concentrations (40 nM) enhanced the process, and high concentrations (0.4 microM) retarded it. Intermediate levels often produced a 'crossover' effect: more reactivation at short intervals, and less at long intervals. The interpretation of these results was that there are two processes which interact to determine the amount of tension produced at short intervals after each contraction: the basal reactivation process and some augmenting mechanism superimposed on it. This mechanism is probably related to other behavioural features of cardiac muscle, such as rate-dependent increases in membrane calcium currents. It is relevant mainly in those cases where tension repriming depends on membrane calcium currents. Further experiments (in the frog atrium) with elevated calcium and with the alpha-adrenergic agonist phenylephrine (both of which slowed down the reactivation process) also support this idea. These agents elevate internal calcium levels, and presumably saturate the augmenting mechanism (by producing maximal tension responses).(ABSTRACT TRUNCATED AT 400 WORDS)     

A method for staining and stabilizing peroxidase activity in polyacrylamide gel electrophoresis

A procedure was developed for a rapid double staining of peroxidase and other proteins in the same polyacrylamide gels using guaiacol and Coomassie blue. The distinguishable colored bands of peroxidase isozymes and proteins are stable for at least 8 months.     

Imaging of Oil/Monoglyceride Networks by Polarizing Near-Field Scanning Optical Microscopy

Polarizing near-field scanning optical microscopy (NSOM) was applied for visualization of lipid coagel structures. The technique ensures obtaining polarization contrast images at micro- and nanoscale resolution. Comparison to the polarizing light microscopy images revealed that the same fractal structural organization persists also at submicron scale, at the level of primary ordered structures creation. Many long birefringent needle-shaped primary crystallites were imaged in the corn oil:monoglyceride samples, and lower amount of smaller oval-shaped primary crystallites—in the olive oil:monoglyceride samples. Unlike atomic force microscopy, polarizing NSOM brought direct evidence on the physical state of specific features. Compared to the polarizing light microscopy, polarizing NSOM provided additional information on the structural organization of oil–monoglyceride coagels at the micro- and submicron scale.     

Gender differences in ANG II levels and action on multiple K+ current modulation pathways in diabetic rats

Gender differences were studied in ventricular myocytes from insulin-deficient (Type 1) diabetic rats. Cells were obtained by enzymatic dispersion of hearts from control male and female rats and from rats made diabetic with streptozotocin (100 mg/kg) 7-14 days before experiments. ANG II content, measured by ELISA, was augmented in diabetic males but unaltered in diabetic females. In diabetic ovariectomized females, ANG II levels were augmented as in males. ANG II affects multiple cellular pathways including activation of protein kinase C (PKC) and several tyrosine kinases as well as inhibition of protein kinase A (PKA). The involvement of these pathways in modulating outward K(+) currents was studied. Transient and sustained outward K(+) currents were measured using the whole cell voltage-clamp method. In males, these currents are attenuated under diabetic conditions but are augmented by the ANG II-converting enzyme inhibitor quinapril. Activation of PKA by 8-bromo-cAMP enhanced both K(+) currents in cells from diabetic males. The augmentation of these currents by quinapril was blocked when PKA inhibition was maintained with the Rp isomer of 3',5'-cyclic monophosphorothioate. Inhibition of tyrosine kinases by genistein also augmented K(+) currents in cells from diabetic males. Action potentials were abbreviated by 8-bromo-cAMP and genistein. However, both genistein and 8-bromo-cAMP had no effect on K(+) currents in cells from diabetic females. In cells from ovariectomized diabetic females, 8-bromo-cAMP and genistein enhanced these K(+) currents as in males. Inhibition of PKC augmented the transient and sustained K(+) currents in cells from diabetic males and females. A contribution of non-ANG II-dependent activation of PKC is suggested. These results describe some of the mechanisms that may underlie gender-specific differences in the development of cardiac disease and arrhythmias.     

Small angle X-ray scattering of resistant starch type III

Starch fraction, which is resistant to enzymatic digestion, is produced during retrogradation. This fraction, termed resistant starch type III (RSIII), has health benefits such as pre-biotic effects, improving lipid and cholesterol metabolism, and reducing the risk of colon cancer. The work presented in this paper is aimed at investigating the colloidal structure of native high amylose corn starch (HACS) and the RSIII polymorphs produced from it using small angle X-ray scattering. Experimental scattering curves were fitted with appropriate theoretical models such as the "modified lamellar model". Our results show that retrogradation at low temperature leads to formation of polymorph B with crystallinity much lower than that of the granular form, but the lamellas are arranged in long-range periodicity. Conversely, retrogradation at high temperature leads to formation of polymorphs A and V with no defined periodicity. The degree of crystallinity is very low, and the system is better described as a dilute particulate system.     

Lst1p and Sec24p cooperate in sorting of the plasma membrane ATPase into COPII vesicles in Saccharomyces cerevisiae

Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of alpha-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles.     

Insulin resistance and the modulation of rat cardiac K(+) currents

K(+) currents were measured using a whole cell voltage-clamp method in enzymatically isolated rat ventricular myocytes obtained from two hyperinsulinemic, insulin-resistant models. Fructose-fed rats as well as genetically obese rats, both of which are resistant to the metabolic effects of insulin, were used. The normal augmentation of a calcium-independent sustained K(+) current was reduced or abolished in insulin-resistant states. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadyl sulfate (3-4 wk treatment or after 5-6 h in vitro) enhanced the sustained K(+) current. The in vitro effect of vanadyl was blocked by cycloheximide. Insulin resistance of the K(+) current was not reversed by vanadyl sulfate. The results show that insulin resistance is expressed in terms of insulin actions on ion channels, in addition to its actions on metabolism. This resistance can be reversed by the insulin-sensitizing drug metformin. Vanadate compounds, which mimic the effects of insulin on metabolism, also mimic the augmenting effects of insulin on a cardiac K(+) current in a manner suggesting synthesis of new channels.