Regulation of cardiac L-type Ca2+ current in Na+-Ca2+ exchanger knockout mice: functional coupling of the Ca2+ channel and the Na+-Ca2+ exchanger

L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.     

Middle cerebral artery blood velocity during intense static exercise is dominated by a Valsalva maneuver.

Lifting of a heavy weight may lead to "blackout" and occasionally also to cerebral hemorrhage, indicating pronounced consequences for the blood flow through the brain. We hypothesized that especially strenuous respiratory straining (a Valsalva-like maneuver) associated with intense static exercise would lead to a precipitous rise in mean arterial and central venous pressures and, in turn, influence the middle cerebral artery blood velocity (MCA V(mean)) as a noninvasive indicator of changes in cerebral blood flow. In 10 healthy subjects, MCA V(mean) was evaluated in response to maximal static two-legged exercise performed either with a concomitantly performed Valsalva maneuver or with continued ventilation and also during a Valsalva maneuver without associated exercise (n = 6). During static two-legged exercise, the largest rise for mean arterial pressure and MCA V(mean) was established at the onset of exercise performed with a Valsalva-like maneuver (by 42 +/- 5 mmHg and 31 +/- 3% vs. 22 +/- 6 mmHg and 25 +/- 6% with continued ventilation; P < 0.05). Profound reductions in MCA V(mean) were observed both after exercise with continued ventilation (-29 +/- 4% together with a reduction in the arterial CO(2) tension by -5 +/- 1 Torr) and during the maintained Valsalva maneuver (-21 +/- 3% together with an elevation in central venous pressure to 40 +/- 7 mmHg). Responses to performance of the Valsalva maneuver with and without exercise were similar, reflecting the deterministic importance of the Valsalva maneuver for the central and cerebral hemodynamic response to intense static exercise. Continued ventilation during intense static exercise may limit the initial rise in arterial pressure and may in turn reduce the risk of hemorrhage. On the other hand, blackout during and after intense static exercise may reflect a reduction in cerebral blood flow due to expiratory straining and/or hyperventilation.     

Separation of heavy metals from water solutions at the laboratory scale

Separation of heavy metals from water may be done by sulphide precipitation. In order to make the metal sulphides more valuable for reuse, they should be as pure as possible. To separate the metals from each other during the precipitation process different parameters as pH, sulphide concentration and redox potential may be controlled. Adjustment of the redox potential to specific values results in separation of copper from cadmium even at the same pH. In the copper precipitation step 98.7 +/- 0.5% was precipitated and the remaining part was transferred to the cadmium precipitation step where 0.7 +/- 0.5% of the copper precipitated. The main part of cadmium (95.6 +/- 1.3%) was precipitated in the second step. These results show that it is possible to separate copper from cadmium by the use of redox potential control.     

Atrial muscle cells from hearts of adult guinea-pigs in culture: a new preparation for cardiac cellular electrophysiology

By means of a modified Langendorff perfusion technique using collagenase and elastase cell suspensions of viable myocytes from atria of adult guinea-pigs can be obtained. If the cell isolation is performed aseptically the myocytes can be kept in long term cell culture. Under these conditions the cells attach to the bottom of the culture dish within 12 to 24 h after plating. Thereafter they round up forming spherical 'cardioballs'. These cardioballs are highly suitable for electrophysiological experiments using different configurations (cell-attached and cell-free) of the patch-clamp technique. They can be employed for these experiments for up to 8 days after isolation. Thereafter they tend to flatten resembling embryonic heart cells in tissue culture.     

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D-erythro-sphingosyl-phosphorylcholine; SPC) and sphingosine 1-phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D-erythro-(2S,3R)-SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure-activity relationship: it requires an unsubstituted amino group because N-acetyl-SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein-coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.     

Fluid or gel phase lipid bilayers to study peptide-membrane interactions?

Since many studies on peptide-membrane interactions are carried out only with fluid phase lipid bilayers (L _α -phase, absence of cholesterol) we have investigated whether this phase is really a suitable model for biological membranes. For this purpose the action of melittin on zwitterionic and negatively charged phospholipid bilayers, in the absence and presence of 30 mol% cholesterol, was investigated by solid state ³¹P-NMR. From the NMR point of view, it appears that systems composed of a single phospholipid best mimic the sterol-containing system a few degrees below the gel-to-fluid phase transition, i. e., in the rippled phase (P _β' ). It is then proposed that a relatively rigid membrane containing local defects, rather than a L _α -bilayer, is required as an appropriate model for natural membranes when probing the action of melittin. Such requirements might be crucial when studying peptide-lipid interactions. Received: 5 February 1996 / Accepted: 1 July 1996     

Spontaneous and experimentally evoked [Ca2+]i-transients in cardiac myocytes measured by means of a fast Fura-2 technique

A setup for dual wavelength-excitation fluorescence measurements is introduced which permits a temporal resolution of up to 1 KHz, using the Ca2(+)-sensitive fluorescent dye Fura-2. The system makes use of a novel technical solution for chopping between two excitation wavelengths which does not move any optical components. Two beams, which are alternatively opened or shut by a rotating chopper wheel, are united by a dichroic mirror and are used for low-noise epifluorescence microscopy. The system includes a device for fast changes of extracellular solution that can be used for studying various components of [Ca2+]i-regulation in excitable and non-excitable cells. Sample recordings of spontaneous and experimentally-evoked [Ca2+]i-transients from cardiac myocytes are presented. Cardiac myocytes are a cell species that produces particularly fast [Ca2+]i-transients and therefore, a high temporal resolution is required in order to study physiological and/or pharmacological properties of these transients.     

Microbial desulfurization of coal and oxidation of pure pyrite byThiobacillus ferrooxidans andAcidianus brierleyi

Summary Thiobacillus ferrooxidans andAcidianus brierleyi were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. First order reactions were assumed in the kinetic analysis performed. For oxidation of pure pyrite the rate constant was higher forA. brierleyi than forT. ferrooxidans. For sulfur removal from coal the values of the rate constants were comparable for the two microorganisms.