Thrombolysis in acute myocardial infarction.

Reperfusion of ischaemic myocardium with thrombolytic agents during the early stages of acute myocardial infarction reduces the mortality rate and can limit infarct size with associated sparing of left ventricular function. Effective and safe regimens are now available and in the absence of contraindications thrombolysis should now form part of the standard management acute myocardial infarction.

The calcium-release channel from cardiac sarcoplasmic reticulum: function in the failing and acutely ischaemic heart.

Junctional SR membrane vesicles have been isolated from chronically failing human hearts explanted at transplant operations. Vesicles have been incorporated into artificial planar phospholipid bilayers and the activity of single calcium-release channels investigated under voltage-clamp conditions. The properties of these channels are similar to those previously reported from normal animal tissue and do not provide evidence that the function of individual calcium-release channels is altered in the failing heart. Using radio-labelled ryanodine binding as a specific marker for the calcium-release channel, we demonstrate that, in the sheep heart, ischaemia results in the degradation of the calcium-release channel. The activation of proteases and oxidant stress in the ischaemic and re-perfused post-ischaemic myocardium are likely mediators of cell injury. Using the protease trypsin and the photosensitisation of rose bengal to generate the reactive oxygen species (ROS) singlet oxygen and superoxide radicals we demonstrate a direct effect on the calcium-release channel in vitro. Exposure of junctional SR vesicles to trypsin or oxidant stress resulted in the progressive loss of specific ryanodine binding and the degradation of high molecular weight proteins identified by polyacrylamide gel electrophoresis. The activity of single channels was also modified during exposure to proteolysis or oxidant stress; an initial increase in channel opening was observed followed by irreversible loss of channel function. Degradation of specific proteins, such as the calcium-release channel, may contribute to contractile dysfunction in the ischaemic and reperfused post-ischaemic myocardium.

A possible molecular mechanism for 'stunning' of the myocardium.

Stunning of the myocardium is mechanical dysfunction that persists after the re-establishment of coronary flow despite the absence of damage and the lack of any evidence for continuing ischaemia. Many mechanisms have been put forward and largely disproved. The most probable mechanism is abnormal function of the sarcoplasmic reticulum due to the generation of oxygen radicals within the myocardium. An hypothesis is proposed based on evidence indicating that the function of the calcium release channel of the sarcoplasmic reticulum is modified by reactive oxygen species before the channel is rendered non-functional.

Phosphodiesterase inhibitors and the cardiac sarcoplasmic reticulum calcium release channel: differential effects of milrinone and enoximone.

STUDY OBJECTIVE: The aim was to investigate possible interactions between milrinone or enoximone and the calcium release channel from cardiac sarcoplasmic reticulum. DESIGN: A membrane preparation enriched with "heavy" sarcoplasmic reticulum vesicles containing the calcium release channel was prepared from sheep myocardium. The incorporation of these vesicles into artificial lipid bilayers permitted investigation of the effects of the drugs on single calcium release channels under voltage clamp conditions. The effects of the drugs on radiolabelled ryanodine binding were also investigated as a functional probe for the activity of large populations of channels. MEASUREMENTS AND MAIN RESULTS: Milrinone (100 microM-2 mM) caused a reversible activation of channel opening when added at the cytoplasmic face of the channel. Lifetime analysis suggests this activation is synergistic with the effects of calcium on the channel. Milrinone also stimulated [3H]ryanodine binding, consistent with the proposition that it is an activating ligand of the calcium release channel. Enoximone (100 microM-1 mM) was without effect on both single channel activity and [3H]ryanodine binding. CONCLUSIONS: Activation of the calcium release channel probably contributes to the positive inotropic action in vivo of milrinone but not enoximone. Other drugs which activate the calcium release channel have been shown to be cardiotoxic, but it is not known whether this is a specific effect of channel activation or a more general result of raising cytoplasmic calcium concentration within the myocyte. Further research is required to determine accurately the mechanism of action of drugs with phosphodiesterase inhibitory activity.

Reactive oxygen species modify the structure and function of the cardiac sarcoplasmic reticulum calcium-release channel.

Restoration of blood flow to the ischemic myocardium prevents continuing cell necrosis, but reperfusion may cause irreversible damage to potentially salvable tissue, possibly through the generation of toxic reactive oxygen species. Intracellular calcium overload, secondary to membrane lipid peroxidation, has been proposed as a general pathogenic mechanism. However, using the photosensitisation of rose bengal to generate singlet oxygen and superoxide radicals, we demonstrate a direct effect of reactive oxygen species on the cardiac sarcoplasmic reticulum calcium-release channel. Exposure of heavy sarcoplasmic reticulum vesicles to reactive oxygen species in vitro resulted in the progressive loss of specific [3H]ryanodine binding and the degradation of high molecular weight proteins identified by polyacrylamide gel electrophoresis. The gating of single channels incorporated into artificial planar phospholipid bilayers was modified during the exposure to reactive oxygen species: an initial increase in open probability being followed by irreversible loss of channel function. Degradation by reactive oxygen species of specific proteins, such as the calcium-release channel, may contribute to in vivo reperfusion injury.

Patterns of interaction between anthraquinone drugs and the calcium-release channel from cardiac sarcoplasmic reticulum.

We have studied the effects of clinically useful anthraquinones on the cardiac sarcoplasmic reticulum calcium-release channel. Micromolar concentrations of doxorubicin and other anthracyclines at the cytosolic face of the channel significantly and reversibly increase the open probability of single channels in artificial phospholipid bilayers. Lifetime analysis shows that anthracyclines and calcium are synergistic activators of the calcium-release channel. Radiolabeled ryanodine binding suggests that all the anthracyclines studied are equally potent as channel activators in vitro. Mitoxantrone, an anthracenedione derivative, variably increases channel open probability at low (1-10 microM) concentrations. Higher concentrations are associated with the appearance of channel currents with lower amplitudes than the fully open state, and normal openings are reduced in frequency. At these concentrations, the interaction of mitoxantrone with the channel reduces the level of ryanodine binding. Abnormal function of the cardiac calcium-release channel will alter calcium handling within the myocyte and may be the basis for anthraquinone-related cardiotoxicity.

Sulmazole (AR-L 115BS) activates the sheep cardiac muscle sarcoplasmic reticulum calcium-release channel in the presence and absence of calcium.

The properties of calcium-release channels of sheep cardiac muscle junctional sarcoplasmic reticulum (SR), have been investigated under voltage-clamp conditions following the fusion of isolated membrane vesicles with planar phospholipid bilayers. In the presence of activating calcium on the cytosolic side of the membrane, additions of the benzimidazole derivative sulmazole (AR-L 115BS) increased the open probability (Po) of the channel reaching saturating values of 1.0 at 3 mM sulmazole. The drug did not affect single-channel conductance and activation was readily reversible. Analysis of channel open and closed lifetimes suggested that low concentrations of sulmazole (0.1 mM) may sensitize the channel to activating calcium, while at higher concentrations (1 mM and above), calcium and sulmazole act synergistically to produce a unique gating scheme for the channel. Millimolar concentrations of sulmazole also stimulate a degree of channel opening at subactivating (60 pM) calcium concentrations. Openings occurring under these conditions show very different kinetics to those of the calcium-activated channel but have an identical single-channel conductance and are modified by ATP, magnesium, ruthenium red and ryanodine in a similar manner to the calcium-activated channel. The release of calcium from the SR following the activation of the calcium-release channel by sulmazole may contribute to the positive inotropic action of this drug on mammalian cardiac muscle.

The cardiac sarcoplasmic reticulum calcium-release channel: modulation of ryanodine binding and single-channel activity.

[3H]Ryanodine binding to a preparation of isolated cardiac sarcoplasmic reticulum has been investigated. A method is reported which produces a very high level of specific binding. Scatchard analysis of binding up to 50 nM ryanodine yields data which infer a single class of binding sites with a Kd of 1.4 nM and a Bmax of 9.7 pmol/mg protein. Micromolar calcium is the principal activating ligand and its effects on binding are modulated by ligands which similarly affect the activity of single calcium-release channels incorporated into artificial planar phospholipid bilayers. The benzimidazole drug, sulmazole, is able to stimulate ryanodine binding in the presence of sub-activating calcium concentrations. Ryanodine binds to the native channel only when it is in its open state and stimulation of maximal ryanodine binding is achieved by ligands which are insufficient to produce full single-channel activation. A model is proposed which relates the modulation of ryanodine binding to the behaviour of single channels.

Developing an environmental assessment for effective clinical laboratory strategic planning: Part II. A five-step process.

The 1990s will pose a unique set of opportunities and challenges for clinical laboratories and the health-care field. Successful positioning of the clinical laboratory requires a well-thought-out strategic plan for the next 3 to 5 years and beyond. To develop an effective strategic plan, you must assess the environment in which your organization operates. This article, the second in a two-part series, focuses on the strategic planning process for the clinical laboratory and on environmental assessment. The first article (1) discussed CLMA's environmental assessment process and its findings. This article will present a step-by-step approach you can use to develop your own clinical laboratory environmental assessment. The future of the clinical laboratory and related industries provides the context within which clinical laboratories must position themselves to effectively meet their clients' needs. These two articles provide a starting point for developing a vision of the future for the clinical laboratory industry and for the individual clinical laboratory.

Single channel recordings from human cardiac sarcoplasmic reticulum.

Native membrane vesicles of human cardiac sarcoplasmic reticulum have been fused with artificial lipid bilayers to obtain recordings of single chloride and calcium release channels. Tissue was obtained from the explanted hearts of patients with end-stage cardiac failure undergoing heart transplantation. Although previous studies of calcium uptake and release have shown that the function of the sarcoplasmic reticulum is abnormal in end-stage cardiac failure, the basic properties of the channels are similar to those reported for equivalent channels from normal animal tissue.

Amino acid transport in the unfertilized and fertilized mouse egg.

The transport of methionine into unfertilized and fertilized mouse eggs appears to involve active transport mechanisms with similar Vmax, Km, substrate specificity and independence from Na+. An exchange diffusion system with a similar amino acid specificity to the uptake system has also been found in both types of egg. An estimate of 6.5 fmol has been made for the size of the total internal pool of exchangeable amino acids.