Epilepsy and respiratory chain defects in children with mitochondrial encephalopathies.

OBJECTIVE: The purpose of this study was to determine the relationship between epilepsy and respiratory chain defects in children with mitochondrial encephalopathies (ME). STUDY DESIGN: We conducted a retrospective review of the medical records of children referred for evaluation of an ME. Only patients assigned a definite diagnosis of ME using modified Walker criteria and with a respiratory chain defect were included. Clinical data pertaining to the ME and epilepsy type were collected. Mitochondria were isolated by subcellular fractionation from a vastus lateralis muscle biopsy and studies were performed using polarographic and spectroscopic techniques for the quantitative determination of NADH and cytochrome components of the respiratory chain. RESULTS: A total of 38 children with ME were identified. Seizures were present in 61%. Sixteen of 23 children with epilepsy (70%) had refractory epilepsy associated with a progressive encephalopathy. Children with epilepsy had a significantly higher incidence of complex I defects than children without epilepsy (p<0.01). Complex III and IV defects were significantly higher in patients without epilepsy (p<0.01 and p<0.05, respectively) than in those with epilepsy. CONCLUSIONS: Epilepsy is an important component of ME. The higher incidence of complex I defects in patients with epilepsy suggests a possible relationship between mitochondrial oxidative stress dysfunction and epileptogenic process.

Mitochondrial activity in Pompe's disease.

Mitochondrial oxidative metabolism was examined in two infants with Pompe's disease. The clinical diagnosis was confirmed by the demonstration of intralysosomal glycogen accumulation and a deficiency of acid alpha-D-glucosidase in muscle biopsies. Light and electron microscopy studies demonstrated a normal number of mitochondria with normal ultrastructure. Spectrophotometric measurements revealed that the specific activities of citrate synthase and the partial reactions of electron transport were markedly elevated in the skeletal muscle homogenates prepared from both infants with Pompe's disease when calculated as micromoles per minute per gram wet weight of tissue. However, when respiratory chain enzyme activities were expressed relative to citrate synthase as a marker mitochondrial enzyme, a different pattern emerged, in which all Pompe muscle respiratory enzymes, except complex IV, were decreased relative to control subjects. These observations demonstrate that caution should be exercised when analyzing and interpreting data obtained from tissue homogenates in general and, in particular, in those prepared from tissues in which the wet weight of tissue may be altered, for example, by pathologic accumulation of carbohydrate or lipid.

Prostaglandin E1-induced deconsolidation of thrombin-activated platelet aggregates I: ultrastructure-computer image analysis.

We have compared, at an ultrastructural-computer image morphometric level, the relaxation induced by Mg-ethylene-bis-oxyethylenenitrilo-tetracetic acid and prostaglandin E1 on a model of a thrombin-activated platelet aggregate. Mg-ethylene-bisoxyethylenenitrilo-tetracetic acid produced a small increase of 5.0% of the intercellular space over the control levels, and a decrease of 10.0+/-1.3% of the cross-sectional area of the platelets, with no apparent cytoskeletal alterations. In contrast, the prostaglandin El-treated preparation shows a 360% increase in the intercellular space and a decrease of the average platelet cross-sectional area of 30.0+/-2.0% with marked cytoskeletal alterations. We use the term "deconsolidation" to describe this effect. The enlargement of the intercellular space allows the observation of two types of contacts: (1) a type S (segmental) complex, of approximately 200-nm length that maintains a narrow interplatelet gap of 20-30 nm, filled with a dense intercellular material, and (2) a type R (reticular) complex, formed by scant focal regions of the plasma membrane from opposing platelets that are connected through a mesh of fibrillar or granular material contained within a variable-size space. We hypothesize that deconsolidation is caused by fluid loss from the platelets into the intercellular space. As a result, platelet volume decreases and intercellular space increases.

Fibrinogen binding is independent of an increase in intracellular calcium concentration in thrombin degranulated platelets.

In a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C (PLC) activation and calcium mobilization in TDP activates by ADP, epinephrine and U46619. ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

A method to prepare degranulated human platelets: use for studies of platelet aggregation and ca(2+) mobilization.

A method for the preparation of a suspension of thrombin-degranulated human platelets is described. Two peptides (RGDS and GPRP) are used to prevent fibrinogen binding and consequent aggregation, and to prevent fibrin polymerization during thrombin activation. A mixture of creatine phosphokinase and creatine phosphate is used to remove ADP. Hirudin and TAMe are used to neutralize thrombin after the platelets have been activated. [(14)C] Serotonin and PF4 release and electron microscopy demonstrate that the preparation is completely degranulated. After all inhibitors are removed and fibrinogen added, the preparation aggregates rapidly to a mixture of agonists composed of ADP, epinephrine and the synthetic analog of prostaglandin H(2)/thromboxane A(2), U46619. ADP and epinephrine when added individually are both able to induce a clearly detectable aggregation, while U46619 induces only a shape change. The preparation is also suitable for intracellular Ca(2+) studies and we find that the mixture of agonists produces an increase in the intracellular calcium concentration to about 1 µM.

Use of Zn-pyrophosphatase in the high-performance liquid chromatographic analysis of cell extracts containing 32P-labelled inositol phosphates.

A simple method is described for eliminating the interference of pyrophosphate and pyrophosphorylated nucleosides in the high-performance liquid chromatographic determination of inositol 1,3,4-triphosphate and inositol 1,4,5-triphosphate of 32P-labelled extracts of cells. Treatment of the extract with pyrophosphatase, but substituting Zn2+ for Mg2+ as the cofactor, converts all nucleoside triphosphates and pyrophosphate to their di- and monoesters. Such change shifts their position in the elution profile, allowing a clear identification and quantification of the inositol phosphates. Typical overall recoveries near 80% or higher of added markers.

(45)Ca efflux and agonist induced changes in force in a model of thrombin activated platelets.

(45)Ca(2+) and (3)H sorbitol were loaded by incubation into a model of thrombin activated, irreversibly aggregated platelets. Total Ca, measured by atomic absorption, was approximately 4.0 nmoles/mg wet weight. 55% of the total Ca(2+) was exchangeable with (45)Ca(2+), 14% was extracellular and 42% cellular, either surface or intracellular. Changes in the efflux of the marker into a buffer containing Mg-EGTA were correlated with the contractile responses of the preparation after addition of agonists. For the contracting agonists tested individually (ADP, epinephrine, and the endoperoxide analogue, U46619) the fractional efflux rate increased in phases, the descending component stabilizing at a rate higher than the basal. When agonists of different classes were added sequentially in supramaximal amounts, the increases in the stable component of the efflux were also additive and correlated well with the increases in the force of contraction. Washout of the agonist returned the efflux to the baseline. Agents increasing cyclic AMP, like prostaglandin E(1), produced a small decrease in the basal level of the efflux of (45)Ca. When contracting agonists were added to the pretreated preparation, a simultaneous decrease in efflux and force generated were found. The inhibition was dose dependent on the relaxing agonist.

The platelet strip. I. A low-fibrin contractile model of thrombin-activated platelets.

The ultrastructure and contractile behavior of a new preparation of thrombin-activated human platelets is described. The preparation is referred to as the "platelet strip" because of its similarities to classical vascular smooth muscle strips. The platelet strip consists of a giant platelet aggregate 10 mm long, 4 mm wide, and 200 micron thick. To facilitate handling, the aggregate has a special high-compliance nylon mesh embedded in its mass. Each strip contains 7.3 X 10(8) platelets. Fibrin contamination is 150-fold lower than in platelet-rich plasma clots. Active isometric forces of up to 100 g/cm2 and 6-10 h viability are easily and reproducibly obtained. Platelet strips remain contracted after thrombin activation. The contraction is tonic and partial. Further small increases in force can be produced by depolarizing solutions or pharmacological agents, e.g., ADP, epinephrine, and endoperoxide analogues. These small increases are reversible on washout of the agents. Full relaxation is induced by agents such as prostaglandin E1 or papaverine, which increase adenosine 3',5'-cyclic monophosphate. However, after washout of these agents, recovery of tension is variable depending on the concentration of the drug and the degree of prestretching of the preparation.

The platelet strip. II. Pharmacomechanical coupling in thrombin-activated human platelets.

A model of contracted, irreversibly aggregated thrombin-activated human platelets relaxes when treated with ethyleneglycol-bis(beta-aminoethylether-N,N'-tetraacetic acid (EGTA) in the presence of Mg2+. Inhibition of the cyclooxygenase or blockade of the thromboxane A2 receptor decreases the tension partially, but EGTA treatment is needed for full relaxation. After a stable relaxation has been achieved (3-4 h), Ca2+ addition in a cumulative manner does not reinduce contraction. Whether in the absence or presence of external Ca2+, the relaxed preparation contracts when stimulated with ADP, epinephrine, thromboxane A2 or its analogues, or thrombin. At supramaximal doses, each of the agonists activates only a partial amount of the total tension capable of being generated. Addition of an agonist of a different class to the partially contracted preparation further increases its force. The contractile responses are reversible on washout, with kinetics dependent on the class of agonist and time of contact with the preparation. The contraction induced by the prolonged simultaneous stimulation with ADP, arachidonate, and thrombin reverts very slowly on washout of the agonists and for all practical purposes reproduces the initial state of irreversible platelet contraction.

Role of myosin phosphorylation in contractility of a platelet aggregate.

The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin-activated, irreversibly aggregated platelets known as the platelet strip. Steady-state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets.

Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.

The internal pH of isolated serotonin containing granules of pig platelets.

The [14C]methylamine distribution method was utilized to measure the internal pH of isolated serotonin containing granules of pig blood platelets under varying conditions. The granules used were isolated by a new protocol which stressed platelet rupture under controlled conditions and preservation of isotonicity throughout the isolation procedure, In a well buffered external medium, pH 6.85, The deltapH was measured as 1.11 with the internal pH being found acidic (pH 5.74). Increasing the external pH produced a corresponding increase in the deltaH. The pH gradient could be collapsed by the addition of ionophores and uncouplers which are known to transport protons across biological membranes. In addition, the deltapH was constant for granules suspended in various ionic media, thus suggesting that the deltaH did not arise secondarily due to the establishment of a Donnan equilibrium. The existence of the acidic intragranular space is discussed with respect to previous ancillary findings. Also, an explication of the possible physiological significance of the deltaH is presented.

An improved method for the isolation of dense storage granules from human platelets.

Pretreatment of human platelets with the metabolic inhibitors rotenone and 2-deoxyglucose, before French press homogenization, has led to the isolation of dense storage granules in an overall yield of about 20%. The concentrations of serotonin, ATP and ADP were estimated in the dense granules. Serotonin was 40--60-fold enriched in the dense granules compared to the platelet homogenate. Stored ATP and ADP were also 40-fold enriched in the dense granules compared to the estimated storage nucleotide pool in intact platelets. The ATP to ADP ratio in the isolated dense granules was 0.68-0.70, the same as the ratio of the secreted ATP and ADP. In platelets prelabeled with [3H]adenine, the specific radioactivities of the ATP and ADP in the isolated dense granules and of the secreted ATP and ADP were both negligible, whereas the estimated specific radioactivity of the metabolically active ATP and ADP was 2,000 cpm/nmol. These results confirm that the ATP and ADP in the isolated dense granules are the same as the secreted ATP and ADP in terms of metabolic inactivity and their ATP to ADP ratios.