Fabry disease: reduced activities of respiratory chain enzymes with decreased levels of energy-rich phosphates in fibroblasts.

UNLABELLED: Fabry disease (FD, MIM 301500) caused by a deficient activity of alpha-galactosidase A is characterized by intralysosomal storage of glycosphingolipids. Main clinical features are paresthesia, hypohidrosis, angiokeratoma, renal insufficiency, and cardiovascular or cerebral complications. The exact pathogenesis is unclear. Beside mechanical storage biochemical factors might play a role. As FD is a multisystemic disorder and mitochondrial dysfunction has been described in patients with neuronal ceroidlipofuscinosis (another lysosomal storage disease) we examined mitochondrial function in fibroblasts from patients with FD. RESULTS: Activities of respiratory chain enzymes I, IV, and V were significantly (p < 0.01) lower in FD-cells. Mitochondrial recovery was unchanged as judged by the activity of the mitochondrial marker enzyme citratesynthase, cellular protein content was not significantly different. CP, ADP, and AMP concentrations were significantly (p < 0.01) lower in FD-cells. ATP was slightly, but not significantly reduced (p = 0.045). CONCLUSION: Organ dysfunction in FD may not only be explained by mechanical storage of glycosphingolipids. As in NCL, lysosomal storage material may lead to mitochondrial dysfunction with a reduction of respiratory chain enzyme activities and a subsequent drop in cellular levels of energy-rich phosphates.

Different effects of gradual vs. acute adenine nucleotide depletion on ATP cost of muscle contraction.

ATP and phosphocreatine (PCr) levels were chronically reduced in rat fast-twitch muscle by 40 and 87%, respectively, after rats were fed a diet containing 1% beta-guanidinopropionate (beta-GPA) for 9 wk. Myosin heavy chain distribution of superficial gastrocnemius muscle changed from 22:78% type IIA/type IIB in control to 39:61% in GPA-treated muscles. ATP cost of contractions was estimated from the PCr changes measured by gated 31P-nuclear magnetic resonance after brief (< 6 s) bursts of contractions. There was no significant change in the ATP cost of either twitch (0.3 mumol.g-1.twitch-1) or tetanic (2.5 mumol.g-1.100 ms tetanus-1) contractions in gastrocnemius muscle after chronic phosphagen depletion due to beta-GPA feeding. In two other groups of rats, ATP of gastrocnemius muscles was acutely depleted to inosine 5'-monophosphate (IMP) by tetanic stimulation. IMP reanimation was inhibited during a subsequent 75-min recovery period in one group by hadacidin treatment (100 mg/kg), resulting in 46% ATP depletion compared with the similarly stimulated and recovered control group. In contrast to gradual ATP depletion due to beta-GPA feeding, this acute ATP depletion treatment was associated with a 39% decrease in ATP cost of twitch contractions.

Adrenergic stimulation of rat hearts with severely reduced cytosolic adenine nucleotide pool and [ATP]/[ADP]ratio.

The effect of severe reduction of cytosolic adenine nucleotide (AdN) pool and [ATP]/[ADP] ratio (by 2-deoxyglucose treatment) on functional and metabolic responses of isovolumic rat heart to increased energy demand induced by coronary flow (CF) rise and isoproterenol (Iso) addition has been investigated. AdN-depleted hearts had reduced phosphocreatine (PCr, by 80%), ATP (by 75%), [ATP]/[ADP] (24 times) and pressure-rate product (PRP, by 60%). An elevation of CF was followed by the increase in PRP in control and AdN-depleted hearts by 40-45% with unchanged metabolic parameters. At increased CF, Iso caused a further rise in PRP in both groups due to elevation of heart rate; however maximal levels of PRP in the AdN-depleted group still remained lower than that of control (by 40%). Only in control experiments was Iso addition accompanied by an increase in the difference between left-ventricular end- and minimal diastolic pressure, cytosolic [Pi] and [ADP], and some decrease in PCr and [ATP]/[ADP]. These data imply that severely reduced cytosolic [ATP]/[ADP] does not prevent acceleration of Ca2+ turnover by Iso in cardiomyocytes, it but restricts maximal force development affecting the myofibrils.

[Changes in the pool and spectrum of adenine nucleotides in the cortex of the solitary kidney in chronic denervation]. / Izmeneniia pula i spektra adeninnukleotidov kory edinstvennoi pochki v usloviiakh khronicheskoi denervatsii.

Chronic energy-++-deficient condition of nephron cortical portions manifested in a two ware pool decrease and spectrum impairment of the adenyl system components was revealed as a result of the long-term studies in tissue contents of ATP, ADP, AMP in the rat renal cortex under conditions of experimental combination of compensatory hypertrophy and chronic nervous decentralization. A negative trophic effect of chronically overloaded cortex tissue de-mediation is responsible for energy metabolism at the early phase while at the later stage, beginning two months after the experiment, the progressing hypoperfusion of the cortical tubular system of a single denervated kidney is.

Diadenosine 5',5'''-p1,p4-tetraphosphate deficiency in blood platelets of the Chédiak-Higashi syndrome.

Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chédiak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.

Clinical disorders of muscle energy metabolism.

The disorders of muscle energy metabolism can be classified into degenerative (myopathic) and dynamic syndromes. Four dynamic syndromes are currently recognized: 1) defective carbohydrate utilization, due to block of glycogenolysis or glycolysis; 2) defective lipid utilization, due to deficiency of the mitochondrial translocation of long-chain fatty acids (carnitine palmityltransferase deficiency); 3) lactic acidosis, due to defects of mitochondrial electron transport enzymes and possibly other unidentified defects; and 4) abnormal adenine nucleotide metabolism, exemplified by adenylate deaminase deficiency. The way in which the response to exercise is affected by impaired muscle energy metabolism is dependent on the type of metabolic defect. Defective carbohydrate metabolism severely limits the ability for high-intensity and ischemic exercise. The ability to perform prolonged exercise is markedly impaired in dynamic disorders of lipid metabolism. Other disorders, including those of adenine nucleotide metabolism also may have important implications for our understanding of the metabolic phenomena involved in exercise and recovery.

Effect of the brachymorphic trait in mice on xenobiotic sulfate ester formation.

Mice carrying the recessive mutation brachymorphic have been shown previously to have a reduced capacity to synthesize 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the required coenzyme in sulfation reactions [K. Sugahara and N. Schwartz, Proc. natn. Acad. Sci. U.S.A. 76, 6615 (1979)]. The capacity of the liver cytosol fractions from brachymorphic (bm/bm) mice or their phenotypically normal littermates (+/+ or +/bm) to catalyze the formation of sulfate esters of [3H]estrone and [14C]p-nitrophenol in vitro was determined. When PAPS was added to the reaction, the rates of sulfate ester formation catalyzed by the two cytosol fractions were similar. In contrast, when PAPS was generated in situ from ATP and SO(4)2-, the rates of sulfate ester formation catalyzed by the brachymorphic cytosol were only 4-22% of the rates catalyzed by the cytosol fraction from normal mice. The hepatic cytosol fraction from brachymorphic mice incorporated less 35SO(4)2- into PAPS than that catalyzed by cytosol of normal mice. [14C]p-Nitrophenol (1.5 mumoles/kg) was eliminated from brachymorphic and normal mice as urinary conjugates; in normal mice, 73% of the urinary radioactivity was p-nitrophenyl sulfate, while in the brachymorphic mice only 33% of the urinary excretion was the sulfate ester. Brachymorphic mice have a reduced capacity for synthesizing sulfate esters of xenobiotics in vitro and in vivo, which is attributable to their reduced synthesis of PAPS.

Prolonged bleeding time in Aleutian mink associated with a cyclo-oxygenase-independent aggregation defect and nucleotide deficit in blood platelets.

A prolonged mean template bleeding time of 13 minutes was present in nine Aleutian mink affected with Chediak-Higashi syndrome (CHS) compared with 4 minutes in dark control mink. The concentrations of blood platelets in normal and affected animals did not differ significantly. However, in mink with CHS, a marked disturbance of platelet response to collagen was present. Administration of aspirin and indomethacin completely blocked CH platelet response to collagen. Blood platelet adenosine triphosphate and adenosine diphosphate values from mink with CHS were significantly less than those of normal mink, and the platelet adenosine triphosphate/adenosine diphosphate ratios were 10.31 in affected mink and 2.74 in normal mink. These findings are consistent with our previous investigations in affectd cattle and persons and indicate that a "storage pool disease" of platelets exist in the mink with CHS.

Serotonin deficiency and prolonged bleeding in beige mice.

Beige mice have been observed to bleed excessively from small wounds. Platelets obtained from these mice were deficient in adenine nucleotides and serotonin and in vivo uptake of labeled serotonin was impaired. Heterozygotes of beige and C57BL/6 mice have adenine nucleotide and serotonin levels comparable to control and do not manifest a bleeding tendency, consistent with the recessive mode of inheritance of the beige mouse syndrome. Morphologic confirmation of the observed biochemical defects was obtained by the demonstration that serotonin storage organelles were not observed in electron photomicrographs of beige mouse platelets. The demonstration that small doses of serotonin were effective in reversing the bleeding tendency in beige mice suggested a causal role for defective storage and release of endogenous serotonin as the basis for the bleeding tendency, acting either independently or synergistically with impaired release of adenine nucleotides.

Labile aggregation stimulating substance (LASS): the factor from storage pool deficient platelets correcting defective aggregation and release of aspirin treated normal platelets.

Mixtures of equal volumes of Hermansky-Pudlak (HP) syndrome (adenine nucleotide storage pool deficient) and aspirin treated (AT) platelets (inhibited release of adenine nucleotides) undergo irreversible aggregation when exposed to collagen or adrenaline, whereas neither alone will do so. The present investigation has explored the basis for the mutual correction. Correction in the mixed system was accompanied by release of significant amounts of [14C]5-hydroxy-tryptamine from the AT platelets, whereas very little isotope was released when AT platelets alone were exposed to collagen. The correction of aggregation and the release of isotope could both be suppressed by pre-treating HP platelets with aspirin. Labile aggregation stimulating substance (LASS) composed of two closely linked intermediates of prostaglandin (PG) E2 and PGF2alpha biosynthesis, produced using a microsomal fraction of HP platelets, could correct the aggregation and secretion defect of AT platelets exposed to collagen. These findings indicate that HP platelets when mixed with AT platelets and exposed to collagen secrete a substance which is responsible for the correction. LASS, identified as the factor involved, acted as an intercellular messenger which mediated the correction by overcoming the influence of aspirin on normal cells.