Intracellular Ca and cell injury: A paradoxical role of Ca in complement membrane attack

Disturbances in intracellular Ca2+ are known to be important in cell injury caused by a wide range of toxic factors. The complement system is a major effector of immune damage in vivo, and is known to be involved in the pathogenesis of many immune diseases. We present here evidence that the potentially lethal membrane attack complex of complement causes a rapid increase in intracellular free Ca2+ concentration before any other detectable biochemical changes in the cell. In nucleated cells the increased intracellular free Ca2+ concentration initially stimulates recovery processes, allowing the cell to escape mild complement attack and also activates the production of inflammatory mediators, which may amplify an ongoing inflammatory response. More severe complement membrane attack causes a more rapid rise in intracellular free Ca2+ concentration allowing a threshold to be breached above which recovery processes are overwhelmed, and cell death occurs. The importance of non-lytic effects and recovery processes mediated by Ca2+, and the molecular basis of these effects are discussed, and the hypothesis proposed that the cell-injuring effects of other "pore-forming" toxins are also caused by increases in intracellular free Ca2+.     

Analysis of fluorescence transients of DCMU-treated leaves of Triticum species to provide estimates of the densities of photosystem II reaction centres

The fluorescence of the chlorophyll associated with photosystem II was studied in seedling and flag leaves of Triticum species. Seedling leaves of the diploid species T. urartu had higher values of t (the normalised area over the fluorescence induction curve of DCMU treated leaves) than those of the other species studied which included hexaploid T. aestivum. However this difference was not evident when leaves were grown in a low light intensity (40 µmol quanta of photosynthetically active radiation m^–2 s^–1). The smaller total number of chlorophyll molecules per photosystem II reaction centre (chl/RCII) in T. urartu (177) as compared with the other species (mean 234) was deduced from the observed differences in t. As a consequence of its lower chl/RCII, despite slightly lower chlorophyll content (mg m^–2), T. urartu had a greater density of reaction centres than the other species (2880 cf 2230 nmol m^–2 of leaf). Consistent with the lower chl/RCII of T. urartu, it had a higher chlorophyll a/b ratio than the other genotypes. Seedling leaves of T. urartu had higher light saturated rates of photosynthesis than those of the other species, when grown at high light, a difference associated with reaction centre density.In flag leaves, when the complications due to variable development and senescence patterns were eliminated, t of the diploid species including T. urartu was lower than that of T. aestivum. The lower apparent chl/RCII of T. urartu arose partly because the molar extinction coefficient of the chlorophyll in the leaves of T. urartu was greater than in T. aestivum. However, the density of PS II reaction centres was slightly lower for the diploid species studied because their chlorophyll contents were lower than the hexaploids.The validity of the method for estimating chl/RCII from fluorescence transients is discussed. The possibility is considered that the difference in apparent chl/RCII of flag and seedling leaves of R. urartu as compared to the other five genotypes is a consequence of its different adaptive response to the spectral quality of the light.     

Chromatographic resolution of chicken phosvitin. Multiple macromolecular species in a classic vitellogenin-derived phosphoprotein.

The 622-residue amino acid sequence of the hydrophilic domain in the porcine NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is reported. The structural data required to complete the sequences published previously [Vogel, Kaiser, Witt & Lumper (1985) Biol. Chem. Hoppe-Seyler 366, 577-587] and to establish the primary structure of the porcine hydrophilic domain have been obtained by sequencing proteolytic subfragments derived from CNBr fragments and by characterizing the overlapping S-[14C]methylmethionine-containing peptides isolated from tryptic digests of the [14C]methyl-labelled hydrophilic domain. The hydrophilic domain displays 91.8% positional identity with that of the corresponding domain in the rat NADPH-cytochrome P-450 reductase. The region Val528-Ser678 in the NADPH-cytochrome P-450 reductase shows a significant homology to the sequence Ile165-Tyr314 in the spinach ferredoxin-NADP+ oxidoreductase. A model for the secondary structure of the hydrophilic domain has been derived by computer-assisted analysis of the amino acid sequence. Cys472 and Cys566 are protected against chemical modification in the NADP+ complex of the NADPH-cytochrome P-450 reductase.     

Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH₄)₂SO₄ to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca²⁺ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca²⁺ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca²⁺ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca²⁺ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca²⁺ uptake without appreciably influencing the apparent affinity of the transport system for Ca²⁺. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca²⁺ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca²⁺ uptake, CPF-I also inhibited the Ca²⁺-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca²⁺-preloaded sarcoplasmic reticulum vesicles did not promote Ca²⁺ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca²⁺ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca²⁺ pump, and therefore in determining the duration of Ca²⁺ signal in slow-contracting muscle fibers.     

Comparison of six rabbit liver cytochrome P-450 isozymes in formation of a reactive metabolite of acetaminophen

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.     

Chelator-mediated iron efflux from reticulocytes

The mechanism by which bipyridine and phenanthroline types of iron chelator inhibit iron uptake from transferrin and iron efflux mediated by pyridoxal isonicotinoyl hydrazone was investigated using rabbit reticulocytes with the aim of providing more information on the normal process of iron uptake by developing erythroid cells. It was shown that the chelators block cellular uptake by chelating the iron immediately after release from transferrin while it is still in the membrane fraction of the cells. The iron-chelator is then released from the cells by a process which is very similar to that of transferrin release with respect to kinetics and sensitivity to incubation temperature and the effects of metabolic inhibitors and other chemical reagents. These results are compatible with the conclusion that both transferrin and the iron-chelators in the cells are mainly present in endocytotic vesicles and are released from the cells by exocytosis. The chelators were also shown to block the pyridoxal isonicotinoyl hydrazone-mediated efflux of iron from cells which had taken up iron in the presence of isoniazid, an inhibitor of haem synthesis, by chelating the iron in the cytosol and the mitochondria. In this case, the iron-chelator complexes were not released from the cells. Measurement of the diethyl ether/water partition coefficients of bipyridine and 1,10-phenanthroline and their iron complexes gave much higher values for the free chelators, supporting the concept that the chelators trap the iron intracellularly because of differences in the lipid solubility and, hence, membrane permeability to the free chelators and their iron complexes.     

Patterns of cancer in geographic and endogamous subdivisions of the Hutterite Brethren of Canada

The Hutterite Brethren comprise a religious isolate and live on communal agricultural farms (colonies) in North America. In 1976 there were approximately 15,000 Canadian Brethren living in 179 colonies of the three endogamous subdivisions, the Dariusleut, Lehrerleut, and Schmiedeleut. Dariusleut and Lehrerleut colonies are located in both Alberta and Saskatchewan, and the Schmiedeleut are in Manitoba. Brethren were identified on population-based cancer registries of the three Prairie Provinces and among death registrations in the vital statistics of Alberta and Saskatchewan. The method of ascertainment was by a search for the 15 contemporary surnames and verification by address. 89 male and 91 female Brethren were identified who had cancer during the period, 1956--1975. The numbers of observed cancers were less than expected from provincial incidence rates for males and females in each province. The largest deficits were for female Brethren in Manitoba and Saskatchewan. There is a marked deficiency of cancer of the uterine cervix among female Brethren. In males there is a significant deficit of lung cancer. The Hutterite way of life contributes to a low risk for cancers of smoking-associated sites. However, there is evidence that male Brethren in Alberta may be at relatively increased risk for stomach cancer and leukemias. The site distribution patterns of cancers among the three endogamous leut are similar.     

Modulation of the alpha 1-adrenergic control of hepatocyte calcium redistribution by increases in cyclic AMP

The Ca2+ content of hepatocytes from juvenile male rats (80-110 g) or adult female rats (135-155 g) displayed a biphasic dose-response curve to epinephrine. Low concentrations (less than or equal to 10(-7) M) caused efflux of Ca2+ from the cells, while higher concentrations (10(-6) M and 10(-5) M) induced net Ca2+ uptake which correlated with a large beta 2-adrenergic-mediated increase in cAMP (Morgan, N. G., Blackmore, P. F., and Exton, J. H. (1983) J. Biol. Chem. 258, 5103-5109). Calcium accumulation could be induced in cells from older male rats (180-230 g) by combining a Ca2+-mobilizing hormone with either exogenous cAMP or glucagon (10(-8) M). Readdition of Ca2+ in the presence of glucagon to cells treated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also resulted in enhanced Ca2+ accumulation compared with controls. Addition of vasopressin plus glucagon to the medium perfusing male rat livers also led to cell Ca2+ accumulation, as evidenced by uptake of Ca2+ from the perfusate. Incubation of hepatocytes with antimycin A, oligomycin, and carbonyl cyanide m-chlorophenylhydrazone prevented net Ca2+ accumulation suggesting that mitochondria play a role in the uptake response. This was confirmed by isolation of mitochondria from cells incubated under conditions which promote Ca2+ accumulation. Within 5 min of incubation, the Ca2+ content of these mitochondria was increased 2-fold relative to controls, an effect which was inhibited by oligomycin. These studies demonstrate that a rise in hepatic cAMP can reverse hormonally induced Ca2+ mobilization and point to a major role for the mitochondria in this effect.