The calcium hypothesis of cystic fibrosis

Data have been presented which suggests that various CF cell types show evidence of alterations in calcium homeostasis. The significance of these observations and the exact nature of the putative calcium defect in CF remains to be elucidated. It must also be determined whether this possible defect is primary, or is secondary or tertiary to some more basic lesion. The data reviewed suggests that altered calcium homeostasis may play some focal role in the aetiology or the pathogenesis of CF.     

Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Dependence of ionophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The effects of the ionophore, X537A, and caffeine on ATP-dependent calcium transport by fragmented sarcoplasmic reticulum were studied in the absence (calcium storage) or presence (calcium uptake) of calcium-precipitating anions. The ionophore caused rapid calcium release after calcium storage, the final level of calcium storage being the same whether a given concentration of X537A was added prior to initiation of the reaction or after calcium storage had reached a steady state. Although 10 to 12 muM X537A caused approximately 90% inhibition of oxalate-supported calcium uptake when added prior to the start of the reaction, this ionophore concentration caused only a small calcium release when added after a calcium oxalate precipitate had formed within the vesicles, and only slight inhibition of calcium uptake velocity when added during the calcium uptake reaction. When low initial calcium loads limited calcium uptake to 0.4 mumol of calcium/mg of protein, subsequent calcium additions in the absence of the ionophore led to renewed calcium uptake. Uptake of the subsequent calcium additions was not significantly inhibited by 10 to 12 muM X537A. These phenomena are most readily understood in terms of constraints imposed by fixed Cai (calcium ion concentration inside the vesicles) on the pump-leak situation in sarcoplasmic reticulum vesicles containing a large amount of an insoluble calcium precipitate, where most of the calcium is within the vesicles and Cai is maintained at a relatively low level. These constraints restrict calcium loss after calcium permeability is increased because calcium release can end when the calcium pump is stimulated by the increased Cao (calcium concentration outside the vesicles) so as to compensate for the increased efflux rate. In contrast, an increased permeability in vesicles that have stored calcium in the absence of a calcium-precipitating ion causes a much larger portion of the internal calcium store to be released. Under these conditions calcium storage capacity is low so that release of stored calcium is less able to raise Cao to levels where the calcium pump can compensate for the increased efflux rate. The constraints imposed by anion-supported calcium uptake explain the finding that more calcium is released by X537A or caffeine when these agents are added at higher levels of Cao, and that more calcium leaves the vesicles in response to a given increase in calcium permeability at higher Cai. Although such calcium release is amplified by increased Cao, the amplification is attributable to the constraints described above and does not represent a "calcium-triggered calcium release."     

Dependence of calcium permeability of sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The ability of sarcoplasmic reticulum vesicles to retain calcium following ATP-supported calcium uptake in the presence of the calcium-precipitating anions oxalate and phosphate depends on Cao (calcium ion concentration outside the vesicles) and Cai (calcium ion concentration within the vesicles). Calcium efflux rates at any level of Cai are accelerated when Cao is increased. Higher Cao at the time that calcium uptake reactions reach steady state is associated with a spontaneous calcium release that reflects this effect of increased Cao. Increasing Cai at any level of Cao causes little or no acceleration of calcium efflux rate so that calcium permeability coefficients, estimated by dividing calcium efflux rates by Cai, the "driving force", are inversely proportional to Cai. Calcium permability coefficients thus correlate, as a first approximation, with the ratio Cai/Cao, decreasing 1000-fold as this ratio increases over a 3000-fold range (Cao = 0.1 to 3.3 muM, Cai =4 to 750 muM). Oscillations in both the calcium content of the vesicles and Cao are seen as calcium uptake reactions approach steady state, suggesting that calcium permeability undergoes time-dependent variations. Sudden reduction of Cao to levels that markedly inhibit calcium influx via the calcium pump unmasks a calcium efflux that decreases slowly over 60 to 90 s.The maximal calcium permeability observed in the present study would allow the calcium efflux rate from the sarcoplasmic reticulum at a Cai of 100 muM to be approximately 10(-10) mol/cm2/s, which is about 1 order of magnitude less than that estimated for the sarcoplasmic reticulum of activated skeletal muscle in vivo. The release of most of the stored calcium in some experiments indicates that the observed permeability changes can occur over a large portion of the surface of the sarcoplasmic reticulum.     

Synthesis of leucine enkephalin derivatives: structure-function studies

Nucleoli isolated from livers of rats injected intraperitoneally with one dose of thioacetamide had a five-fold increase in the rate of RNA synthesis $\text{in vitro}$ when compared with livers of rats treated with saline or CCl₄. The stimulation was maximal 24 hours after treatment and decreased to control values 73 hours after treatment. The enhanced level of nucleolar activity was maintained at that level when thioacetamide was injected daily. Along with the increase in the endogenous activity there was a 7-fold increase in the “free” RNA polymerase I activity determined by blocking the bound enzyme with actinomycin D (7). The nucleoli of the thioacetamide-treated rats offer a useful model of modulation of ribosomal gene function.     

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle.

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37 degrees C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 +/- 0.002 (S.E. of mean) muM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 +/- 0.02 (S.E. of mean) muM Ca2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 +/- 0.01 (S.E. of mean) muM Ca2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 +/- 0.03 (S.E. of mean) muM Ca2+ concentration. Kinetic analysis of the Ca2+ activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.     

Restriction endonuclease and nucleotide sequence analyses of molecularly cloned unintegrated avian tumor virus DNA: structure of large terminal repeats in circle junctions

Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322. Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction endonuclease mapping and by DNA sequencing. The results of these studies indicate that (i) the two large terminal repeats (LTRs) present in PATV-6, are different sizes, (ii) pATV-8 and pATV-9 contain only one LTR, (iii) pATV-7 contains an inversion of 0.6 kilobase in the env gene and a deletion of the U3 region and the src gene, and (iv) the src gene is deleted in pATV-6 and pATV-9. Circle formation from linear molecules was also examined in several of the clones by DNA sequencing through the circle joint. pATV-6 is an example of one class of circular molecules and contains a partially repeated LTR similar to that reported by Ju and Skalka (Cell 22:379-386, 1980). A second class of circles was exemplified by pATV-8 and pATV-9, which contain a single copy of the LTR with no base changes or deletions. This is in contrast to a class of circles containing a complete double LTR structure described by Swanstrom et al. (Proc. Natl. Acad. Sci. U.S.A. 78:124-128, 1981) and suggests that circles containing a single intact LTR may be formed by a homologous recombinational event in which an entire LTR or complementary regions from both LTRs are removed from the linear DNA molecule during circularization.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

Hypothalamic sarcoidosis and hypopituitarism

4 patients with presumed pituitary hypothalamic sarcoidosis are described. 3 had histological diagnoses compatible with sarcoidosis and in the other this diagnosis was strongly suspected from chest X-rays. 2 patients presented with diabetes insipidus. ACTH reserve was diminished in 3 out of 4 and growth hormone reserve was diminished in the 3 who were tested. All 4 patients developed secondary amenorrhea. 3 patients had hypothalamic hypothyroidism. Prolactin dynamics were intact. Tomograms of the sella turcica in all 4 and computerized tomography of the hypothalamic area in 2 patients failed to reveal any abnormality.