Developmental control of cAMP-induced Ca2+-influx by cGMP: influx is delayed and reduced in a cGMP-phosphodiesterase D deficient mutant of Dictyostelium discoideum.

It was previously shown that cGMP enhances cAMP-induced Ca2+-influx in Dictyostelium discoideum. This finding is based on experiments done with strains defective in cGMP-hydrolysis, the streamer F cells. In this work, we show that these chemically mutagenized cells display different properties in their cAMP-induced light-scattering response and cAMP-induced Ca2+-influx compared with a cGMP-phosphodiesterase knock-out strain, pdeD KO, generated by homologous recombination. PdeD KO cells possess a reduced Ca2+-influx that is developmentally regulated. This finding contradicts the result of streamer F cells, where cAMP-induced Ca2+-influx is prolonged and elevated. Both mutants, however, showed a three to four-fold delayed response to cAMP at 3-4h of starvation. Thus, the consequence of an elevated cGMP concentration is a delay and an inhibition of Ca2+-influx and not an enhancement. Results obtained with streamer F cells should therefore be interpreted with caution because the mutation(s) responsible for the divergent phenotype to pdeD KO cells has not been identified. We show by the use of membrane-permeant cGMP-analogues in wild type (wt) cells, permeabilized cells and measurements on isolated vesicles that the cause for the reduced Ca2+-influx seems to be due to developmentally regulated Ca2+-channel inhibition by cGMP.

Back pain as a secondary disability in persons with lower limb amputations.

OBJECTIVE: To evaluate the frequency, duration, intensity, and interference of back pain in a sample of persons with lower limb amputations. DESIGN: Retrospective, cross-sectional survey. SETTING: Community-based survey from clinical databases. PARTICIPANTS: Participants who were 6 or more months post lower limb amputation (n = 255). INTERVENTION: An amputation pain survey that included several standardized pain measures. MAIN OUTCOME MEASURES: Frequency, duration, intensity, and interference of back pain. RESULTS: Of the participants who completed the survey (return rate, 56%), 52% reported experiencing persistent, bothersome back pain. Of these, 43% reported average back pain intensity in the mild range (1-4 on 0-10 rating scale) and 25% reported pain of moderate intensity (5-6 on 0-10 scale). Most respondents with back pain rated the interference of their pain on function as none to minimal. However, nearly 25% of those with back pain described it as frequent, of severe intensity (>or=7 on 0-10 scale), and as severely interfering with daily activities including social, recreational, family, and work activities. CONCLUSIONS: Back pain may be surprisingly common in persons with lower limb amputations, and, for some who experience it, may greatly interfere with function.

Canadian beef quality audit 1998-99.

The second beef quality audit was conducted in Canada in 1998-99 to determine the prevalence of quality defects in slaughtered cattle and to monitor changes since the first audit in 1995. Approximately 0.6% of the number of cattle processed annually in Canada were evaluated. Brands were observed on 49% and tag was observed on 43% of the hides. Both brands and tag had increased from 1995. Seventy percent of the cattle were polled and 5% had full horns; thus, the number of horned cattle had decreased from 1995. Bruises were found on 54% of the carcasses, which was a decrease from 78% in 1995. Sixty-eight percent of the bruises were minor, 28% major, and 4% critical in severity. The distribution of bruises on the carcass was 17% on the chuck, 36% on the rib, 30% on the loin, and 16% on the round. Grubs were observed on 0.008% of the carcasses, and surface injection site lesions were observed on 0.2% of the whole carcasses, a decrease from the 1.3% seen in 1995. Seventy-two percent of the livers were passed for human food and 14% for pet food; 14% were condemned. Approximately 64% of the liver losses were due to abscesses. Five percent of the heads and tongues and 0.3% of the whole carcasses were condemned. The hot carcass weight was highly variable in all cattle, averaging 353 kg (s = 43). The average ribeye area was 90 cm2 (s = 13). Both hot carcass weight and ribeye area had increased from 1995. The average grade fat was 9 mm (s = 5), ranging from 0 mm to 48 mm. Lean meat yield averaged 58.8% (s = 4.6). One percent of the carcasses were devoid of marbling, 17% were Canada A, 49% were Canada AA, 32% were Canada AAA, and 1% were Canada Prime, which was an increase in marbling from 1995. Dark cutters were found in 1% of all carcasses; 1% of steers, 0.5% of heifers, 3% of cows, and 14% of bulls. Three percent of the carcasses were underfinished and 13% were overfinished. The number of overfinished carcasses had increased from 1995. Stages, steers with bullish traits, were infrequently observed in 0.5% of the steers, and 0.2% of the steers and 0.3% of the heifers had poor conformation. Yellow fat was not observed in any steers or heifers, but it was found on 65% of the cow carcasses. Only 0.6% of the heifers had an aged carcass, based on skeletal maturity. Based on August 1998 to July 1999 prices, it was estimated that the Canadian beef industry lost $82.62 per head processed, or $274 million annually, from quality nonconformities, which was an increase from 1995. Additional improvements in management, feeding, handling, genetics, marketing, and grading are needed in the beef industry to reduce quality defects.

Mechanism of cAMP-induced H(+)-efflux of Dictyostelium cells: a role for fatty acids.

Aggregating Dictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H(+) ATPase inhibitor concanamycin A and with the plasma membrane H(+) ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H(+) ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H(+) ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca2(+)-fluxes involves phospholipase A2 activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca2(+)-efflux. Here we show that arachidonic acid elicited H(+)-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca2(+)-release channel.

Correlations with patients' perspectives of the result of lower-extremity amputation.

BACKGROUND: Patients' perceptions of the result of lower-extremity amputation vary widely, yet the factors associated with this variability are not well understood. Our objective was to identify important correlations with the perceived result that may help to indicate the factors that deserve particular emphasis in the management of patients who have had an amputation. METHODS: In this retrospective study, 148 patients who had had a major lower-extremity amputation completed a standardized questionnaire designed to assess the demographic characteristics, comorbidities, amputation characteristics, prosthetic function, and social function at a mean of seven years after surgery. We correlated each of these variables with four result metrics: general satisfaction, quality of life, freedom from frustration, and walking distance. RESULTS: The four result metrics were significantly and strongly correlated with (1) the comfort of the residual limb; (2) the condition of the contralateral limb; (3) the comfort, function, and appearance of the prosthesis; (4) social factors; and (5) the ability to exercise recreationally (p < 0.0001). Interestingly, the level and laterality of the amputation were not significantly correlated with the patients' perceived result. CONCLUSIONS: The perceived result of amputation is not associated with the amount of the limb that was amputated but rather with factors that may be optimized by surgical, prosthetic, and social management.

A Xestospongin C-sensitive Ca(2+) store is required for cAMP-induced Ca(2+) influx and cAMP oscillations in Dictyostelium.

Xestospongin C (XeC) is known to bind to the inositol 1,4, 5-trisphosphate (IP(3))-sensitive store in mammalian cells and to inhibit IP(3)- and thapsigargin-induced Ca(2+) release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca(2+) uptake into purified vesicle fractions and induced Ca(2+) release. This suggests that, in the case of Dictyostelium, XeC opens rather than plugs the IP(3) receptor channel as was proposed for mammalian cells (Gafni, J., Munsch, J. A. , Lam, T. H., Catlin, M. C., Costa, L. G., Molinski, T. F., and Pessah, I. N. (1997) Neuron 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca(2+) store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca(2+)](i) and inhibited cAMP-induced Ca(2+) influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP signaling.

Fatty acids induce release of Ca2+ from acidosomal stores and activate capacitative Ca2+ entry in Dictyostelium discoideum.

cAMP-induced Ca2+ fluxes in Dictyostelium discoideum largely depend on phospholipase A2 activity generating non-esterified fatty acids [Schaloske and Malchow (1997) Biochem. J. 327, 233-238]. In the present study the effect of fatty acids on Ca2+ homoeostasis in D. discoideum was investigated. Cytosolic free Ca2+ concentration ([Ca2+]i) was analysed by digital imaging of single fura2-dextran-loaded cells. Arachidonic acid and linoleic acid induced a transient increase in [Ca2+]i. The concentration of arachidonic acid determined the percentage of responding cells, with the mean height of the increase being dose-independent. In nominally Ca2+-free medium or in the presence of bis-(o-aminophenoxy)ethane-N, N,N',N'-tetra-acetic acid (BAPTA), no [Ca2+]i transient was detectable. In spite of this, we found that (1) arachidonic acid induced Ca2+ release from permeabilized cells and from vesicular fractions at concentrations that elicited Ca2+ influx in intact cells and (2) Ca2+ entry was inhibited by inhibitors of Ca2+-transport ATPases and V-type H+-ATPase, indicating that intracellular Ca2+ release precedes Ca2+ entry. Inhibition studies and mutant analysis point to the acidosomal Ca2+ stores as a target of fatty acids. Although fatty acids can substitute fully for cAMP with respect to Ca2+ influx in wild-type cells, experiments with a mutant strain revealed that cAMP also sensitizes the Ca2+-entry mechanism: cAMP-induced Ca2+ influx was normal in a phospholipase C knockout mutant but influx was fairly insensitive to arachidonic acid in this strain. This defect could be overcome by higher doses of arachidonic acid which cause sufficient Ca2+ to be released from the stores to trigger extracellular Ca2+ entry.

Mechanism of cAMP-induced Ca2+ influx in Dictyostelium: role of phospholipase A2.

cAMP-induced Ca2+ influx in Dictyostelium follows two pathways: a G-protein-dependent pathway where influx is reduced by 50-70% in Galpha2 and Gbeta-negative strains and a heterotrimeric G-protein-independent pathway. Using a pharmacological approach, we found that phospholipase A2 (PLA2) is the target of both pathways. The products of PLA2 activity, arachidonic acid (AA) and palmitic acid, induced Ca2+ influx to a similar extent as cAMP. Half-maximal activation occurred at 3 microM AA and saturation at 10 microM AA. The response to AA was quantitatively similar throughout early differentiation and thus independent of cAMP-receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The PLA2-activating peptide, a peptide with sequence similarity to the G-protein beta-subunit, activated Ca2+ influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C and other kinases, suggesting that tyrosine kinase may directly or indirectly activate PLA2 in this case.

An increase in cytosolic Ca2+ delays cAMP oscillations in Dictyostelium cells.

We have shown that calmidazolium (R24571) causes a transient increase in the cytosolic free Ca2+ concentration ([Ca2+]i) in Dictyostelium discoideum [Schlatterer and Schaloske (1996) Biochem. J. 313, 661-667]. Here we have used R24571 to artifically increase [Ca2+]i during light-scattering oscillations and have found that, depending on the time of addition during the oscillatory cycle, R24571 suppressed cAMP synthesis and delayed the next spike for several minutes. Addition of Ca2+ to the medium, which also elevates [Ca2+]i, induced phase delays and resulted in a similar phase response curve as R24571. The magnitude of the phase delay was correlated with the point during the oscillatory cycle at which Ca2+ was added, indicating that an artificial increase in [Ca2+]i also resets the phase of the intrinsic oscillator.

Calmodulin-antagonists inhibit vesicular Ca2+ uptake in Dictyostelium.

Chemotactic stimulation by cAMP elicits Ca2+ entry across the plasma membrane and uptake into intracellular Ca2+ stores. In order to better understand Ca2+ regulation in Dictyostelium we measured 45Ca2+ uptake in homogenates of aggregation competent cells. Besides the InsP3-sensitive store the acidosomes are responsible for Ca2+ transport. About 50% of the vesicular 45Ca2+ accumulation was inhibited by the calmodulin antagonist W-7 and 14% by the less efficacious analogue W-5. Half maximal inhibition by W-7 occurred at 37 microM concentration. Calmodulin antagonised the activity of W-7, and a monoclonal antibody against Dictyostelium calmodulin inhibited Ca2+ sequestration as did calmodulin antagonists of different classes. 100 microM BHQ-a SERCA-type Ca2+ transport ATPase blocker-inhibited most of the W-7 sensitive compartment and oxalate increased Ca2+ uptake into this compartment indicating that intracellular Ca2+ stores are the target of W-7. Ca2+/calmodulin thus seems to provide for a feedback regulation of Ca2+ sequestration.

On the role of calcium during chemotactic signalling and differentiation of the cellular slime mould Dictyostelium discoideum.

Transient cytosolic calcium elevations are required for chemotaxis and differentiation of Dictyostelium discoideum since Ca2+ chelating buffers introduced into the cells by scrape loading inhibited motility as well as orientation in a Ca2+ specific manner. Ca2+ changes are provided either by intrinsic cytosolic Ca2+ oscillations that can be determined as periodic Ca2+ efflux, or by receptor-mediated Ca2+ liberation from the InsP3-sensitive store and Ca2+ influx. Cytosolic Ca2+ homeostasis as well as oscillations seem to be regulated by two different Ca2+ stores, the acidosomes and the InsP3-sensitive store, both of which are dependent on Ca2+ pumps and V-type H+ ATPases. Ca2+ transients are sensed by calmodulin-binding proteins. The latter have been detected in Dictyostelium by 125I-calmodulin labeling. A calmodulin-dependent protein phosphatase, calcineurin A, was cloned, sequenced, purified and characterized biochemically. Overproduction of calcineurin A as well as antisense constructs will help to the elucidation of its function in signal transduction. Surprisingly, protein synthesis is also controlled by Ca2+/calmodulin. An integral ribosomal protein of the 60S subunit, L19, proved to be a calmodulin-binding protein and calmodulin antagonists of different classes, inhibited in vitro translation of Dictyostelium and wheat germ extracts.

The role of calcium in aggregation and development of Dictyostelium.

Changes in cytosolic Ca2+ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca2+]i) at a low level in the face of steep gradients of extracellular and sequestered Ca2+. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca2+ into the cytosol and elevation of [Ca2+]i. After such stimulation Ca2+ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca2+]i to its basal level. Such responses to elevation of [Ca2+]i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.

Stimulation of calcium influx by platelet activating factor in Dictyostelium.

Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 minutes. PAF showed a reduced response in the G protein beta- strain LW14 and was unable to induce Ca2+ influx in the G alpha 2- strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx, albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of G alpha 2, we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this, NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store.

Prosthetic history, prosthetic charges, and functional outcome of the isolated, traumatic below-knee amputee.

PURPOSE: To review the prosthetic history, prosthetic charges, and functional status of traumatic, isolated, unilateral below-knee amputees at select intervals following amputation. METHODS: This descriptive study was conducted among patients admitted to Harborview Medical Center between 1980 and 1987 who survived initial trauma, and who required an isolated, below-knee amputation. Hospital and prosthetist records were abstracted to calculate the number of prostheses fabricated and the prosthetic charges since initial amputation. Functional outcomes were determined by personal interview and self-administration of the SF-36 Health Status Profile. RESULTS: The average age of patients was 36 with the age range extending from 19 to 59 years. The prosthetic history and prosthetic charges were determined from the medical record and the billing records of the prosthetist. Exact charges were determined for 15 of the 20 patients. During the first 3 years, the mean number of prostheses acquired per patient was 3.4 (range 1-5), with average total prosthetic charges of $10,829 (range $2,558-$15,700). Over the first 5 years the mean number of prostheses acquired per patient was 4.4 (range 2-8), with average total prosthetic charges of $13,945 (range $6,203-$20,070). The SF-36 Health Status Profile scores were significantly decreased from published normal aged-matched scores in the categories of physical function and role limitations because of physical health problems and pain. Scores were not significantly different from published normal aged-matched scores in the other five categories: role limitations due to emotional problems, social functioning, mental health, energy/fatigue, and health perception.

Evidence for two intracellular calcium pools in Dictyostelium: the cAMP-induced calcium influx is directed into a NBD-Cl- and 2,5-di-(tert-butyl)1,4-hydroquinone-sensitive pool.

Signal transduction in Dictyostelium for oriented movement and differentiation involves a fine tuning of the cytosolic Ca2+ concentration. We have previously shown that cAMP binding to the cell surface receptor elicits two cellular events: (i) to enhance Ca2+ entry across the plasma membrane; (ii) to increase Ca2+ uptake into Ca(2+)-sequestering organelles. Here we used permeabilised cells to show that cAMP-induced Ca2+ uptake in these cells was sensitive to the Ca2+ transport ATPase blocker 2,5-di-(tert-butyl)-1,4-hydroquinone (BHQ) and the vacuolar H(+)-ATPase inhibitor NBD-Cl. By contrast, bafilomycin A1 and vanadate, inhibitors of Ca2+ uptake into acidosomes in Dictyostelium, did not reduce the cAMP-induced Ca2+ uptake of permeabilised cells. GTP gamma S served as a tool to measure Ins(1,4,5)P3- (InsP3)-sensitive Ca2+ release. Following NBD-Cl or BHQ treatment Ca2+ release was reversibly inhibited. We conclude that the cAMP-controlled Ca2+ influx is directed into a NBD-Cl and BHQ-sensitive compartment, which comprises the InsP3-releasable pool. The acidosomal Ca2+ store seems to provide for additional Ca2+ if required.

Intercellular guanosine-5'-0-(3-thiotriphosphate) blocks chemotactic motility of Dictyostelium discoideum amoebae.

Starving amoebae of the cellular slime mold Dictyostelium discoideum react chemotactically towards the attractant cAMP. In this study, the effect of nonhydrolyzable analogs of GTP and GDP on the chemotactic behavior was analyzed with light microscopic techniques. Guanosine-5'-0-(2-thiotriphosphate) (GTP gamma S) or guanosine-5'-0-(2-thiodiphosphate) (GDP beta S) was scrape-loaded into the cytoplasm of cells, together with a fluorescent marker. Stimulation with a cAMP-filled glass capillary revealed a reduced capacity of loaded cells to migrate towards the capillary tip. Most cells still protruded filopods in the direction of the capillary tip, but full extension of pseudopods was inhibited in a dose-dependent and reversible manner. This indicates that in the presence of the analogs, chemotactic sensing still occurs, and that a more distal step of the cascade of events leading to the formation of the pseudopod is impaired. In cells loaded with the analogs together with the calcium indicator fura-2, stimulation with 10 microM cAMP led to a transient change in the intracellular free calcium concentration ([Ca2+]i), which was detectable in 28% of the cells. Furthermore, large vacuoles were found containing high amounts of calcium. On the other hand, clamping of [Ca2+]i at low levels with 1,2-bis(2-aminophenoxy) ethane N,N,N',N'-tetraacetic acid (BAPTA) also inhibited motility, with neither filopods nor pseudopods formed. The data suggest that chemotactic migratory activity involves GTP-dependent processes that participate in the regulation of the Ca2+ homeostasis of the cell and in the regulation of membrane traffic that contributes to the directed locomotion.

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Intracellular calcium during chemotaxis of Dictyostelium discoideum: a new fura-2 derivative avoids sequestration of the indicator and allows long-term calcium measurements.

During stimulation of Dictyostelium discoideum amoebae with the chemoattractant cAMP, extracellular calcium is taken up by the cells. The aim of this study was to determine the cytosolic free calcium concentration ([Ca++]i) during chemotaxis of Dictyostelium cells. In contrast to most vertebrate cells, three major drawbacks were encountered: 1) the indicator fura-2 could not be introduced into the cells by incubation with the ester form, 2) once loaded, the dye was rapidly sequestered into vesicles, 3) the organic anion transport blocker probenecid was not suitable to block sequestration. These problems were met by introducing the indicator into the cells with the scrape-loading technique adapted for use with Dictyostelium and the construction of a new fura-2 derivative, fura-2-dextran. Scrape-loading of Dictyostelium yielded up to 40% of labeled, vital cells. Fura-2-dextran fulfilled the following criteria: 1) it remained homogeneously distributed in the cytoplasm of motile Dictyostelium cells, 2) it retained the fluorescence intensity of fura-2 and the affinity for calcium binding, 3) it was very well suitable to demonstrate changes of [Ca++]i in serum-stimulated fibroblasts. [Ca++]i-measurements with fura-2-dextran in chemotactically active D. discoideum amoebae revealed that the large decrease in the extracellular calcium concentration is not accompanied by an overall change in [Ca++]i. Chemotaxis in this organism occurs in the absence of global changes in [Ca++]i. However, we cannot exclude either short-lived or local changes just beneath the plasma membrane.