ABSTRACT
Purification of DNA from fixed, paraffin-embedded tissue (PET) requires the removal of paraffin, proteins, and, in some cases, heavy metal ions. The resulting DNA is not generally suitable for Southern blot analysis. However, the polymerase chain reaction (PCR) can be used to amplify short regions of this DNA. In this appendix, the basic protocol can be performed rapidly and involves only a small number of manipulations, thus minimizing the possibility of contamination. An alternate protocol involves additional steps including extractions with xylenes and phenol/chloroform. This procedure is recommended as the primary approach when tissue has not been fixed in an optimal manner or as a secondary approach when the basic protocol has failed to produce satisfactory DNA.
Subject(s)
Animals, Laboratory , Animals , Blotting, Southern , Cricetinae , DNA/chemistry , DNA/isolation & purification , Mice , Paraffin Embedding , Polymerase Chain Reaction , RatsABSTRACT
Although for most experiments lymphoid tissue is removed from the freshly sacrificed animal, there are several circumstances which require the surgical removal, under anesthesia, of either the thymus or spleen, and the recovery of the animal for further investigation. This unit describes survival surgery for removal of the adult spleen as well as the adult and neonatal thymus of the mouse.
Subject(s)
Splenectomy/methods , Thymectomy/methods , Anesthesia , Animals , Animals, Newborn , Mice , Spleen/cytology , Spleen/immunology , Survival Rate , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
Although for most experiments lymphoid tissue is removed from the freshly sacrificed animal, there are several circumstances which require the surgical removal, under anesthesia, of either the thymus or spleen, and the recovery of the animal for further investigation. This unit describes survival surgery for removal of the adult spleen as well as the adult and neonatal thymus of the mouse.
Subject(s)
Lymph Node Excision/methods , Lymph Nodes/surgery , Spleen/surgery , Splenectomy/methods , Thymectomy/methods , Thymus Gland/surgery , Animals , Animals, Newborn , MiceABSTRACT
Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).
Subject(s)
Ceramides/pharmacology , Sodium-Calcium Exchanger/drug effects , Sphingosine/pharmacology , Amino Acid Sequence , Animals , CHO Cells , Calcium/metabolism , Cricetinae , Molecular Sequence Data , Sodium-Calcium Exchanger/chemistry , Sodium-Calcium Exchanger/metabolismABSTRACT
Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger were subjected to two periods of 5 and 3 min, respectively, during which the extracellular Na(+) concentration ([Na(+)](o)) was reduced to 20 mm; these intervals were separated by a 5-min recovery period at 140 mm Na(+)(o). The cytosolic Ca(2+) concentration ([Ca(2+)](i)) increased during both intervals due to Na(+)-dependent Ca(2+) influx by the exchanger. However, the peak rise in [Ca(2+)](i) during the second interval was only 26% of the first. The reduced rise in [Ca(2+)](i) was due to an inhibition of Na(+)/Ca(2+) exchange activity rather than increased Ca(2+) sequestration since the influx of Ba(2+), which is not sequestered by internal organelles, was also inhibited by a prior interval of Ca(2+) influx. Mitochondria accumulated Ca(2+) during the first interval of reduced [Na(+)](o), as determined by an increase in fluorescence of the Ca(2+)-indicating dye rhod-2, which preferentially labels mitochondria. Agents that blocked mitochondrial Ca(2+) accumulation (uncouplers, nocodazole) eliminated the observed inhibition of exchange activity during the second period of low [Na(+)](o). Conversely, diltiazem, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger, increased mitochondrial Ca(2+) accumulation and also increased the inhibition of exchange activity. We conclude that Na(+)/Ca(2+) exchange activity is regulated by a feedback inhibition process linked to mitochondrial Ca(2+) accumulation.
Subject(s)
Calcium/metabolism , Mitochondria/metabolism , Sodium-Calcium Exchanger/metabolism , Animals , CHO Cells , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cattle , Cricetinae , Cytosol/metabolism , Diltiazem/pharmacology , Feedback , Kinetics , Mitochondria/drug effects , Myocardium/metabolism , Recombinant Proteins/metabolism , Sodium/metabolism , TransfectionABSTRACT
The effects of the protein phosphatase inhibitors calyculin A and okadaic acid on Na(+)/Ca(2+) exchange activity were examined in transfected Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger. Incubating the cells for 5-10 min with 100 nM calyculin A reduced exchange-mediated (45)Ca(2+) uptake or Ba(2+) influx by 50-75%. Half-maximal inhibition of (45)Ca(2+) uptake was observed at 15 nM calyculin A. The nonselective protein kinase inhibitors K252a and staurosporine provided partial protection against the effects of calyculin A. Okadaic acid, another protein phosphatase inhibitor, nearly completely blocked exchange-mediated Ba(2+) influx. Chinese hamster ovary cells expressing a mutant exchanger in which 420 out of 520 amino acid residues were deleted from the central hydrophilic domain of the exchanger remained sensitive to the inhibitory effects of calyculin A and okadaic acid. Surprisingly, Na(o)(+)-dependent Ca(2+) efflux appeared to be only modestly inhibited, if at all, by calyculin A or okadaic acid. We conclude that protein hyperphosphorylation during protein phosphatase blockade selectively inhibits the Ca(2+) influx mode of Na(+)/Ca(2+) exchange, probably by an indirect mechanism that does not involve phosphorylation of the exchanger itself.
Subject(s)
Phosphoprotein Phosphatases/antagonists & inhibitors , Sodium-Calcium Exchanger/antagonists & inhibitors , Animals , Barium/metabolism , CHO Cells , Cattle , Cricetinae , Enzyme Inhibitors/pharmacology , Marine Toxins , Okadaic Acid/pharmacology , Oxazoles/pharmacology , Phosphorylation , Sodium-Calcium Exchanger/genetics , Sodium-Calcium Exchanger/metabolism , Spectrometry, Fluorescence , TransfectionABSTRACT
In this report, we have demonstrated that Na+/Ca2+ exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+ exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+ exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+ exchanger could make platelets especially sensitive to daily fluctuations in salt intake.
Subject(s)
Blood Platelets/metabolism , Sodium-Calcium Exchanger/metabolism , Calcium/metabolism , Cell Line , Humans , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/genetics , Retinal Rod Photoreceptor Cells/metabolism , Sodium-Calcium Exchanger/geneticsABSTRACT
The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.
Subject(s)
Calcium/metabolism , Sodium-Calcium Exchanger/metabolism , Animals , Biological Transport , CHO Cells/drug effects , Calcium-Transporting ATPases/antagonists & inhibitors , Cattle , Cricetinae , Cricetulus , Egtazic Acid/metabolism , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/metabolism , Fura-2/metabolism , Ionomycin/pharmacology , Ionophores/pharmacology , Sodium/metabolism , Sodium-Calcium Exchanger/drug effects , Sodium-Calcium Exchanger/genetics , Thapsigargin/pharmacology , TransfectionABSTRACT
Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+ exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+ concentration was clamped with gramicidin at approximately 20 mM. [Ca2+]i was varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+ influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+ regulation between whole cells and membrane patches is unknown.
Subject(s)
Calcium/metabolism , Sodium-Calcium Exchanger/metabolism , Animals , Barium/pharmacokinetics , CHO Cells , Calcium Chloride/metabolism , Cattle , Cricetinae , Cytosol/metabolism , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Homeostasis , Ionomycin/pharmacology , Kinetics , Myocardium/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sodium-Calcium Exchanger/biosynthesis , TransfectionABSTRACT
The Na+/Ca2+ exchange system is the primary Ca2+ efflux mechanism in cardiac myocytes, and plays an important role in controlling the force of cardiac contraction. The exchanger protein contains 11 transmembrane segments plus a large hydrophilic domain between the 5th and 6th transmembrane segments; the transmembrane regions are responsible for mediating ion translocation while the hydrophilic domain is responsible for regulation of activity. Exchange activity is regulated in vitro by interconversions between an active state and either of two inactive states. High concentrations of cytosolic Na+ or the absence of cytosolic Ca2+ promote the formation of the inactive states; phosphatidylinositol-(4,5)bisphosphate (or other negatively charged phospholipids) and cytosolic Ca2+ counteract the inactivation process. The importance of these mechanisms in regulating exchange activity under normal physiological conditions is uncertain. Exchanger function is also dependent upon cytoskeletal interactions, and the exchanger's location with respect to intracellular Ca2+-sequestering organelles. An understanding of the exchanger's function in normal cell physiology will require more detailed information on the proximity of the exchanger and other Ca2+-transporting proteins, their interactions with the cytoskeleton, and local concentrations of anionic phospholipids and transported ions.
Subject(s)
Calcium/metabolism , Homeostasis , Sodium-Calcium Exchanger/physiology , Sodium/metabolism , Actins/physiology , Animals , Calcium/pharmacology , Calcium/physiology , Cells, Cultured , Cytoskeleton/physiology , Models, Biological , Myocardial Contraction , Sodium-Calcium Exchanger/chemistryABSTRACT
BACKGROUND: In the twelve months following the announcement of the UK pit closure programme in October 1992, 22,500 miners were made redundant. In 1994 we undertook a cross-sectional survey to determine whether the mental and physical health of men who had been employed in the Nottinghamshire mining industry differed from that of the general population. METHODS: A postal questionnaire was designed incorporating the General Health Questionnaire (GHQ-12), and six domains from SF-36. Questionnaires were sent to 1064 miners and ex-miners and 2097 other men in Nottinghamshire. Non-responders were sent two reminders. RESULTS: The final response rate was 51%. The percentage of responders with GHQ-12 scores of three or more (suggesting psychological disorder) was 46% for those still employed in the mining industry, 52% for unemployed former miners and 22% for working non-miners (odds ratios: 3.0 [95% C.I. 2.2-4.1] for current miners and 3.9 [95% C.I. 2.6-5.7] for unemployed miners compared with working non-miners). The miners and ex-miners also had lower scores (suggesting greater morbidity) for each of the SF-36 domains tested. When stratifying for age in respondents of social classes IIIM-V the scores of current miners were significantly lower than those of working non-miners (P < 0.01). CONCLUSIONS: This study suggests that when surveyed in 1994, men who had been employed in three Nottinghamshire collieries in 1992 were psychologically and physically disadvantaged compared with working non-miners. Whether these findings are a result of pit closures is uncertain. However, significant potential health needs have been demonstrated.
Subject(s)
Coal Mining , Health Status , Mental Health , Unemployment , Adolescent , Adult , Aged , Cross-Sectional Studies , Employment/psychology , Health Surveys , Humans , Male , Middle Aged , Morbidity , Odds Ratio , Surveys and Questionnaires , Unemployment/psychology , United KingdomABSTRACT
Inhibitors of mitochondrial oxidative metabolism have been proposed to interfere with Ca2+ influx mediated by store-operated channels (SOC), secondary to their effects on ATP production. We assessed SOC activity by 45Ca2+ influx and fluorimetric measurements of free Ca2+ or Mn2+ quench in thapsigargin-treated Chinese hamster ovary cells and Jurkat T-cells, and additionally by electrophysiological measurements of the Ca2+-release-activated Ca2+ current (Icrac) in Jurkat T-cells. Various mitochondrial antagonists were confirmed to inhibit SOC. However, the following evidence supported the proposal that oligomycin, in particular, exerts an inhibitory effect on SOC in addition to its known actions on mitochondria and Na+-pump activity: (i) the concentrations of oligomycin required to inhibit SOC-mediated Ca2+ influx or Icrac (half-inhibitory concentration approximately 2 microM) were nearly 50-fold higher than the concentrations that blocked mitochondrial ATP production; (ii) the rank order of potency of oligomycins A, B and C for decreasing SOC-mediated Ca2+ influx or Icrac differed from that known for inhibition of mitochondrial function; (iii) oligomycin blocked Icrac under voltage clamp and with intracellular Na+ and K+ concentrations fixed by dialysis from the patch pipette, arguing that the effect was not secondary to membrane polarization or pump activity; and (iv) fixing the cytosolic ATP concentration by dialysis from the patch pipette attenuated rotenone- but not oligomycin-mediated inhibition of Icrac. Oligomycin also blocked volume-activated Cl- currents, a profile common to some other known blockers of SOC that are not known mitochondrial inhibitors. These findings raise the possibility that oligomycin interacts directly with SOC, and thus may extend the known pharmacological profile for this type of Ca2+-influx pathway.
Subject(s)
Adenosine Triphosphate/metabolism , Ion Channels/drug effects , Mitochondria/drug effects , Oligomycins/pharmacology , Animals , CHO Cells , Calcium/metabolism , Cricetinae , Humans , Ion Transport , Jurkat Cells , Manganese/metabolism , Mitochondria/metabolismABSTRACT
We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-mediated 45Ca2+ uptake with a Ki approximately 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20-40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles. Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as-Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25-50 nM We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+.
Subject(s)
Barium/metabolism , CHO Cells/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Myocardium/metabolism , Transfection , Animals , CHO Cells/physiology , Cattle , Cricetinae , Intracellular Membranes/metabolism , Organelles/metabolism , Osmolar Concentration , Sodium/metabolism , Sodium-Calcium ExchangerABSTRACT
The P2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca2+]i due to Ca2+ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca2+ release was accompanied by a Ca(2+)-dependent regulatory activation of Na+/Ca2+ exchange activity: Addition of extracellular Ca2+ (0.7 mM) 0-1 min after ATP evoked a dramatic rise in [Ca2+]i in Na(+)-free media (Li+ substitution) compared to Na(+)-containing media; no differences between Na(+)- and Li(+)-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na+ and Ca2+, the ATP-evoked rise in [Ca2+]i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca2+]i which eventually came to exceed the declining [Ca2+]i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba2+ influx, consistent with regulatory activation of the Na+/Ca2+ exchanger. The acceleration of Ba2+ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na+ or when the Ca2+ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca2+]i under Na(+)-free conditions, but did not inhibit the ATP-evoked stimulation of Ba2+ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca2+ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca2+ release from intracellular stores through regulatory activation by increased [Ca2+]i. In the absence of extracellular Ca2+, the stimulation of exchange activity is short-lived and follows the time course of the [Ca2+]i transient; in the presence of extracellular Ca2+, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca2+ entry.
Subject(s)
Adenosine Triphosphate/pharmacology , CHO Cells/metabolism , CHO Cells/physiology , Calcium/metabolism , Carrier Proteins/metabolism , Transfection , Animals , Barium/metabolism , CHO Cells/drug effects , Carrier Proteins/genetics , Cattle , Cricetinae , Intracellular Membranes/metabolism , Osmolar Concentration , Protein Kinase C/metabolism , Sodium/pharmacology , Sodium-Calcium ExchangerABSTRACT
Our experiments with transfected cells provide new insights into the role of Na-Ca exchange activity in Ca homeostasis and emphasize the role of local interactions in determining exchanger function. Thus, the effects of ATP depletion and cytochalasin D highlight the influence of the actin cytoskeleton in regulating exchange activity. Cytoskeletal interactions could provide a mechanism for modulating exchange activity by mechanical stretch and might constitute a novel feedback mechanism for regulating contractile activity in the heart. The effects of Na on Ca entry during SDCI in the transfected cells suggest that local gradients of [Ca]i are important determinants of exchanger function. The surface distribution of exchanger proteins in relation to that of Ca channels therefore represents another area in which interactions with the cytoskeleton may be a central element in understanding the physiological function(s) of the exchange activity. At present, it seems likely that the exchanger's central hydrophilic domain mediates the connection between the exchanger and the cytoskeleton. This provides a rationale for understanding the importance of tissue-specific alterations in the exchanger's hydrophilic domain, which appear to have little affect on the kinetic behavior of the exchanger. Future work in our laboratory will be directed toward clarifying the role of cytoskeletal interactions in exchanger function.
Subject(s)
Calcium/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Homeostasis , Transfection , Amino Acid Sequence , Animals , CHO Cells , Carrier Proteins/chemistry , Cricetinae , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , Sodium/metabolism , Sodium-Calcium ExchangerABSTRACT
The effects of extracellular Na+ on store-dependent Ca2+ influx were compared for transfected Chinese hamster ovary cells expressing the bovine cardiac Na+-Ca2+ exchanger (CK1.4 cells) and vector-transfected control cells. Store-dependent Ca2+ influx was elicited by depletion of intracellular Ca2+ stores with ionomycin, thapsigargin, or extracellular ATP, a purinergic agonist. In each case, the rise in [Ca2+]i upon the addition of extracellular Ca2+ was reduced in CK1.4 cells compared with control cells at physiological [Na+]o. When Li+ or NMDG was substituted for Na+, the CK1.4 cells showed a greater rise in [Ca2+]i than control cells over the subsequent 3 min after the addition of Ca2+o. Under Na+-free conditions, SK&F 96365 (50 microM), a blocker of store-operated Ca2+ channels, nearly abolished the thapsigargin-induced rise in [Ca2+]i in the control cells but only partially inhibited this response in the CK1.4 cells. We conclude that in the CK1.4 cells, Ca2+ entry through store-operated channels was counteracted by Na+o-dependent Ca2+ efflux at physiological [Na+]o, whereas Ca2+ entry was enhanced through Na+i-dependent Ca2+ influx in the Na+-free medium. We examined the effects of thapsigargin on Ba2+ entry in the CK1.4 cells because Ba2+ is transported by the Na+-Ca2+ exchanger, but it enters these cells only poorly through store-operated channels, and it is not sequestered by intracellular organelles. Thapsigargin treatment stimulated Ba2+ influx in a Na+-free medium, consistent with an acceleration of Ba2+ entry through the Na+-Ca2+ exchanger. We conclude that organellar Ca2+ release induces a regulatory activation of Na+-Ca2+ exchange activity.
Subject(s)
Calcium/metabolism , Carrier Proteins/metabolism , Myocardium/metabolism , Sodium/metabolism , Animals , Barium/metabolism , Biological Transport/drug effects , CHO Cells , Calcium Channel Blockers/pharmacology , Calcium-Transporting ATPases/antagonists & inhibitors , Carrier Proteins/biosynthesis , Cattle , Clone Cells , Cricetinae , Culture Media , Enzyme Inhibitors/pharmacology , Genetic Vectors , Imidazoles/pharmacology , Kinetics , Organelles/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Sodium/pharmacology , Sodium-Calcium Exchanger , Terpenes/pharmacology , Thapsigargin , TransfectionABSTRACT
Mouse hybridomas were isolated that produce monoclonal antibodies (MAbs) to several regions of the HIV envelope, gp120, which may be used to map immunogenic regions in animal and human immunity. One series of MAbs was generated by repeated immunization with recombinant gp120 (rgp120) from a single isolate, and a second series by sequential immunization with rgp120 from three isolates. All MAbs bound to rgp120IIIB, but only one bound well to cell surface-expressed gp160. Synthetic peptides spanning much of the length of gp120 were used to map MAb reactivity. Two MAbs were mapped to the C1 region, one to the C2 region, and three to the C5 region by this approach. Six distinct epitopes were detected by competitive binding analysis. One MAb binding the C1 region blocks binding of CD4 to rgp120IIIB, binds by ELISA to rgp120MN (an isolate not used during immunization), and binds to cell surface-expressed gp160. These hybridomas will be made available to the scientific community through a hybridoma culture facility.
Subject(s)
AIDS Vaccines/immunology , Antibodies, Monoclonal/biosynthesis , HIV Envelope Protein gp120/immunology , HIV-1/isolation & purification , Animals , Antibodies, Monoclonal/chemistry , Binding Sites, Antibody , CD4 Antigens/immunology , CD4 Antigens/metabolism , Cell Fusion/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Gene Products, env/analysis , HIV Envelope Protein gp160 , Humans , Mice , Mice, Inbred BALB C , Protein Precursors/analysisABSTRACT
Chinese hamster ovary cells expressing the bovine cardiac Na/Ca exchanger were treated with ouabain to increase [Na+]i and stimulate Ca2+ influx by Na/Ca exchange. Depletion of cellular ATP inhibited 45Ca uptake by 40% or more and reduced the half-maximal Na+ concentration for inhibition of 45Ca uptake from 90 to 55 mM. ATP depletion also reduced the rate of rise in [Ca2+]i when [Na+]o was reduced and inhibited the decline in [Ca2+]i when high [Na+]o was restored. The effects of ATP depletion were either absent or reduced in cells expressing a mutant exchanger missing most of the cytosolic hydrophilic domain. We were unable to detect a phosphorylated form of the exchanger in immunoprecipitates from 32P-labeled cells. ATP depletion caused a breakdown in the actin cytoskeleton of the cells. Treatment of the cells with cytochalasin D mimicked the effects of ATP depletion on the [Na+] inhibition profile for 45Ca uptake. Thus, ATP depletion inhibits both the Ca2+ influx and Ca2+ efflux modes of Na/Ca exchange, and may alter the competitive interactions of extracellular Na+ and Ca2+ with the transporter. The latter effect appears to be related to changes in the actin cytoskeleton.
Subject(s)
Adenosine Triphosphate/physiology , Calcium/metabolism , Carrier Proteins/physiology , Sodium/metabolism , Actins/metabolism , Animals , CHO Cells , Cattle , Cricetinae , Cytochalasin D/pharmacology , Hydrogen-Ion Concentration , Myocardium/metabolism , Sodium-Calcium Exchanger , TransfectionABSTRACT
Differences in cation transport have been observed between African Americans and whites. These differences may underlie the increased predisposition of African Americans to essential hypertension. To further explore these racial differences, we used platelets as a cellular model for calcium regulation. We measured 45Ca fluxes in platelets from 21 African American and 25 white men. Additionally, using fura 2, we measured cytosolic free calcium levels in resting platelets and platelets treated with ouabain and thrombin. Platelet 45Ca uptake was described by two exchangeable pools: a small, rapidly exchangeable pool and a larger, slowly exchangeable pool. Both pools were larger in platelets from African Americans than from whites (263 versus 185 pmol per 1 x 10(8) platelets for the rapidly exchangeable pool, P < .05; 744 versus 532 pmol per 1 x 10(8) platelets for the slowly exchangeable pool, P < .01). 45Ca washout was described by a rapidly exchangeable pool and a static pool. The former was also higher in platelets from African Americans than from whites (246 versus 202 pmol per 1 x 10(8) platelets, P < .01). The cytosolic free calcium concentrations in resting platelets were lower in African Americans than in whites. After treatment with ouabain and thrombin, the sustained posttransient levels of cytosolic free calcium increased to a greater extent in platelets from African Americans (46.7 nmol/L) than from whites (34.5 nmol/L, P = .033). Platelets from African Americans demonstrate higher intracellular calcium stores than platelets from whites. This racial difference could explain the sensitivity of African Americans to vasoactive agents acting through calcium mobilization from intracellular stores and cytosolic calcium.