Adenine and hypoxanthine metabolism in phythohemagglutinin-stimulated and unstimulated human lymphocytes

Summary The uptake and subsequent metabolism of adenine and hypoxanthine in phytohemagglutinin-stimulated and unstimulated peripheral human blood lymphocytes, freshly prepared or cultured, were studied. To investigate the initial step of nucleic acid metabolism the incorporation of¹⁴C-purines into the acid soluble material was examined. No preferential uptake of adenine or hypoxanthine was observed in freshly prepared and cultured lymphocytes during an incubation of 1 h. However, cultured cells utilized approximately 1/3 of the purines compared to freshly drawn cells. Within the cells 2/3 of adenine and 1/2 of hypoxanthine were metabolized to nucleotides (mainly AMP and ADP). Incubation of lymphocytes with PHA for 1 h produced in the freshly prepared cells an increase of adenine- and hypoxanthine-uptake to 191% and 153%, in 48 h stimulated cells to 158% and 132%. There was, however, no change in the relative rates of the metabolic routes though the intracellular concentrations of nucleotides formed increased with adenine as substrate to 152% and with hypoxanthine to 161% during a 1 h stimulation. In contrast no enhanced formation of acid soluble nucleotide formation could be observed with PHA stimulation during 48 h. The increased rates of purine uptake and metabolism apparent 1 h after addition of mitogen may be due to an altered transport mechanism at the beginning of the transformation as an adaptive response to the increased requirements for the synthetic processes soon to follow. Once the lymphocytes are transformed no demand of purines is necessary and the uptake and metabolism is switched off.This work has been supported by grant No. 9796 of the Fonds zur Förderung der wissenschaftlichen Forschung     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Swelling of normal,preneoplastic and neoplastic liver mitochondria

Summary The swelling of rat and mouse liver mitochondria, isolated in 0.30 M sucrose and incubated in sucrose-Tris of p_H 7.4 at 22±1°C, in response to succinate, adenine nucleotides (10^–5 M), malonate, thyroxine, and DNP, separately or in various combinations, was measured.It was found that both the spontaneous and thyroxine-induced swelling of fresh mitochondria were counteracted by succinate and, to a still greater extent, by succinate plus adenine nucleotide (ADPATP or > AMP). The protection was abolished by malonate or DNP. The protection against swelling afforded by succinate to fresh mitochondria was followed by a phase of rapid swelling. Progressive ageing or frequent washing of the particles, prior to their incubation, led to a reduction or abolition of the protective effect. It was concluded that mitochondrial swelling was governed by the degree of intrinsic stability of the isolated particles.

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Zusammenfassung Die Schwellung von in 0,30 M Saccharose isolierten und in Saccharose-Tris von p_H 7,4 bei 22±1°C inkubierten Mitochondrien aus Mäuse- und Rattenlebern wurde gemessen nach Zusatz von Succinat, Adeninnucleotiden (10^–5 M), Malonat, Thyroxin und DNP, allein und in verschiedenen Kombinationen. Sowohl die spontane wie die thyroxininduzierte Schwellung frischer Mitochondrien wird durch Succinat und noch mehr durch Succinat und Adeninnucleotide (ADPATP oder > AMP) gehemmt. Die Hemmung wird aufgehoben durch Malonat oder DPN. Auf die Hemmung der Schwellung von frischen Mitochondrien durch Succinat folgt eine Phase schneller Schwellung. Allmähliche Alterung oder wiederholte Waschungen von den Mitochondrien vor der Inkubation führen zu einer Verringerung oder Aufhebung des hemmenden Effektes des Succinats.Daraus wird gefolgert, daß die mitochondriale Schwellung von dem Grade der Eigenstabilität der isolierten Partikel abhängig ist.

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Abbreviation used O.D.₅₂₀ optical density at 520 m - EDTA ethylenediamine tetra-acetate - AMP, ADP and ATP adenosine mono-,-di- and-triphosphates - Tris 2-amino-2-hydroxymethylpropane-1:3-diolchloride - DPN diphosphopyridine nucleotide - DNP 2:4-dinitrophenol     

Untersuchungen über Malignolipin

Zusammenfassung Eine experimentelle und theoretische Überprüfung der Kosakischen Arbeiten in bezug auf den von ihm isolierten Stoff Malignolipin wurde unternommen.Bei einer Anzahl von 56 krebskranken Patienten wurde der Malignolipin-Blut-Test nach der Kosakischen Methode durchgeführt. Bei 46 Personen (82%) wurde papierchromatographisch ein Stoff nachgewiesen, welcher den Angaben von Kosaki über Malignolipin entsprach.Aus operativ gewonnenen und experimentell erzeugten Tumoren wurde ebenfalls Malignolipin als Pikrat isoliert oder papierchromatographisch nachgewiesen.Eine Modifikation des Malignolipin-Blut-Testes wurde eingeführt für die Trennung des Malignolipins von anderen Ninhydrin-positiven Verbindungen mit Hilfe eines Aminosäure-Analysators.

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Investigations on malignolipin

Summary Experimental investigations have been undertaken to prove the authenticity of Malignolipin test for the early diagnosis of cancer (Kosaki and co-workers).Of 56 patients with malignant tumours, 46 showed a positive malignolipin reaction (82%), using the paper chromatographic technic.A malignolipin-like compound has been isolated from ovarian-cancer, Yoshida-Sarcom^a, Ehrlich-Mouse-Ascites-Carcinom^a, Plasmocytom^a, and Benzpyrene-Sarcom^a with the method of Kosaki.Since the method for malignolipin detection is complicated, mistakes may be made during the isolation of malignolipin from blood and tumours that could decrease the validity of the test for cancer diagnosis. A modification of the method of Kosaki consists in the further separation of the malignolipin fraction with an Amino Acid-Analyser. Two ninhydrin positive absorption peaks were found in the blood of control healthy persons; besides these two peaks, a third peak was observed in the blood of patients bearing malignant tumours.Radioactive malignolipin was obtained after intraperitoneal injection of ³²P-sodium orthophosphate to EMAC animals. After i. p. administration of ¹⁴C-putrescin several radioactive matabolic products were isolated by means of paper chromatography. Some of these compounds were identify as spermine, spermidine, ethanolamine etc.

     

Uptake and metabolism of radiolabelled GM1-ganglioside in skin fibroblasts from controls and patients with GM1-gangliosidosis

Summary The uptake and metabolism of [3-³H-sphingosine]GM1-ganglioside was measured in cultured skin fibroblasts from controls and patients with infantile, juvenile and adult GM1-gangliosidosis. When dissolved in medium with phosphatidylserine, GM1-ganglioside was efficiently taken up by cultured skin fibroblasts and transferred into lysosomes. A linear increase in GM1-ganglioside endocytosis was shown with phosphatidylserine concentrations of up to 40m/ml. A pulse-chase study revealed that [³H]GM1-ganglioside was metabolized to GM2-ganglioside, GM3-ganglioside, ceramide dihexoside, ceramide monohexoside, ceramide and sphingosine. Sphingosine was recycled to sphingomyelin. In a 20-h pulse study, cell lines from patients with GM1-gangliosidosis of infantile, juvenile and adult types hydrolysed 2–5%, 20–44% and 54–58% of the total endocytosed GM1-ganglioside respectively. These values were lower than in control cells (64.17 ± 5.43% (n=10)). The hydrolysis rates of exogenous [³H]GM1-ganglioside in cultured fibroblasts from patients with various types of GM1-gangliosidosis closely reflected the clinical severity.Abbreviations GM1 Gal13GalNAc14(NeuAc23)Gal14Galc-1-ceramide - GM2 GalNAc14(NeuAc23)Gal14Glc-1-ceramide - GM3 NeuAc23Gal14Glc-1-ceramide - CDH Gal14Glc-1-ceramide     

β-Adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1

Aims/hypothesis Brown adipocytes provide a potentially important model system for understanding AMP-activated protein kinase (AMPK) regulation, where adrenergic stimulation leads to mitochondrial uncoupling through uncoupling protein-1 (UCP1) activity. AMPK is a sensor of energy homeostasis and has been implicated in glucose and lipid metabolism in several insulin-sensitive tissues. The aim of this study was to characterise the potential role of AMPK in adrenergically mediated glucose uptake and to find out whether UCP1 is involved in the adrenergic activation of AMPK.Methods We used primary brown adipocytes differentiated in culture and measured AMPK phosphorylation and glucose uptake following adrenergic activation.Results Treatment of adipocytes with noradrenaline (norepinephrine) caused phosphorylation of AMPK via -adrenoceptors and not ₁- or ₂-adrenoceptors. This effect was not ₃-adrenoceptor specific, since responses remained intact in adipocytes from ₃-adrenoceptor knock-out mice. These effects were also mimicked by forskolin and cAMP analogues. Treatment of cells with adenine 8--d-arabinofuranoside, an AMPK inhibitor, partially blocked -adrenoceptor-mediated increases in glucose uptake. Brown adipocytes are characterised by the production of UCP1, which can uncouple the mitochondria. Using adipocytes from Ucp1^+/+ and Ucp1^–/– mice, we showed that noradrenaline-mediated phosphorylation of AMPK does not require the presence or activity of UCP1.Conclusions/interpretation These results suggest a pathway where increases in cAMP mediated by -adrenoceptors leads to activation of AMPK in brown adipocytes, which contributes in part to -adrenoceptor-mediated increases in glucose uptake, an effect independent of the presence or function of UCP1.     

Anoxia inhibits guanosine salvage in cardiac myocytes

The adult heart depends largely on salvage synthesis to supply its 5-nucleotide needs. Previous work from this laboratory established that guanosine is metabolized into guanine 5-nucleotides in heart cells, but that salvage rates are very slow as compared to adenosine. The author hypothesized that guanosine salvage is regulated according to the needs of the cell for guanine nucleotides. This hypothesis was tested using cardiac myocytes which were rendered anoxic for 0–60 min. During this anoxic period, guanine nucleotides were depleted about 50%. At 0, 30, and 60 min, aliquots were removed for cell counting and nucleotide analysis; 50 M³H-guanosine was then added and the incubation continued for 1 min. The cells were then extracted and assayed for radioactivity in the guanine nucleotide products. Anoxia for 60 min, depressed GTP levels by 89%, total guanine nucleotides by 50%, and short-term guanosine salvage by 48% over aerobic controls. Reoxygenation of the myocytes after 30 min of anoxia returned guanosine salvage rates to nearly normal (87% of control). Preincubation of the myocytes with 5 mM ribose for times up to 1 hour modestly increased salvage rates of guanosine in aerobic cells. These results suggest that guanosine salvage in cardiac myocytes is not regulated by the size of the guanine nucleotide pool (that is, not sensitive to the demand for guanine nucleotides). Instead, salvage of guanosine is probably limited by cytosolic levels of ATP or phosphoribosylpyrophosphate, the production of which are dependent on adequate oxygen supplies.     

Abnormal expansions of granular lymphocytes: Reactive lymphocytosis or chronic leukemia? case report and literature review

Summary A case of chronic lymphoproliferative disorder is presented, wherein a morphologically homogeneous population of lymphoid cells displayed properties similar to those described for large granular lymphocytes (LGL). Besides their LGL-like phenotype (VEP 13⁺, OKM 1⁺, OKT 10⁺ Fc-IgG-receptor⁺, OKT 3^–), the proliferating cells were cytotoxic to NK targets as well as to antibody-coated target cells. Clinically, our patient presented low-grade lymphocytosis, splenomegaly, neutropenia, hyperimmunoglobulinemia and recurrent infections. Based upon this and 32 similar cases reported in the literature, we conclude that lymphoproliferative disorders involving GL encompass a variety of clinical entities, ranging from reactive GL lymphocytoses to overt lymphocytic malignancies.Abbreviations GL granular lymphocytes - LGL large granular lymphocytes - PBL peripheral blood lymphocytes - NK natural killer - ADCC antibody-dependent cellular cytotoxicity - APh acid phosphatase - PAS periodic acid-Schiff - -Glu -glucuronidase - ANBE alpha-naphtyl-butyrat-esterase - ANAE acid esterase - POX peroxidase - PTA parallel tubular arrays - E-R surface membrane receptor for sheep erythrocytes This work was financially supported by the Austrian fund Zur Förderung der wissenschaftlichen Forschung project no. 4578 and by the Jubiläumsfond of the Austrian National Bank     

Alterations in the heart sarcolemmal Ca2+ transport activity by some β-adrenergic antagonists

Summary The effects of some -adrenergic antagonists such as acebutolol, propranolol, pindolol and oxprenolol (1–1000 M) were studied on the rat heart sarcolemmal Ca²⁺ transport activities. Pindolol enhanced sarcolemmal ATP-dependent Ca²⁺ binding and Ca²⁺-stimulated ATPase whereas acebutolol had no effect. Both propranolol and oxprenolol had biphasic actions on the sarcolemmal Ca²⁺ pump activities; the lower concentrations (1 and 10 M) were stimulatory, but the higher concentrations (100 and 1000 M) were inhibitory. None of the drugs used in this study had any effect on Mg²⁺ ATPase and non-specific Ca²⁺ binding activities of heart sarcolemma except that 1000 M propranolol decreased Mg²⁺ ATPase activity significantly. Mitochondrial and microsomal ATP-dependent Ca²⁺ binding activities were unaffected by these drugs (1–1000 M), except that 1000 M propranolol was inhibitory. These results suggest differences among various -adrenergic blocking drugs with respect to their actions on sarcolemmal Ca²⁺ pump in the myocardium.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Summary Antineoplastic activity of tiazofurin (2--D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC₅₀ of 19.2 M. TAD content in K 562 cells (1.3 nmol/10⁹/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.Abbreviations ALL acute lymphoblastic leukemia - ANLL acute non lymphocytic leukemia - CML chronic myelogenous leukemia - GMP, GDP, GTP guanosine 5-mono-, di-, triphosphate - HPLC high pressure liquid chromatography - IMP inosine 5-monophosphate - IMPD IMP dehydrogenase - NAD nicotinamide-adenine-dinucleotide - NCI National Cancer Institute; - PBS phosphate buffered saline - TAD thiazole-4-carboxamide-adenine dinucleotide - TCA trichloracetic acid - TRMP tiazofurin 5-monophosphate - TRTP tiazofurin 5-triphosphate. The research work outlined in this paper was supported by United States Public Health Service grant R 35 CA 42510 awarded by the National Cancer Institute, Department of Health and Human Services, USA to G. Weber. The results will be presented at the 79th annual meeting of the American Association for Cancer Research in New Orleans, May 25–28, 1988     

Increased uptake of low density lipoprotein by SV40-transformed smooth muscle cells

We compared the metabolism of low density lipoprotein (LDL) in SV40-transformed smooth muscle cells (TSMCs) to that in nontransformed smooth muscle cells (SMCs). When SMCs were incubated in medium with 100 g/ml LDL for 24 hours, they did not accumulate sudanophilic lipid droplets. On the other hand, when TSMCs were incubated in medium containing more than 100 g/ml LDL, they accumulated a large amount of lipid droplets in their cytoplasm. When cells were incubated with 200 g/ml LDL for 24 hours, cholesteryl ester levels significantly increased in TSMCs (18.3±3.53 g/mg protein), as compared with SMCs (2.40±0.85 g/mg protein). However, there was no difference in the cellular level of free cholesterol between the TSMCs and SMCs. Although the TSMCs and SMCs had a similar number of binding sites for LDL, the TSMCs demonstrated a markedly higher uptake of LDL labeled with 1,1-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine perchlorate (Dil-LDL), compared with the SMCs. SMCs that had been pretreated with 100 g/ml of unlabeled LDL for 24 hours showed a decreased uptake of Dil-LDL. In contrast, TSMCs incorporated Dil-LDL independently of the preincubation with 100 g/ml LDL. The presence of brefeldin A, which may block the transport of glycoproteins from the ER to Golgi apparatus, had less of an effect on the uptake of LDL in the TSMCs than in the SMCs. These results suggest that SV40-transformed smooth muscle cells show an increased uptake of LDL independent of the cellular cholesterol level, which may induce the accumulation of lipid droplets in their cytoplasm. A LDL receptor-independent pathway may be related to the increased uptake of LDL in SV40-transformed smooth muscle cells.     

Locoregional administration of 5-fluoro-2′-deoxyuridine (FdUrd) in Novikoff hepatoma in the rat: effects of dose and infusion time on tumor growth and on FdUrd metabolite levels in tumor tissue as determined by19F-NMR spectroscopy

Summary The influence of infusion time and dose on the anticancer efficacy of 5-fluoro-2-deoxyuridine (FdUrd) was investigated using a locoregional therapy model: Novikoff hepatoma transplanted i.m. into the thigh of Wistar rats and FdUrd infusion via a catheter implanted in the femoral artery. In experiment A the FdUrd dose (five daily doses of 12, 19 and 30 mg/kg) and the duration of administration (bolus, 1 h, 5 h, and 24 h) were varied. The change in tumor volume following treatment and the number of rats showing regression vs progression served as indicators of therapy response. The results showed a clear dose dependence, and for each infusion time the 30 mg/kg dose was the most effective, without any signs of general toxicity. At this dose the longest infusion time (24 h) was less effective (regression in three of six rats) compared with 1-h or 5-h treatments (four of five in regression). In experiment B either one or five daily FdUrd doses (15, 30, 60 mg/kg) were administered i.a. for the same infusion times used in experiment A. After treatment, tumors were explanted ex vivo and approximately 1-g tissues samples were immediately frozen in liquid nitrogen for storage.¹⁹F-NMR spectroscopy at 11.7 T was used to quantify FdUrd metabolites [5-fluorouracil (FUra),-fluoro--alanine (FAla.), 5-fluorouracil nucleosides and nucleotides (F-Nuc)] in the solid tumor tissue samples (maintained at 4° C) with a detection threshold of about 5 nmol/g. The metabolite signal pattern indicated that FdUrd is first converted to FUra, followed by anabolism primarily to nucleotides in the oxy form (e.g. FUTP). The total amount of fluorine detected in tumor tissue increased with dose and decreased with infusion time. For all treatments FNuc could be detected, even after 24 h infusion, and their levels showed a good linear correlation with the total F. The major catabolite FAla was present in tumor at low levels that correlated poorly with total F, indicating recirculation from other organs (e.g. liver) as the main source. Thus, the NMR method can provide detailed information regarding the efficiency of locoregional treatment (catheter function, drug uptake and metabolism). Initial results of non-invasive in vivo NMR experiments are also presented.Abbreviations FUra 5-fluorouracil - FNuc all 5-fluorouracil nucleosides e.g. FUrd, FdUrd - FNucP _n all 5-fluorouracil nucleotides e.g. FUMP, FdUMP, FUTP - FAla -fluoro--alanine - total F sum of all fluorine-containing metabolites - T ₁ nuclear spin lattice relaxation time - FNuc(P _n) all FUra anabolites This work was supported in part by a grant from the Tumorzentrum Heidelberg/Mannheim     

Effects of granulocyte colony-stimulating factor on neutrophils and inflammatory cytokines in the early stage of severe acute pancreatitis in rats

Background The high mortality rate of severe acute pancreatitis (SAP) is closely associated with secondary infections of pancreatic and peripancreatic tissues. It was reported that granulocyte colony-stimulating factor (G-CSF) increased the number of leukocytes and enhanced their functions. However, an inflammatory response may be enhanced by an increased number of leukocytes. Our purpose was to study the roles of G-CSF in peritoneal-exudate neutrophils and inflammatory cytokines in the early stage of experimental SAP.Methods SAP was induced by injecting 0.2ml of 3% taurocholate acid into the biliopancreatic duct in male Wistar rats. G-CSF (90µg/kg body weight) or saline was administered 1h before the SAP induction. The number of neutrophils and their phagocytic and bactericidal activities were evaluated, and the concentrations of tumor necrosis factor (TNF)-, interleukin (IL)-6, and IL-1 in plasma and ascitic fluid were measured 1h and 3h after the SAP induction.Results The number of peritoneal-exudate neutrophils (PENs) at 3h was increased by G-CSF administration (81 ± 50 × 10⁵ cells/total exudate), as compared with that shown with saline administration (28 ± 13 × 10⁵ cells/total exudate; P < 0.05). The numbers of phagocytic and bactericidal neutrophils were also elevated by G-CSF administration. G-CSF administration did not increase the concentrations of TNF-, IL-6, and IL-1 in the plasma and ascitic fluid.Conclusions G-CSF increases the numbers of neutrophils and enhances their functions against bacteria, but it does not enhance intraabdominal and systemic inflammatory responses in the early stage of SAP.     

Prognostic relevance of histological findings on bone marrow biopsy in myelodysplastic syndromes

Summary Bone marrow biopsy (BMB) has aroused growing interest as a possible aid in the diagnostic and prognostic evaluation of myelodysplastic syndromes (MDS). Previous reports have pointed out that MDS patients with blastic aggregates or severe bone marrow (BM) fibrosis are characterized by a worse clinical outcome. BMBs of 106 MDS patients were retrospectively reviewed, and relationships among the different histological parameters as well as clinicopathological correlations were looked for. Three patterns of BM blastic infiltration (diffuse, cluster, and large) were recognized. Overt leukemic transformation and overall survival were selected as prognostic end points. BM infiltration was diffuse in 18, cluster in 48, and large in 40 cases. RAEB-t patients accounted for about half of the large cases, and none had a diffuse pattern (p<0.01). Nineteen patients showed extensive BM fibrosis; most of them were characterized by cluster blastic infiltration and megakaryocyte hyperplasia. Leukemic transformation occurred in 67% of large cases (p<0.001) and in none of the cluster cases with severe BM fibrosis (p<0.01); however, survival was equally poor in these two groups because of early leukemic transformation (large cases) and BM failure (cluster cases). The FAB classification did not significantly correlate with prognosis. Patients with cluster BM infiltration and severe fibrosis can be regarded as a true separate MDS subset characterized by unique clinicopathological and prognostic features. Because of the subacute clinical behavior of most cases, and the poor performance status of many elderly patients, there is still controversy as to the best therapeutic approach in MDS. Histological analysis allowed two groups of MDS patients to be identified, both characterized by poor life expectancy, who could benefit from early aggressive chemotherapy.     

DNA adducts by N-nitroso compounds

Summary Some unsolved problems in DNA alkylation by N-nitroso compounds are discussed in this overview. (1) Does O⁶ alkylation of guanine represent the initiating event exclusively or are O⁴ alkylation of thymidine and phosphate triester formation also involved in the initiating process? (2) Does the formation of rearranged DNA alkylation products by longer chained alkylnitroso compounds have any significance for the carcinogenic effects of these compounds? The concept of hard and soft acids and bases (HSAB principle) as a qualitative model can predict the changes in the DNA alkylation pattern by branched carbenium ions.Presented at the SEK workshop DNA Adducts and Chemical Carcinogenesis, Tübingen, February 28–March 1, 1986     

TCR1+ large granular lymphocyte proliferation in rheumatoid arthritis

The T-lymphoproliferative syndrome is characterized by a proliferation of large granular lymphocytes (LGL). It is often associated with neutropenia, and in 30% of cases with rheumatoid arthritis (RA). Phenotypic analysis has demonstrated that in most cases of RA with T-proliferative disease, the LGL represent T cells with a clonal rearrangement of the / T cell receptor (TCR2). Here, three patients with / TCR1⁺ LGL proliferation suffering from long-standing arthritis and neutropenia are described. The first patient with RA showed an expansion of a heterogeneous CD2⁺ CD16⁺ CD56^- LGL population, of which 30% coexpressed TCR1 with V1 rearrangement. The second patient with ankylosing spondylitis and RA was suffering from proliferation of TCR1⁺ (V9^-, V1^-), CD2⁺ CD16^- CD56^- LGL with low coexpression of CD8. The third patient with RA was suffering from a proliferation of TCR1⁺ (V1⁺, V9^-) CD4^- CE8^- CD16^- CD56^- lymphocytes. On the basis of these unusual findings, the pathogenetic role of TCR1⁺ T cells in RA is discussed.     

α2-Adrenergic stimulation counteracts glucose-induced rise of sodium in pancreatic islets exposed to ouabain

The effects of ₂-adrenergenic activation by clonidine on sodium handling were analysed in beta-cell-rich pancreatic mouse islets. In the steady-state situation, clonidine (1 M) amplified lowering of sodium induced by 20 mM glucose, while the content remained unchanged in 3 mM glucose. The loss of sodium in Na⁺-deficient medium was stimulated by glucose but was not affected by clonidine. This agonist also did not influence the ouabain-induced uptake of sodium at 3 mM glucose but partially counteracted additional uptake in response to 20 mM glucose. Although lacking effects of its own, 5 M yohimbine completely counteracted the action of clonidine. The glucose amplification of the ouabain-induced uptake of sodium was suppressed also by 10 M of the Ca²⁺-channel blockers methoxyverapamil and diltiazem. Both tolbutamide (100 M) and dibutyryl cyclic AMP (1 mM) mimicked the action of glucose by promoting clonidine-sensitive uptake of sodium in the presence of ouabain. It is concluded that activation of ₂-adrenoceptors has profound effects on the sodium handling of pancreatic beta-cells exposed to glucose and other stimulators of insulin release.     

Effect of metoprolol on activity of β-adrenoceptor coupled to guanine nucleotide binding regulatory proteins in adriamycin-induced cardiotoxicity

Summary Prevention of cardiotoxicity without interfering with the therapeutic efficacy of adriamycin is a very crucial question. We have investigated the activity of -adrenoceptor coupled to guanine nucleotide binding regulatory proteins (G-proteins) and Ca²⁺-ATPase activity in experimental adriamycin-induced cardiotoxicity and the influence of metoprolol treatment on these variables. Adriamycin was administered to rats intravenously as a single dose of 6 mg/kg, and metoprolol was continuously given by means of implanted osmotic pumps. -adrenoceptor characteristics were measured by radioligand-binding experiments and by basal and stimulated adenylyl cyclase activity. Northern blot and dot blot analysis was used to quantify G-protein mRNA. It was shown that adriamycin did not induce any change in the total -adrenoceptor density, nor did the high affinity agonist binding to -adrenoceptor change. Adriamycin did not induce any alteration in the amount of mRNA encoding for stimulatory (Gs) or inhibitory (Gi) G-proteins. Also, basal and stimulated adenylyl cyclase activities were identical in the different experimental groups. In contrast, the Ca²⁺-ATPase was shown to increase in adriamycin-treated rats compared to control rats (45 ± 3.8 versus 23 ± 1.2 mol Pi/mg/h, P < .01). Metoprolol was shown to normalize this increase (29 ± 2.1 mol Pi/mg/h). Thus, it may be concluded that in experimental adriamycin-induced cardiotoxicity, despite Ca²⁺-overloading, the -adrenoceptor-G protein-adenylyl cyclase system remains intact. Metoprolol seems to prevent Ca²⁺-overloading independently of the -adrenoceptors studied here.     

Detection of coronary arterial microvascular disorders using <Superscript>99m</Superscript>Tc-tetrofosmin uptake increase during exercise and coronary blood flow velocity patterns obtained by magnetic resonance imaging

This study reports an evaluation of coronary arterial blood flow patterns in patients with diabetes mellitus and healthy subjects using magnetic resonance coronary angiography (MRCA). Twenty patients with diabetes mellitus (DM group) and 20 healthy subjects (N group) were studied using MRCA and myocardial SPECT images using ^99mTc-tetrofosmin (TF). The rate of change in myocardial TF uptake was measured during a 1-day protocol of exercise and rest. Initial and delayed exercise single photon emission computed tomography (SPECT) images were acquired 30min and 3h after injection (370MBq of TF) (TF1 and TF2, respectively). Thereafter, 740MBq of TF was administered intravenously, again, and resting SPECT images (TF3) were acquired 30min later. The myocardial counts of these three points of acquisition were defined, and the rate of change of myocardial TF uptake between exercise and rest was determined. The % increase in uptake was significantly lower in the DM group than in the N group in all myocardial segments. The average coronary arterial diastolic velocity determined using MRCA was slightly lower in the DM group than in the N group, and the average systolic peak velocity (ASPV) was slightly greater in the DM group than in the N group, although these values were not statistically significant. The diastolic/systolic velocity ratio (DSVR) in the DM group was significantly lower than that in the N group (P 0.05). There was a significant correlation between DSVR and % uptake increase (r = 0.605, P 0.05). These results indicate that the measurements made using MRCA and the % uptake increase measured using TF myocardial scintigraphy represent a potentially useful noninvasive method for diagnosing microvascular dysfunction in diabetic patients.     

Release of adenine nucleotide metabolites by toxic concentrations of cardiac glycosides

Summary In isolated perfused guinea-pig hearts the effect of toxic concentrations of cardiac glycosides on the release of the adenine nucleotide metabolites adenosine, inosine, hypoxanthine, xanthine, and uric acid was investigated. Digoxin concentrations of 0.03–1 mol · l^–1 produced moderate to severe tachyarrhythmias. Large amounts of metabolites were released by concentrations of 0.1 mol · l^–1, and higher. Occurrence of glycoside-induced ventricular fibrillation was associated with a particularly high release. Metabolite release was also obtained when fibrillation was elicited electrically in normal control hearts, or in hearts receiving simultaneously a marginally toxic digoxin concentration (0.03 mol · l^–1). Digoxin-induced tachyarrhythmias and metabolite release were almost completely prevented by a high potassium concentration in the coronary perfusion fluid (8.1 mmol · l^–1). The antiarrhythmic effect was also obtained with lidocaine (60 mol · l^–1), but the release was only partially antagonized. Similar results concerning arrhythmias and metabolite release as with digoxin were obtained with ouabain. The findings suggest that the decrease in myocardial ATP observed in glycoside-intoxicated heart preparations is partly due to the loss of nucleotide precursor substances. Moreover, it appears likely that liberated adenosine in the interstitium of severely intoxicated heart preparations reaches pharmacologically effective concentrations.