Diagnostic utility of fine needle aspiration (FNA) cytology in HIV-infected patients with lymphadenopathy

reid a. j. c., miller r. f. and kocjan g. i. (1998) Cytopathology 9, 230–239
Diagnostic utility of fine needle aspiration (FNA) cytology in HIV-infected patients with lymphadenopathy
Sixty-five FNA cytology procedures were performed on lymph nodes in 52 HIV⁺ patients. Cervical lymph nodes were the commonest site of FNA cytology investigation (54%). The diagnoses were persistent generalized lymphadenopathy (38%), infection (17%), and malignancy (11%). Diagnosis could not be rendered in 25% of FNA cytology due to inadequate sampling. Of those with infection, mycobacterial disease was the commonest cause (91%), the diagnosis of which was enhanced by concurrent microbiological examination. Non-Hodgkin's lymphoma was the commonest malignancy. Sixteen lymph node FNA cytologies had subsequent tissue biopsy. There were two false-positive and four false-negative FNA cytologies. FNA cytology in HIV⁺ patients is most useful in the diagnosis of infection, obviating the need for tissue biopsy and allowing prompt initiation of treatment.     

Ca2+- and Mg2+-Modulated Lipolysis in Neonatal Rat Brain Slices Observed by One- and Two-Dimensional NMR

Abstract: Superfused cortical brain slices from neonatal rats demonstrated large increases in levels of NMR-detectable lipids after sample preparation and perfusion with standard artificial CSF. These increases were reduced by an average of 58% by perfusion with buffer with low (no added) Ca²⁺ or by perfusion in Ca²⁺-free buffer. Perfusion with buffer with elevated MgSO₄ (10 mmol/L) reduced the lipid changes by 47%. A reduction of 88% was observed in samples perfused in buffer with both low Ca²⁺ and high Mg²⁺, suggesting a role for Mg²⁺ in reducing lipolysis distinct from its known ability to block Ca²⁺ influx.     

Impairment of Na+,K+-ATPase activity following enterotoxigenic Campylobacter jejuni infection: changes in Na+, Cl− and 3-O-methyl-D-glucose transport in vitro, in rat ileum

Abstract Unidirectional fluxes of Na⁺, Cl⁻ and 3-O-methyl-D-glucose (3-MG) were measured in vitro across Campylobacter jejuni live culture-infected and control rat ileal short-circuited tissues by the Using Chamber technique. Net secretion of Na⁺ and enhanced secretion of Cl⁻ ions was observed in the infected animals ( P < 0.001, n =6) as compared to the net absorption of Na⁺ and marginal secretion of Cl⁻ ions in the control animals. There was a significant decrease in the mucosal-to-serosal fluxes of 3-MG in C. jejuni -infected rat ileum. The specific Na⁺,K⁺-ATPase activity when measured biochemically in the membrane-rich fraction of enterocytes was found to be significantly lower (58%) in the infected group as compared to the control group ( P < 0.001). Our results therefore suggest that infection with an enterotoxigenic C. jejuni inhibits the Na⁺,K⁺-ATPase activity in rat enterocytes. The impairment of Na⁺,K⁺-ATPase activity thus appears to induce a secondary change in Na⁺,Cl⁻ and 3-MG transport in vitro in rat ileum.     

Ca2+ and Reactive Oxygen Species in Staurosporine-Induced Neuronal Apoptosis

Abstract: Staurosporine (0.03–0.5 µ M ) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 µ M ) or the cell cycle inhibitor mimosine (100 µ M ). To investigate the role of Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D_28K, a Ca²⁺ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D_28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 m M K⁺, suggesting that Ca²⁺ influx via voltage-sensitive Ca²⁺ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µ M ) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 µ M ) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1–10 µ M ) and N -acetylcysteine (100 µ M ) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis.     

Repeatedly nondiagnostic thyroid fine-needle aspirations do not modify malignancy risk

Objective: Thyroid nodules with nondiagnostic (ND) fine-needle aspirations (FNA) typically undergo repeat sampling. While repeat FNA is often diagnostic, little is known regarding the significance of repeatedly ND aspirates. Limited data suggest there is very low, if any, risk of malignancy for repeatedly ND FNAs. Study Design: We performed a retrospective analysis of ND thyroid FNAs over a nearly 6-year period at our institution to further address this question. Results: There were 834 ND thyroid FNAs, representing 694 distinct thyroid nodules. Repeat FNA was performed after an initial ND aspirate in 52% of cases (363/694); 19% (70/363) had at least one additional ND diagnosis on repeat FNA. Surgical follow-up was available for 57 cases. Malignancy was identified histologically in 21% (9/42) of nodules after a single ND FNA and in 20% (3/15) of nodules with 2 or more repeatedly ND aspirates. Accounting for all benign cytologic follow-up, the overall risk of malignancy was 4% [12/303; 3.5% (9/255) following a single ND FNA and 6.3% (3/48) after repeated ND FNAs]. Conclusion: We observed no modification of malignancy risk when repeated FNAs were ND. Clinical management for an ND aspirate should remain repeat aspiration along with clinical and sonographic correlation.     

Hypoxia-Induced Catecholamine Release and Intracellular Ca2+ Increase via Suppression of K+ Channels in Cultured Rat Adrenal Chromaffin Cells

Abstract: Hypoxia (5% O₂) enhanced catecholamine release in cultured rat adrenal chromaffin cells. Also, the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increased within 3 min in ∼50% of the chromaffin cells under hypoxic stimulation. The increase depended on the presence of extracellular Ca²⁺. Nifedipine and ω-conotoxin decreased the population of the cells that showed the hypoxia-induced [Ca²⁺]_i increase, showing that the Ca²⁺ influx was attributable to L- and N-type voltage-dependent Ca²⁺ channels. The membrane potential was depolarized during the perfusion with the hypoxic solution and returned to the basal level following the change to the normoxic solution (20% O₂). Membrane resistance increased twofold under the hypoxic condition. The current-voltage relationship showed a hypoxia-induced decrease in the outward K⁺ current. Among the K⁺ channel openers tested, cromakalim and levcromakalim, both of which interact with ATP-sensitive K⁺ channels, inhibited the hypoxia-induced [Ca²⁺]_i increase and catecholamine release. The inhibitory effects of cromakalim and levcromakalim were reversed by glibenclamide and tolbutamide, potent blockers of ATP-sensitive K⁺ channels. These results suggest that some fractions of adrenal chromaffin cells are reactive to hypoxia and that K⁺ channels sensitive to cromakalim and glibenclamide might have a crucial role in hypoxia-induced responses. Adrenal chromaffin cells could thus be a useful model for the study of oxygen-sensing mechanisms.     

Interaction of divalent metal ions with the NADP+-malic enzyme from maize leaves

The effect of several metal ions on NADP⁺-malic enzyme (EC 1.1.1.40) purified from Zea mays L. leaves was studied Mg²⁺, Mn²⁺, Co²⁺ and Cd²⁺ were all active metal cofactors. The malic enzyme from maize has a moderately high intrinsic preference for Mn²⁺ relative to Mg²⁺ at pH 7.0 and 8.0 Negative cooperativity detected in the binding of Mg²⁺ at pH 7.0 and 8.0 and in the binding of Mn²⁺ at pH 7.0 suggests the existence of at least two binding sites with different affinity. All of the activating metal ions have preference for octahedral coordination geometry and have ionic radii of 0.86–1.09 Å. The ions that act as inhibitors are outside this range and/or are incapable of octahedral coordination. Ba²⁺, Sr²⁺, Cd²⁺, Ca²⁺, Be²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Co²⁺, Hg²⁺ showed mixed-type inhibition. The reciprocal of their K₁ values follow the order of their apparence in the Irving-Williams series of stability that derives in part from size effects. It is suggested that the size of the ions may play a partial role in determining the strength of the metal interaction.     

Vaccination with SIVmac239Δnef activates CD4+ T cells in the absence of CD4+ T-cell loss

Background  Pathogenic HIV and SIV infections characteristically deplete central memory CD4⁺ T cells and induce chronic immune activation, but it is controversial whether this also occurs after vaccination with attenuated SIVs and whether depletion or activation of CD4⁺ T-cell play roles in protection against wild-type virus challenge.
Methods  Rhesus macaques were vaccinated with SIVmac239Δnef and quantitative and phenotypic polychromatic flow cytometry analyses were performed on mononuclear cells from blood, lymph nodes and rectal biopsies.
Results  Animals vaccinated with SIVmac239Δnef demonstrated no loss of CD4⁺ T cells in any tissue, and in fact CCR5⁺ and CD28⁺CD95⁺ central memory CD4⁺ T cells were significantly increased. In contrast, CD4⁺ T-cell numbers and CCR5 expression significantly declined in unvaccinated controls challenged with SIVmac239. Also, intracellular Ki67 increased acutely as much as 3-fold over baseline in all tissues after SIVmac239Δnef vaccination then declined following primary infection.
Conclusion  We demonstrated in this study that SIVmac239Δnef vaccination did not deplete CD4⁺ T cells but transiently activated and expanded the memory cell population. However, increases in numbers and activation of memory CD4⁺ T cells did not appear to influence protective immunity.     

Histological examination of clots from cytological specimens: is this a worthwhile procedure?

A comparison of conventional cytology and clot histology was made on 174 serous fluids, fine needle aspirates (FNA) and other non‐gynaecological specimens. In eight cases (4.5%) this clot material contained malignant cells or cells suspicious of malignancy despite the absence of suspicious or malignant cells in conventionally prepared smears from the same specimen. In 11 cases (6.3%) the clot was negative, although conventional smears contained malignant or suspicious cells. A χ² test showed that there was no statistically significant difference between the number of positive diagnostic scores in each group with χ² of 0.223 (degrees of freedom=1), P=0.637. Examination of clot material from serous fluids and FNA aspirates is as effective as examination of conventional cytological preparations. Processing of clots from cytological aspirates for histological examination should be more widely adopted, and is applicable in all cytopathology laboratories.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Puccinellia tenuiflora maintains a low Na+ level under salinity by limiting unidirectional Na+ influx resulting in a high selectivity for K+ over Na+

Puccinellia tenuiflora is a useful monocotyledonous halophyte that might be used for improving salt tolerance of cereals. This current work has shown that P. tenuiflora has stronger selectivity for K⁺ over Na⁺ allowing it to maintain significantly lower tissue Na⁺ and higher K⁺ concentration than that of wheat under short- or long-term NaCl treatments. To assess the relative contribution of Na⁺ efflux and influx to net Na⁺ accumulation, unidirectional ²²Na⁺ fluxes in roots were carried out. It was firstly found that unidirectional ²²Na⁺ influx into root of P. tenuiflora was significantly lower (by 31–37%) than in wheat under 100 and 150 m m NaCl. P. tenuiflora had lower unidirectional Na⁺ efflux than wheat; the ratio of efflux to influx was similar between the two species. Leaf secretion of P. tenuiflora was also estimated, and found the loss of Na⁺ content from leaves to account for only 0.0006% of the whole plant Na⁺ content over 33 d of NaCl treatments. Therefore, it is proposed that neither unidirectional Na⁺ efflux of roots nor salt secretion by leaves, but restricting unidirectional Na⁺ influx into roots with a strong selectivity for K⁺ over Na⁺ seems likely to contribute to the salt tolerance of P. tenuiflora .     

Effects of amiodarone on K+, internal pH and Ca2+ homeostasis in Saccharomyces cerevisiae

In this study, amiodarone, at very low concentrations, produced a clear efflux of K⁺. Increasing concentrations also produced an influx of protons, resulting in an increase of the external pH and a decrease of the internal pH. The K⁺ efflux resulted in an increased plasma membrane potential difference, responsible for the entrance of Ca²⁺ and H⁺, the efflux of anions and the subsequent changes resulting from the increased cytoplasmic Ca²⁺ concentration, as well as the decreased internal pH. The Δ tok1 and Δ nha1 mutations resulted in a smaller effect of amiodarone, and Δ trk1 and Δ trk2 showed a higher increase of the plasma membrane potential. Higher concentrations of amiodarone also produced full inhibition of respiration, insensitive to uncouplers and a partial inhibition of fermentation. This phenomenon appears to be common to a large series of cationic molecules that can produce the efflux of K⁺, through the reduction of the negative surface charge of the cell membrane, and the concentration of this cation directly available to the monovalent cation carriers, and/or producing a disorganization of the membrane and altering the functioning of the carriers, probably not only in yeast.     

Is a five‐category reporting scheme for thyroid fine needle aspiration cytology accurate? Experience of over 18 000 FNAs reported at the same institution during 1998–2007

S. Piana, A. Frasoldati, M. Ferrari, R. Valcavi, E. Froio, V. Barbieri, C. Pedroni and G. Gardini Is a five‐category reporting scheme for thyroid fine needle aspiration cytology accurate? Experience of over 18 000 FNAs reported at the same institution during 1998–2007 Objective: Fine needle aspiration (FNA) has long been recognized as an essential technique for the evaluation of thyroid nodules. Although specific cytological patterns have been recognized, a wide variety of reporting schemes for thyroid FNA results have been adopted. This study reports our experience with a five‐category reporting scheme developed in‐house based on a numeric score and applied to a large series of consecutive thyroid FNAs. It focuses mainly on the accuracy of thyroid FNA as a preoperative test in a large subset of histologically distinct thyroid lesions. Methods: During the 1998–2007 period, 18 359 thyroid ultrasound‐guided FNAs were performed on 15 269 patients; FNA reports were classified according to a C1–C5 reporting scheme: non‐diagnostic (C1), benign (C2), indeterminate (C3), suspicious (C4), and malignant (C5). Results: Non‐diagnostic (C1) and indeterminate (C3) FNA results totalled 2 230 (12.1%) and 1 461 (7.9%), respectively, while suspicious (C4) and malignant (C5) results totalled 238 (1.3%) and 531 (2.9%), respectively. Histological results were available in 2 047 patients, with thyroid malignancy detected in 840. Positive predictive value of FNA was 98.1% with a 49.0 likelihood ratio (LR) of malignancy in patients with a C4/C5 FNA report. Conclusions: This five‐category scheme for thyroid FNA is accurate in discriminating between the virtual certainty of malignancy associated with C5, a high rate (92%) of malignancy associated with C4, and a 98% probability of a histological benign diagnosis associated with C2. Further sub‐classifications of C3 may improve the accuracy of the diagnostic scheme and may help in recognizing patients eligible for a ‘wait and see’ management.     

A2A Adenosine Receptors Inhibit ATP-Induced Ca2+ Influx in PC12 Cells by Involving Protein Kinase A

Abstract: The regulatory role of A_2A adenosine receptors in P₂ purinoceptor-mediated calcium signaling was investigated in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with 2- p -(2-carboxyethyl)-phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680), a specific agonist of the A_2A adenosine receptor, the extracellular ATP-evoked rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) was inhibited by 20%. Both intracellular calcium release and inositol 1,4,5-trisphosphate production evoked by ATP were not affected by CGS-21680 treatment. However, ATP-evoked Ca²⁺ influx was inhibited following CGS-21680 stimulation. The CGS-21680-mediated inhibition occurred independently of nifedipine-induced inhibition of the [Ca²⁺]_i rise. The CGS-21680-induced inhibition was completely blocked by reactive blue 2. The CGS-21680 effect was mimicked by forskolin and dibutyryl-cyclic AMP and blocked by Rp -adenosine 3',5'-cyclic monophosphothioate, a protein kinase A inhibitor, or by staurosporine, a general kinase inhibitor. The data suggest that in PC12 cells activation of A_2A adenosine receptors leads to inhibition of P₂ purinoceptor-mediated Ca²⁺ influx through ATP-gated cation channels and involves protein kinase A.     

ENERGETICS OF AMINO ACID TRANSPORT INTO BRAIN SLICES: EFFECTS OF K+ DEPLETION AND Rb+ OR Cs+ SUBSTITUTION ON AMINO ACID UPTAKE

Abstract— Mouse brain slices were depleted of K⁺ by three 10-min incubations-in oxygenated HEPES-buffered medium lacking glucose and K⁺. Addition of K⁺ or Rb⁺ (or Cs⁺, to a smaller degree) with glucose, or with succinate, malate, and pyruvate (SMP) before incubation at 37°C with ¹⁴C-amino acids restored active low-affinity transport of d -Glu, α-aminoisobutyrate (AIB), GABA, Gly, His, Val, Leu, Lys, and Orn. Ouabain at 1–2μ m with Rb⁺ was more inhibitory with SMP than with glucose, suggesting that the glycoside may affect specific energy coupling to transport. Valinomycin, in contrast, showed no specificity of inhibition of amino acid uptake with glucose or SMP and K⁺ or Rb⁺. Cs⁺ partially restored amino acid uptake, but Li⁺ was less effective than Cs ⁺. NaF at 10 m m with SMP + Rb⁺, or SMP + K⁺ did not inhibit amino acid uptake. Therefore, it was possible to dissociate glycolysis and Na⁺, K ⁺ -ATPase activity from amino acid transport. The ion replacements for K ⁺ that supported active amino acid transport indicate that the specificity of ions in possible ionic gradients for transport energetics should be reexamined.     

Cation-stimulated H+ efflux by intact roots of barley

Abstract. Rates of proton extrusion and potassium (⁸⁶Rb) influx by intact roots of barley ( Hordeum vulgare cvs . Fergus, Conquest and Betzes) plants were simultaneously measured in short-term (15min) experiments. The nature and extent of apparent coupling between these ion fluxes was explored by manipulating conditions of temperature, pH and cation composition and concentration during flux determinations. In addition, the influence of salt status upon these fluxes was examined. At low K⁺ concentrations (0.01 to 1 mol m⁻³), H⁺ efflux and K⁺ influx were strongly correlated in both low- and high-K⁺ roots, although K⁺: H⁺ exchange stoichiometries were almost consistently greater than 2:1. At higher concentrations (1 to 5 mol m⁻³), H⁺ efflux was either reduced or remained unchanged while K⁺ influxes increased. In the presence of Na₂SO₄, rates of H⁺ extrusion demonstrated similar cation dependence, although below 10 mol m⁻³ Na₂SO₄, H⁺ fluxes were generally 50% lower than in equivalent concentrations of K₂SO₄. These observations are considered in the context of current hypotheses regarding the mechanisms of k⁺/H⁺ exchange.     

Ontogenesis of Ca++ and Mg++ Contents of Embryonic Chick Heart

The Ca⁺⁺ and Mg⁺⁺ contents of embryonic chick heart were studied by atomic absorption spectrophotometry during a period from 48 h of foetal development until 2-3 days post-hatching. The hearts were isolated and incubated for 40 min at 22°C in three different media aerated with 95% 0₂-5% C0₂. The media included: normal Ringer's; Ca⁺-free Ringer's with 3 mM EGTA; and Ca⁺⁺-free Ringer's with 3 mM EDTA. At 48 h, the tubular myocardium contained 7-3 mM Ca⁺⁺ per wet weight which decreased rapidly to 1-2 mM by 10 days of development and remained between 0-9 and 1-1 mM until hatching. The Ca⁺⁺ content paralleled the changes in Na⁺ content reported earlier. Treatment with excess chelators, EGTA or EDTA, resulted in removal of 65-75% of the Ca⁺⁺ content throughout development until the time of hatching, when 50% of the Ca⁺⁺ became firmly bound. In contrast to the results with Ca⁺⁺, myocardial Mg⁺⁺ content rose rapidly from an initial value of 3.2 mM at 48 h to 6.7 mM by the 5th day of development, and then gradually declined throughout the remaining foetal development to 4.8 mM 2-3 days post-hatching. The Mg⁺⁺ contents closely paralleled changes in K⁺ content during development, which were reported earlier. Treatment with EGTA and EDTA removed 13-22% and 19-28% of the myocardial Mg⁺⁺, respectively, during development until just prior to hatching, when only 10-12% could be removed by chelation.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.