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PubMed2005

Divalent cations influence colon cancer cell adhesion in a murine transplantable tumor model.

BACKGROUND: Cancer cells adhere principally by integrins, matrix receptors that may be influenced by divalent cations. Surgical wound fluid is high in Mg2+ and low in Ca2+. We hypothesized that Mg+ and Mn2+ promote perioperative adhesion of shed cancer cells to surgical sites and that washing surgical wounds with Ca2+ inhibits implantation. METHODS: We tested our hypothesis in a murine colon 26 adenocarcinoma model. We added 10 mmol/L CaCl2, 0.25 mmol/L MgCl2, or 0.5 mmol/L MnCl2 to suspended murine colon 26 cancer cells and placed these suspensions into wounds in anesthetized mice. After 30 minutes, we washed away nonadherent cells. In some studies, we 51Cr-labeled the cells and assayed tumor adhesion by wound radioactivity. In parallel studies, we closed the wounds and observed the mice for 90 days. RESULTS: Mg2+ increased adhesion to 188% +/- 15% of control (n = 10, P < .001) and Mn2+ to 130% +/- 6% (n = 7, P < .001). However, Ca2+ inhibited adhesion to 61% +/- 12% (n = 7, P = .006) of control. Seventy-two percent of survival controls developed tumors during follow-up. Mg2+ and Mn2+ stimulated tumor formation to 96% and 92%, respectively, but adding Ca2+ to the wounds reduced subsequent tumor formation to 56% without altering serum Ca2+. The survival curves each differed significantly by log-rank test (P < .01 each). All pair-wise multiple comparisons were significant (Holm-Sidak, P < .05 each). CONCLUSION: Thus, the high Mg2+ in endogenous wound fluid may potentiate tumor cell adhesion. However, 10 micromol/L Ca2+ inhibits cancer cell adhesion to murine wounds and subsequent tumor development. Irrigating with dilute CaCl2 could decrease local tumor recurrence by inhibiting the adhesion of shed tumor cells.

Adenocarcinoma↗

PubMed1999

Comparison of the effects of divalent cations on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells.

1. The facilitatory effects of external Ca2+, Sr2+ and Ba2+ (Ca2+o, Sr2+o and Ba2+o) on the noradrenaline-evoked non-selective cation current (Icat) were compared in rabbit portal vein smooth muscle cells using patch pipette techniques. 2. All divalent cations tested potentiated the amplitude of Icat and the potency sequence was Ca2+o > Sr2+o > Ba2+o. Ca2+o and Sr2+o increased the amplitude of Icat by about eight times whereas Ba2+o produced only a threefold facilitation. 3. The current-voltage relationship of Icat was not changed by Ca2+o, Sr2+o or Ba2+o. 4. From noise analysis the single channel conductance (gamma) was approximately 10 pS in divalent cation-free solution but was about 20 pS with Ca2+o, Sr2+o and Ba2+o. 5. From noise and voltage-jump experiments it was apparent that at least three kinetically resolvable channel states are associated with Icat in divalent cation-free solution. Ca2+o and Sr2+o produced marked changes in the characteristics of the power spectrum and relaxations of Icat in response to voltage steps, consistent with a shift in the equilibrium between the channel states, whereas Ba2+o produced minimal effects. 6. The data show that Ca2+o, Sr2+o and Ba2+o increase the amplitude of Icat, which results in part from an increase in the single channel conductance. In addition the results suggest that Ca2+o and Sr2+o alter the kinetic behaviour of the single channels whereas Ba2+o has little effect on the equilibrium between the channel states.

Animals↗

PubMed1995

Effects of a non-divalent cation binding mutant of myosin regulatory light chain on tension generation in skinned skeletal muscle fibers.

Each myosin molecule contains two heavy chains and a total of four low-molecular weight light chain subunits, two "essential" and two "regulatory" light chains (RLCs). Although the roles of myosin light chains in vertebrate striated muscle are poorly understood at present, recent studies on the RLC have suggested that it has a modulatory role with respect to Ca2+ sensitivity of tension and the rate of tension development, effects that may be mediated by Ca2+ binding to the RLC. To examine possible roles of the RLC Ca2+/Mg2+ binding site in tension development by skeletal muscle, we replaced endogenous RLC in rabbit skinned psoas fibers with an avian mutant RLC (D47A) having much reduced affinity for divalent cations. After replacement of up to 80% of the endogenous RLC with D47A RLC, maximum tension (at pCa 4.5) was significantly reduced compared with preexchange tension, and the amount of decrease was directly related to the extent of D47A exchange. Fiber stiffness changed in proportion to tension, indicating that the decrease in tension was due to a decrease in the number of tension-generating cross-bridges. Decreases in both tension and stiffness were substantially, although incompletely, reversed after reexchange of native RLC for D47A. RLC exchange was also performed using a wild-type RLC. Although a small decrease in tension was observed after wild-type RLC exchange, the decrease was not proportional to the extent of RLC exchange and was not reversed by reexchange of the native RLC. D47A exchange also decreased the Ca2+ sensitivity of tension and reduced the apparent cooperativity of tension development. The results suggest that divalent cation binding to myosin RLC plays an important role in tension generation in skeletal muscle fibers.

Animals↗

PubMed1982

Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations.

The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions.

Blood Platelets↗

PubMed1986

Barbiturates block divalent cation action potentials in leech nociceptive cells.

Phenobarbital (PNB), pentobarbital (PTB) and methohexital (MTX) decreased the maximum rate of depolarization Vmax and duration of divalent cation action potentials elicited in leech nociceptive neurons in Na+-free solutions containing the K+-channel blocker TEA, without significantly affecting resting membrane potential or conductance. The block of the divalent cation action potentials was reversible and dose-dependent, ED50 for inhibition of Vmax being 560 microM for MTX, 800 microM for PTB and 3000 microM for PNB. This order of potency correlated well with the ratio of unchanged/charged form of the drugs at physiological pH suggesting that in leech, as in other preparations, the non-ionized form was the active one. In Na+-containing Ringer, the 3 barbiturates depolarized and decreased membrane resistance in the lateral nociceptive cells, but not the medial nociceptive cells. These results provide additional information regarding the newly described pharmacological differences among closely related neurons. These membrane actions may be related to some of the excitatory properties described for other barbiturates in invertebrate and mammalian preparations.

Action Potentials↗

PubMedDate not supplied

Centrosome organization and centriole architecture: their sensitivity to divalent cations.

The centrosome plays a major role in the spatial organization of the microtubular network and has a controlled cycle of duplication, the two duplicated centrosomes functioning as mitotic poles during subsequent cell division. However, a comprehensive description of the overall organization of the centrosome in animal cells is lacking. In order to integrate the various pieces contributing to the centrosome structure and to optimize the quality of the data, we have undertaken an extensive ultrastructural study of centrosomes isolated from human lymphoblasts, which involved (i) orientation of centrosomes by sedimentation before embedding and sectioning, (ii) ultrathin serial sectioning, (iii) digitalization of micrographs to obtain quantitative data, and finally, (iv) comparison between two methods of isolation, which differ by the presence or absence of EDTA. Using this strategy, we have unambiguously described the pericentriolar organization of two distinct sets of appendages (distal and subdistal) about the so-called parental centriole. New structures have been also observed in association with the microtubule sets in this study: (i) external columns, which are dense structures localized at the basis of the subdistal appendages and (ii) internal columns, which are made of globular subunits integrated in a more luminal and probably helical structure. We have also observed that removal of divalent cations by the EDTA during the isolation procedure could affect the centrosomal structure at different levels (subdistal appendages, internal and external columns, pericentriolar matrix), including a significant variation in centriole diameter. A scheme of the overall organization of the centrosome from animal cells and of its modulation by divalent cations can be drawn from this study. Our data gives a view of the centrosome as an organelle displaying a complex and possibly dynamic structural organization.

Calcium↗

PubMed1994

The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations.

The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.

Amino Acid Sequence↗

PubMed1985

Effects of divalent cation ionophore A23187 on cardiac contractile parameters.

The divalent cation ionophore A23187, when added to guinea pig papillary muscle, produced contractile effects that were similar to those produced by isoproterenol or histamine, but the ionophore's effects did not appear to result from the release of endogenous transmitters or prostaglandin production. Optimally effective concentrations of A23187 (6 microM) and isoproterenol (1 microM) more than doubled the peak contractile force and the rates of force development and relaxation and caused a 25% decrease in the duration of the contraction. Both A23187 and isoproterenol decreased the resting tension by approximately 0.15 g and significantly diminished the magnitude of a potassium-induced contracture. The positive inotropic effect of A23187 was prevented by incubating the tissue in calcium-depleted medium and antagonized by D 600, a blocker of sarcolemmal calcium influx, and acetylcholine. The contractile effects of A23187 appear to be related, in part, to its ability to increase the movement of calcium across the sarcolemma electroneutrally, since no change of the action potential occurred. In addition, possible intracellular actions of this ionophore may produce contractile effects that resemble those produced by isoproterenol and that reflect an increased sequestration of calcium within the myocardial cell.

Acetylcholine↗

PubMed1986

Spontaneous tumor cytolysis mediated by inflammatory neutrophils: dependence upon divalent cations and reduced oxygen intermediates.

The role of divalent cations and reactive products of the respiratory burst were investigated in spontaneous tumor lysis mediated by inflammatory neutrophils (PMNs). Murine peritoneal PMNs, obtained five hours after intraperitoneal injection of bacteria, conjugated and lysed teratocarcinoma cells in chromium release and single-cell cytotoxicity assays. The presence of extracellular magnesium was required and was sufficient for tumor cell binding to PMNs. Postbinding lytic events depended upon the simultaneous presence of extracellular calcium and magnesium. Catalase and superoxide dismutase inhibited postbinding lytic events, indicating that production of reduced oxygen moieties was important. Scavengers of hydroxyl radicals could inhibit tumor cell binding, but none could affect postbinding lytic events. Neither could inhibitors of myeloperoxidase decrease tumor lysis. The ability of conjugating PMNs to lyse their bound targets correlated with their reduction of nitro blue tetrazolium (NBT). Optimal concentrations of phorbol myristate acetate (PMA) markedly increased the NBT positivity of PMNs and the killing of bound tumor cells. Even with optimal stimulation of the respiratory burst, however, there was still a significant number (19%) of bound targets that escaped lysis, suggesting active resistance to oxygen-mediated tumor cell injury.

Animals↗

PubMed1987

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons.

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.

Animals↗

PubMed1998

Divalent cations inhibit IsK/KvLQT1 channels in excised membrane patches of strial marginal cells.

The IsK/KvLQT1 K+ channel in the apical membrane of strial marginal cells and vestibular dark cells is an essential ion transport pathway for the secretion of K+ into the endolymph of the inner ear. Study of this control point has been impeded by rundown of channel activity upon excision into commonly used cytosolic solutions. This paper describes conditions under which patches of apical membrane of strial marginal cells and vestibular dark cells from gerbil containing this channel can be excised, retaining its characteristic voltage dependence, kinetic properties, ion permeability sequence and pharmacological sensitivity, similar to those found during on-cell and perforated-patch whole cell recordings (Shen et al., Audit. Neurosci. 3 (1997) 215-230). Those excised-patch conditions include removal of Mg2+ from the cytosolic solution and use of a K+-rich pipette electrolyte. The inhibition of channel activity by Mg2+ was found to be a general feature of divalent cations; the channel was also inhibited by Ca2+, Ba2+ and Sr2+. The concentrations causing 50% inhibition of IsK/KvLQT1 channel current were 7 x 10(-5) M, 6 x 10(-6) M, 3 x 10(-4) M and 7 x 10(-5) M, respectively. It was also found that a chemical cross-linking agent, 3,3'-dithio-bis(sulfosuccinimidyl propionate) (DTSSP), which was previously shown to persistently activate IsK/KvLQTI channels expressed in Xenopus oocytes, maintained in excised patches channel activity which retained voltage dependence and pharmacological sensitivity. These data demonstrate that (1) the channel complex is inhibited by Ca2+, Mg2+ and other divalent cations, (2) the activation by Ca2+ observed previously in whole-cell preparations was due to action via other cellular pathways. These findings must be taken into account when considering the action of receptors which alter the cytosolic Ca2+ activity.

Animals↗

PubMed1999

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Amino Acid Sequence↗

PubMed1999

Divalent cation selectivity is a function of gating in native and recombinant cyclic nucleotide-gated ion channels from retinal photoreceptors.

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.

Ambystoma↗

PubMed1991

Regulation of smooth muscle phosphatase-II by divalent cations.

Smooth Muscle Phosphatases II (SMP-II) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg2+ and Mn2+ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg2+ is 1.5-2.5-fold higher than the maximal Mn2+ activation. However, the latter is achieved at a lower concentration than the maximal Mg2(+)-activation. Furthermore, at low cation concentrations (less than or equal to 2 mM), the Mn2(+)-activated activity is higher than the Mg2(+)-activated activity. In the presence of both cations, the effect of Mn2+ predominates suggesting that the affinity of the enzyme for Mn2+ is greater than for Mg2+. In contrast to Mg2+ and Mn2+, Ca2+ does not activate SMP-II but it was observed to antagonize the effects of Mg2+ and Mn2+. Ca2+ acts as a competitive inhibitor of Mg2+. However, the inhibitory effect at high Ca2+ concentrations is not completely reversed by increasing the Mg2+ concentration. Mn2+ activation is also inhibited by Ca2+ but to a lesser extent. Ca2+ cannot completely inhibit Mn2(+)-activation suggesting that SMP-II has greater affinity for Mn2+ than for Ca2+. The finding that Ca2+ inhibits the activation of SMP-II raises the possibility that Ca2+ may be a regulator of SMP-II in vivo.

Animals↗

PubMedDate not supplied

[Interaction of natural and synthetic polynucleotides with liposomes in the presence of divalent cations].

The formation of complexes of polynucleotides (DNA, poly A.poly U) with liposomes from egg lecithins, L-alpha-phosphatidylcholine, dimirystoyl and other lipids in the presence of divalent cations was studied by differential scanning microcalorimetry circular dichroism and turbidimetry. It was shown that the secondary structure of polynucleotides (double or triple helix) was necessary for the formation of these complexes. This structure was partially destroyed during formation of complexes. It was shown, that three main types of lipids, i.e. phosphatidylcholine, phosphatidylethanolamine and sphingomyelin participate in interactions between liposomes, polynucleotides and Mg2+.

Calorimetry, Differential Scanning↗

PubMed1983

Detectability of pituitary PRL and GH by immunoassay is increased by thiols and suppressed by divalent cations.

The amount of pituitary PRL detected by RIA in extracts of tissue and of secretory granules was augmented by thiols or EDTA in the extractant, and diminished by divalent cations or cysteamine. GH was affected by thiols and cations similarly, but to a lesser extent, and was not influenced by EDTA or cysteamine. For full immunologic detection of tissue PRL and GH, thiol-dependent mechanisms appear to be required to unmask immunoreactive sites from the poorly immunoreactive oligomeric granule storage forms. Interpretation of studies which rely on measurement of tissue PRL and GH content by RIA, immunoprecipitation, or electrophoretic detection should be reassessed in light of these observations.

Animals↗

PubMed1994

Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification.

1. By use of a 'grease-gap' technique, the depolarizing effects of adenosine 5'-triphosphate (ATP) and ATP analogues on the rat isolated vagus nerve were determined in normal and in Ca2+/Mg(2+)-free (+ 1 x 10(-3) M ethylenediamine tetraacetic acid) physiological salt solution (PSS). 2. In normal PSS, ATP produced concentration-dependent depolarization responses but the concentration-effect curve to ATP was incomplete and a maximum effect was not achieved. The threshold concentration for depolarization was 1 x 10(-5) M and at the highest concentration tested (1 x 10(-3) M) the peak amplitude of the response to ATP only amounted to 71% of the depolarization produced by a near maximal response to 5-hydroxytryptamine (5-HT, 1 x 10(-5) M). 3. In Ca2+/Mg(2+)-free PSS, ATP produced depolarization responses at much lower concentrations and of markedly larger amplitude. Under these conditions, the threshold concentration for depolarization was 1-3 x 10(-7) M and the maximal response to ATP amounted to 526% of the response to 5-HT (1 x 10(-5) M) in normal PSS. The concentration-effect curve to ATP was sigmoid, with a defined maximum effect and a mean EC50 value of 1.2 x 10(-6) M. 4. In contrast to the effects on responses to ATP, the absence of divalent cations in the PSS did not modify the effective concentrations of either alpha, beta-methylene ATP or 5-HT. However, the maximum responses to both alpha, beta-methylene ATP and 5-HT were significantly increased in Ca2+/Mg(2+)-free PSS. 5. The depolarizing effects of several analogues of ATP were determined in Ca2+/Mg2+-free PSS.ATP-gamma-S and 2-methylthioATP were of similar potency to ATP (respective equi-effective molar ratios(EMRs) of 1.9 and 1.3, where ATP = 1) and similar maximum responses were obtained. Alpha, beta-MethyleneATP, beta, gamma-methylene ATP and ,beta, gamma-imido ATP were considerably less potent than ATP, analysis yielding mean EMRs of 48.9, 85.0 and 60.0, respectively. Maximum responses to these latter three agonists were not obtained at the highest concentrations tested (1 x 10-4-3 X 10- M). Benzoyl ATP, adenosine 5'-0-(2-thiodiphosphate) and adenosine diphosphate produced only small depolarizing responses at high concentrations (>1 x 10-4 M). Adenosine monophosphate, adenosine and uridine S'-triphosphate each had little or no depolarizing effect in Ca2+/Mg2+-free PSS.6. These data demonstrate that in the absence of divalent cations the excitatory actions of some, but not all, purine nucleotides in the rat vagus nerve are markedly potentiated. In Ca2+/Mg2+-free PSS, the rank order of agonist potencies was ATP = 2-methylthioATP = ATP-gamma-S>> alpha,beta-methylene ATP = beta, gamma imido ATP = P,y-methylene ATP. These findings are in stark contrast to our previous observations in normal PSS where the rank order of agonist potencies for these nucleotides was alpha,beta-methyleneATP> ATP-gamma-S > beta,gamma-imido ATP = beta,gamma-methylene ATP> 2-methylthioATP> ATP.7. We suggest that the two different rank orders of potency can be explained by differential metabolism involving Ca2+/Mg2+-dependent ectonucleotidases. If so, these data indicate that ATP and 2-methylthioATP are inherently more potent than alpha,beta-methylene ATP as agonists at neuronal P2X purinoceptors in the rat vagus nerve. The possible implications of these findings to the present system for subclassifying P2 purinoceptors are profound.

Adenosine Triphosphate↗

PubMed2003

Divalent cation hinder the solubilization of a tubulin kinase activity from Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes were extracted under various conditions in order to examine the role of divalent cations in the solubilization of microtubule proteins. When epimastigotes were homogenized in the presence of 5 mM Mg+2 and 5 mM Ca+2, a protein kinase responsible for phosphorylating tubulin, as well as the tubulin that became phosphorylated, remained tightly associated with the parasite particulate and detergent-resistant fractions. On the contrary, tubulin kinase and its substrate were predominantly released into the parasite cytosolic and detergent-soluble fractions, when epimastigotes were extracted in the presence of 5 mM EDTA and 5 mM EGTA. These evidences demonstrated a divalent cation-dependent solubilization of the enzyme responsible for the phosphorylation of tubulin in T. cruzi epimastigotes and suggested a tight association between tubulin and this kinase. Under all conditions tested, tubulin kinase activity in epimastigote extracts was lower than the addition of the corresponding value in the parasite cytosolic and membranous fractions, suggesting the presence of a kinase inhibitor or regulatory subunit which also seemed to be modulated by divalent cations. Additionally, inhibition experiments in the presence of heparin, 2,3-bisphosphoglycerate and GTP established that the parasite tubulin kinase corresponded to a protein kinase CK2.

Animals↗