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Notes: Abstract: Modes of Ca2+ activation by bradykinin, serotonin, and ATP and the possible receptor cross-talk were investigated in mouse neuroblastoma × rat glioma hybrid cells (108CC15) by monitoring fura-2 fluorescence in single cells. A transient rise of cytosolic Ca2+ activity was induced by short pulses of the hormones. Brief exposure of cells to ionomycin, which depletes intracellular Ca2+ stores, reduced the size of subsequent responses to bradykinin or ATP, but not to serotonin. Superfusion of the cells with Ca2+-free medium abolished the Ca2+ response to serotonin, whereas the responses to bradykinin and to ATP were only slightly reduced. This indicates that ATP, like bradykinin, Induces the release of Ca2+ from intracellular stores. Serotonin, in contrast, activates Ca2+ entry from the extracellular space. To investigate whether ATP releases Ca2+ from the same stores as bradykinin, we examined the interaction of the hormones by applying them consecutively. When ATP was applied after bradykinin, the nucleotide did not evoke any response, irrespective of the presence or absence of extracellular Ca2+. The application of ATP before that of bradykinin reduced the size of a following bradykinin-induced Ca2+ response in Ca2+-free medium, but not in Ca2+-containing medium. This suggests that bradykinin may interact with the ATP-activated mechanism by cross-desensitization. Possibly, bradykinin receptors are coupled to additional Ca2+ stores not accessible to ATP that are refilled by extracellular Ca2+. Cyclic AMP and cyclic GMP apparently do not affect the Ca2+ responses to bradykinin and serotonin, as shown by the lack of influence of preincubation of the cells with forskolin or sodium nitroprusside.

Notes: Three rare-gas halide (RgX−) anions, KrBr−, XeBr−, and KrCl−, and the corresponding neutral, open-shell van der Waals complexes are studied with anion zero electron kinetic energy spectroscopy. The spectra for each system reveal well-resolved progressions in the low frequency vibrations of the anion and one or more of the three neutral electronic states accessed by photodetachment, providing a detailed spectroscopic probe of the Rg–X− and Rg–X interaction potentials. In the case of KrBr−, transitions to all three of the "covalent" neutral electronic states (the X1/2, I3/2, and II1/2 states) were observed. For XeBr−, transitions to the X1/2 and II1/2 neutral states were observed. For KrCl−, only the X1/2 state could be studied. From our data, we construct model potentials for the anion and each observed neutral state, and these are compared with other experimental and theoretical potentials. © 1998 American Institute of Physics.

Notes: Abstract: The mechanism by which cyclic GMP synthesis is activated through a nucleotide receptor was studied in mouse neuroblastoma × rat glioma hybrid cells [108CC15 (NG 108-15)]. The transient increase in cyclic GMP level induced by ATP reached its maximum at 20 s and lasted for ∼1 min. The maximal rise in cyclic GMP level achieved was highest for ATP and decreased in the following order: ATP = adenosine 5′-(γ-thio)triphosphate 〉 UTP = 2-methylthio-ATP 〉 ADP ≫ CTP, AMP, α,β-methylene-ATP, 2′- and 3′-O-(4-benzoylbenzoyl)ATP. The EC50 of 1 ± 0.2 µM for UTP was significantly lower than that for ATP (14 ± 8 µM) and for all the other nucleotides tested. The rank order of potency is consistent with the pharmacology of a P2u receptor. At submaximal concentrations of the nucleotides ATP and UTP, the rise in cyclic GMP level was inhibited by suramin (IC50 = 40–60 µM) or the pyridoxal phosphate analogue pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (IC50 = 20–30 µM). Pretreatment of cells with the Ca2+ ionophore ionomycin or with 2,5-di(tert-butyl)-1,4-benzohydroquinone, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, a maneuver to deplete internal Ca2+ stores, suppressed the ATP- or UTP-induced stimulation of cyclic GMP synthesis. Similarly, loading of the cells with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid inhibited cyclic GMP formation by ATP. Preincubation with forskolin to raise the cyclic AMP level potentiated the ATP-induced rise in cyclic GMP level by 60%. The cyclic GMP response caused by ATP was suppressed either by arginine analogues (IC50 for nitroarginine = 1 µM) or by hemoglobin (IC50 = 2 µM). This indicates that ATP/UTP via a P2-receptor causes formation of nitric oxide, which activates guanylate cyclase. The synthesis of nitric oxide depends on a preceding rise in cytosolic Ca2+ level, mostly due to release of Ca2+ from internal stores. Bradykinin induces a rise in cyclic GMP level with an amplitude and time course comparable to that caused by ATP. Therefore, we studied cross-desensitization between ATP and bradykinin receptors. Pretreatment with bradykinin completely suppressed a subsequent response to ATP. However, stimulation with ATP reduced a following response to bradykinin by ∼40% only. This indicates a heterologous cross-desensitization predominantly in one direction (bradykinin ⇒ ATP).

Notes: Zero electron kinetic energy (ZEKE) spectroscopy has been utilized to study the 40Ar35Cl− anion and the X1/2, I3/2 and II1/2 electronic states of neutral ArCl. Well-resolved progressions in the low-frequency vibrations of the anion and the neutral complexes are observed in the ZEKE spectra. From our spectroscopic data we construct model potential functions for the anion and three neutral states. This yields refined values for the neutral state splittings and the first accurate experimental ArCl− anion potential. Absolute uncertainties for Rm and ε in all potentials are estimated to be ±0.08 Å and ±0.6 meV, respectively. © 1999 American Institute of Physics.

Notes: Two-photon, two-color (1+1') zero-kinetic-energy (ZEKE) photoelectron spectra are presented for the 1:1 phenol-water complex, a prototype system for hydrogen bonding between an aromatic molecule and a simple solvent. ZEKE spectra via different (intermolecular) vibrational intermediate S1 levels of the fully protonated complex (C6H5OH–H2O, h3) as well as the ZEKE spectrum via the vibrationless S1 state of the threefold deuterated complex (C6H5OD–D2O, d3) have been recorded. The spectra are rich in structure, which is mainly attributable to intermolecular vibrations of the ionic complex. Progressions of the intermolecular stretch vibration (240 cm−1) in combination with different intermolecular and intramolecular vibrational levels are the dominant feature of all ZEKE spectra obtained and indicate a large change in the complex geometry along the hydrogen-bond coordinate on ionization. Comparison between the spectrum of the d3 complex and the spectra via different intermediate intermolecular levels of the h3 complex has allowed a more detailed analysis of the intermolecular features compared to previously reported results. Finally, the vibrational assignments obtained are compared with ab initio results for the phenol-water cation reported in the following paper in this issue.

Notes: Rotationally resolved "zero kinetic energy (ZEKE)'' electron spectra of ammonia obtained in a 2+1 photon two-color pump–probe experiment are reported. The rovibronic states with J'=3, K'=1 (ortho-NH3) and J'=3, K'=2 (para-NH3) in the B˜ (1E‘) v2=2 state are used as intermediate resonances. Rotational energy levels of the NH+3 ion in two vibrational states of the X˜+ (2A‘2) electronic ground state, v+2 =1 and 2, are observed in the ZEKE spectra. The rotational constants are: B+=10.19±0.03 cm−1 and C+=5.30±0.04 cm−1 for v+2 =1, and B+ =9.77±0.04 cm−1 and C+ =5.39±0.05 cm−1 for v+2 =2. The adiabatic ionization energies are determined as 83 062.5±1 cm−1 for X˜+ v2=1 and 84 002.9±1 cm−1 for X˜+ v2=2, with respect to the lowest (unoccupied) v2=0+, J‘=0, K‘=0, Γevr=A1 rotational state of the X˜ (1A'1) electronic ground state of NH3. The observed propensity rules for the change in K quantum number ion←neutral in the rotational ZEKE transitions are explained from "near symmetry''conservation rules in electronically allowed transitions.

Notes: Rotationally resolved zero kinetic energy-pulsed field ionization (ZEKE-PFI) photoelectron spectra of NH3 were obtained by nonresonant two-photon ionization from the X˜(1A1') electronic ground state. The ZEKE spectra were recorded up to an internal ion energy of 9500 cm−1 by pulsed field ionization of long-lived Rydberg states of very high principal quantum numbers. Rovibronic bands of the NH3+ cation with v2+=1–9 (umbrella mode), v4+=1, and for the first time the mode ν1 in the combination band v+=1121 of the X˜+(2A2‘) electronic ground state are observed in the ZEKE spectra. Rotational constants and band origins are determined in the analysis. The adiabatic ionization energy obtained by a recent ZEKE measurement is confirmed at 82 159±1 cm−1.

Notes: Abstract: The voltage-dependent Na+ ionophore of various neuronal cells is permeable not only to Na+ ions but also to guanidinium ions. Therefore, the veratridine-(or aconitine-) stimulated influx of [14C]guanidinium in neuroblastoma × glioma hybrid cells was measured to characterize the Na+ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 μM veratridine. At 1 mM guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 mM Li+, Na+, or K+ and by a few millimolar Mn2+, Co2+, or Ni2+. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine (Ki= 50 to 500 nM) and atropine (Ki= 5 to 30 μM) and the adrenergic antagonists phentolamine (Ki= 5 μM) and propranolol (Ki= 60 μM). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.

Notes: Proteins which recognize the two messengers phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3), a membrane lipid, and inositol 1,3,4,5-tetrakisphosphate (InsP4), a water-soluble ligand, play important roles by integrating external stimuli, which lead to differentiation, cell death or survival. p42IP4, a PtdInsP3/InsP4-binding protein, is predominantly expressed in brain. The recently described centaurin α2 of similar molecular mass which is 58% identical and 75% homologous to the human p42IP4 orthologue, is expressed rather ubiquitously in many tissues. Here, elucidating the gene structure for both proteins, we found the human gene for centaurin α2 located on chromosome 17, position 17q11.2, near to the NF1 locus, and human p42IP4 on chromosome 7, position 7p22.3. The two isoforms, which both have 11 exons and conserved exon/intron transitions, seem to result from gene duplication. Furthermore, we studied binding of the two second messengers, PtdInsP3 and InsP4, and subcellular localization of the two proteins. Using recombinant baculovirus we expressed centaurin α2 and p42IP4 in Sf9 cells and purified the proteins to homogeneity. Recombinant centaurin α2 bound both InsP4 and PtdInsP3 equally well in vitro. Furthermore, fusion proteins of centaurin α2 and p42IP4, respectively, with the green fluorescent protein (GFP) were expressed in HEK 293 cells to visualize subcellular distribution. In contrast to p42IP4, which was distributed throughout the cell, centaurin α2 was concentrated at the plasma membrane already in unstimulated cells. The protein centaurin α2 was released from the membrane upon addition of wortmannin, which inhibits PI3-kinase. p42IP4, however, translocated to plasma membrane upon growth factor stimulation. Thus, in spite of the high homology between centaurin α2 and p42IP4 and comparable affinities for InsP4 and PtdInsP3, both proteins showed clear differences in subcellular distribution. We suggest a model, which is based on the difference in phosphoinositide binding stoichiometry of the two proteins, to account for the difference in subcellular localization.