A role for endothelial NO synthase in LTP revealed by adenovirus- mediated inhibition and rescue

Science

Volumn 274, Issue 5293, 1996, Pages 1744-1748

  Kantor, David B ^a   Lanzrein, Markus ^a   Stary, S Jennifer ^a   Sandoval, Gisela M ^a   Smith, W Bryan ^a   Sullivan, Brian M ^a   Davidson, Norman ^a   Schuman, Erin M ^a  

^a CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANTIVIRUS AGENT; NITRIC OXIDE; NITRIC OXIDE SYNTHASE; VIRUS VECTOR;

ADENOVIRUS; ANIMAL CELL; ARTICLE; CHO CELL; CONTROLLED STUDY; ENZYME DEFECT; EXTRACELLULAR SPACE; LONG TERM POTENTIATION; MYRISTYLATION; NONHUMAN; PRIORITY JOURNAL; VIRUS INHIBITION; VIRUS RECOMBINANT;

ADENOVIRIDAE; ANIMALS; CELL MEMBRANE; CHO CELLS; CRICETINAE; CYTOSOL; ENDOTHELIUM; GENETIC VECTORS; HIPPOCAMPUS; LONG-TERM POTENTIATION; MICE; MYRISTIC ACID; MYRISTIC ACIDS; NEURONS; NITRIC OXIDE SYNTHASE; RECOMBINANT FUSION PROTEINS; SYNAPTIC TRANSMISSION; TRANSFECTION;

ADENOVIRIDAE; ANIMALIA;

EID: 0029826368     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5293.1744     Document Type: Article

References (39)

1. G. A. Böhme, C. Bon, J.-M. Stutzmann, A. Doble, J.-C. Blanchard, Eur. J. Pharmacol. 199, 379 (1991).
2. E. M. Schuman and D. V. Madison, Science 254, 1503 (1991).
3. T. J. O'Dell, R. J. Hawkins, E. R. Kandel, O. Arancio, Proc. Natl. Acad. Sci. U.S.A. 88, 11285 (1991).
4. J. E. Haley, G. L. Wilcox, P. F. Chapman, Neuron 8, 211 (1992).
5. E. M. Schuman and D. V. Madison, Science 263 532 (1994).
6. However, several groups (D. M. Chetkovich, E. Klann, J. D. Sweatt, NeuroReport 4, 919 (1993); V. K. Gribkoff and J. T. Lum-Ragan, J. Neurophysol. 68, 639 (1992); J. E. Haley, P. L. Malen, P. F. Chapman, Neurosci. Lett 160, 85 (1993); J. H. Williams et al., Neuron 11, 877 (1993); J. A. Cummings, S. M. Nicola, R. C. Malenka, Neurosci. Lett. 176, 110 (1994)] have shown that NO-independent forms of LTP can be elicited with different stimulation protocols.
7. However, several groups (D. M. Chetkovich, E. Klann, J. D. Sweatt, NeuroReport 4, 919 (1993); V. K. Gribkoff and J. T. Lum-Ragan, J. Neurophysol. 68, 639 (1992); J. E. Haley, P. L. Malen, P. F. Chapman, Neurosci. Lett 160, 85 (1993); J. H. Williams et al., Neuron 11, 877 (1993); J. A. Cummings, S. M. Nicola, R. C. Malenka, Neurosci. Lett. 176, 110 (1994)] have shown that NO-independent forms of LTP can be elicited with different stimulation protocols.
8. However, several groups (D. M. Chetkovich, E. Klann, J. D. Sweatt, NeuroReport 4, 919 (1993); V. K. Gribkoff and J. T. Lum-Ragan, J. Neurophysol. 68, 639 (1992); J. E. Haley, P. L. Malen, P. F. Chapman, Neurosci. Lett 160, 85 (1993); J. H. Williams et al., Neuron 11, 877 (1993); J. A. Cummings, S. M. Nicola, R. C. Malenka, Neurosci. Lett. 176, 110 (1994)] have shown that NO-independent forms of LTP can be elicited with different stimulation protocols.
9. However, several groups (D. M. Chetkovich, E. Klann, J. D. Sweatt, NeuroReport 4, 919 (1993); V. K. Gribkoff and J. T. Lum-Ragan, J. Neurophysol. 68, 639 (1992); J. E. Haley, P. L. Malen, P. F. Chapman, Neurosci. Lett 160, 85 (1993); J. H. Williams et al., Neuron 11, 877 (1993); J. A. Cummings, S. M. Nicola, R. C. Malenka, Neurosci. Lett. 176, 110 (1994)] have shown that NO-independent forms of LTP can be elicited with different stimulation protocols.
10. However, several groups (D. M. Chetkovich, E. Klann, J. D. Sweatt, NeuroReport 4, 919 (1993); V. K. Gribkoff and J. T. Lum-Ragan, J. Neurophysol. 68, 639 (1992); J. E. Haley, P. L. Malen, P. F. Chapman, Neurosci. Lett 160, 85 (1993); J. H. Williams et al., Neuron 11, 877 (1993); J. A. Cummings, S. M. Nicola, R. C. Malenka, Neurosci. Lett. 176, 110 (1994)] have shown that NO-independent forms of LTP can be elicited with different stimulation protocols.
11. T. J. O'Dell et al., Science 265, 542 (1994).
12. P. L. Huang, T. M. Dawson, D. S. Bredt, S. H. Snyder, M. C. Fishman, Cell 75, 1273 (1993).
13. J. L. Dinerman, T. M. Dawson, M. J. Schell, A. Snowman, S. H. Snyder, Proc. Natl. Acad. Sci U.S.A. 91, 4214 (1994).
14. J. Pollock, V. Klinghofer, U. Forstermann, F. Murad, FEBS Lett. 309, 402 (1992).
15. J. Liu and W. C. Sessa, J. Biol. Chem. 269, 11691 (1994).
16. L. Busconi and T. Michel, ibid. 268, 8410 (1993).
17. R. Helm, A. B. Cubitt, R. Y. Tsien, Nature 373, 663 (1995).
18. M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, D. C. Prasher, Science 263, 802 (1994).
19. 2 (G2A)] in pBluescript SK was excised at the 5′ Cla I site and the 3′ Xba I site. In a three-way ligation, the CD8 and eNOSG2A fragments were subcloned into pBluescript SK. Orientation was confirmed by restriction analysis, and the junction sequence of the CD8-eNOS G2A fusion was confirmed by DNA sequencing. The fusion cDNA was then excised with Eco RI, subcloned into pAC CMV, and screened for orientation. Membrane localization was confirmed by immunocytochemistry with a fluorescein-conjugated monoclonal antibody to human CDS (Sigma). Recombinant adenoviruses were prepared by standard transfection methods into HEK 293 cells, with the use of the experimental plasmids plus the right arm of the AdSpAC I virus digested with Xba I and Cla I. This virus contains a 2.7-kb deletion in the E3 region, thus providing adequate cloning capacity for all inserts.
20. 5 cells per dish. A thin glass cover slip was embedded in the bottom of the plastic dish to enable the use of oil immersion lenses in confocal microscopy. CHO cells were transfected with pGreenlantern-1 (Gibco-BRL, Gaithersburg, MD), pACeNOS-GL, or pACCD8-eNOS (2 ng of plasmid DNA per dish), or were cotransfected with pACeNOS-GL and pACTeNOS at a ratio of 1:3 (8 μg of plasmid DNA per dish). When added, HMA was at a final concentration of 100 μM and ethanol was at a final concentration of 0.25%. Transfections were carried out with lipotectamine (Gibco-BRL) according to the manufacturer's instructions. After transfection (20 to 24 hours), the cells were analyzed with an inverted-stage laser confocal microscope system (BioRad MRC-600) using an argon laser, an excitation wavelength of 488 nm, and a X63 objective. Contrast and brightness settings were constant between experiments. Images were imported and displayed in Adobe Photoshop 3.0.
21. W.C. Sessa et al., J. Biol. Chem. 270, 17641 (1995).
22. 2 atmosphere. One-half of the volume of the medium was exchanged every 12 hours. β-Gal was detected with X-Gal staining [J. R. Sanes, J. L. R. Rubenstein, J.-F. Nicolas, EMBO J. 5, 3133 (1986)]. In 50-μm sections of infected slices, we counted the number of neurons containing β-Gal, and estimated that 68 to 95% of neurons in the injected region were infected, depending on whether central or peripheral regions of virus injection were scrutinized. Stained slices were photographed (Zeiss Axiophot, X2.5 and × 10 objectives) and imported into Adobe Photoshop 3.0 for graphic presentation.
23. 2. fEPSPs measured independently in SR or SO at a depth of 100 to 150 μm below the slice surface were evoked by two different stimulating electrodes activating the Schaffer collateral-commissural afferents (once every 15 s); the initial (1- to 2-ms) slope was measured. LTP was induced by four trains of highfrequency stimulation (100 Hz for 1 s) separated by 30-s intervals. To quantify I/O relations, the slopes of the regression lines for each experiment were compared. All electrophysiology experiments, with the exception of those noted in (22), were conducted with the experimenter being unaware of the experimental condition of the slice The percents of baseline measurements indicated in the text were taken 50 to 60 min after tetanus. Ensemble average plots represent group means of each EPSP slope, for all experiments, aligned with respect to the time of LTP induction. To assess statistical significance, paired or independent t tests were performed. P values greater than 0.05 are designated as NS.
24. 14C]citrulline in hippocampal homogenates made from infected CA1 regions of hippocampal slices [D. S. Bredt and S. H. Snyder, Proc. Natl. Acad. Sci. U.S.A. 86, 9030 (1989)]. (The NOS activity of Ad-CD8-eNOS was assessed in CHO cells with the same method.) We found that Ad-TeNOS inhibited NOS activity by 14.4 ± 32% when compared with Ad-lacZ-infected slices (n = 6 experiments, 3 slices per experiment, for both Ad-lacZ and Ad-TeNOS). This modest but significant inhibition may reflect the relatively small contribution of eNOS to the total NOS activity detected in the hippocampus (7) and may account for the observed block of LTP in SR by TeNOS, although alternative mechanisms may also contribute.
25. J. E. Haley and D. V. Madison, Learn. Mem., in press.
26. We combined the simultaneous SR-SO experiments shown in Fig. 3 with an earlier blind study examining LTP in Ad-lacZ- versus TeNOS-infected slices at SR synapses only, because the two sets of SR data were statistically indistinguishable from one another.
27. R. A. J. McIlhinney and K. MGlone, J. Neurochem. 54, 110 (1990).
28. M. J. S. Nadler, M. L. Harrison, C. L. Ashendel, J. M. Cassady, R. L. Geahlen, Biochemistry 32, 9250 (1993).
29. L. A. Paige, G. Zheng, S. A. De Frees, J. M. Cassady, R. L. Geahlen, ibid. 29, 10566 (1990).
30. L. Busconi and T. Michel, J. Biol. Chem. 269, 25016 (1994).
31. Slices were treated with 100 μM HMA dissolved in ethyl alcohol (EtOH) and bovine serum albumin (BSA) (final EtOH concentration. 0.25%; final BSA concentration, 0.25 mg/ml) or with vehicle alone and were maintained in the same MEM described above for 20 to 26 hours before electrophysiological recording. Adjacent slices from one hippocampus were exposed to either experimental or control conditions, and only data from pairs in which the control slice expressed LTP were considered. For experiments in which HMA was applied acutely, a two-pathway design was implemented. HMA dissolved in EtOH plus BSA was added directly to the superfusate at the indicated times, and during the control recording period, slices were perfused with Ringer's containing EtOH plus BSA. In experiments using a NOS inhibitor, L-N-monomethyl-arginine (100 μM; Sigma) was applied to slices for at least 3 hours before recording.
32. The half-life of eNOS has been reported to be approximately 20 hours [L. J. Robinson, L. Busconi, T. Michel, J. Biol. Chem. 270, 995 (1995); D. J. Stuehr and O. W. Griffith, Adv. Enzymol. Relat. Areas Mol. Biol. 65, 287 (1992)].
33. The half-life of eNOS has been reported to be approximately 20 hours [L. J. Robinson, L. Busconi, T. Michel, J. Biol. Chem. 270, 995 (1995); D. J. Stuehr and O. W. Griffith, Adv. Enzymol. Relat. Areas Mol. Biol. 65, 287 (1992)].
34. 3H]myristoyl coenzyme A was generated enzymatically [D. Towler and L Glaser, Proc. Natl. Acad. Sci. U.S.A. 83, 2812 (1986)], and the NMT activity assay was performed on 50 μg of homogenate protein. Data are expressed as adjusted counts per minute (cpm).
35. 3H]myristoyl coenzyme A was generated enzymatically [D. Towler and L Glaser, Proc. Natl. Acad. Sci. U.S.A. 83, 2812 (1986)], and the NMT activity assay was performed on 50 μg of homogenate protein. Data are expressed as adjusted counts per minute (cpm).
36. P. Sukhatme, K. C. Sizer, A. C. Vollmer, T. Hunkapiller, J. R. Parnes, Cell 40, 591 (1985).
37. D. B. Kantor and E. M. Schuman, unpublished observations.
38. T. Sakoda et al., Mol. Cell. Biochom. 152, 143 (1995).
39. We thank T. Michel for sharing various eNOS cDNAs and unpublished results, B. Seed for CD8 cDNA, A. Berk and L. Wu for Ad-lacZ, and G. Laurent for technical assistance and discussion. Supported by European Molecular Biology Organization grant ALTF 168-1996 (M.L.), National Institute of Mental Health grant 49176 (N.D.), NIH grant NS37292 (E.M.S.), and a Beckman Young Investigator award (E.M.S.).