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The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate (NMDA) and glutamate were highly efficacious agonists, whereas alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-801. Cyclic GMP accumulations induced by NMDA and non-NMDA agonists alike were Ca(2+)-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L-NG-nitroarginine, a highly potent component (IC50 = 6 nM) evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under "basal" conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation.

Selected aminothiazolyl-oxime cephalosporin congeners substituted at C-3' with a catechol moiety were used to probe the basis of the enhanced antibacterial activity against Escherichia coli K-12 often associated with chemical modifications of this type. Evidence is presented for a tonB-dependent illicit transport of the compounds across the outer membrane of E. coli K-12, the process involving jointly and specifically the Fiu and Cir iron-regulated outer membrane proteins. Thus, both tonB and flu cir mutants showed a comparably reduced susceptibility to the probe compounds, whereas mutants singularly lacking any one of the six iron-regulated outer membrane proteins (Fiu, FepA, FecA, FhuA, FhuE, and Cir) or lacking any combination of any two of these proteins (except Fiu plus Cir) did not show this resistance. Mutants devoid of all six iron-regulated outer membrane proteins were no more resistant to the probe compounds than fiu cir or tonB strains. In addition to the latter genes, the products of the exbB and possibly the exbC loci were necessary for maximal antibacterial potency. A dependence of antibacterial activity on the level of expression of the uptake system components was noted. Comparison of penicillin-binding protein target affinity with antibacterial activity suggested a possible periplasmic accumulation of active compounds by E. coli K-12. Free vicinal hydroxyl groups of the catechol residue were a primary chemical requirement for recognition by the uptake pathway and thus for high antibacterial activity.Recent reports have described the enhanced antibacterial potency against members of the family Enterobacteriaceae and pseudomonads of ,B-lactam antibiotics variously substituted with catechol, derivatized catechol, and related moieties (2, 6, 29-31, 33, 34, 45). Using cephalosporin E-0702 (featuring a catechol derivative at C-7), Watanabe et al. (45) reported the isolation of spontaneous Escherichia coli K-12 mutants specifically resistant to this compound owing to a defect at the tonB locus. From the known functionality of the tonB gene product in the transport of iron-chelated siderophores by E. coli K-12, it was hypothesized that the potency of E-0702 resulted from the incorporation of the compound by a tonB-dependent siderophore transport route (45). Unpublished studies in this laboratory have shown that such (tonB) mutants of E. coli K-12 can be isolated irrespective of the chemical diversity of the catechol-substituted cephalosporin (catechol-cephalosporin) used. These mutants, like tonB mutants independently isolated as multiply colicintolerant variants of sensitive strains (3,24,25), are specifically cross resistant to catechol-cephalosporins as a chemical class (unpublished observations and this report).In view of the pivotal importance of the tonB gene product in determining the sensitivity of E. coli K-12 to these agents, we attempted to identify whether any of the known tonBdependent uptake processes of E. coli K-12 could be implicated in catechol-cephalosporin activity.The tonB gene pro...

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