Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis . In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain . Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels . " 1P; is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin . OS-IS take up and release "Ca" by Na'-, Ca`-, and IBMX-sensitive mechanisms. Illumination causes release of "Ca", which confirms retinal studies by other groups using Ca'-sensitive electrodes . Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina . Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation .

Frog rod outer segments contain o00.25 mol of GTP and 0.25 mol of ATP per tool of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per tool of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X l02 and 5 • l0 e rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10 -s M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10 -6 and 10 -s M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.

Light decreases GTP and ATP levels in purified suspensions of physiologically active frog rod outer segments still attached to their inner segment ellipsoids (OS-IS). (a) The GTP decrease is slower in OS-IS (t1/2= 40 s) than in isolated outer segments (hi2 = 7 s), which suggests there is more effective buffering in OS-IS. (b) The GTP decrease becomes detectable only at intensities greater than those required to saturate the photoresponse. As the intensity of a continuous light is increased over 4 log units, GTP levels decrease linearly with log intensity by as much as 60% . GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity. (c) At levels of illumination bleaching >0.003% of the rhodopsin, a decrease in ATP levels becomes detectable. (d) Following a flash, GTP levels fall and-then rise with a recovery time dependent on the intensity of the flash. (e) After both 0.2 and 2% flash bleaches, the recovery of GTP levels parallels the recovery of light sensitivity, which is slower than the recovery of the dark current. This raises the possibility of a link between GTP levels and light sensitivity.

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Two approaches to the synthesis of boron-substituted pyrimidines and borazaroquinazolines (1) have been explored First, (dibutoxyboryl)ethene and bromomalononitrile were converted to l,l-dicyano-3-bromo-3-(dibutoxyboryl)propane (2), which was reduced with triphenyltin hydride to l,l-dicyano-3-(dibutoxyboryl)propane (3), which condensed with thiourea to yield 2-mercapto-4,6-diamino-5-(2-dihydroxyborylethyl)pyrimidine (4a). However, conversion of 4a to a borazaroquinazoline was not attempted because the development of boron-substituted carbanion chemistry promised a more direct and efficient approach. This second method involved condensation of 4,6-dichloro-5-formylpyrimidine (5) with the carbanion from tetrakis(trimethylenedioxyboryl)methane to form 4,6-dichloro-5-[2,2-bis(trimethylenedioxyboryl)vinyl]pyrimidine (6a), which on treatment with ammonia at 25 °C yielded 4-chloro-6-trimethylenedioxyboryl-7-hydroxy-7,8-dihydro-7,8-borazaroquinazoline (7a), which reacted with ammonia at 75 °C to form the 4-amino derivative 8. Improved yields were obtained in a similar sequence starting from tetrakis(ethylenedioxyboryl)methane. Characterization of the amino-substituted borazaroquinazolines was aided by 13 *C NMR correlations.Substitution of a boron atom for a carbon in a biochemically significant molecule might lead to antimetabolite ac-