Spinal nerve ligation does not alter the expression or function of GABA(B) receptors in spinal cord and dorsal root ganglia of the rat.

Loss of GABA-mediated inhibition in the spinal cord is thought to mediate allodynia and spontaneous pain after nerve injury. Despite extensive investigation of GABA itself, relatively little is known about how nerve injury alters the receptors at which GABA acts. This study examined levels of GABA(B) receptor protein in the spinal cord dorsal horn, and in the L4 and L5 (lumbar designations) dorsal root ganglia one to 18 weeks after L5 spinal nerve ligation. Mechanical allodynia was maximal by 1 week and persisted at blunted levels for at least 18 weeks after injury. Spontaneous pain behaviors were evident for 6 weeks. Western blotting of dorsal horn detected two isoforms of the GABA(B(1)) subunit and a single GABA(B(2)) subunit. High levels of GABA(B(1a)) and low levels of GABA(B(1b)) protein were present in the dorsal root ganglia. However, GABA(B(2)) protein was not detected in the dorsal root ganglia, consistent with the proposed existence of an atypical receptor composed of GABA(B(1)) homodimers. The levels of GABA(B(1a)), GABA(B(1b)), and GABA(B(2)) protein in the ipsilateral dorsal horn were unchanged at any time after injury. Immunohistochemical staining also did not detect a change in GABA(B(1)) or GABA(B(2)) subunits in dorsal horn segments having a robust loss of isolectin B4 staining. The levels of GABA(B(1a)) protein were also unchanged in the L4 or L5 dorsal root ganglia at any time after spinal nerve ligation. Levels of GABA(B(2)) remained undetectable. Finally, baclofen-stimulated binding of guanosine-5'-(gamma-O-thio)triphosphate in dorsal horn did not differ between sham and ligated rats. Collectively, these results argue that a loss of GABA(B) receptor-mediated inhibition, particularly of central terminals of primary afferents, is unlikely to mediate the development or maintenance of allodynia or spontaneous pain behaviors after spinal nerve injury.

Catabolism of porphobilinogen by etiolated barley leaves.

When [2,4-(14)C]porphobilinogen (PBG) or [2 (aminomethyl),5-(14)C]PBG is administered to etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness, label becomes incorporated into CO(2), organic and amino acids, sugars, lipids, and proteins during a 4-hour incubation. Less than 1% of the label, however, is incorporated into porphyrins. The rate of (14)CO(2) evolution from leaves fed [2,4-(14)C]PBG is strongly inhibited by anaerobiosis but is unaffected by aminooxyacetic acid, while the rate of (14)CO(2) evolution from [2(aminomethyl),5-(14)C]PBG is strongly inhibited by aminooxyacetic acid but is not affected by anaerobiosis.THESE RESULTS SUGGEST THAT: (a) exogenous PBG is taken up and metabolized by etiolated barley leaves; (b) PBG is not metabolized exclusively to porphyrins but can be converted to a variety of intermediary metabolites; (c) this metabolism involves reactions which are partially dependent upon O(2) and pyridoxal phosphate.

Catabolism of 5-Aminolevulinic Acid to CO(2) by Etiolated Barley Leaves.

The in vivo oxidation of the C(4) and C(5) of 5-aminolevulinic acid (ALA) to CO(2) has been studied in etiolated barley (Hordeum vulgare L. var. Larker) leaves in darkness. The rate of (14)CO(2) evolution from leaves fed [4-(14)C]ALA is strongly inhibited by aminooxyacetate, anaerobiosis, and malonate. The rate of (14)CO(2) evolution from leaves fed [5-(14)C]ALA is also inhibited by these treatments but to a lesser extent. These results suggest that (a) one step in ALA catabolism is a transamination reaction and (b) the C(4) is oxidized to CO(2) via the tricarboxylic acid cycle to a greater extent than is the C(5).

Catabolism of [1-C]levulinic Acid by etiolated and greening barley leaves.

Levulinic acid (LA), a competitive inhibitor of delta-aminolevulinic acid (ALA) dehydratase (EC 4.2.1.24), has been used extensively in the study of ALA formation during greening. When [1-(14)C]LA is administered to etiolated barley (Hordeum vulgare L. var. Larker) shoots in darkness, (14)CO(2) is evolved. This process is accelerated when such tissues are incubated with 2 millimolar ALA or placed under continuous illumination. Label from the C-1 of LA becomes incorporated into organic acids, amino acids, sugars, lipids, and proteins during a 4-hour incubation in darkness or in the light. This metabolism is discussed in relation to the use of LA as a tool in the study of chlorophyll synthesis in higher plants.

Studies with 4,6-dioxoheptanoic Acid on etiolated and greening barley leaves.

4,6-Dioxoheptanoic acid (DA), an inhibitor of 5-aminolevulinic acid (ALA) dehydratase (EC 4.3.1.24), causes ALA to accumulate at the expense of chlorophyll when applied to greening leaves of Hordeum vulgare L. var. Larker. Preincubating etiolated leaves with DA in darkness eliminates the lag phase in ALA accumulation during a subsequent exposure to illumination. More than 50% of the DA taken up during a 2-hour incubation disappeared during a subsequent 4-hour incubation. These results suggest that barley leaves can metabolize DA, and the products of this metabolism may enhance the capacity for ALA synthesis.

The effects of levulinic Acid and 4,6-dioxoheptanoic Acid on the metabolism of etiolated and greening barley leaves.

Application of levulinic acid (LA), a competitive inhibitor of delta-aminolevulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of [(14)C]amino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of (14)CO(2) evolution from labeled precursors, including amino and organic acids. Inhibition of (14)CO(2) evolution by these compounds is more effective in greening leaves than in etiolated leaves when [4-(14)C]ALA or [1-(14)C]glutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of (32)Pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed.

Reversal of alpha,alpha'-Dipyridyl-induced Porphyrin Synthesis in Etiolated and Greening Red Kidney Bean Leaves.

The chemical induction of porphyrin synthesis has been investigated in etiolated and greening leaves of Phaseolus vulgaris L. var. Red Kidney. When these leaves are incubated in darkness with solutions of transition metal ion chelators such as alpha,alpha'-dipyridyl, 1,10-phenanthroline, pyridine-2-aldoxime, or other related aromatic heterocyclic nitrogenous bases, they synthesize large amounts of protochlorophyllide and Mg protoporphyrins. Greening leaves produce more porphyrin than do etiolated leaves under such conditions. If the leaves are then transferred to 1 millimolar solutions of various transition metal salts such as Fe(2+), Zn(2+), or Co(2+) (but not Mn(2+) or Mg(2+)), Mg protoporphyrin (monomethyl ester) synthesis immediately ceases and the pigment(s) rapidly disappear(s); protochlorophyllide synthesis gradually diminishes during 4 to 8 hours of treatment. The loss in Mg protoporphyrin(s) can be accounted for by a simultaneous increase in protochlorophyllide in partially greened leaves but not in etiolated leaves. In the latter, the decline in Mg protoporphyrin(s) initiated by the application of Zn(2+) is retarded by low temperature and anaerobiosis but not by respiratory inhibitors. Cycloheximide inhibits the loss of Mg protoporphyrin(s) but does not affect their conversion to protochlorophyllide.THESE RESULTS INDICATE THAT: (a) greening leaves have a greater capacity to synthesize delta-aminolevulinic acid than do etiolated leaves; (b) alpha,alpha'-dipyridyl induction of porphyrin synthesis in etiolated and greening leaves can be blocked by application of certain transition metal salts; (c) in greening leaves the accumulated Mg protoporphyrin(s) are stoichiometrically converted to protochlorophyllide upon treatment with these salts whereas in etiolated leaves the accumulated Mg protoporphyrin(s) are labile and are not quantitatively converted to protochlorophyllide upon such treatment; (d) in etiolated leaves the accumulated Mg protoporphyrin(s) are destroyed via a light-independent, probably enzymic process which requires cytoplasmic protein synthesis.

Protoheme extraction from plant tissue.

A method for reproducibly estimating the protoheme content of plant tissues has been developed. The tissue sample is homogenized in 80% acetone to remove pigments and lipids; protoheme is then extracted from the tissue residue with 2% HCl in acetone and quantitatively transferred into diethyl ether. After evaporation of the ether, the residue is dissolved in alkaline pyridine, and the protoheme concentration is estimated from a dithionite-reduced-minus-ferricyanide-oxidized spectrum. When compared to some other methods, this procedure gives consistently higher yields.

Characterization of protoheme levels in etiolated and greening plant tissues.

The protoheme content of etiolated, greening, and fully greened bean (Phaseolus vulgaris L. var. Light Red Kidney) leaves has been studied. The protoheme level in etiolated and fully greened leaf tissue stays relatively constant from age 7 to 14 days. In agreement with the studies reported for barley (Castelfranco and Jones 1975 Plant Physiol 55: 485-490), the protoheme content of greening bean and barley (Hordeum vulgare var. Larker) leaves does not change appreciably during the first 9 hours of illumination, but the level rises significantly by the 24th hour of illumination (cf. Hendry and Stobart 1977 Phytochemistry 16: 1545-1548). This increase also occurs in seedlings returned to the dark for 24 to 48 hours following a 10-minute pulse of light. These results demonstrate a limited correlation with previous studies on the development of b-type cytochromes during greening of these tissues (Gregory and Bradbeer 1973; Planta 109: 317-326).

Induction of porphyrin synthesis in etiolated bean leaves by chelators of iron.

Primary leaves of 7- to 9-day-old etiolated seedlings of Phaseolus vulgaris L. var. Red Kidney infiltrated in darkness with aqueous solutions of alpha, alpha'-dipyridyl, o-phenanthroline, pyridine-2-aldoxime, pyridine-2-aldehyde, 8-hydroxyquinoline, or picolinic acid synthesize large amounts of magnesium protoporphyrin monomethyl ester and lesser amounts of magnesium protoporphyrin, protoporphyrin, and protochlorophyllide. Pigment formation proceeds in a linear manner for up to 21 hours after vacuum infiltration with 10 mm alpha, alpha'-dipyridyl. Etiolated tissues of Zea mays L., Cucumis sativus L., and Pisum sativum L. respond in the same way to dipyridyl treatment. Compounds active in eliciting this response are aromatic heterocyclic nitrogenous bases which also act as bidentate chelators and form extremely stable complexes with iron; other metal ion chelators, such as ethylenediaminetetraacetic acid, salicylaldoxime, and sodium diethyldithiocarbamate, do not elicit any pigment synthesis. The ferrous, ferric, cobaltous, and zinc chelates of alpha, alpha'-dipyridyl are similarly ineffective. If levulinic acid is supplied to etiolated bean leaves together with alpha, alpha'-dipyridyl, porphyrin production is inhibited and delta-aminolevulinic acid accumulates in the tissue. Synthesis of porphyrins proceeds in the presence of 450 micrograms per milliliter chloramphenicol or 50 micrograms per milliliter cycloheximide with only partial diminution. We propose that heme or an iron-protein complex blocks the action of the enzyme(s) governing the synthesis of delta-aminolevulinic acid in etiolated leaves in the dark and that iron chelators antagonize this inhibition, leading to the biosynthesis of delta-aminolevulinic acid and porphyrins.

The Conversion of Photoinactive Protochlorophyllide(633) to Phototransformable Protochlorophyllide(650) in Etiolated Bean Leaves Treated with delta-Aminolevulinic Acid.

The relationship of phototransformable protochlorophyllide to photoinactive protochlorophyllide has been studied in primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Various levels of photoinactive protochlorophyllide, absorbing at 633 nm in vivo, were induced by administering delta-aminolevulinic acid to the leaves in darkness. Phototransformable protochlorophyllide, absorbing at 650 nm in vivo, was subsequently transformed to chlorophyllide by a light flash, and the regeneration of the photoactive pigment was followed by monitoring the absorbance increase at 650 nm in vivo. A small increase in the level of protochlorophyllide(633) causes a marked increase in the extent of regeneration of protochlorphyllide(650) following a flash. High levels of the inactive pigment species, however, retard the capacity to reform photoactive protochlorophyllide. A nonstoichiometric and kinetically complex decrease in absorbance at 633 nm in vivo accompanied the absorbance increase at 650 nm. The half-time for protochlorophyllide(650) regeneration in control leaves was found to be three times longer than the half-time for conversion of chlorophyllide(678) to chlorophyllide(683) at 22 C. The results are consistent with the hypothesis that protochlorophyllide(633) is a direct precursor of protochlorophyllide(650) and that the protein moiety of the protochlorophyllide holochrome acts as a "photoenzyme" in the conversion of protochlorophylide to chlorophyllide.

A Reversible Conversion of Phototransformable Protochlorophyll(ide)(656) to Photoinactive Protochlorophyll(ide)(656) by Hydrogen Sulfide in Etiolated Bean Leaves.

The relationship of phototransformable protochlorophyll-(ide) to photoinactive protochlorophyll(ide) has been studied in the primary leaves of 7- to 9-day-old dark-grown bean (Phaseolus vulgaris L. var. Red Kidney) seedlings. Subjecting the leaves to an atmosphere of H(2)S causes an immediate loss of phototransformable protochlorophyll(ide)(650) and a simultaneous increase in photoinactive protochlorophyll(ide)(633). When such leaves are returned to air or N(2), the absorbance at 650 nm increases, whereas the absorbance at 633 nm decreases and photoactivity is restored. The reversion of protochlorophyll-(ide)(633) to protochlorophyll(ide)(650) is one-half complete in 3 minutes at 22 C in 8-day-old leaves. Ninety-five per cent recovery of protochlorophyll(ide)(650) is obtained when exposure to H(2)S is less than 3 minutes in duration; longer periods reduce the reversion capacity proportionately. The leaves are relatively undamaged by brief exposures to H(2)S, as judged by electron microscopy and by their ability to synthesize chlorophyll under continuous illumination. Hydrogen sulfide has no immediate effect upon the absorption properties of a partially purified preparation of the protochlorophyll(ide) holochrome, an etioplast suspension, or leaves subjected to freezing and thawing. Compounds such as HCN and HN(3) cause an irreversible conversion of protochlorophyll(ide)(650) to protochlorophyll(ide)(633) with total loss of photoactivity. Sulfhydryl agents, such as beta-mercaptoethanol and cysteine, cause a slow, irreversible transformation of the photoactive pigment to the photoinactive form and inhibit the ability of the leaves to synthesize chlorophyll under continuous illumination. The results obtained suggest that H(2)S may alter the interaction between the source of hydrogens on the protein moiety of the holochrome and the chromophore in vivo by reducing a disulfide bond in the protein, thereby causing a reversible conformational change in the complex.

Rapid regeneration of protochlorophyllide(650).

The rate of regeneration of protochlorophyllide(650) was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding delta-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides(635) but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous delta-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0 degrees , suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides(650) holochrome.

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris.

An enzyme catalyzing the formation of delta-aminolevulinic acid by transamination of gamma,delta-dioxovaleric acid with l-alpha-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in delta-aminolevulinic acid metabolism in plants is not clearly understood.

Control of chlorophyll production in rapidly greening bean leaves.

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of delta-amino-levulinic acid partially relieves the inhibition by chloramphenicol.It is suggested that light regulates chlorophyll synthesis by controlling the availability of delta-aminolevulinic acid, possibly by mediating the formation of an enzyme of delta-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

Studies on the regeneration of protochlorophyllide after brief illumination of etiolated bean leaves.

The effects of various inhibitors of nucleic acid and protein synthesis on protochlorophyllide synthesis in dark-grown Phaseolus vulgaris var. Red Kidney have been studied. Actinomycin D, chloramphenicol, and puromycin inhibit the regeneration of protochlorophyllide holochrome (detected as a 650 mmu absorption peak) in vivo in darkness after photoconversion of endogenous protochlorophyllide a to chlorophyllide a; this inhibition does not occur in similarly treated leaves supplied with delta-aminolevulinic acid.These data suggest that the regeneration of protochlorophyllide results from the synthesis of RNA and enzymes required for the production of delta-aminolevulinate.