Inositol hexakisphosphate and sulfonylureas regulate beta-cell protein phosphatases

In human type 2 diabetes, loss of glucose-stimulated insulin exocytosis from the pancreatic beta-cell is an early pathogenetic event. Mechanisms controlling insulin exocytosis are, however, not fully understood. We show here that inositol hexakisphosphate (InsP(6)), whose concentration transiently increases upon glucose stimulation, dose-dependently and differentially inhibits enzyme activities of ser/thr protein phosphatases in physiologically relevant concentrations. None of the hypoglycemic sulfonylureas tested affected protein phosphatase-1 or -2A activity at clinically relevant concentrations in these cells. Thus, an increase in cellular phosphorylation state, through inhibition of protein dephosphorylation by InsP(6), may be a novel regulatory mechanism linking glucose-stimulated polyphosphoinositide formation to insulin exocytosis in insulin-secreting cells.     

ATP-sensitive potassium channel regulates astrocytic gap junction permeability by a Ca2+-independent mechanism

Using the scrape-loading technique in cultured astrocytes, we show that sulfonylureas such as tolbutamide and glybenzcyclamide, which inhibit the ATP-sensitive K+ channel, prevent the inhibition of gap junction permeability caused by several structurally unrelated uncouplers such as oleic acid, arachidonic acid, endothelin-1, octanol, and alpha-glycyrrhetinic acid. When the intracellular level of Ca2+ was diminished, all the uncouplers tested were still able to inhibit gap junction communication, indicating that their inhibitory effect was not mediated by Ca2+. In addition, tolbutamide and glybenzcyclamide prevented the inhibitory effect of these uncouplers in Ca(2+)-depleted astrocytes, suggesting that the inhibition of the ATP-sensitive K+ channel increases gap junction permeability through a Ca(2+)-independent mechanism. The activation of the ATP-sensitive K+ channel caused by potassium channel openers such as diazoxide and pinacidil led to the inhibition of gap junction communication and overcame the effect of sulfonylureas. These results suggest that the ATP-sensitive K+ channel regulates gap junctional permeability.     

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum)

Summary Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 151 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25–260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha^-1 chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6–4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha^-1) and full (4.5 g ha^-1) rates of metsulfuron methyl.     

Genetic and biochemical characterization of corn inbred lines tolerant to the sulfonylurea herbicide primisulfuron

Summary Inbred lines of corn (Zea mays L.) have been characterized, which exhibit differential sensitivity to the sulfonylurea herbicide primisulfuron (2-[3-(4,6-bis(di-fluoromethoxy) pyrimidin-2-yl)-ureidosulfonyl]-benzoic acid methylester). When treated postemergence with 160 g a.i. per hectare, inbred 4CO exhibited complete tolerance while inbred 4N5 was killed. The F₁ hybrid 4C0 x 4N5 was uniformly tolerant indicating dominance of the tolerance trait. The field observations correlated with laboratory tests in which seedling root growth was measured. Based on IC₅₀, inbred 4CO was more than ten times more tolerant than inbred 4N5. In the F₂ and F₃ generations, a 31 segregation of tolerant and sensitive individuals was observed, consistent with tolerance being inherited as a single dominant trait. Backcrosses of heterozygous F₁ plants with the sensitive parent (4N5) yielded progeny that segreated at the expected 11 ratio. Backcrosses with 4C0 yielded tolerant offspring only. Inhibition characteristics of acetohydroxyacid synthase (AHAS; E.C. 4.1.3.18) were determined. The enzymes from both inbreds and their F₁ hybrid were equally sensitive and strongly inhibited by primisulfuron (IC₅₀: 7 nM). The fate of ¹⁴C-labeled primisulfuron in seedling tissues of inbred 4C0 and the hybrid, 4C0 x 4N5, indicated rapid metabolism with a half-life (t _1/2) of approximately 3 h. On the other hand, the herbicide-sensitive inbred 4N5 was considerably slower to metabolize primisulfuron (t _1/2 >24 h). These data indicate that differential metabolism is the mechanism of tolerance to the sulfonylurea herbicide primisulfuron in tolerant corn.Deceased     

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

Summary The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3 end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.     

Acetohydroxyacid synthase from Mycobacterium avium and its inhibition by sulfonylureas and imidazolinones

Tuberculosis (TB) remains one of the world's leading causes of death from infectious disease. It is caused by infection with Mycobacterium tuberculosis or sometimes, particularly in immune-compromised patients, Mycobacterium avium. The aim of this study was to create a tool that could be used in the search for new anti-TB drugs that inhibit branched-chain amino acid (BCAA) biosynthesis, as these are essential amino acids that are not available to a mycobacterium during growth in an infected organism. To this end, we cloned, overexpressed, purified and characterised for the first time an acetohydroxyacid synthase (AHAS), a key enzyme in the pathway to the biosynthesis of the BCAAs, from the genus Mycobacterium. Nine commercial herbicides of the sulfonylurea and imidazolinone classes were tested for their influence on this enzyme. Four of the sulfonylureas were potent inhibitors of the enzyme. The relative potency of the different inhibitors towards the M. avium enzyme was unlike their potency towards other AHASs whose inhibitor profile has been reported, emphasising the advantage of using a mycobacterial enzyme as a tool in the search for new anti-TB drugs.     

Carbamazepine as a Novel Small Molecule Corrector of Trafficking-impaired ATP-sensitive Potassium Channels Identified in Congenital Hyperinsulinism

ATP-sensitive potassium (K_ATP) channels consisting of sulfonylurea receptor 1 (SUR1) and the potassium channel Kir6.2 play a key role in insulin secretion by coupling metabolic signals to β-cell membrane potential. Mutations in SUR1 and Kir6.2 that impair channel trafficking to the cell surface lead to loss of channel function and congenital hyperinsulinism. We report that carbamazepine, an anticonvulsant, corrects the trafficking defects of mutant K_ATP channels previously identified in congenital hyperinsulinism. Strikingly, of the 19 SUR1 mutations examined, only those located in the first transmembrane domain of SUR1 responded to the drug. We show that unlike that reported for several other protein misfolding diseases, carbamazepine did not correct K_ATP channel trafficking defects by activating autophagy; rather, it directly improved the biogenesis efficiency of mutant channels along the secretory pathway. In addition to its effect on channel trafficking, carbamazepine also inhibited K_ATP channel activity. Upon subsequent removal of carbamazepine, however, the function of rescued channels was recovered. Importantly, combination of the K_ATP channel opener diazoxide and carbamazepine led to enhanced mutant channel function without carbamazepine washout. The corrector effect of carbamazepine on mutant K_ATP channels was also demonstrated in rat and human β-cells with an accompanying increase in channel activity. Our findings identify carbamazepine as a novel small molecule corrector that may be used to restore K_ATP channel expression and function in a subset of congenital hyperinsulinism patients.     

Photoaffinity Labeling of the Cerebral Sulfonylurea Receptor Using a Novel Radioiodinated Azidoglibenclamide Analogue

Abstract: In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145–150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [³H]glibenclamide or a ¹²⁵I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate ( K _D = 0.13 n M ) and solubilized ( K _D = 0.56 n M ) sulfonylurea receptor from cerebral cortex. The novel probe showed >100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a K _D value of 0.38 n M . Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed K _D values for these sulfonylureas of 0.06 n M , 1.6 n M , and 1.2 µ M , respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.     

Identification of a cytochrome P450 hydroxylase, CYP81A6, as the candidate for the bentazon and sulfonylurea herbicide resistance gene, Bel, in rice

Bentazon and sulfonylureas have been used for selective control of broadleaf weeds and sedges in rice fields for more than 20 years. A bentazon and sulfonylurea susceptible mutant, bel, was previously identified for the purpose of allowing these herbicides to be used for removing false hybrids from hybrid rice. While this mutation has been used successfully in rice breeding, the genetic nature of bel is not known. Using 1,776 susceptible plants from a population of 10,000 F₂ individuals, we constructed a fine map for the Bel locus and delimited it to a 36-kb DNA fragment between two restriction fragment length polymorphism markers, L5 and P17. Bioinformatic analysis indicated that there are five genes within this interval, an ethylene-responsive OsER33 gene and four tandem repeats of cytochrome P450 genes designated as CYP81A5, CYP81A6, CYP81A7, and CYP81A8. Comparative sequencing could not find any differences in the coding regions of the OsER33, CYP81A5, CYP81A7, and CYP81A8 genes between the mutant bel and its wild-type progenitor W6154S, but did identify a single base guanine deletion at position +1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contains an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylurea herbicides in rice. A gene-specific cleaved amplified polymorphic sequence marker and tightly linked flanking markers were developed that will be very useful for selection of the bel allele when transferred to photoperiod-/thermo-sensitive genic male sterility and CMS lines in hybrid rice breeding programs.