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The vesicular glutamate transport (VGLUT) system selectively mediates the uptake of l-glutamate into synaptic vesicles. Uptake is linked to an H⁺-ATPase that provides coupling among ATP hydrolysis, an electrochemical proton gradient, and glutamate transport. Substituted quinoline-2,4-dicarboxylic acids (QDCs), prepared by condensation of dimethyl ketoglutaconate (DKG) with substituted anilines and subsequent hydrolysis, were investigated as potential VGLUT inhibitors in synaptic vesicles. A brief panel of substituted QDCs was previously reported (Carrigan et al. Bioorg. Med. Chem. Lett. 1999, 9, 2607−2612)¹ and showed that certain substituents led to more potent competitive inhibitors of VGLUT. Using these compounds as leads, an expanded series of QDC analogues were prepared either by condensation of DKG with novel anilines or via aryl-coupling (Suzuki or Heck) to dimethyl 6-bromoquinolinedicarboxylate. From the panel of almost 50 substituted QDCs tested as inhibitors of the VGLUT system, the 6-PhCH&dbd;CH-QDC (Ki = 167 μM), 6-PhCH2CH2-QDC (Ki = 143 μM), 6-(4‘-phenylstyryl)-QDC (Ki = 64 μM), and 6-biphenyl-4-yl-QDC (Ki = 41 μM) were found to be the most potent blockers. A preliminary assessment of the key elements needed for binding to the VGLUT protein based on the structure−activity relationships for the panel of substituted QDCs is discussed herein. The substituted QDCs represent the first synthetically derived VGLUT inhibitors and are promising templates for the development of selective transporter inhibitors.

Na+ uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater 22Na+ uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K+ permeability and high Cl- permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity (Ki = 9 +/- 1.8 nM), accounting for 85% of the uptake, and one with a low affinity (Ki = 2.2 +/- 0.71 microM), accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K+ and Cl- indicate that most of the 22Na+ uptake in membranes derived from treated rats is through a Na+-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37 degrees C in Ca2+-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable 22Na+ uptake compared with vesicles derived from cells maintained at 0 degrees C. The addition of Ca2+, but not of Mg2+, to the incubation solution markedly reduced this temperature-dependent enhancement in 22Na+ uptake. The uptake of 22Na+ into vesicles from normal rats was unaffected by preincubation at 37 degrees C or the addition of Ca+ to the incubation solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

The potency of several amiloride analogues to inhibit electrogenic Na+ transport in colon from dexamethasone-treated rats was compared. Short-circuit current (Isc) across the colonic mucosa and 22Na+ uptake into membrane vesicles derived from colonic enterocytes was determined in dexamethasone-treated rats. Kinetic analysis of inhibition of Isc and 22Na+ uptake revealed the presence of a high- and low-affinity amiloride pathway. One pathway had a high affinity [(Ki-Isc; Ki uptake] to benzamil (15.5 nM; 5.4 nM), phenamil (19.4 nM; 7.0 nM), 3',4'-dichlorobenzamil (29.0 nM; 25.2 nM), and amiloride (115 nM; 12.4 nM) but a much lower affinity to 5-(N-ethyl-N-isopropyl)amiloride (EIPA) (greater than 100 microM; greater than 9.9 microM) and 5-(N-propyl-N-butyl)-2'-4'-dichlorobenzamil (PBDCB) (greater than microM; greater than 32.8 microM). The high-affinity pathway accounted for 75-83% of the transport of Na+. The second pathway had nearly the same low affinity for each of the analogues (e.g., amiloride Ki-Isc 1 microM; Ki uptake 4 microM) and accounted for only 15-25% of the transport of Na+. The results demonstrate that the structure-inhibitory pattern of these amiloride analogues for the high-affinity pathway is the pattern observed in other electrogenic Na+-transporting epithelia and that this pharmacological profile is preserved in membrane vesicles derived from colonic enterocytes. In addition, the potency of EIPA and benzamil to inhibit electroneutral Na+ transport across the colon from normal rats (i.e., not treated with dexamethasone) was also investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin enhanced the absorption of water and Na+ across everted sacs of rat colon descendens but had no effect on absorption across the colon ascendens. The short‐circuit current (Isc) and open‐circuit potential difference (p.d.) across the colon descendens were dose‐dependently decreased by vasopressin. Isc and p.d. across the colon ascendens were not altered by vasopressin. In the colon descendens the decrease in Isc and p.d. was significant at 1 microu. vasopressin/ml and reached a maximum at 1 mu./ml. Propranolol and phentolamine or naloxone did not alter the decrease in Isc and p.d. to a submaximal dose of vasopressin. Vasopressin increased the mucosal to serosal flux of Na+ and Cl‐ and decreased the serosal to mucosal flux of Cl‐ across short‐circuited colon descendens. Consequently these changes increased the net flux of Na+ and Cl‐. Adenylate cyclase activity in homogenates of the colon descendens was not altered by vasopressin. Omission of Ca2+ from the serosal bathing solution reversibly decreased Isc and p.d. and increased Na+ and Cl‐ absorption across the colon descendens in a similar way as did vasopressin. The results suggest that the effect of vasopressin on the colon descendens may be due to a decrease in intracellular Ca2+ activity.