Synthetic modification of prostaglandin f(2alpha) indicates different structural determinants for binding to the prostaglandin F receptor versus the prostaglandin transporter.
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Several principles governing the binding of E series prostaglandins to EP receptors have emerged in recent years. The C-1 carboxyl group binds electrostatically to a conserved arginine residue in the seventh transmembrane segment of the receptor. Prostaglandin E analogs involving bioisosteric replacements of the carboxyl group, such as acylsulfonamide, are also active. In addition, structurally similar esters may also exhibit similar affinity, presumably by virtue of hydrogen bonding. Other regions of the substrate molecule appear to bind to other domains of EP receptors, either via hydrophobic interactions or by hydrogen bonding. Less information is available about the structural requirements for substrate binding to FP receptors. Prostanoids also bind to the prostaglandin transporter PGT. In this case, a conserved C-1 carboxyl group is critically important, since C-1 esters exhibit little affinity. Here we examined the binding of chemically diverse PGF(2alpha) structural analogs to the FP receptor and compared these with binding by the PG transporter. PGT recognized a wide range of anionic substituents. In contrast, the carboxylic acid group was essential for optimal binding to the FP receptor, since replacement by larger moieties with a similar pK(a), such as acylsulfonamide and tetrazole, substantially decreased binding affinity. Interestingly, insertion of cyclic substituents in the omega chain increased binding to the FP receptor but reduced affinity for PGT, and substitution for the 15-hydroxyl group produced only a modest reduction in FP receptor binding, but eliminated binding by PGT. Because extracellular PGF(2alpha) may compete for binding between FP receptors and PGT, these findings have implications for designing PGF(2alpha) analogs for treating disease states. (+info)
Impaired solute accumulation in inner medulla of Clcnk1-/- mice kidney.
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The CLC-K1 chloride channel is a kidney-specific CLC chloride channel expressed in the thin ascending limb of Henle's loop (tAL). Recently, we determined that Clcnk1-/- mice show nephrogenic diabetes insipidus (NDI). To investigate the pathogenesis of impaired urinary concentrating ability, we analyzed renal functions of Clcnk1-/- mice in more detail. The osmolar clearance-to-creatinine clearance ratio was not significantly different between Clcnk1+/- and Clcnk1+/+ mice. Fractional excretion of sodium, chloride, and urea was also not significantly affected in Clcnk1-/- mice. These results indicate that the polyuria observed in Clcnk1-/- mice was water diuresis and not osmotic diuresis. The papillary osmolarity in Clcnk1-/- mice was significantly lower than that in Clcnk1+/+ mice under a hydrated condition, and it did not increase even after water deprivation. Sodium and chloride contents in the inner medulla in Clcnk1-/- mice were at about one-half the levels observed in Clcnk1+/+ mice. Furthermore, the accumulation of urea was also impaired in Clcnk1-/- mice, suggesting that the overall countercurrent system was impaired by a defect of its single component, chloride transport in the tAL. The aldose reductase mRNA abundance in Clcnk1-/- mice was decreased, further evincing that inner medullary tonicity is decreased in Clcnk1-/- mice. We concluded that NDI in Clcnk1-/- mice resulted from an impairment in the generation of inner medullary hypertonicity by a dysfunction of the countercurrent systems. (+info)
Correlated spiking activity and associated Ca(2+) waves in the developing retina are important in determining the connectivity of the visual system. Here, we show that GABA, via GABA(B) receptors, regulates the temporal characteristics of Ca(2+) waves occurring before synapse formation in the embryonic chick retina. Blocking ionotropic GABA receptors did no affect these Ca(2+) transients. However, when these receptors were blocked, GABA abolished the transients, as did the GABA(B) agonist baclofen. The action of baclofen was prevented by the GABA(B) antagonist p-3-aminopropyl-p-diethoxymethyl phosphoric acid (CGP35348). CGP35348 alone increased the duration of the transients, showing that GABA(B) receptors are tonically activated by endogenous GABA. Blocking the GABA transporter GAT-1 with 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid (SKF89976A) reduced the frequency of the transients. This reduction was prevented by CGP35348 and thus resulted from activation of GABA(B) receptors by an increase in external [GABA]. The effect of GABA(B) receptor activation persisted in the presence of activators and blockers of the cAMP-PKA pathway. Immunocytochemistry showed GABA(B) receptors and GAT-1 transporters on ganglion and amacrine cells from the earliest times when Ca(2+) waves occur (embryonic day 8). Patch-clamp recordings showed that K(+) channels on ganglion cell layer neurons are not modulated by GABA(B) receptors, whereas Ca(2+) channels are; however, Ca(2+) channel blockade with omega-conotoxin-GVIA or nimodipine did not prevent Ca(2+) waves. Thus, the regulation of Ca(2+) waves by GABA(B) receptors occurs independently of N- and L-type Ca(2+) channels and does not involve K(+) channels of the ganglion cell layer. GABA(B) receptors are likely to be of key importance in regulating retinal development. (+info)
Transgenic mice ubiquitously overexpressing murine gamma-aminobutyric acid transporter subtype I were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. This preliminary finding indicates that the inappropriate level of gamma-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity. (+info)
A family of yeast proteins mediating bidirectional vacuolar amino acid transport.
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Seven genes in Saccharomyces cerevisiae are predicted to code for membrane-spanning proteins (designated AVT1-7) that are related to the neuronal gamma-aminobutyric acid-glycine vesicular transporters. We have now demonstrated that four of these proteins mediate amino acid transport in vacuoles. One protein, AVT1, is required for the vacuolar uptake of large neutral amino acids including tyrosine, glutamine, asparagine, isoleucine, and leucine. Three proteins, AVT3, AVT4, and AVT6, are involved in amino acid efflux from the vacuole and, as such, are the first to be shown directly to transport compounds from the lumen of an acidic intracellular organelle. This function is consistent with the role of the vacuole in protein degradation, whereby accumulated amino acids are exported to the cytosol. Protein AVT6 is responsible for the efflux of aspartate and glutamate, an activity that would account for their exclusion from vacuoles in vivo. Transport by AVT1 and AVT6 requires ATP for function and is abolished in the presence of nigericin, indicating that the same pH gradient can drive amino acid transport in opposing directions. Efflux of tyrosine and other large neutral amino acids by the two closely related proteins, AVT3 and AVT4, is similar in terms of substrate specificity to transport system h described in mammalian lysosomes and melanosomes. These findings suggest that yeast AVT transporter function has been conserved to control amino acid flux in vacuolar-like organelles. (+info)
Transport of L-carnitine in isolated cerebral cortex neurons.
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The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxyli c acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nalecz, D. Dymna, J. E. Mroczkowska, A. Broer, S. Broer, M. J. Nalecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells. (+info)
GABA uptake and heterotransport are impaired in the dentate gyrus of epileptic rats and humans with temporal lobe sclerosis.
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In vivo dialysis and in vitro electrophysiological studies suggest that GABA uptake is altered in the dentate gyrus of human temporal lobe epileptics characterized with mesial temporal sclerosis (MTLE). Concordantly, anatomical studies have shown that the pattern of GABA-transporter immunoreactivity is also altered in this region. This decrease in GABA uptake, presumably due to a change in the GABA transporter system, may help preserve inhibitory tone interictally. However, transporter reversal can also occur under several conditions, including elevations in [K(+)]o, which occurs during seizures. Thus GABA transporters could contribute to seizure termination and propagation through heterotransport. To test whether GABA transport is compromised in both the forward (uptake) and reverse (heterotransport) direction in the sclerotic epileptic dentate gyrus, the physiological effects of microapplied GABA and nipecotic acid (NPA; a compound that induces heterotransport) were examined in granule cells in hippocampal slices from kainate (KA)-induced epileptic rats and patients with temporal lobe epilepsy (TLE). GABA- and NPA-induced responses were prolonged in granule cells from epileptic rats versus controls (51.3 and 31.3% increase, respectively) while the conductance change evoked with NPA microapplication was reduced by 40%. Furthermore the ratio of GABA/NPA conductance, but not duration, was significantly >1 in epileptic rats but not controls, suggesting a compromise in transporter function in both directions. Similar changes were observed in tissue resected from epileptic patients with medial temporal sclerosis but not in those without the anatomical changes associated with MTLE. These data suggest that the GABA transporter system is functionally compromised in both the forward and reverse directions in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. This alteration may enhance inhibitory tone interically yet be permissive for seizure propagation due to a decreased probability for GABA heterotransport during seizures. (+info)
Overexpression of gamma-aminobutyric acid transporter subtype I leads to susceptibility to kainic acid-induced seizure in transgenic mice.
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Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters. (+info)