(1/92) Evolutionary analysis of TATA-less proximal promoter function.
Many molecular studies describe how components of the proximal promoter affect transcriptional processes. However, these studies do not account for the likely effects of distant enhancers or chromatin structure, and thus it is difficult to conclude that the sequence variation in proximal promoters acts to modulate transcription in the natural context of the whole genome. This problem, the biological importance of proximal promoter sequence variation, can be addressed using a combination of molecular and evolutionary analyses. Provided here are molecular and evolutionary analyses of the variation in promoter function and sequence within and between populations of Fundulus heteroclitus for the lactate dehydrogenase-B (Ldh-B) proximal promoter. Approximately one third of the Ldh-B proximal promoter contains interspersed regions that are functionally important: (1) they bind transcription factors in vivo, (2) they effect a change in transcription as assayed by transient transfection into two different fish cell lines, and (3) they bind purified transcription factors in vitro. Evolutionary analyses that compare sequence variation in these functional regions versus the nonfunctional regions indicate that the changes in the Ldh-B proximal promoter sequences are due to directional selection. Thus, the Ldh-B proximal promoter sequence variations that affect transcriptional processes constitute a phenotypic change that is subject to natural selection, suggesting that proximal promoter sequence variation affects transcription in the natural context of the whole genome. (+info)
(2/92) Morphological adaptation to thermal stress in a marine fish, Fundulus heteroclitus.
Populations of Fundulus heteroclitus (Cyprinodontidae), a coastal marine fish, were studied in control and artificially heated environments on the north shore of Long Island to determine patterns of variation in morphology and the extent to which this variation reflected adaptation to environmental characteristics. Principal components and discriminant function analyses were used to analyze variation in and among seventeen morphological characters. Fishes living in water artificially heated by a power plant exhibited marked divergence from control populations in head morphology, and convergence with a population sampled at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Greater morphological variation is detected at the heated locality than at control localities, and this may be partially due to a breakdown in developmental homeostasis, and partially due to selection favoring phenotypes that are rare in this environment. (+info)
(3/92) Active lucifer yellow secretion in renal proximal tubule: evidence for organic anion transport system crossover.
Recent studies show that organic anion secretion in renal proximal tubule is mediated by distinct sodium-dependent and sodium-independent transport systems. Here we investigated the possibility that organic anions entering the cells on one system can exit into the lumen on a transporter associated with the other system. In isolated rat kidneys perfused with 10 microM lucifer yellow (LY, a fluorescent organic anion) plus 100 micrograms/ml inulin, the LY-to-inulin clearance ratio averaged 1.6 +/- 0.2, indicating net tubular secretion. Probenecid significantly reduced both LY clearance and LY accumulation in kidney tissue. In intact killifish proximal tubules, confocal microscopy was used to measure steady-state LY uptake into cells and secretion into the tubular lumen. Probenecid, p-aminohippurate, and ouabain nearly abolished both uptake and secretion. To this point, the data indicated that LY was handled by the sodium-dependent and ouabain-sensitive organic anion transport system. However, leukotriene C4, an inhibitor of the luminal step for the sodium-independent and ouabain-insensitive organic anion system, reduced luminal secretion of LY by 50%. Leukotriene C4 did not affect cellular accumulation of LY or the transport of fluorescein on the sodium-dependent system. A similar inhibition pattern was found for another fluorescent organic anion, a mercapturic acid derivative of monochlorobimane. Thus, both organic anions entered the cells on the basolateral transporter for the classical, sodium-dependent system, but about half of the transport into the lumen was handled by the luminal carrier for the sodium-independent system, which is most likely the multidrug resistance-associated protein. This is the first demonstration that xenobiotics can enter renal proximal tubule cells on the carrier associated with one organic anion transport system and exit into the tubular lumen on multiple carriers, one of which is associated with a second system. (+info)
(4/92) Localization of an organic anion transporter-GFP fusion construct (rROAT1-GFP) in intact proximal tubules.
The organic anion transporter, rROAT1, is a dicarboxylate/organic anion exchanger, a function associated with the basolateral membrane in rat proximal tubule. To directly establish the subcellular localization of rROAT1 in renal epithelia, we made a rROAT1-green fluorescent protein (GFP) fusion construct (rROAT1-GFP). Plasma membrane-associated fluorescence was observed in rROAT1-GFP-expressing Xenopus oocytes examined by confocal microscopy. Uptake of 3H-labeled p-aminohippurate (PAH) increased 2. 5-fold in rROAT1-GFP-expressing Xenopus oocytes, and this increase was abolished by 1 mM probenecid. Thus the construct was capable of specific organic anion transport. Cultured renal epithelial cell lines (MDCK and LLC-PK1) transfected with the vector pEGFP-C3 showed a diffuse, evenly distributed cytoplasmic signal. However, when transfected with pEGFP-C3/rROAT1 (vector coding for rROAT1-GFP), both cell lines showed predominantly plasma membrane fluorescence. The expression and distribution of rROAT1-GFP in intact renal proximal tubules was also investigated. Isolated killifish (Fundulus heteroclitus) renal tubules transfected with pEGFP-C3/rROAT1 showed marked basal and lateral membrane-associated fluorescence, but no detectable signal in the nucleus or the apical pole of tubule cells. Tubules transfected with pEGFP-C3 showed diffuse cytoplasmic fluorescence. Function of the rROAT1-GFP construct was demonstrated in transfected killifish tubules by fluorescein transport assay. These results demonstrate the basolateral subcellular localization of rROAT1 in polarized renal epithelia and validate a new technique for localizing cloned transporters within intact renal tubules. (+info)
(5/92) Interactions of HIV protease inhibitors with ATP-dependent drug export proteins.
We used renal proximal tubules from a teleost fish (killifish; Fundulus heteroclitus), fluorescent substrates and confocal microscopy to study the interactions between human immunodeficiency virus protease inhibitors and drug-transporting ATPases. Both saquinavir and ritonavir inhibited luminal accumulation of a fluorescent cyclosporin A derivative (a substrate for P-glycoprotein) and of fluorescein methotrexate [a substrate for multidrug resistance-associated protein 2 (Mrp2)]. Of the two protease inhibitors, ritonavir was the more potent inhibitor of transport by a factor of at least 20. Ritonavir was at least as good an inhibitor of P-glycoprotein- and Mrp2-mediated transport as cyclosporin A and leukotriene C4, respectively. Inhibition of P-glycoprotein- and Mrp2-mediated transport was not due to toxicity or impaired metabolism, because neither saquinavir nor ritonavir inhibited transport of fluorescein on the renal organic anion system. Experiments with a fluorescent saquinavir derivative showed strong secretion into the tubular lumen that was inhibited by verapamil, leukotriene C4, saquinavir, and ritonavir. Together, the data demonstrate that saquinavir, and especially ritonavir, are potent inhibitors of P-glycoprotein- and Mrp2-mediated transport. The experiments with the fluorescent saquinavir derivative suggest that these protease inhibitors may also be substrates for both P-glycoprotein and Mrp2. (+info)
(6/92) Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule.
In the kidney, endothelins (ETs) are important regulators of blood flow, glomerular hemodynamics, and sodium and water homeostasis. They have been implicated in the pathophysiology of acute ischemic renal failure, nephrotoxicity by cyclosporine, cisplatin and radiocontrast agents, and vascular rejection of kidney transplants. Here, we used intact killifish renal proximal tubules, fluorescent substrates for Mrp2 (fluorescein-methotrexate, FL-MTX) and P-glycoprotein (a fluorescent CSA derivative, NBD-CSA), and confocal microscopy to reveal a new role for renal ET: regulation of ATP-driven drug transport in proximal tubule. Subnanomolar to nanomolar concentrations of ET-1 rapidly reduced the cell-to-tubular lumen transport of both fluorescent compounds. These effects were prevented by an ET(B) receptor antagonist but not by an ET(A) receptor antagonist. Immunostaining with an antibody to mammalian ET(B) receptors showed specific localization to the basolateral membrane of the fish tubular epithelial cells. ET-1 effects on transport were blocked by protein kinase C-selective inhibitors, implicating protein kinase C in ET-1 signaling. Finally, the nephrotoxic radiocontrast agent iohexol reduced cell-to-lumen FL-MTX and NBD-CSA transport, and these effects were abolished by an ET(B) receptor antagonist. These are the first results linking ET to the control of xenobiotic transport and the first demonstrating control of renal multidrug resistance-associated protein 2 and P-glycoprotein by a hormone. (+info)
(7/92) P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule.
1. Transepithelial transport of a fluorescent derivative of octreotide (NBD-octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus). 2. Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD-octreotide into the tubular lumen was demonstrated and exhibited the all characteristics of specific and energy-dependent transport. Steady state luminal fluorescence averaged about five times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. NBD-octreotide secretion was inhibited in a concentration-dependent manner by unlabelled octreotide, verapamil and leukotriene C(4) (LTC(4)). Conversely, unlabelled octreotide reduced in a concentration dependent manner the p-glycoprotein (Pgp)-mediated secretion of a fluorescent cyclosporin A derivative (NBDL-CS) and the mrp2-mediated secretion of fluorescein methotrexate (FL-MTX). 4. This inhibition was not due to impaired metabolism or toxicity since octreotide had no influence on the active transport of fluorescein (FL), a substrate for the classical renal organic anion transport system. 5. The data are consistent with octreotide being transported across the brush border membrane of proximal kidney tubules by both Pgp and mrp2. (+info)
(8/92) Control of epithelial Cl(-) secretion by basolateral osmolality in the euryhaline teleost Fundulus heteroclitus.
Euryhaline teleost fish adapt rapidly to salinity change and reduce their rate of ion secretion on entry to fresh water. Killifish (Fundulus heteroclitus) transferred from full-strength sea water to fresh water showed large reductions in plasma [Na(+)] and osmolality at 6 h which were corrected by 24 h. To mimic this in vitro, a hypotonic shock of 20-70 mosmol kg(-)(1) was applied on the basolateral side of opercular epithelia. This hypotonic shock reversibly reduced the short-circuit current (I(sc), equivalent to the rate of secretion of Cl(-)) in a dose-dependent fashion, with a 40 mosmol kg(-)(1) hypotonic shock reducing I(sc) by 58+/-4.6 % in 40 min. Similar reductions in [NaCl], but with added mannitol to maintain osmolality, were without effect, indicating that the effect was purely osmotic. Hypotonic inhibition of I(sc) was accompanied by reductions in epithelial conductance (G(t)) but no significant change in transepithelial potential (V(t)). The hypotonic inhibition was apparently not Ca(2+)-mediated because Ca(2+)-depleted salines, thapsigargin and ionomycin all failed to block the reduction in I(sc) produced by hypotonic shock. The inhibition was not mediated via a reduction in intracellular cyclic AMP level because cyclic AMP levels, measured by radioimmunoassay, were unchanged by hypotonic shock and by 1.0 micromol l(-)(1) clonidine (which inhibits I(sc) by changing intracellular [Ca(2+)]) but were increased markedly by 1.0 micromol l(-)(1) isoproterenol, a positive control. The protein tyrosine kinase inhibitor genistein (100 micromol l(-)(1)), but not its inactive analogue daidzein, inhibited I(sc) in normal osmolality but produced a stimulation of I(sc) after hypotonic shock (and after clonidine treatment). The inhibitory effects of genistein and hypotonicity were not additive, suggesting that the same portion of the I(sc) was inhibited by both treatments. These data are consistent with a model for Cl(-) transport regulation involving tyrosine phosphorylation in cell-swelling-induced inhibition of Cl(-) secretion when euryhaline teleosts adapt to fresh water. (+info)