Locally formed 5-hydroxytryptamine stimulates phosphate transport in cultured opossum kidney cells and in rat kidney.
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ABSTRACT:
SUBMITTER: Hafdi Z
PROVIDER: S-EPMC1217974 | biostudies-other | 1996 Dec
REPOSITORIES: biostudies-other
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Project description:We studied with biochemical and immunofluorescent techniques the interactions between the actin microfilament and tubulin microtubule cytoskeleton and Na+/P1 co-transport in opossum kidney cells, a line with proximal tubular characteristics. On brief (5 min) incubation of the cells with a low (0.1 mM) concentration of Pi, a rapid F-actin depolymerization takes place, which fails to occur in cells incubated under similar conditions with 1 mM Pi. The disassembly of actin microfilaments could be quantitatively expressed as a 33% increase in the ration of monomeric G-actin to polymerized F-actin (G/F-actin ration from 0.80 +/- 0.03 to 1.06 +/- 0.06, n = 28, P<0.01), owing to a significant decrease in the latter. Under these conditions microfilaments were also markedly destabilized, as shown by their diminished resistance to graded cytochalasin B concentrations. In addition, incubation of opossum kidney cells with low Pi concentrations (0.1 mM) resulted within 5 min in a substantial depolymerization of microtubules, shown by immunofluorescence microscopy and measured as a 70.9 +/- 6.9% (n = 11, P<0.01) decrement by immunoblot analysis. These changes, which occur only when extracellular Pi concentrations are kept low, seem to be related to a significant increase within 5 min in the rate of cellular Pi uptake by 25.5% under these conditions. The shifts in the dynamic equilibria between monomeric and polymerized actin and tubulin in response to cellular Pi uptake were transient, being fully reversible within 30 min. Moreover, the effect of Pi seemed to be specific because inhibition of its uptake by phosphonoformic acid blunted microtubular disassembly markedly. In contrast, measurement of Pi uptake in the presence of agents known to stabilize cytoskeletal structures showed a substantial decrease with phallacidin, which stabilized microfilaments, whereas the microtubule stabilizer taxol had no apparent effect. These results indicate that acute alterations in the polymerization dynamics and stability of both microfilaments and microtubules are involved in the modulation of Na+/Pi co-transport and suggest important cytoskeletal participation in proximal tubular transport functions.
| S-EPMC1217177 | biostudies-other
Project description:This study deals with the effect of 5-hydroxytryptamine (5-HT; serotonin) on glucose transport in isolated rat cardiac myocytes. In these cells, 5-HT (10-300 microns), as well as tryptamine, 5-methoxytryptamine and dopamine, elicited a 3-5 fold increase in glucose transport, as compared with control. This effect was maximal after 90 min, and was concomitant with a 1.8- and 1.5-fold increase in the amounts of glucose transporters GLUT1 and GLUT4 at the cell surface of the cardiomyocytes, as determined by using the photoaffinity label 3H-2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-manno s-4-yl) propyl-2-amine (3H-ATB-BMPA). In contrast, 3-3000 microM of the selective 5-HT receptor agonists 5-carboxyamido-tryptamine, alpha-methyl-serotonin, 2-methyl-serotonin or renzapride failed to stimulate glucose transport. The effect of 5-HT was not affected by (i) the 5-HT receptor antagonists methysergide (1 microM), ketanserin (1 microM), cyproheptadine (1 microM), MDL 72222 (1 microM) or ICS 205-930 (3 microM), nor by (ii) the adrenergic receptor antagonists prazosin (1 microM), yohimbine (1 microM) or propranolol (5 microM), nor by (iii) the dopaminergic antagonists SCH 23390 (1 microM) or haloperidol (1 microM). The monoamine oxidase inhibitors clorgyline (1 microM) and tranylcypromine (1 microM) completely suppressed the effect of 5-HT, whereas the control and insulin-stimulated rates of glucose transport were unaffected. Addition of catalase or glutathione diminished the 5-HT-dependent stimulation of glucose transport by 50%; these two factors are known to favour the degradation of H2O2 (which can be formed during the deamination of amines by monoamine oxidases). Glutathione also depressed the stimulatory action of exogenously added H2O2 (20 microM) by 30%. Furthermore, in cells treated with 5_HT, a time-dependent accumulation of 5-hydroxy-1H-indol-3-ylacetic acid (a product of 5-HT metabolism via monoamine oxidases) was observed, which paralleled the changes in glucose transport. In conclusion, the stimulation of glucose transport by 5-HT in cardiomyocytes is not mediated by a 5-HT1, 5-HT2, 5-HT3 or 5-HT4 receptor, nor by an adrenergic or dopaminergic receptor, but is likely to occur through the degradation of by a monoamine oxidase and concomitant formation of H2O2.
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Project description:Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca(2+) and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL) expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca(2+) excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5), calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b), whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a) and type 3 (PIT2) were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca(2+)/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.
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Project description:The involvement of dopamine (DA) receptor subtypes in regulation of renal phosphate transport by DA, either exogenous or locally synthesized from L-dihydroxyphenylalanine (L-dopa) was evaluated in opossum kidney (OK) cells with proximal tubular phenotype. DA synthesis from L-dopa by OK cells was abolished by carbidopa and benserazide, two dissimilar inhibitors of aromatic L-amino acid decarboxylase. L-Dopa stimulated cyclic AMP generation and inhibited Na-dependent Pi uptake, and these effects were abolished by carbidopa and benserazide. The effects of L-dopa or DA on cyclic AMP generation and on Na-Pi co-transport were mimicked by SKF 38393, a DA1 receptor agonist, and were potentiated by S-sulpiride, a DA2 receptor antagonist. Bromocriptine, a DA2 receptor agonist, blunted in a pertussis toxin-dependent manner parathyroid hormone (PTH)-induced cyclic AMP generation and inhibition of Pi uptake. In contrast with PTH, neither L-dopa nor DA affected significantly the cytosolic calcium concentration. These results support the involvement of DA1 and DA2 receptors, positively and negatively coupled into adenylate cyclase respectively, in modulation of renal phosphate transport.
| S-EPMC1136280 | biostudies-other
Project description:The effect of albumin on the release of [3H]lysophosphatidylcholine from cultured rat hepatocytes prelabelled with [Me-3H]choline was studied. In the absence of serum and albumin from the medium, the cells released essentially no [3H]lysophosphatidylcholine. Albumin stimulated this process dramatically, and it reached a plateau at 2 mg/ml. After an initial lag of 30 min, the release of [3H]lysophosphatidylcholine was linear for at least 4 h. At low concentrations, albumin slightly stimulated [3H]phosphatidylcholine release. The albumin had no measurable effect on the metabolism of cellular [3H]phosphatidylcholine, [3H]lysophosphatidylcholine or [3H]glycerophosphocholine. In addition, albumin did not alter the release of 3H-labelled water-soluble compounds, including [3H]glycerophosphocholine, into the medium. The possibility that the [3H]lysophosphatidylcholine was arising from catabolism of [3H]phosphatidylcholine in the medium by secreted enzymes was excluded. The effect on [3H]lysophosphatidylcholine secretion was also observed when the cells were incubated with alpha-cyclodextrin, a cyclic polysaccharide that has the ability to bind lysophosphatidylcholine. The albumin-released lysophosphatidylcholine was enriched in unsaturated fatty acids. Alteration of the fatty acid composition of cellular phosphatidylcholine gave rise to parallel changes in phosphatidylcholine and lysophosphatidylcholine in the medium. It is concluded that phosphatidylcholine is constantly being degraded in the rat hepatocyte to lysophosphatidylcholine which is released into the medium only when a suitable acceptor is present.
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Project description:The transport of amino acids across cell membranes is believed to be mediated by integral membrane proteins with distinct substrate specificities. Using expression cloning in Xenopus oocytes and assaying for the uptake of 14C-labeled cystine, we isolated a 2.3-kilobase cDNA (D2) from a rat kidney library. D2 is expressed specifically in kidney and intestine and induces the transport of both neutral and cationic amino acids. The deduced amino acid sequence predicts a 78-kDa protein with a single transmembrane domain, a structure not typical of the known membrane transport proteins, which generally have multiple membrane-spanning regions. The putative extracellular region is highly similar to the 4F2 heavy-chain cell surface antigen and to a family of alpha-glucosidases, which raises the possibility that D2 encodes a transport activator or regulatory subunit.
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Project description:Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.
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Project description:We investigated the effects of purified histone H4 on glucose transport activity in rat soleus and flexor digitorum brevis muscles. Histone H4, at concentrations up to 11.8 microM, increased 2-deoxyglucose (2-DG) uptake in a dose-dependent fashion. However, at concentrations higher than 11.8 microM, H4 caused a decrease in 2-DG uptake from the maximum, suggesting a secondary inhibitory action of this compound. The maximal effect of H4 on 2-DG uptake was not additive to the maximal effect of insulin. Moreover, 2-DG uptake in the presence of both H4 and insulin was significantly lower than the 2-DG uptake in the presence of insulin alone. The maximal effect of H4 on stimulation of 2-DG uptake was neither additive nor inhibitory to the maximal effects of the intracellularly acting insulin mimetics sodium vanadate or H2O2. It was, on the other hand, additive to the maximal effects of muscle contractions. Also, in contrast with the effects of H4 on insulin-stimulated 2-DG uptake, H4 did not inhibit insulin-like growth factor-I (IGF-I)-stimulated 2-DG uptake, as the maximal effects of H4 and IGF-I were additive. Scatchard analysis of the binding of 125I-insulin in the absence or presence of histone H4 revealed that H4 increased the specific binding of insulin without affecting receptor affinity. These data suggest that H4 interacts with the insulin, rather than the hypoxia/contraction, pathway for activation of glucose transport in muscle tissue, and that H4 acts either directly or indirectly to increase the number of insulin receptors at the surface of the muscle cell. This interaction does not appear to occur with the similar, although distinct, IGF-I receptor. These studies may provide additional insight into the complex signal-transduction systems of insulin action.
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Project description:WNK1 belongs to a unique family of Ser/Thr kinases that have been implicated in the control of blood pressure. Intronic deletions in the WNK1 gene result in its overexpression and lead to pseudohypoaldosteronism type II, a disease with salt-sensitive hypertension and hyperkalemia. How overexpression of WNK1 leads to Na(+) retention and hypertension is not entirely clear. Similarly, there is no information on the hormonal regulation of expression of WNK kinases. There are two main WNK1 transcripts expressed in the kidney: the originally described "long" WNK1 and a shorter transcript that is specifically expressed in the kidney (KS-WNK1). The goal of this study was to determine the effect of aldosterone, the main hormonal regulator of Na(+) homeostasis, on the transcription of WNK1 isoforms in renal target cells, by using an unique mouse cortical collecting duct cell line that stably expresses functional mineralocorticoid receptors. Our results demonstrate that aldosterone, at physiological concentrations, rapidly induces the expression of the KS-WNK1 but not that of the long-WNK1 in these cells. Importantly, stable overexpression of KS-WNK1 significantly increases transepithelial Na(+) transport in cortical collecting duct cells. Similarly, coexpression of KS-WNK1 and the epithelial Na(+) channel in Fischer rat thyroid epithelial cells also stimulates Na(+) current, suggesting that KS-WNK1 affects the subcellular location or activity but not the expression of epithelial Na(+) channel. These observations suggest that stimulation of KS-WNK1 expression might be an important element of aldosterone-induced Na(+) retention and hypertension.
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