RESUMEN
Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.
Asunto(s)
Endocitosis/fisiología , Túbulos Renales Colectores/enzimología , Orgánulos/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Túbulos Renales Colectores/ultraestructura , Masculino , Microscopía Electrónica , Orgánulos/ultraestructura , Ratas , Ratas EndogámicasRESUMEN
Filtered inorganic phosphate (Pi) is largely reabsorbed in the proximal tubule. Na-Pi cotransport, with a stoichiometry of at least 2:1, mediates uphill transport at the apical membrane; at the basolateral membrane different types of transport systems can be involved in efflux and uptake of Pi from the interstitium. Regulation of transcellular Pi flux involves alteration of the apical Na-Pi cotransport; at least three different cellular control/sensing systems seem to participate in this regulation and are exemplified by parathyroid hormone (PTH)-dependent inhibition, Pi deprivation-dependent increase, and insulin-like growth factor I (IGF-I)-dependent increase in Na-Pi cotransport. For PTH inhibition, recent evidence suggests a role of the phospholipase C/protein kinase C-dependent regulatory cascade in inhibition of Na-Pi cotransport, at least at low PTH concentrations. In addition, an endocytic mechanism seems to be involved in this PTH action. Little is known of the cellular mechanisms in Pi deprivation-dependent and/or IGF-I-dependent increases in Na-Pi cotransport; they are dependent on de novo protein synthesis. Recent experiments involving an expression in Xenopus laevis oocytes led to the identification of an approximately 50 kDa membrane protein that is a good candidate for being involved in brush-border membrane Na-Pi cotransport activity.
Asunto(s)
Túbulos Renales Proximales/fisiología , Fosfatos/metabolismo , Simportadores , Absorción , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Modelos Biológicos , Hormona Paratiroidea/farmacología , Proteínas Cotransportadoras de Sodio-FosfatoRESUMEN
In normal pituitary cells somatostatin (SRIF) blocks the spontaneous oscillations in [Ca++]i by inhibiting the generation of action potentials. This is sufficient to explain the inhibitory effect on basal, but not entirely that on stimulated pituitary hormone secretion. In insulin secreting cells, which, in contrast to pituitary cells, only fire action potentials on stimulus-evoked depolarization, SRIF hyperpolarization and lowering of [Ca++]i is only transient. The marked inhibition of insulin secretion is suggested to be due to a coordinated action of SRIF on membrane potential and [Ca++]i as well as a direct interference with late secretory events.
Asunto(s)
Calcio/metabolismo , Hipófisis/fisiología , Tasa de Secreción/fisiología , Somatostatina/fisiología , Animales , Células Cultivadas , Citosol/metabolismo , Páncreas/fisiología , Ratas , Somatostatina/farmacologíaRESUMEN
Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C. Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system. This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line. To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis. Long term exposure of the cells to PTH induced changes in apical protein concentration. Four proteins were found to be decreased and one protein was found to be increased in its concentration. Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins. In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h. Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours. The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system. It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport.
Asunto(s)
Membrana Celular/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Hormona Paratiroidea/farmacología , Animales , Membrana Celular/efectos de los fármacos , Células Cultivadas , Electroforesis en Gel Bidimensional , Proteínas de la Membrana/aislamiento & purificación , Peso Molecular , Zarigüeyas , Fosfatos/metabolismo , Fosfoproteínas/biosíntesis , Fosfoproteínas/aislamiento & purificación , Fosforilación , Sodio/metabolismoRESUMEN
Using an established renal epithelial cell line (OK cells) the effect of the amino-acid side-chain modifying reagent N-acetylimidazole (NAI) upon the sodium-dependent transport of phosphate (Pi) was investigated. After an incubation with 10 mM NAI for 20 min, cellular Na+/Pi uptake was inhibited by 70%. The presence of 5 mM Pi protected this transport function from being affected by NAI by 80 to 100%. Since the presence of sulfate was unable to protect the Na+/Pi transport inactivation by NAI and since the presence of Pi did not affect NAI inhibition of other transport systems, it is suggested that NAI interacts with the Pi transporter directly. The protective effect of Pi was used as a criterion to identify Pi-protectable [3H]NAI labelling of OK cell plasma membrane proteins. Pi protection was observed in four molecular mass regions: 31, 53, 104 and 176 kDa. Since the incorporation of [3H]NAI into these proteins was also affected by parathyroid hormone at 10(-10) M, it is concluded that the identified proteins represent possible candidates for the renal Na+/Pi cotransporter.
Asunto(s)
Proteínas Portadoras/fisiología , Imidazoles/farmacología , Riñón/metabolismo , Fosfatos/metabolismo , Sodio/metabolismo , Simportadores , Animales , Proteínas Portadoras/antagonistas & inhibidores , Fraccionamiento Celular , Línea Celular , Membrana Celular/fisiología , Técnicas In Vitro , Zarigüeyas , Proteínas Cotransportadoras de Sodio-Fosfato , Sulfatos/farmacología , Tirosina/farmacologíaRESUMEN
Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Fluorometría , Hipófisis/metabolismo , Animales , Benzofuranos/metabolismo , Fluorometría/instrumentación , Fura-2 , Masculino , Microquímica/instrumentación , Hipófisis/citología , RatasRESUMEN
The cytosolic free calcium concentration, [Ca2+]i, was monitored in single rat lactotrophs in primary culture with the fluorescent probe Fura 2. It was found that lactotrophs are very heterogeneous in their [Ca2+]i response to TRH and dopamine, the major physiological regulators of PRL secretion. While in most lactotrophs TRH raises [Ca2+]i, the kinetics of this rise and the magnitude of its first and second phases vary considerably. For dopamine two clearly divergent response types can be observed. In part of the lactotrophs dopamine causes a lowering of [Ca2+]i from elevated levels, whereas in about 40% of the lactotrophs dopamine leads to a transient rise of [Ca2+]i. The present study reveals subclasses of lactotrophs with distinct [Ca2+]i response characteristics. It is suggested that such response type heterogeneity is a means of optimizing the secretory response to the complex regulatory influences on the pituitary.
Asunto(s)
Calcio/metabolismo , Dopamina/farmacología , Adenohipófisis/metabolismo , Prolactina/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Animales , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Cinética , Masculino , Adenohipófisis/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
Electrical activity in non-neuronal cells can be induced by altering the membrane potential and eliciting action potentials. For example, hormones, nutrients and neurotransmitters act on excitable endocrine cells. In an attempt to correlate such electrical activity with regulation of cell activation, we report here direct measurements of cytosolic free Ca2+ changes coincident with action potentials. This was achieved by the powerful and novel combination of two complex techniques, the patch clamp and microfluorimetry using fura 2 methodology. Changes in intracellular calcium concentration were monitored in single cells of the pituitary line GH3B6. We show that a single action potential leads to a marked transient increase in cytosolic free calcium. The size of these short-lived maxima is sufficient to evoke secretory activity. The striking kinetic features of these transients enabled us to identify oscillations in intracellular calcium concentration in unperturbed cells resulting from spontaneous action potentials, and hence provide an explanation for basal secretory activity. Somatostatin, an inhibitor of pituitary function, abolishes the spontaneous spiking of free cytosolic Ca2+ which may explain its inhibitory effect on basal prolactin secretion. Our data therefore demonstrate that electrical activity can stimulate Ca2+-dependent functions in excitable non-neuronal cells.
Asunto(s)
Potenciales de Acción , Calcio/fisiología , Hipófisis/fisiología , Benzofuranos , Línea Celular , Citosol/fisiología , Fura-2 , Canales Iónicos/fisiología , Periodicidad , Hipófisis/citología , Somatostatina/farmacología , Factores de TiempoRESUMEN
Pertussis toxin, PT, abolishes inhibitory regulation of adenylate cyclase by cell surface receptors. Inhibitors of adenylate cyclase in GH3 cells, namely somatostatin and the muscarinic cholinergic agonist carbachol, lower the cytosolic free Ca2+ concentration. [Ca2+]i and cause hyperpolarization. These responses are selectively abolished by PT. It is concluded that the effects of somatostatin and carbachol to lower [Ca2+]i and to hyperpolarize are secondary to their inhibitory action on adenylate cyclase. In contrast, PT does not impair the TRH induced rise in [Ca2+]i in GH3 cells demonstrating that the coupling of TRH receptors to Ca2+ mobilization is not mediated by a PT substrate.
Asunto(s)
Toxinas Bacterianas/farmacología , Calcio/metabolismo , Citosol/metabolismo , Neoplasias Hipofisarias/metabolismo , Somatostatina/farmacología , Toxina de Adenilato Ciclasa , Adenilil Ciclasas/metabolismo , Animales , Carbacol/farmacología , Línea Celular , Toxina del Pertussis , Ratas , Hormona Liberadora de Tirotropina/farmacología , Péptido Intestinal Vasoactivo/farmacología , Factores de Virulencia de BordetellaRESUMEN
Muscarinic cholinergic agonists have been shown to inhibit PRL secretion in normal and tumor-derived pituitary cells. Evidence from experiments with the fluorescent Ca2+ probe quin 2 shows that carbachol, acting through muscarinic acetylcholine receptors, lowers the cytosolic free Ca2+ concentration ([Ca2+]i), in GH3 cells. A decrease in [Ca2+]i is observed rapidly after carbachol addition, the lowered steady state [Ca2+]i is maintained, and upon the addition of atropine [Ca2+]i returns to the initial basal value. The lowering from a basal [Ca2+]i, averaging 110 +/- 2 nM (+/- SEM, n = 9), to a steady state [Ca2+]i of 63 +/- 4 nM (+/- SEM, n = 5) at 10 micron carbachol is dose dependent, a significant decrease from basal [Ca2+]i being observed at 0.1 micron. Carbachol does not prevent TRH-induced mobilization of Ca2+ but attenuates the resulting rise in [Ca2+]i. The lowering of steady state [Ca2+]i and the attenuation of the rise in [Ca2+]i provoked by stimulators of PRL secretion could explain the inhibition of both basal and stimulated PRL secretion. Concomitantly with the action on [Ca2+]i, carbachol causes hyperpolarization of GH3 cells. Together with the established inhibition of adenylate cyclase by muscarinic cholinergic agonists, these findings suggest a relation between changes in trans-membrane Ca2+ fluxes and cAMP generation.
Asunto(s)
Calcio/metabolismo , Carbacol/farmacología , Células Clonales/metabolismo , Hipófisis/efectos de los fármacos , Neoplasias Hipofisarias/metabolismo , Animales , Atropina/farmacología , Línea Celular , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Potenciales de la Membrana , Hipófisis/metabolismo , Potasio/farmacología , Ratas , Receptores Colinérgicos/metabolismo , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
Changes in the cytosolic free Ca2+ concentration, [Ca2+]i, have been proposed to mediate the regulation of the secretion of pituitary hormones by hypothalamic peptides. Using an intracellularly trapped fluorescent Ca2+ probe, quin2, [Ca2+]i was monitored in GH3 cells. Somatostatin lowers [Ca2+]i in a dose dependent manner from a prestimulatory level of 120 +/- 4 nM (SEM, n = 13) to 78 +/- 9 nM (n = 5) at 10(-7)M; the effect is half maximal at 2 X 10(-9) M somatostatin. The decrease in [Ca2+]i occurs rapidly after somatostatin addition and a lowered steady state [Ca2+]i is maintained for several minutes. Somatostatin does not inhibit the rapid rise in [Ca2+]i elicited by thyrotropin releasing hormone (TRH) and can still cause a decrease in [Ca2+]i in the presence of TRH (10(-7)M). Concomitantly with its action on [Ca2+]i somatostatin causes hyperpolarization of GH3 cells assessed with the fluorescent probe bis-oxonol. The lowering of [Ca2+]i by somatostatin is however not only due to reduced Ca2+ influx through voltage dependent Ca2+ channels, since it persists in the presence of the channel blocker verapamil. These results suggest that somatostatin may exert its inhibitory action on pituitary hormone secretion by decreasing [Ca2+]i.