Regulation of the renal type IIa Na/Pi cotransporter by cGMP.

Inhibition of proximal tubular phosphate (Pi) reabsorption involves, as far as we know, brush border membrane retrieval of the type IIa Na/Pi-cotransporter. The aim of the present study was to analyze whether intracellular cGMP-mediated regulation of Pi reabsorption also involves retrieval of the type IIa Na/Pi-cotransporter, as previously shown for cAMP. Atrial natriuretic peptide (ANP) and nitric oxide (NO) were used to stimulate guanylate cyclase. In vivo perfusion of mice kidneys with either ANP or NO donors resulted in a downregulation of type IIa Na/Pi-cotransporters on the brush border membranes of proximal tubules. These effects were mimicked by activation of protein kinase G with 8Br-cGMP. In in-vitro-perfused mice proximal tubules, ANP was effective when added either to the apical or basolateral perfusate, suggesting the presence of receptors on both membrane sites. The effects of ANP and NO were blocked by the protein kinase G inhibitor LY 83553. Parallel experiments in OK cells, a renal proximal tubule model, provided similar information. Our findings document that cGMP-mediated regulation (ANP and NO) of type IIa Na/Pi-cotransporters also takes place via internalization of the transporter protein.

Electrophysiology of betaine transport in isolated perfused straight proximal tubule.

Betaine is accumulated in proximal renal tubular cells as an organic osmolyte. In theory, concentrative cellular uptake of betaine can be accomplished by the Na+, Cl-, betaine, GABA-cotransport system (BGT-1) cloned from MDCK cells. The carrier mediates the Na(+)-coupled electrogenic transport of organic osmolytes. Cl- may be transported together with Na+ and organic substrate but it is not an obligatory substrate. The expression of BGT is upregulated by osmotic cell shrinkage. In the present study, isolated perfused straight proximal tubules of the mouse were studied using electrophysiological techniques to elucidate the effects of betaine and gamma-aminobutyric acid (GABA) on the potential difference across the basolateral cell membrane (PDbl). Betaine and to a lesser extent GABA added to the bath led to the rapid depolarization of PDbl. The betaine-induced depolarization was virtually abolished in the absence of extracellular Na+ and blunted in the absence of extracellular Cl-. Inhibition of K+ channels by Ba2+ did not significantly modify betaine-induced depolarization. Increases of extracellular osmolarity enhanced, and decreases of extracellular osmolarity blunted, the betaine-induced depolarization. In conclusion, betaine transport across the basolateral cell membrane of straight proximal tubules from the mouse kidney is similar to the transport by BGT in terms of Na+ and Cl- sensitivity but not the apparent affinity for GABA. The sensitivity to ambient osmolarity indicates that betaine transport in proximal tubules is regulated rapidly and nongenomically.

Luminal and contraluminal action of 1-34 and 3-34 PTH peptides on renal type IIa Na-P(i) cotransporter.

Parathyroid hormone (PTH) inhibits proximal tubular reabsorption of P(i) by retrieval of type IIa Na-P(i) cotransporters (NaPi-IIa) from the brush-border membrane (BBM). We analyzed by immunohistochemistry whether PTH analogs, signaling through either protein kinase A (PKA) and C (PKC; 1-34 PTH) or only PKC (3-34 PTH), elicit in rat kidney in vivo or in the perfused murine proximal tubule in vitro a retrieval of NaPi-IIa and whether pharmacological agonists or inhibitors of these kinases are able to either mimic or interfere with these PTH effects. Treatment with either 1-34 or 3-34 PTH downregulated NaPi-IIa in rat kidney. In isolated murine proximal tubules 1-34 PTH was effective when added to either the apical or basolateral perfusate, whereas 3-34 PTH acted only via the luminal perfusate. These effects were mimicked by an activation of PKA with 8-bromoadenosine 3',5'-cyclic monophosphate or PKC with 1, 2-dioctanoylglycerol. The luminal action of both PTH peptides was blocked by inhibition of the PKC pathway (calphostin C), whereas the basolateral effect of 1-34 PTH was completely abolished by inhibiting both pathways (H-89 and calphostin C). These results suggest that 1) NaPi-IIa can be internalized by cAMP-dependent and -independent signaling mechanisms; 2) functional PTH receptors are located in both membrane domains; and 3) apical PTH receptors may preferentially initiate the effect through a PKC-dependent mechanism.

The diversity of volume regulatory mechanisms.

Mammalian cells utilize a wide variety of cell volume regulatory mechanisms. For rapid adjustment of cell volume cells release or accumulate ions through respective channels and transport systems across the cell membrane. The most widely used mechanisms of cell volume regulatory ion release include ion channels and KCl symport. Ion uptake is most frequently mediated by Na+ channels, Na+, K+, 2Cl- cotransport, and Na+/H+ exchange. Chronic adjustment of cell osmolarity is accomplished by the formation or accumulation of organic osmolytes, molecules specifically designed to create intracellular osmolarity without interfering with cellular function. The most widely occurring osmolytes are sorbitol, inositol, glycerophosphorylcholine, betaine, taurine, and amino acids. The osmolytes are either synthesized by or transported into shrunken cells. During cell swelling osmolytes can be rapidly degraded or released. Any given cell may utilize several volume-regulatory mechanisms. Moreover, different mechanisms are utilized in different tissues. The diversity of cell volume regulatory mechanisms allows the cells to defend the constancy of cell volume against a myriad of challenges with relatively little impairment of cellular function.

Dynamics of surfactant release in alveolar type II cells.

Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, fluorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1-43 resulted in bright fluorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1-43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca2+]i, but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.

Functional significance of cell volume regulatory mechanisms.

To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.

Nitric oxide donors inhibit spontaneous depolarizations by L-type Ca2+ currents in alveolar epithelial cells.

L2 cells, a cloned pneumocyte-derived cell line, express voltage-dependent L-type Ca2+ channels, causing transient depolarizing spikes of the membrane potential (Vm) [P. Dietl, T. Haller, B. Wirleitner, H. Völkl, F. Friedrich, and J. Striessing. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L873-L883, 1995]. In this study, we examined the effect of nitric oxide (NO)- and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent cell signaling on the activity of L-type Ca2+ channels. Using conventional microelectrodes, spontaneous depolarizations (SD) of Vm by activation of these channels are regularly seen in the presence of 10 mM bath Sr2+. The NO donors sodium nitroprusside (SNP; 1 mM), 3-morpholinosydnonimine (SIN-1; 100 microM), as well as S-nitroso-N-acetyl-D,L-penicillamine (SNAP; 10 microM) caused a significant reduction of the frequency of Sr(2+)-induced SD. These effects were completely reversed by 6-anilino-5,8-quinolinequinone (10 microM), an inhibitor of the soluble guanylyl cyclase, and could be mimicked by 8-bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP; 100 microM). Perforated patch-clamp experiments revealed that 8-BrcGMP exerted a significant decrease of the depolarization-induced L-type Sr2+ current in the majority of tested cells. Consistent with the dependency of these NO-mediated effects on cGMP, incubation of L2 cells with SNP, SIN-1, and SNAP lead to a pronounced increase of cellular cGMP concentration. We conclude that the NO donors inhibit the activity of L-type Ca2+ channels in L2 cells via a cGMP-dependent pathway. In the alveoli, this might occur under conditions associated with the release of NO.

Neopterin and 7,8-dihydroneopterin induce apoptosis in the rat alveolar epithelial cell line L2.

The neopterin derivatives, neopterin and 7,8-dihydroneopterin, modulate the cellular oxidant-antioxidant balance as well as the expression of the inducible nitric oxide synthase (iNOS) gene. Since apoptosis can be induced by reactive oxygen intermediates and nitric oxide (NO) we investigated whether these neopterin derivatives induce apoptotic cell death. As model we selected the rat alveolar epithelial cell line L2. 24 h incubation of neopterin (1-1000 microM) as well as 7,8-dihydroneopterin (1-1000 microM) resulted in a significant increase of percent apoptotic cells (measured by FACS analysis). Coincubation of both pteridines with the cytomix (interferon-gamma plus tumor necrosis factor-alpha) led to a significantly higher apoptosis than the cytomix alone. In contrast to the cytomix, no iNOS gene expression and no NO release could be detected after incubation with neopterin or 7,8-dihydroneopterin. We conclude that neopterin and 7,8-dihydroneopterin are per se inducers of apoptosis which is not mediated by nitric oxide. This may be of importance in inflammatory pulmonary diseases associated with an activation of the cellular immune system.

The lysosomal Ca2+ pool in MDCK cells can be released by ins(1,4,5)P3-dependent hormones or thapsigargin but does not activate store-operated Ca2+ entry.

In several cell types, Ca2+ release from intracellular Ca2+ stores by Ins(1,4,5)P3 elicits Ca2+ influx from the extracellular space into the cytoplasm, termed store-operated Ca2+ entry (SOCE). In MDCK cells, the Ins(1,4,5)P3-sensitive Ca2+ store giving rise to SOCE essentially overlaps with the thapsigargin (TG)-sensitive store. Recent evidence suggests that in MDCK cells lysosomes form a Ca2+ pool that is functionally coupled with the Ins(1,4,5)P3-sensitive Ca2+ store: Ca2+ can be selectively released from lysosomes by glycyl-L-phenylalanine naphthylamide, an agent inducing lysosomal swelling with subsequent and reversible permeabilization of the vesicular membranes. This compartment is also depleted by Ins(1,4,5)P3-dependent agonists or TG, indicating that it is part of a larger, Ins(1,4,5)P3-sensitive Ca2+ pool. Here we show that whereas SOCE is triggered by Ca2+ release from the entire Ins(1,4,5)P3-sensitive Ca2+ pool, selective Ca2+ release from lysosomes alone is unable to trigger SOCE. This finding is consistent with measurements of the store-operated cation current, a direct parameter for store-operated Ca2+ and Na+ entry into MDCK cells. Hence it is proposed that the Ins(1,4,5)P3-sensitive Ca2+ pool is composed of different intracellular compartments that do not uniformly stimulate Ca2+ entry into the cell.

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release.

To test for a possible role of lysosomes in intracellular Ca2+ homeostasis, the effects of glycyl-L-phenylalanine-beta-naphthylamide (GPN), known to permeabilize these organelles by osmotic swelling, were studied in single MDCK cells. Fluorescence of acridine orange, rhodol green dextran, lysotracker green and FITC-dextran indicated that GPN (0.2 mmol/l) elicited a reversible permeabilization of lysosomes. Cytosolic Ca2+ ([Ca2+]i) as determined by Fura-2 fluorescence increased from 60 +/- 11 to 534 +/- 66 nmol/l (n = 41) in the presence of GPN. Whereas only a single intracellular Ca2+ release could be induced by GPN in a Ca(2+)-free perfusate, repetitive release could be evoked in Ca2+ containing solutions suggesting reuptake of Ca2+ into lysosomal stores. GPN-induced Ca2+ release was blunted after pretreatment with thapsigargin (TG), an inhibitor of Ca(2+)-ATPase, or repeated applications of ATP inducing Ca2+ release from inositol trisphosphate (InsP3) sensitive Ca2+ stores. The effect of ATP on Ca2+ release was, however, not abolished by preceding GPN treatment. GPN-induced Ca2+ release from lysosomes was independent of InsP3 formation or Ca(2+)-induced Ca2+ release, since it was unaffected by the phospholipase C inhibitor U-73, 122 or by caffeine and ruthenium red. These results suggest that Ca2+ largely accumulates in lysosomal vesicles. Moreover, these organelles seem to be part or functionally coupled with InsP3-sensitive Ca2+ stores.

Calcium entry stimulated by swelling of Madin-Darby canine kidney cells.

Cell swelling in Madin-Darby canine kidney (MDCK) cells by reduction of extracellular osmolarity (omission of 70 ad 150 mmol/l mannitol, respectively) leads to the activation of anion of channels and Ca2+ sensitive K+ channels. The K+ channel activation leads to an initial transient hyperpolarization of the cell membrane potential (PD) followed by a sustained depolarization due to activation of anion channels. The present study elucidates the role of intracellular calcium (Ca2+i) in regulatory cell volume decrease (RVD) of MDCK cells. While reduction of extracellular osmolarity by omitting 70 mmol/l mannitol did not lead to a detectable change in Ca2+i, severe cell swelling by omitting 150 mmol/l mannitol led to a transient rise in Ca2+i. PD changes, on the other hand, were not different under either condition. In addition, the response of PD to cell swelling was not altered by treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate diester, pertussis toxin or cholera toxin. In the nominal absence of extracellular Ca2+, reduction of extracellular osmolarity did not lead to an increase in Ca2+i and no initial transient hyperpolarization was observed, whereas addition of 10 mumol/l ATP still led to a significant hyperpolarization. Omission of extracellular Ca2+ was followed by a strong decrease in cell membrane resistance (Rm) due to activation of a depolarizing cation conductance. Subsequent readdition of Ca2+ caused a marked increase in Ca2+i due to Ca2+ influx. This Ca2+ entry was further stimulated by cell swelling. RVD was significantly blunted in the absence of extracellular Ca2+. The results suggest that cell swelling stimulates a Ca2+-permeable pathway in the cell membrane favoring Ca2+ entry into the cell with subsequent activation of Ca2+-sensitive K+ channels.

Activation of L-type Ca2+ channels after purinoceptor stimulation by ATP in an alveolar epithelial cell (L2).

In the alveolar epithelium, ATP increases the intracellular Ca2+ concentration ([Ca2+]i) and stimulates the secretion of surfactant. We investigated the effects of extracellular ATP on the membrane potential (Vm), the whole cell current, and [Ca2+]i in a cloned rat alveolar epithelial cell line (L2). In microelectrode experiments, ATP caused a sustained depolarization of Vm, resulting from the activation of cation and Cl- conductances, as revealed by ion replacements. The depolarizing phase of the Vm shift was superimposed by Ca(2+)-dependent depolarizing spikes. Spikes were also induced by depolarizing Vm with charybdotoxin or maitotoxin. Replacement of bath Ca2+ with Ba2+ or Sr2+ also evoked repetitive spikes. Ca2+ (Ba2+, Sr2+)-induced spikes were unaffected by pretreatment with ionomycin or thapsigargin. They were, however, completely abolished by (+)-isradipine (100 nM) and stimulated by BAY K 8644 (100 nM). Whole cell L-type Ca2+ (Ba2+, Sr2+) currents were similarly abolished by (+)-isradipine and enhanced by BAY K 8644. L-type Ca2+ channels were further confirmed by demonstrating high-affinity dihydropyridine receptors stereoselectively labeled by (+)-[3H]-isradipine, apparent dissociation constant < 1 nM. In fura 2 experiments, ATP evoked a transient elevation of [Ca2+]i in the absence of Ca2+ and a biphasic sustained elevation in the presence of Ca2+, indicating intracellular Ca2+ release and Ca2+ entry. The ATP-induced fura 2 signals were unaffected by (+)-isradipine. We conclude that in L2 cells, L-type Ca2+ channels are activated after purinoceptor stimulation by ATP. The overall [Ca2+]i response is, however, mediated by Ca2+ entry through and (+)-isradipine-insensitive mechanism and by intracellular Ca2+ release.

Effect of amiloride and emopamil on intracranial pressure.

The authors sought to determine whether amiloride or emopamil could reduce intracranial pressure in experimental brain edema of the rat. For this purpose the rats functionally nephrectomized and brain edema of the cytotoxic type induced by infusion of 100 ml aqua bidest/kg body weight. After the end of the infusion 10 or 20 ml mM amiloride/kg body weight or 50 microliters mM (s)-emopamil/kg body weight in 10 ml 150 mM NaCl/kg body weight or 10 ml isotonic saline/kg body weight were injected followed by continued recording of intracranial pressure (ICP) and systemic arterial pressure for at least 3 hours. The values of the ICP for the amiloride and s-emopamil treated animals are significantly (p < 0.05, Student's t-test for unpaired data) lower at any point after the injection of amiloride or (s)-emopamil. Amiloride and (s)-emopamil prevent the rise in ICP seen after the saline injection in the control group.

Involvement of microtubules in the link between cell volume and pH of acidic cellular compartments in rat and human hepatocytes.

Cell swelling is shown to induce an increase in acridine orange fluorescence intensity, an effect pointing to the alkalinization of acidic vesicles. Since autophagic hepatic proteolysis is accomplished by pH-sensitive proteinases within acidic lysosomes, this effect may contribute to the well-known inhibitory effect of cell swelling on proteolysis. In the present study, the role of microtubules in volume-dependent alterations of pH in acidic vesicles of rat and human hepatocytes was studied. Colcemid and colchicine were used to depolymerize microtubules and vesicular pH was monitored using two different fluorescent dyes, fluorescein isothiocyanate conjugated-dextran and acridine orange. Colcemid and colchicine, but not the inactive stereoisomer gamma-lumicolchicine, blunted the increase of pH during osmotic cell swelling. The alkalinization of acidic vesicles by NH4Cl was not significantly modified by colcemid or colchicine, indicating that the vesicles were still sensitive to alkalinizing procedures other than cell swelling. Further, colchicine, but not gamma-lumicolchicine, inhibited the antiproteolytic action of osmotic cell swelling. The present observations point to an involvement of the microtubule network in the link of cell volume, lysosomal pH, and proteolysis.

Maitotoxin activates a nonselective cation channel and stimulates Ca2+ entry in MDCK renal epithelial cells.

We examined the mechanisms of maitotoxin (MTX), a water-soluble polyether from the marine dinoflagellate Gambierdiscus toxicus, in stimulation of Ca2+ entry into Mardin-Darby canine kidney cells. In the presence of bath Ca2+, MTX (3 nM) caused an elevation of the intracellular calcium concentration ([Ca2+]i), which was partially inhibited by SK&F 96365 (25 microM) or La3+ (100 microM). A stimulation of Ca(2+)-dependent K+ channels in cell-attached membrane patches coincided with this rise in [Ca2+]i and was also partially inhibited by SK&F 96365. Before the rise in [Ca2+]i, a nonselective cation current (Ins), studied by the whole-cell patch-clamp technique, was irreversibly activated. Ins poorly discriminated between Na+, K+, and Cs+, was unaffected by replacement of Cl- with gluconate-, and was not voltage gated. MTX-induced Ins was partially blocked by La3+ ions (100 microM) but not by SK&F 96365 (25 microM) or nifedipine (10 microM). SK&F 96365 by itself induced a small but significant stimulation of Ins and a rise in [Ca2+]i. The activation of Ins by MTX was instantaneous and depended on the presence of extracellular Ca2+ ions. In the absence of other cations, the inward current of Ins was dependent on the bath Ca2+ concentration. Cell-attached and excised single-channel measurements revealed that MTX activated a SK&F 96365-insensitive, approximately 40-pS, nonselective cation channel from the outside. We conclude that the initial action of MTX is the stimulation of a nonselective cation channel, which requires the presence of extracellular Ca2+ ions. The subsequent rise in [Ca2+]i is at least in part caused by another, SK&F 96365-sensitive, Ca2+ entry pathway, which may be activated as a result of or independently of Ins.

Regulation of proximal renal tubular K+ conductance by intracellular pH.

Conventional electrophysiology and 2', 7'-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein fluorescence have been applied to elucidate the effects of metabolic acidosis on straight proximal tubules of the mouse kidney. Reduction of extracellular bicarbonate concentration from 20 to 10 mmol/l leads to a decline of intracellular pH from 7.00 +/- 0.06 to 6.85 +/- 0.05, a depolarization of the cell membrane (PDbl) from -72 +/- 1 to -59 +/- 2 mV, a decrease of the basolateral transference number for potassium (tK) from 0.80 +/- 0.01 to 0.54 +/- 0.03, an increase of the basolateral transference number for bicarbonate (tb) from 0.16 +/- 0.02 to 0.42 +/- 0.03 and an increase of the fractional resistance of the basolateral over the luminal cell membrane (Rb/Ra) by 64 +/- 8%. Upon return to 20 mmol/l bicarbonate after a 5-min exposure to 10 mmol/l bicarbonate, the intracellular pH approached a more alkaline value (7.28 +/- 0.08) than before exposure to acidosis. Despite the intracellular alkalosis, PDbl (-67 +/- 1 mV) and tK (0.73 +/- 0.02) remained significantly below, and tb (0.26 +/- 0.02) and Rb/Ra (32 +/- 8%) significantly above the respective values before induction of acidosis. Even transient exposure of the tubules to 40 mmol/l extracellular bicarbonate did not restore the original electrophysiological properties of the tubule cells. It is concluded that both a rapidly reversible and a long-lasting decrease of proximal tubular K+ conductance follows cellular acidosis.

Alkalinization of acidic cellular compartments following cell swelling.

Osmotic swelling of rat hepatocytes increases fluorescence of Acridine orange and of fluorescein isothiocyanate (FITC)-dextran, both indicative of alkalinization of acidic intracellular vesicles. Similar to osmotic cell swelling, insulin and glutamine lead to an increase in Acridine orange fluorescence, an effect virtually abolished upon osmotic reversal of glutamine-induced cell swelling. Barium, which blocks K+ channels in the plasma membrane, similarly leads to cell swelling and increase of Acridine orange fluorescence. Since proteolysis is governed by lysosomal pH, these observations indicate that the anti-proteolytic action of osmotic cell swelling is mediated by lysosomal alkalinization. Thereby, insulin, glutamine and barium probably exert their anti-proteolytic action by cell swelling and subsequent lysosomal alkalinization.