Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996     

Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C

The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca⁺⁺ ([Ca⁺⁺] _bl ), and closing them by returning [Ca⁺⁺] _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca⁺⁺] _bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca⁺⁺ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca⁺⁺ being necessary to allow TJ recovery. A step rise of apical Ca⁺⁺ concentration ([Ca⁺⁺] _ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca⁺⁺ entering the open TJs. The specific condition of having Ca⁺⁺ only in the apical solution and the TJs located midway between the Ca⁺⁺ source (apical solution) and the Ca⁺⁺-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca⁺⁺] _ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca⁺⁺. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca⁺⁺ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca⁺⁺ removal but induces a prompt blockade of TJ oscillations induced by apical Ca⁺⁺, oscillations which reappear again when H7 is removed. Received: 9 May 2000/Revised: 30 August 2000     

Regulation of Tight Junction Resistance in T84 Monolayers by Elevation in Intracellular Ca2+: A Protein Kinase C Effect

Elevation in intracellular Ca²⁺ acting via protein kinase C (PKC) is shown to regulate tight junction resistance in T₈₄ cells, a human colon cancer line and a model Cl⁻ secretory epithelial cell. The Ca²⁺ ionophore A23187, which was used to increase the intracellular Ca²⁺ concentration, caused a decrease in tight junction resistance in a concentration- and time-dependent manner. Dual Na⁺/mannitol serosal-to-mucosal flux analysis performed across the T₈₄ monolayers treated with 2 μm A23187 revealed that A23187 increased both fluxes and that in the presence of ionophore there was a linear relationship between the Na⁺ and mannitol fluxes with a slope of 56.4, indicating that the decrease in transepithelial resistance was due to a decrease in tight junction resistance. Whereas there was no effect of 0.1 μm A23187, 1 or 2 μm produced a 55% decrease in baseline resistance in 1 hr and 10 μm decreased resistance more than 80%. The A23187-induced decrease in tight junction resistance was partially reversible by washing 3 times with a Ringer's-HCO₃ solution containing 1% BSA. The A23187 effect on resistance was dependent on intracellular Ca²⁺; loading the T₈₄ cells with the intracellular Ca²⁺ chelator BAPTA significantly reduced the decrease in tight junction resistance caused by A23187. This intracellular Ca²⁺ effect was mediated by protein kinase C and not calmodulin. While the protein kinase C antagonist H-7 totally prevented the action of A23187 on tight junction resistance, the Ca²⁺/calmodulin inhibitor W13 did not have any effect. Sphingosine, another inhibitor of PKC, partially reduced the A23187-induced decline in tight junction resistance. The PKC agonist PMA mimicked the A23187 effect on resistance, although the effect was delayed up to 1 hr after exposure. In addition, however, PMA also caused an earlier increase in resistance, indicating it had an additional effect in addition to mimicking the effect of elevating Ca²⁺. The effects of a phospholipase inhibitor (mepacrine) and of inhibitors of arachidonic acid metabolism (indomethacin for the cyclooxygenase pathway, NDGA for the lipoxygenase pathway, and SKF 525A for the epoxygenase pathway) on the A23187 action were also examined. None of these agents altered the A23187-induced decrease in resistance. Monolayers exposed to 2 μm A23187 for 1 hr were stained with fluorescein conjugated phalloidin, revealing that neighboring cells did not part one from another and that A23187 did not have a detectable effect on distribution of F-actin in the perijunctional actomyosin ring. The results indicate that elevation in intracellular Ca²⁺ decreases tight junction resistance in the T₈₄ monolayer, acting through protein kinase C by a mechanism which does not involve visible changes in the perijunctional actomyosin ring. Received: 14 July 1995/Revised: 25 September 1995     

Effects of Hyperglycemia and Protein Kinase C on Connexin43 Expression in Cultured Rat Retinal Pigment Epithelial Cells

Previous results demonstrated that the intercellular communication mediated by gap junctions in retinal pigment epithelial (RPE) cells from the healthy Long Evans (LE) rat strain is higher than that from the dystrophic Royal College of Surgeons (RCS) rat strain. We examined connexin (Cx) expression in both cell types. At the mRNA level, a qualitatively similar expression pattern was found whereby Cx26, Cx32, Cx36, Cx43, Cx45 and Cx46 were all expressed. At the protein level, only Cx43 and Cx46 were detected. Expression of both isoforms was higher in LE-RPE as compared to RCS-RPE by a factor of 1.25 and 2 respectively. Phosphorylation of Cx43 was increased upon activation of protein kinase C (PKC) by 1 μM phorbol 12-myristate 13-acetate (PMA). The phosphorylation status was not changed in hyperglycemic conditions, but this treatment strongly decreased total Cx43 levels to about 75 and 40% (in LE-RPE and RCS-RPE cells respectively) of the control level in LE-RPE cells. This decrease could be overcome by PKC downregulation. These results demonstrate that PKC activation and hyperglycemic conditions have different effects on Cx43 and that PKC is involved in the metabolic pathway induced by hyperglycemic conditions. Received: 21 July 2000/Revised: 19 January 2001     

Lithium and Protein Kinase C Modulators Regulate Swelling-Activated K-Cl Cotransport and Reveal a Complete Phosphatidylinositol Cycle in Low K Sheep Erythrocytes

K-Cl cotransport (COT), a ouabain-insensitive, Cl-dependent bidirectional K flux, is ubiquitously present in all cells, plays a major role in ion and volume homeostasis, and is activated by cell swelling and a variety of chemical interventions. Lithium modulates several cation transport pathways and inhibits phospholipid turnover in red blood cells (RBCs). Lithium also inhibits K-Cl COT by an unknown mechanism. To test the hypothesis whereby Li inhibits swelling-activated K-Cl COT by altering either its osmotic response, its regulation, or by competing with K for binding sites, low K (LK) sheep (S) RBCs were loaded with Li by Na/Li exchange or the cation ionophore nystatin. K-Cl COT was measured as the Cl-dependent, ouabain-insensitive K efflux or Rb influx. The results show that Li altered the cell morphology, and increased both cell volume and diameter. Internal (Li _i ) but not external (Li _o ) Li inhibited swelling-activated K-Cl COT by 85% with an apparent K _i of ∼7 mm. In Cl, Li _i decreased K efflux at relative cell volumes between 0.9 and 1.2, and at external pHs between 7.2 and 7.4. Li _i reduced the V _max and increased the K _m for K efflux in Cl. Furthermore, Li _i increased the production of diacylglycerol in a bimodal fashion, without significant effects on the phosphatidylinositol concentration, and revealed the presence of a complete PI cycle in LK SRBCs. Finally, phorbol ester treatment and PD89059, an inhibitor of mitogen-activated protein kinase (ERK2) kinase, caused a time-dependent inhibition of K-Cl COT. Hence, Li _i appears to inhibit K-Cl COT by acting at an allosteric site on the transporter or its putative regulators, and by modulation of the cellular phospholipid metabolism and a PKC-dependent regulatory pathway, causes an altered response of K-Cl COT to pH and volume. Received: 1 November 1999/Revised: 6 June 2000     

Phylogenetic Position of the Hexactinellida Within the Phylum Porifera Based on the Amino Acid Sequence of the Protein Kinase C from Rhabdocalyptus dawsoni

Recent analyses of genes encoding proteins typical for multicellularity, especially adhesion molecules and receptors, favor the conclusion that all metazoan phyla, including the phylum Porifera (sponges), are of monophyletic origin. However, none of these data includes cDNA encoding a protein from the sponge class Hexactinellida. We have now isolated and characterized the cDNA encoding a protein kinase C, belonging to the C subfamily (cPKC), from the hexactinellid sponge Rhabdocalyptus dawsoni. The two conserved regions, the regulatory part with the pseudosubstrate site, the two zinc fingers, and the C2 domain, as well as the catalytic domain were used for phylogenetic analyses. Sequence alignment and construction of a phylogenetic tree from the catalytic domains revealed that the yeast Saccharomyces cerevisiae and the protozoan Trypanosoma brucei are at the base of the tree, while the hexactinellid R. dawsoni branches off first among the metazoan sequences; the other two classes of the Porifera, the Calcarea (the sequence from Sycon raphanus was used) and the Demospongiae (sequences from Geodia cydonium and Suberites domuncula were used), branch off later. The statistically robust tree also shows that the two cPKC sequences from the higher invertebrates Drosophila melanogaster and Lytechinus pictus are most closely related to the calcareous sponge. This finding was also confirmed by comparing the regulatory part of the kinase gene. We suggest, that (i) within the phylum Porifera, the class Hexactinellida diverged first from a common ancestor to the Calcarea and the Demospongiae, which both appeared later, and (ii) the higher invertebrates are more closely related to the calcareous sponges. Received: 6 August 1997 / Accepted: 24 October 1997     

Short-Term Regulation of the Proximal Tubule Na+,K+-ATPase: Increased/Decreased Na+,K+-ATPase Activity Mediated by Protein Kinase C Isoforms

In different species and tissues, a great variety of hormones modulate Na⁺,K⁺-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na⁺,K⁺-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na⁺,K⁺-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na⁺,K⁺-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.     

Regulation of Epithelial Na+ Permeability by Protein Kinase C is Tissue Specific

Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to stimulate Na⁺ transport across frog skin epithelium by increasing the apical Na⁺ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC. In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species. The PKC activator TPA uniformly increased the transepithelial Na⁺ transport (measured as amiloride-sensitive short-circuit current, I _SC, across skins from Rana temporaria and Bufo marinus, and inhibited I _SC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced stimulation of I _SC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone. We conclude that the effect of PKC on epithelial Na⁺ channels is organ, and not species specific. The stimulation of Na⁺ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme of PKC previously detected in frog skin. Received: 19 January 1996/Revised: 10 April 1996     

Protein Kinase A Activation Phosphorylates the Rat ClC-2 Cl− Channel but Does Not Change Activity

Phosphorylation-dependent events have been shown to modulate the activity of several members of the mammalian CLC Cl⁻ channel gene family, including the inward rectifier ClC-2. In the present study we investigated the regulation of rat ClC-2 expressed in the TSA-201 cell line (a transformed HEK293 cell line that stably expresses the SV40 T-antigen) by protein kinases. Protein kinase A activation phosphorylated ClC-2 in vivo, whereas stimulation of protein kinase C with phorbol 12-myristate 13-acetate did not. In vitro labeling studies confirmed that protein kinase A could directly phosphorylate ClC-2, and that protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II did not. Nevertheless, protein kinase A-dependent phosphorylation of CLC-2 failed to regulate either the magnitude or the kinetics of the hyperpolarization-activated Cl⁻ currents. Considered together, we demonstrate that protein kinase A activation results in the phosphorylation of rat ClC-2 in vivo, but this event is independent of Cl⁻ channel activity. Received: 20 November 2000/Revised: 28 March 2001     

Regulation of Intestinal Epithelial Brush Border Na+/H+ Exchanger Isoforms, NHE2 and NHE3, in C2bbe Cells

Until recently, studies to characterize the intestinal epithelial Na⁺/H⁺ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na⁺/H⁺ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure NHE activity. Because changes in pH _i only approximate Na⁺/H⁺ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation, we have used ²²[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition by cAMP and Ca⁺⁺ involves changes to both K _Na and V _max . In contrast, the same two second messengers inhibit NHE3 by a decrease in V _max exclusively. Phorbol ester activation of protein kinase C alters both V _max and K _Na of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active and are differentially regulated by signal transduction pathways. Received: 28 January 1999/Revised: 18 May 1999     

Protein Kinase Inhibitors and the Dynamics of Tight Junction Opening and Closing in A6 Cell Monolayers

This study focuses, in A6 cell monolayers, on the role of protein kinases in the dynamics of tight junction (TJ) opening and closing. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺-switch assay (FCSA), which consisted of opening the TJs by removing basolateral Ca⁺⁺ (Ca⁺⁺ _bl), and closing them by returning Ca⁺⁺ _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the evaluation of the effects of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in response to the FCSA followed single-exponential time courses. A rise of apical Ca⁺⁺ (Ca⁺⁺ _ap) causes a reduction of TJ opening rate in an FCSA or even a partial recuperation of G, an effect that is interpreted as mediated by Ca⁺⁺ _ap entering the open TJs. Protein kinase C (PKC) inhibition by H7 at low concentrations caused a reduction of the rate of junction opening in response to Ca⁺⁺ _bl removal, without affecting junction closing, indicating that PKC in this preparation is a key element in the control of TJ opening dynamics. H7 at 100 μm completely inhibits TJ opening in response to Ca⁺⁺ _bl withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase, a process that can be halted again by H7 reintroduction into the bathing solution. Differently from the condition in which Ca⁺⁺ is absent from the apical solution, in which H7 halts the process of G increase in response to a FCSA, when Ca⁺⁺ is present in the apical solution, addition of H7 during G increase in an FCSA not only induces a halt of the G increase but causes a marked recuperation of the TJ seal, indicated by a drop of G, suggesting a cooperative effect of Ca⁺⁺ and H7 on the TJ sealing process. Staurosporine, another PKC inhibitor, differently from H7, slowed both G increase and G decrease in an FCSA. Even at high concentrations (400 nm) staurosporine did not completely block the effect of Ca⁺⁺ withdrawal. These discrepancies between H7 and staurosporine might result from distinct PKC isoforms participating in different steps of TJ dynamics, which might be differently affected by these inhibitors. Immunolocalizations of TJ proteins, carried out in conditions similar to the electrophysiological experiments, show a very nice correlation between ZO-1 and claudin-1 localizations and G alterations induced by Ca⁺⁺ removal from the basolateral solution, both in the absence and presence of H7. Received: 18 April 2001/Revised: 16 July 2001     

Phosphorylation is Involved in the Regulation of the Taurine Influx Via the β-system in Ehrlich Ascites Tumor Cells

The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by an increased level of cAMP or following addition of N⁶,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially stimulates taurine transport when the β-system is in State II. Received: 8 September/Revised: 9 November 1995     

Role of Protein Phosphatase in the Regulation of Na+-K+-ATPase by Vasopressin in the Cortical Collecting Duct

In the cortical collecting duct (CCD), arginin vasopressin (AVP) has been shown to increase the number and activity of basolateral Na⁺-K⁺-ATPase by recruiting or activating a latent pool of pumps. However, the precise mechanism of this phenomenon is still unknown. The aim of this study was to investigate whether this AVP-induced increase in basolateral Na⁺-K⁺-ATPase could depend on a dephosphorylation process. To this purpose, the effect of protein serine/threonine phosphatase (PP) inhibitors was examined on both the specific ³H-ouabain binding (to evaluate the number of pumps in the basolateral membrane) and the ouabain-dependent ⁸⁶Rb uptake (to evaluate pump functionality) in the presence or absence of AVP. In addition, the activity of two PP, PP1 and PP2A, was measured and the influence of AVP was examined on both enzymes. Experiments have been performed on mouse CCD isolated by microdissection. Results show that inhibition of PP2A prevents the AVP-induced increase in the number and activity of Na⁺-K⁺-ATPases, independent of an effect on the apical cell sodium entry. In addition, AVP rapidly increased the activity of PP2A without effect on PP1. These data suggest that PP2A is implied in the regulation of Na⁺-K⁺-ATPase activity by AVP in the CCD and that the AVP-dependent increase in the number of Na⁺-K⁺-ATPases is mediated by a PP2A-dependent dephosphorylation process. Received: 22 March 1996/Revised: 21 June 1996     

Protein Kinase C and Regulatory Volume Decrease in Mudpuppy Red Blood Cells

This study examined whether protein kinase C (PKC) stimulates K⁺ efflux during regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The limit of osmotic fragility increased with the general protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 μm), but not with the cyclic nucleotide-dependent kinase antagonists N-(2′-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 10 μm) and N-2-(methylamino)ethyl-5-isoquinoline-sulfonamide (H-8, 5 μm). Consistent with these results, osmotic fragility also increased with the PKC antagonists bisindolylmaleimide I (GF-109203X or bis I, 100 nm), bisindolylmaleimide II (bis II, 100 nm), and chelerythrine (10 μm). The effect of these three antagonists and H-7 was reversed with gramicidin (5 μm in a choline Ringer), indicating PKC was linked to K⁺ efflux (gramicidin is a cationophore that was used to ensure a high K⁺ permeability). We also measured cell volume recovery from hypotonic shock (0.5× Ringer) with a Coulter counter and estimated cell volume from the hematocrit. The percent RVD compared to control decreased with H-7 (10 μm), sphingosine (100 nm), chelerythrine (10 μm), bis I (100 nm), and bis II (100 nm), but not with HA-1004 (10 μm) nor H-8 (5 μm). Inhibition of RVD by H-7, chelerythrine, bis I, and bis II was reversed with gramicidin (5 μm). Furthermore, using the patch clamp technique, we found H-7 (10 μm) reduced a whole cell conductance that was activated during cell swelling. In addition, a conductance responsible for K⁺ efflux during cell swelling was inhibited by bis I (100 nm) and bis II (100 nm). These results indicate that a conductive pathway mediating K⁺ loss during RVD is regulated, at least in part, by protein kinase C. Received: 20 January 1998/Revised: 2 September 1998     

Regulation of Renal Na-HCO3 Cotransporter: VIII. Mechanism of Stimulatory Effect of Respiratory Acidosis

We examined the effect of respiratory acidosis on the Na-HCO₃ cotransporter activity in primary cultures of the proximal tubule of the rabbit exposed to 10% CO₂ for 5 min, 2, 4, 24 and 48 hr. Cells exposed to 10% CO₂ showed a significant increase in Na-HCO₃ cotransporter activity (expressed as % of control levels, 5 min: 142 ± 6, 2 hr: 144 ± 13, 4 hr: 145 ± 11, 24 hr: 150 ± 15, 48 hr: 162 ± 24). The increase in activity was reversible after 48 hr. The role of protein kinase C (PKC) on the stimulatory effect of respiratory acidosis on the cotransporter was examined in presence of PKC inhibitor calphostin C or in presence of PKC depletion. Both calphostin C and PKC depletion prevented the effect of 10% CO₂ for 5 min or 4 hr to increase the activity of the cotransporter. 10% CO₂ for 5 min or 4 hr increased total and particulate fraction PKC activity. To examine the role of phosphotyrosine kinase (PTK) on the increase in cotransporter activity we studied the effect of two different inhibitors, 2-hydroxy-5-(2,5-dihydroxylbenzyl) aminobenzoic acid (HAC) and methyl 2,5-dihydroxycinnamate (DHC) which inhibit phosphotyrosine kinase in basolateral membranes. Cells were pretreated either with vehicle or HAC or DHC and then exposed to 10% CO₂ for 5 min or 4 hr. In cells treated with vehicle, 10% CO₂ significantly increased cotransporter activity as compared to control cells exposed to 5% CO₂. This stimulation by 10% CO₂ was completely prevented by HAC or DHC at 5 min (5% CO₂: 1.8 ± 0.2, 10% CO₂: 2.6 ± 0.2, 10% CO₂+ HAC: 1.6 ± 0.2, 10% CO₂: +DHC: 2.0 ± 0.3 pH unit/min) and also at 4 hr. The protein synthesis inhibitors actinomycin D and cycloheximide appear to prevent the effect of 10% CO₂ for 4 hr on the cotransporter. Our results show that early respiratory acidosis stimulates the Na-HCO₃ cotransporter through PKC and PTK-dependent mechanisms and the late effect appears to be mediated through protein synthesis. Received: 28 March 1997/Revised: 22 December 1997     

Role of Calmodulin and Protein Kinase C in Astrocytic Cell Volume Regulation

We investigated the role of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume. Calmodulin (CaM) antagonists were used to inhibit CaM and thus Ca2+/CaM-dependent protein kinase. The effect of these inhibitors as well as activators and inhibitors of protein kinase C (PKC) on astrocytic volume was measured in response to hypoosmotic stress and under isoosmotic conditions. In conditions of hypoosmolarity, CaM antagonists had no effect on swelling, but inhibited the regulatory volume decrease. PKC activation facilitated the swelling induced by hypoosmotic stress. PKC inhibitors induced cell shrinkage and inhibited the initial phase of regulatory volume decrease, whereas PKC down-regulation caused pronounced swelling and partial inhibition of regulatory volume decrease. In isoosmotic conditions, CaM antagonists and PKC activation did not affect astrocytic volume, but PKC inhibitors caused shrinking and PKC down-regulation led to swelling of these cells. These studies indicate the importance of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume.     

Regulation of Lens rCx46-formed Hemichannels by Activation of Protein Kinase C, External Ca2+ and Protons

Rodent lens connexin46 (rCx46) formed active voltage-dependent hemichannels when expressed in Xenopus oocytes. Time-dependent macroscopic currents were evoked upon depolarization. The observed two activation time constants were weakly voltage-dependent and in the order of hundreds of milliseconds and seconds, respectively. Occasionally, the macroscopic steady-state current and the corresponding current-voltage curve showed inactivation at high depolarizing voltages (>+50 mV). To account for the fast recovery from inactivation (<2 msec) favored by hyperpolarization, a four-state kinetic model (C _{1 closed} ↔C _{2 closed} ↔O _open ↔I _inactivated ) is proposed. In the absence of inactivation, the macroscopic conductance decreased and inactivation became visible at voltages positive of +50 mV when the rCx46-expressing oocytes were treated with the protein-kinase-C-activators OAG or TPA, high external concentrations of Ca²⁺ or H⁺. However, the underlying mechanisms of OAG, H⁺ or Ca²⁺ action were different. While OAG did not alter the voltage-dependent activation of the rCx46-hemichannels, an increase in the external Ca²⁺ or H⁺ level shifted the voltage threshold for activation to more positive voltages. In contrast to Ca²⁺, protons were not effective in the physiological concentration range. We propose that under physiological conditions only external Ca²⁺ and intracellular PKC-dependent processes regulate rCx46 in the lens. Received: 30 March 1999/Revised: 18 September 1999     

Evolution of the PP2C Family in Caenorhabditis: Rapid Divergence of the Sex-Determining Protein FEM-2

To investigate the causes and functional significance of rapid sex-determining protein evolution we compared three Caenorhabditis elegans genes encoding members of the protein phosphatase 2C (PP2C) family with their orthologs from another Caenorhabditis species (strain CB5161). One of the genes encodes FEM-2, a sex-determining protein, while the others have no known sex-determining role. FEM-2's PP2C domain was found to be more diverged than the other PP2C domains, supporting the notion that sex-determining proteins are subjected to selective pressures that allow for or cause rapid divergence. Comparison of the positions of amino acid substitutions in FEM-2 with a solved three-dimensional structure suggests that the catalytic face of the protein is highly conserved among C. elegans, CB5161, and another closely related species C. briggsae. However, the non-conserved regions of FEM-2 cannot be said to lack functional importance, since fem-2 transgenes from the other species were unable to rescue the germ-line defect caused by a C. elegans fem-2 mutation. To test whether fem-2 functions as a sex-determining gene in the other Caenorhabditis species we used RNA-mediated interference (RNAi). fem-2 (RNAi) in C. elegans and C. briggsae caused germ-line feminization, but had no noticeable effect in CB5161. Thus the function of fem-2 in CB5161 remains uncertain. Received: 11 April 2001 / Accepted: 6 August 2001     

P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration-dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2-methylthioadenosine 5′-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin-insensitive G_q protein. Short-term (10 min) treatment of cells with 1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of UTP- or ATP-stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF₄^?, but not Ca²⁺ ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of PKCε in the P2Y receptor/G_q protein/phospholipase Cβ pathway might be the G_q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor-mediated phosphoinositide turnover.