PI 3-kinase regulation of dopamine uptake

The magnitude and duration of dopamine (DA) signaling is defined by the amount of vesicular release, DA receptor sensitivity, and the efficiency of DA clearance, which is largely determined by the DA transporter (DAT). DAT uptake capacity is determined by the number of functional transporters on the cell surface as well as by their turnover rate. Here we show that inhibition of phosphatidylinositol (PI) 3-kinase with LY294002 induces internalization of the human DAT (hDAT), thereby reducing transport capacity. Acute treatment with LY294002 reduced the maximal rate of [(3) H]DA uptake in rat striatal synaptosomes and in human embryonic kidney (HEK) 293 cells stably expressing the hDAT (hDAT cells). In addition, LY294002 caused a significant redistribution of the hDAT from the plasma membrane to the cytosol. Conversely, insulin, which activates PI 3-kinase, increased [(3)H]DA uptake and blocked the amphetamine-induced hDAT intracellular accumulation, as did transient expression of constitutively active PI 3-kinase. The LY294002-induced reduction in [(3)H]DA uptake and hDAT cell surface expression was inhibited by expression of a dominant negative mutant of dynamin I, indicating that dynamin-dependent trafficking can modulate transport capacity. These data implicate DAT trafficking in the hormonal regulation of dopaminergic signaling, and suggest that a state of chronic hypoinsulinemia, such as in diabetes, may alter synaptic DA signaling by reducing the available cell surface DATs.     

Membrane cholesterol extraction decreases Na+ transport in A6 renal epithelia

In this study, we have investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 renal epithelia. We continuously monitored short-circuit current (I_sc), transepithelial conductance (G_T), and transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with hyposmotic shock, oxytocin, and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1 h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters; in contrast, marked reductions were observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin, and adenosine. Analysis of the blocker-induced fluctuation in I_sc demonstrated that apical mCD treatment decreased the epithelial Na⁺ channel (ENaC) open probability (P_o) in the steady state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment as a result of reduced Na⁺/K⁺ pump activity. On the basis of the findings in this study, we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing ENaC P_o. epithelial Na⁺ channel; Na⁺-K⁺-ATPase activity; short-circuit current; methyl--cyclodextrin; channel open probability     

Na+ transport and impedance properties of cultured renal (A6 and 2F3) epithelia

Previous impedance analysis studies of intact epithelia have been complicated by the presence of connective tissue or smooth muscle. We now report the first application of this method to cultured epithelial monolayers. Impedance analysis was used as a nondestructive method for deducing quantitative morphometric parameters for epithelia grown from the renal cell line A6, and its subclonal cell line 2F3. The subclonal 2F3 cell line was chosen for comparison to A6 because of its inherently higher Na+ transport rate. In agreement with previous results, 2F3 epithelia showed significantly higher amiloride-sensitive short-circuit currents (Isc) than A6 epithelia (44 +/- 2 and 27 +/- 2 microA/cm2, respectively). However, transepithelial conductances (GT) were similar for the two epithelia (0.62 +/- 0.04 mS/cm2 for 2F3 and 0.57 +/- 0.04 mS/cm2 for A6) because of reciprocal differences in cellular (Gc) and paracellular (Gj) conductances. Significantly lower Gj and higher Gc values were observed for 2F3 epithelia than A6 (Gj = 0.23 +/- 0.02 and 0.33 +/- 0.04 mS/cm2 and Gc = 0.39 +/- 0.16 and 0.26 +/- 0.10 mS/cm2, respectively). Nonetheless, the cellular driving force for Na+ transport (Ec) and the amount of transcellular Na+ current under open-circuit conditions (Ic) were similar for the two epithelia. Three different morphologically-based equivalent circuit models were derived to assess epithelial impedance properties: a distributed model which takes into account the resistance of the lateral intercellular space and two models (the "dual-layer" and "access resistance" models), which corrected for impedance of small fluid-filled projections of the basal membrane into the underlying filter support. Although the data could be fitted by the distributed model, the estimated value for the ratio of apical to basolateral membrane resistances was unreasonably large. In contrast, the other models provided statistically superior fits and reasonable estimates of the membrane resistance ratio. The dual-layer model and access resistance models also provided similar estimates of apical and basolateral membrane conductances and capacitances. In addition, both models provided new information concerning the conductance and area of the basolateral protrusions. Estimates of the apical membrane conductance were significantly higher for 2F3 (0.79 +/- 0.23 mS/cm2) than A6 epithelia (0.37 +/- 0.07 mS/cm2), but no significant difference could be detected for apical membrane capacitances (1.4 +/- 0.04 and 1.2 +/- 0.1 microF/cm2 for 2F3 and A6, respectively) or basolateral membrane conductances (3.48 +/- 1.67 and 2.95 +/- 0.40 mS/cm2). The similar basolateral membrane properties for the two epithelia may be explained by their comparable transcellular Na+ currents under open-circuit conditions.     

The role of PI 3-kinase in EGF-stimulated jejunal glucose transport

Epidermal growth factor (EGF) rapidly increases jejunal glucose transport. Signal transduction mechanisms mediating EGF-induced alterations in jejunal glucose transport remain to be determined. New Zealand White rabbit (1 kg) jejunal tissue was stripped and mounted in short-circuited Ussing chambers. The transport of tritiated 3-O-methylglucose was measured in the presence of the PKC agonist 1,2-dioctanoyl-sn-glycerol (1,2-DOG) or the inactive analog 1,3-dioctanoyl-sn-glycerol (1,3-DOG). Additional experiments examined the effect of the PKC inhibitor chelerythrine, the PLC inhibitor U73122, the MAPK inhibitor PD 98059, the G-protein inhibitor GDP-betaS, the PI 3-kinase inhibitor LY294002, or the microtubule inhibitor colchicine on EGF-induced jejunal glucose transport. Net jejunal 3-O-methylglucose absorption was significantly increased following specific activation of PKC. A PKC antagonist inhibited the EGF-induced increase in net 3-O-methylglucose transport, while PI 3-kinase inhibition completely blocked the EGF-induced transport increase. Inhibition of PLC, MAPK, G-proteins, and microtubules had no effect on EGF-stimulated increases in jejunal transport. We conclude that the effect of EGF on jejunal glucose transport is mediated at least in part by PKC and PI 3-kinase.     

Hyposmolality stimulates Na(+)/H(+) exchange and HCO(3)(-) absorption in thick ascending limb via PI 3-kinase

The signal transduction mechanisms that mediateosmotic regulation of Na⁺/H⁺ exchange are notunderstood. Recently we demonstrated that hyposmolality increasesHCO₃ absorption in the renal medullary thickascending limb (MTAL) through stimulation of the apical membraneNa⁺/H⁺ exchanger NHE3. To investigate themechanism of this stimulation, MTALs from rats were isolated andperfused in vitro with 25 mM HCO₃-containingsolutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitorswortmannin (100 nM) and LY-294002 (20 µM) blocked completely thestimulation of HCO₃ absorption by hyposmolality. Intissue strips dissected from the inner stripe of the outer medulla, theregion of the kidney highly enriched in MTALs, hyposmolality increasedPI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-inducedPI 3-K activation. Further studies examined the interaction betweenhyposmolality and vasopressin, which inhibits HCO₃absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO₃ absorption, due to aneffect of cAMP to inhibit hyposmolality- induced activation of PI 3-K.In contrast to their effects to block stimulation by hyposmolality, PI3-K inhibitors and vasopressin have no effect on inhibition of apicalNa⁺/H⁺ exchange (NHE3) andHCO₃ absorption by hyperosmolality. These resultsindicate that hyposmolality increases NHE3 activity andHCO₃ absorption in the MTAL through activation of aPI 3-K-dependent pathway that is inhibited by vasopressin and cAMP.Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediatedthrough different signal transduction mechanisms.

     

Redox regulation of PI 3-kinase signalling via inactivation of PTEN

The tumour suppressor PTEN is a PtdIns(3,4,5)P(3) phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P(3) levels and activation of the downstream PtdIns(3,4,5)P(3) target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P(3) and oxidants are produced concurrently.     

Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis

We have investigated the mechanisms of leading edge formation in chemotaxing Dictyostelium cells. We demonstrate that while phosphatidylinositol 3-kinase (PI3K) transiently translocates to the plasma membrane in response to chemoattractant stimulation and to the leading edge in chemotaxing cells, PTEN, a negative regulator of PI3K pathways, exhibits a reciprocal pattern of localization. By uniformly localizing PI3K along the plasma membrane, we show that chemotaxis pathways are activated along the lateral sides of cells and PI3K can initiate pseudopod formation, providing evidence for a direct instructional role of PI3K in leading edge formation. These findings provide evidence that differential subcellular localization and activation of PI3K and PTEN is required for proper chemotaxis.     

Tissue-specific regulation of IRS-2/PI 3-kinase association in aged rats

We have examined the insulin-stimulated IRS-2 association with PI 3-kinase and the phosphorylation of AKT/PKB, which is functionally located downstream of the PI 3-kinase, in aged (obese) rats. The IRS-2 protein levels were similar in 2 and 20 month-old rats in both tissues, liver and muscle. There were reductions in insulin-induced IRS-2 tyrosine phosphorylation in liver and muscle, accompanied by a decrease in IRS-2/PI 3-kinase association and in AKT/PKB phosphorylation only in muscle tissue of aged rats. This regulation may be important in the altered glucose metabolism observed in aged (obese) rats.     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

Ran-binding protein 3 phosphorylation links the Ras and PI3-kinase pathways to nucleocytoplasmic transport

The major participants of the Ras/ERK and PI3-kinase (PI3K) pathways are well characterized. The cellular response to activation of these pathways, however, can vary dramatically. How differences in signal strength, timing, spatial location, and cellular context promote specific cell-fate decisions remains unclear. Nuclear transport processes can have a major impact on the determination of cell fate; however, little is known regarding how nuclear transport is regulated by or regulates these pathways. Here we show that RSK and Akt, which are activated downstream of Ras/ERK and PI3K, respectively, modulate the Ran gradient and nuclear transport by interacting with, phosphorylating, and regulating Ran-binding protein 3 (RanBP3) function. Our findings highlight an important link between two major cell-fate determinants: nuclear transport and the Ras/ERK/RSK and PI3K/Akt signaling pathways.     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Role of PKCalpha in feedback regulation of Na(+) transport in an electrically tight epithelium

It has long been known thatNa⁺ channels in electrically tight epithelia are regulatedby homeostatic mechanisms that maintain a steady state and allow newlevels of transport to be sustained in hormonally challenged cells.Little is known about the potential pathways involved in theseprocesses. In addition to short-term effect, recent evidence alsoindicates the involvement of PKC in the long-term regulation of theepithelial Na⁺ channel (ENaC) at the protein level(40). To determine whether stimulation of ENaC involvesfeedback regulation of PKC levels, we utilized Western blot analysis todetermine the distribution of PKC isoforms in polarized A6 epithelia.We found the presence of PKC isoforms in the conventional ( and), novel (, , and ), and atypical (, , and) groups. Steady-state stimulation of Na⁺ transport withaldosterone was accompanied by a specific decrease of PKC proteinlevels in both the cytoplasmic and membrane fractions. Similarly,overnight treatment with an uncharged amiloride analog (CDPC), aprocedure that through feedback regulation causes a stimulation ofNa⁺ transport, also decreased PKC levels. These effectswere additive, indicating separate mechanisms that converge at thelevel of PKC. These effects were not accompanied by changes ofPKC mRNA levels as determined by Northern blot analysis. We proposethat this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na⁺ transport.

     

Negative regulation of PI 3-kinase by Ruk, a novel adaptor protein

Class I(A) phosphatidylinositol 3-kinase (PI 3-kinase) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, Ruk(l), which forms complexes with the PI 3-kinase holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of Ruk and the SH3 domain of the p85 alpha regulatory subunit of the class I(A) PI 3-kinase. In contrast to many other adaptor proteins that activate PI 3-kinase, interaction with Ruk(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of Ruk(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of PI 3-kinase or its downstream effector PKB/Akt. Our data provide evidence for the existence of a negative regulator of the PI 3-kinase signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between Ruk, CIN85 and CD2AP/CMS suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.     

LY294002-geldanamycin heterodimers as selective inhibitors of the PI3K and PI3K-related family

Several LY294002-GM heterodimers were synthesized with the intent of modulating their activity in the presence of hsp90 and thereby creating selective inhibitors of PI3K and PI3K-related family.     

TNFalpha elicits association of PI 3-kinase with the p60TNFR and activation of PI 3-kinase in adherent neutrophils

Tumor necrosis factor (TNFalpha) is an incomplete secretagogue in neutrophils and requires the engagement of beta integrins to trigger secretion of superoxide anion (O(-)(2)). The p60 TNF receptor (p60TNFR) is responsible for signal transduction for activation of O(-)(2) generation. Activation of TNFalpha-triggered O(-)(2) generation in neutrophils adherent to fibrinogen-coated surfaces involves the beta2 integrin receptor CD11b/CD18. Phosphoinositide 3-kinase (PI 3-kinase) is essential for activation of O(-)(2) generation; wortmannin, an inhibitor of PI 3-kinase, inhibited TNFalpha-elicited O(-)(2) generation. p60TNFR immunoprecipitated from neutrophils was associated with immunoreactivity to PI 3-kinase in adherent neutrophils exposed to TNFalpha, but not in TNFalpha-treated neutrophils in suspension. In addition, PI 3-kinase immunoprecipitated from TNFalpha-activated neutrophils showed enhanced activity in adherent but not in nonadherent neutrophils. These findings suggest that synergism between CD11b/CD18 and p60TNFR in the presence of TNFalpha is required to elicit assembly of a signaling complex involving association of p60TNFR with PI 3-kinase, activation of PI 3-kinase, and generation of O(-)(2).