Regulation of membrane trafficking and endocytosis by protein kinase C: emerging role of the pericentrion, a novel protein kinase C-dependent subset of recycling endosomes.

The protein kinase C (PKC) family of isoenzymes has been shown to regulate a variety of cellular processes, including receptor desensitization and internalization, and this has sparked interest in further delineation of the roles of specific isoforms of PKC in membrane trafficking and endocytosis. Recent studies have identified a novel translocation of PKC to a juxtanuclear compartment, the pericentrion, which is distinct from the Golgi complex but epicentered on the centrosome. Sustained activation of PKC (longer than 30 min) also results in sequestration of plasma membrane lipids and proteins to the same compartment, demonstrating a global effect on endocytic trafficking. This review summarizes these studies, particularly focusing on the characterization of the pericentrion as a distinct PKC-dependent subset of recycling endosomes. We also discuss emerging insights into a role for PKC as a central hub in regulating vesicular transport pathways throughout the cell, with implications for a wide range of pathobiologic processes, e.g. diabetes and abnormal neurotransmission or receptor desensitization.

Flocking regimes in a simple lattice model.

We study a one-dimensional lattice flocking model incorporating all three of the flocking criteria proposed by Reynolds [Computer Graphics 21, 4 (1987)]: alignment, centering, and separation. The model generalizes that introduced by O. J. O'Loan and M. R. Evans [J. Phys. A. 32, L99 (1999)]. We motivate the dynamical rules by microscopic sampling considerations. The model exhibits various flocking regimes: the alternating flock, the homogeneous flock, and dipole structures. We investigate these regimes numerically and within a continuum mean-field theory.

G protein-coupled receptors desensitize and down-regulate epidermal growth factor receptors in renal mesangial cells.

Different types of plasma membrane receptors engage in various forms of cross-talk. We used cultures of rat renal mesangial cells to study the regulation of EGF receptors (EGFRs) by various endogenous G protein-coupled receptors (GPCRs). GPCRs (5-hydroxytryptamine(2A), lysophosphatidic acid, angiotensin AT(1), bradykinin B(2)) were shown to transactivate EGFRs through a protein kinase C-dependent pathway. This transactivation resulted in the initiation of multiple cellular signals (phosphorylation of the EGFRs and ERK and activation of cAMP-responsive element-binding protein (CREB), NF-kappaB, and E2F), as well as subsequent rapid down-regulation of cell-surface EGFRs and internalization and desensitization of the EGFRs without change in the total cellular complement of EGFRs. Internalization of the EGFRs and the down-regulation of cell-surface receptors in mesangial cells were blocked by pharmacological inhibitors of clathrin-mediated endocytosis and in HEK293 cells by transfection of cDNA constructs that encode dominant negative beta-arrestin-1 or dynamin. Whereas all of the effects of GPCRs on EGFRs were dependent to a great extent on protein kinase C, those initiated by EGF were not. These studies demonstrate that GPCRs can induce multiple signals through protein kinase C-dependent transactivation of EGFRs. Moreover, GPCRs induce profound desensitization of EGFRs by a process associated with the loss of cell-surface EGFRs through clathrin-mediated endocytosis.

Bradykinin B2 receptors activate Na+/H+ exchange in mIMCD-3 cells via Janus kinase 2 and Ca2+/calmodulin.

We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein-coupled receptor, the bradykinin B2 receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein fluorescence measuring the accelerated rate of pH(i) recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B2 receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kinase C and phosphatidylinositol 3'-kinase. Immunoprecipitation studies showed that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. CaM appears to be a direct substrate for phosphorylation by Jak2 as measured by an in vitro kinase assay. We propose that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of CaM and most likely by increasing the binding of CaM to NHE-1.

Multiplicity of mechanisms of serotonin receptor signal transduction.

The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

A functional angiotensin II receptor-GFP fusion protein: evidence for agonist-dependent nuclear translocation.

We constructed an expression vector for a fusion protein [ANG II type 1a receptor-green fluorescent protein (AT(1a)R-GFP)] consisting of enhanced GFP attached to the COOH terminus of the rat AT(1a)R. Chinese hamster ovary (CHO) cells transfected with AT(1a)R-GFP demonstrated specific, high-affinity (125)I-labeled ANG II binding (IC(50) 21 nM). ANG II exposure stimulated sodium-proton exchange and cytoplasmic calcium release to a similar extent in cells transfected with AT(1a)R or AT(1a)R-GFP; these responses were desensitized by prior exposure to ANG II and were sensitive to the AT(1)R blocker losartan. ANG II-driven internalization of AT(1a)R-GFP in transfected CHO cells was demonstrated both by radioligand binding and by laser scanning confocal microscopy. Colocalization of GFP fluorescence with that of the nuclear stain TOTO-3 in confocal images was increased more than twofold after 1 h of ANG II exposure. We conclude that AT(1a)R-GFP exhibits similar pharmacological behavior to that of the native AT(1a)R. Our observations also support previous evidence for the presence of AT(1a)R in the nucleus and suggest that the density of AT(1a)R in the nucleus may be regulated by exposure to its ligand.

Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-kappaB in primary astrocytes.

The present study underlines the importance of p21(ras) in regulating the inducible nitric oxide synthase (iNOS) in primary astrocytes. Bacterial lipopolysaccharides induced the GTP loading of p21(ras), and the expression of a dominant-negative mutant of p21(ras) (Deltap21(ras)) inhibited lipopolysaccharide-induced GTP loading in rat primary astrocytes. To delineate the role of p21(ras) in the induction of iNOS, we examined the effect of Deltap21(ras) on the expression of iNOS and the production of nitric oxide. It is interesting that expression of Deltap21(ras) markedly inhibited the production of nitric oxide and the expression of iNOS in lipopolysaccharide- and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta; interferon-gamma)-stimulated rat and human primary astrocytes. Inhibition of iNOS promoter-derived chloramphenicol acetyltransferase activity by Deltap21(ras) suggests that p21(ras) is involved in the transcription of iNOS. As activation of nuclear factor-kappaB (NF-kappaB) is necessary for the transcription of iNOS, we examined the effect of Deltap21(ras) on the activation of NF-kappaB. Expression of Deltap21(ras) inhibited the DNA binding as well as the transcriptional activity of NF-kappaB in activated astrocytes, suggesting that Deltap21(ras) inhibits the expression of iNOS by inhibiting the activation of NF-kappaB. These studies also suggest that inhibitors of p21(ras) may be used as therapeutics in nitric oxide- and cytokine-mediated neuroinflammatory diseases.

Expression of a constitutively active form of phosphatidylinositol 3-kinase inhibits the induction of nitric oxide synthase in human astrocytes.

The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in attenuating the induction of nitric oxide synthase (iNOS) in human astrocytes. Proinflammatory cytokines induced the production of nitric oxide (NO) and the expression of iNOS in human U373MG astrocytoma cells and primary astrocytes. Expression of a catalytically active p110 subunit (p110*) of PI 3-kinase but not that of a kinase-deficient mutant of p110 (p110-kd) induced an increase in PI 3-kinase activity and inhibited cytokine-induced production of NO and expression of iNOS. However, expression of p110* had no effect on the activation of NF-kB, suggesting that p110* inhibits the expression of iNOS without inhibiting the activation of NF-kB.

5-HT(2A) receptors stimulate mitogen-activated protein kinase via H(2)O(2) generation in rat renal mesangial cells.

Serotonin (5-HT) stimulates mitogenesis in rat renal mesangial cells through a G protein-coupled 5-HT(2A) receptor. We tested the hypothesis that oxidants might be involved in the signal transduction pathway linking the receptor to extracellular signal-regulated protein kinase (ERK). 5-HT rapidly increased the activity and phosphorylation of ERK. These effects were blocked by the 5-HT(2A) receptor antagonist ketanserin. The peak effect was noted at 5-10 min, and half-maximal stimulation was achieved at 10-30 nM 5-HT. Chemical inhibitor and activator studies supported the involvement of phospholipase C, protein kinase C (PKC), and reactive oxygen species (ROS, i.e., H(2)O(2) and superoxide) generated by an NAD(P)H oxidase-like enzyme in the ERK activation cascade. Mapping studies supported a location for the NAD(P)H oxidase enzyme and the ROS downstream from PKC. Our studies are most consistent with an ERK activation pathway as follows: 5-HT(2A) receptor --> G(q) protein --> phospholipase C --> diacylglycerol --> classical PKC --> NAD(P)H oxidase --> superoxide --> superoxide dismutase --> H(2)O(2) --> mitogen-activated extracellular signal-regulated kinase --> ERK. These studies demonstrate a role for the 5-HT(2A) receptor in rapid, potent, and efficacious activation of ERK in rat renal mesangial cells. They support a role for oxidants in conveying the stimulatory signal from 5-HT, because 1) chemical antioxidants attenuate the 5-HT signal, 2) oxidants and 5-HT selectively activate ERK to a similar degree, 3) 5-HT produces superoxide and H(2)O(2) in these cells, and 4) a specific enzyme [NAD(P)H oxidase] has been implicated as the source of the ROS, which react selectively downstream of classical PKC.

5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts.

The hypothesis of this work is that the 'serotonin' or 5-hydroxytryptamine (5-HT)(1A) receptor, which activates the extracellular signal-regulated kinase (ERK) through a G(i)betagamma-mediated pathway, does so through the intermediate actions of reactive oxygen species (ROS). Five criteria were shown to support a key role for ROS in the activation of ERK by the 5-HT(1A) receptor. (1) Antioxidants inhibit activation of ERK by 5-HT. (2) Application of cysteine-reactive oxidant molecules activates ERK. (3) The 5-HT(1A) receptor alters cellular redox properties, and generates both superoxide and hydrogen peroxide. (4) A specific ROS-producing enzyme [NAD(P)H oxidase] is involved in the activation of ERK. (5) There is specificity both in the effects of various chemical oxidizers, and in the putative location of the ROS in the ERK activation pathway. We propose that NAD(P)H oxidase is located in the ERK activation pathway stimulated by the transfected 5-HT(1A) receptor in Chinese hamster ovary (CHO) cells downstream of G(i)betagamma subunits and upstream of or at the level of the non-receptor tyrosine kinase, Src. Moreover, these experiments provide confirmation that the transfected human 5-HT(1A) receptor induces the production of ROS (superoxide and hydrogen peroxide) in CHO cells, and support the possibility that an NAD(P)H oxidase-like enzyme might be involved in the 5-HT-mediated generation of both superoxide and hydrogen peroxide.

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species.

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.

The recombinant 5-HT1A receptor: G protein coupling and signalling pathways.

The 5-hydroxytryptamine 5-HT1A receptor was one of the first G protein coupled receptors whose cDNA and gene were isolated by molecular cloning methods. Transfection of the cDNA of this receptor into cells previously bearing no 5-HT receptors has resulted in the acquisition of large amounts of information regarding potential signal transduction pathways linked to the receptor, correlations of receptor structure to its various functions, and pharmacological properties of the receptor. Transfection studies with the 5-HT1A receptor have generated critical new information that might otherwise have been elusive. This information notably includes the discovery of unsuspected novel signalling linkages, the elucidation of the mechanisms of receptor desensitization, the refinement of models of the receptor pharmacophore, and the development of silent receptor antagonists, among others. The current review summarizes the most important studies of the recombinant 5-HT1A receptor in the decade since the identification of its cDNA.

Serotonin 5-HT2A receptor induces TGF-beta1 expression in mesangial cells via ERK: proliferative and fibrotic signals.

We examined the links between fibrotic and proliferative pathways for the 5-HT2A receptor in rat mesangial cells. Serotonin (5-hydroxytryptamine, 5-HT) induced transforming growth factor-beta1 (TGF-beta1) mRNA in a concentration-dependent (peak at 30 nM 5-HT) and time-dependent fashion. For 10 nM 5-HT, the effect was noticeable at 1 h and maximal by 6 h. Inhibition of 1) protein kinase C (PKC), 2) mitogen- and extracellular signal-regulated kinase kinase (MEK1) with 2'-amino-3'-methoxyflavone (PD-90859), and 3) extracellular signal-regulated kinase (ERK) with apigenin attenuated this effect. The effect was blocked by antioxidants, N-acetyl-L-cysteine (NAC) and alpha-lipoic acid, and mimicked by direct application of H2O2. TGF-beta1 mRNA induction was also blocked by diphenyleneiodonium and 4-(2-aminoethyl)-benzenesulfonyl fluoride, which inhibit NAD(P)H oxidase, a source of oxidants. 5-HT increased the amount of TGF-beta1 protein, validating the mRNA studies and demonstrating that 5-HT potently activates ERK and induces TGF-beta1 mRNA and protein in mesangial cells. Mapping studies strongly supported relative positions of the components of the signaling cascade as follow: 5-HT2A receptor --> PKC --> NAD(P)H oxidase/reactive oxygen species --> MEK --> ERK --> TGF-beta1 mRNA. These studies demonstrate that mitogenic signaling components (PKC, MEK, and oxidants) are directly linked to the regulation of TGF-beta1, a key mediator of fibrosis. Thus a single stimulus can direct both proliferative and fibrotic signals in renal mesangial cells.

Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP.

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.

Agonist-induced translocation of Gq/11alpha immunoreactivity directly from plasma membrane in MDCK cells.

Both Gsalpha and Gqalpha are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) alpha-subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11alpha immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 microM) caused Gq/11alpha, but not Gialpha, to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11alpha immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11alpha immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11alpha is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity.

Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1beta induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1beta-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85alpha, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-kappaB in LPS- or IL-1beta-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1beta alone to induce the expression of iNOS, both LPS and IL-1beta individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-kappaB. Wortmannin alone was unable to activate MAP kinase and NF-kappaB. Moreover, wortmannin had no effect on LPS- or IL-1beta-mediated activation of MAP kinase and NF-kappaB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1beta-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-kappaB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-kappaB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-kappaB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Activation of protein kinase Calpha couples cell volume to membrane Cl- permeability in HTC hepatoma and Mz-ChA-1 cholangiocarcinoma cells.

Physiological increases in liver cell volume lead to an adaptive response that includes opening of membrane Cl- channels, which is critical for volume recovery. The purpose of these studies was to assess the potential role for protein kinase C (PKC) as a signal involved in cell volume homeostasis. Studies were performed in HTC rat hepatoma and Mz-ChA-1 human cholangiocarcinoma cells, which were used as model hepatocytes and cholangiocytes, respectively. In each cell type, cell volume increases were followed by: 1) translocation of PKC from cytosolic to particulate (membrane) fractions; 2) a 10- to 40-fold increase in whole-cell membrane Cl- current density; and 3) partial recovery of cell volume. In HTC cells, the volume-dependent Cl- current response (-46 +/- 5 pA/pF) was inhibited by down-regulation of PKC (100 nmol/L phorbol 12-myristate 13-acetate for 18 hours [PMA]; -1.97 +/- 1.5 pA/pF), chelation of cytosolic Ca2+ (2 mmol/L EGTA; -5.3 +/- 4.0 pA/pF), depletion of cytosolic adenosine triphosphate (ATP) (3 U/mL apyrase; -12.58 +/- 1. 45 pA/pF), and by the putative PKC inhibitor, chelerythrine (25 micromol/L; -7 +/- 3 pA/pF). In addition, PKC inhibition by chelerythrine and calphostin C (500 nmol/L) prevented cell volume recovery from swelling. Similar results were obtained in Mz-ChA-1 biliary cells. These findings indicate that swelling-induced activation of PKC represents an important signal coupling cell volume to membrane Cl- permeability in both hepatic and biliary cell models.

Inhibition of Na+/H+ exchange stimulates CCK secretion in STC-1 cells.

It has been demonstrated that K+ channel regulation of membrane potential is critical for control of CCK secretion. Because certain K+ channels are pH sensitive, it was postulated that pH affects K+ channel activity in the CCK-secreting cell line STC-1 and may participate in regulating CCK secretion. The present study examines the role of electroneutral Na+/H+ exchange on extracellular acidification and hormone secretion. Treatment of STC-1 cells with the amiloride analog ethylisopropyl amiloride (EIPA) to inhibit Na+/H+ exchange inhibited Na+-dependent H+ efflux and increased basal CCK secretion. Substituting choline for NaCl in the extracellular medium elevated basal intracellular Ca2+ concentration and stimulated CCK release. Stimulatory effects on hormone secretion were blocked by the L-type Ca2+ channel blocker diltiazem, indicating that secretion was dependent on the influx of extracellular Ca2+. To determine whether the effects of EIPA and Na+ depletion were due to membrane depolarization, we tested graded KCl concentrations. The ability of EIPA to increase CCK secretion was inhibited by depolarization induced by 10-50 mM KCl in the bath. Maneuvers to lower intracellular pH (pHi), including reducing extracellular pH (pHo) to 7.0 or treatment with sodium butyrate, significantly increased CCK secretion. To examine whether pH directly affects membrane K+ permeability, we measured outward currents carried by K+, using whole cell patch techniques. K+ current was significantly inhibited by lowering pHo to 7.0. These effects appear to be mediated through changes in pHi, because intracellular dialysis with acidic solutions nearly eliminated current activity. These results suggest that Na+/H+ exchange and membrane potential may be functionally linked, where inhibition of Na+/H+ exchange lowers pHi and depolarizes the membrane, perhaps through inhibition of pH-sensitive K+ channels. In turn, K+ channel closure and membrane depolarization open voltage-dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ and CCK release. The effects of pHi on K+ channels may serve as a potent stimulus for hormone secretion, linking cell metabolism and secretory functions.

Regulation by serotonin of tooth-germ morphogenesis and gene expression in mouse mandibular explant cultures.

Serotonin (5-HT) stimulates tooth-germ development in embryonic mouse mandibular explant cultures, but it is not clear whether this is due to a direct action on epithelial-mesenchymal interactions, or whether development was stimulated indirectly by serotonergic regulation of other morphoregulatory molecules. A calcium-binding protein, S-100beta, and the extracellular-matrix molecule, tenascin, two molecules thought to be important in craniofacial development, together with cartilage proteoglycan core protein, a marker for chondrogenesis, are modulated by serotonergic ligands in mandibular micromass cultures. Here, it was demonstrated that 5-HT stimulates expression of cartilage proteoglycan core protein, and inhibits expression of S-100beta and tenascin in mandibular explants. Further, ondansetron (Zofran), a 5-HT3 receptor antagonist, and NAN-190, a 5-HT1A antagonist, reversed the serotonergic stimulation of core protein and tooth germ development. In contrast serotonergic modulation of S-100beta and tenascin expression was not reversed by any of the 5-HT receptor antagonists tested, although the 5-HT uptake inhibitor, fluoxetine, did reverse the effect of 5-HT on S-100beta expression, as well as tooth-germ development. These results support previous work suggesting that 5-HT plays an important part in craniofacial development, especially in dentinogenesis and chondrogenesis. However, the possibility that tenascin or S-100beta mediate the effects of 5-HT on tooth-germ development is not supported. Rather, these results raise the possibility that 5-HT may exert effects directly on tooth-germ morphogenesis mediated by intracellular uptake of 5-HT and/or activation of 5-HT1A and 5-HT3 receptors.