Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl--dependent mechanism

The Na-K-2Clcotransporter NKCC1 is an important volume-regulatory transporter thatis regulated by cell volume and intracellular Cl. Thisregulation appears to be mediated by phosphorylation of NKCC1, althoughthere is evidence for additional, cytoskeletal regulation via myosinlight chain (MLC) kinase. NKCC1 is also activated by growth factors andmay contribute to cell hypertrophy, but the mechanism is unknown. Inaortic endothelial cells, NKCC1 (measured as bumetanide-sensitive⁸⁶Rb⁺ influx) was rapidly stimulated by serum,lysophosphatidic acid, and fibroblast growth factor, with the greateststimulation by serum. Serum increased bumetanide-sensitive influxsignificantly more than bumetanide-sensitive efflux (131% vs. 44%),indicating asymmetric stimulation of NKCC1, and produced a 17%increase in cell volume and a 25% increase in Cl contentover 15 min. Stimulation by serum and hypertonic shrinkage wereadditive, and serum did not increase phosphorylation of NKCC1 or MLC,and did not decrease cellular Cl content. When cellularCl was replaced with methanesulfonate, influx via NKCC1increased and was no longer stimulated by serum, whereas stimulation by hypertonic shrinkage still occurred. Based on these results, we proposea novel mechanism whereby serum activates NKCC1 by reducing itssensitivity to inhibition by intracellular Cl. Thisresetting of the Cl set point of the transporter enablesthe cotransporter to produce a hypertrophic volume increase.

     

Role of PLCgamma and Ca(2+) in VEGF- and FGF-induced choroidal endothelial cell proliferation

Although bothvascular endothelial growth factor (VEGF) and fibroblast growth factor(FGF) receptors have been shown to be important in the regulation ofvascular endothelial cell growth, the roles of phospholipase C (PLC)and Ca²⁺ in their downstream signaling cascades are stillnot clear. We have examined the effects of VEGF and FGF on PLCphosphorylation and on changes in intracellular Ca²⁺ levelsin primary endothelial cells. VEGF stimulation leads to PLCactivation and increases in intracellular Ca²⁺, which arecorrelated with mitogen-activated protein (MAP) kinase (MAPK)activation and cell growth. Inhibition of Ca²⁺ increases bythe Ca²⁺ chelator1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid(BAPTA)-AM resulted in marked inhibition of MAPK activation, which wasshown to be linked to regulation of cell growth in these cells. Incontrast, FGF stimulation did not lead to PLC activation or tochanges in intracellular Ca²⁺ levels, although MAPKphosphorylation and stimulation of cell proliferation were observed.Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on theseFGF-stimulated responses. These data demonstrate a direct role forPLC and Ca²⁺ in VEGF-regulated endothelial cell growth,whereas this signaling pathway is not linked to FGF-mediated effects inprimary endothelial cells. Thus endothelial cell-specific factorsregulate the ability of VEGF receptors and FGF receptors to couple tothis signaling pathway.

     

Nicotine stimulates DNA synthesis and proliferation in vascular endothelial cells in vitro

Nicotine is a major component of cigarette smokeand has been postulated to play an important role in atherogenesis andmalignancy. Endothelial cell growth may be regulated by nicotine, yetoperative mechanisms at the endothelial level are poorly understood. We studied the effects of nicotine(10¹⁴-10⁴M) on endothelial DNA synthesis, DNA repair, proliferation, and cytotoxicity by using cultures of bovine pulmonary artery endothelial cells. Assays were performed on cells incubated with nicotine in thepresence and absence of hydroxyurea (an inhibitor of scheduled DNAsynthesis), serum, human platelet-poor plasma, and platelet-derived growth factor and endothelial cell growth factor (PDGF andPDECGF, respectively). Nicotine significantly stimulatedendothelial cell DNA synthesis and proliferation at concentrationslower than those obtained in blood after smoking(<10⁸ M). The stimulatoryeffects of nicotine were enhanced by serum (0.5%) and PDECGF and wereblocked by the nicotinic-receptor antagonist hexamethonium. Theresponse to nicotine was bimodal because cytotoxicity was observed athigher concentrations(>10⁶ M). This study hasimplications for understanding cellular mechanisms of nicotine action.The results may be important in tumor angiogenesis, atherogenesis, andvascular dysfunction in smokers.

     

Separate taurine and chloride efflux pathways activated during regulatory volume decrease

Organic osmolyte and halide permeability pathways activated inepithelial HeLa cells by cell swelling were studied by radiotracer efflux techniques and single-cell volume measurements. The replacement of extracellular Cl byanions that are more permeant through the volume-activated Cl channel, as indicated byelectrophysiological measurements, significantly decreasedtaurine efflux. In the presence of less-permeant anions, an increase intaurine efflux was observed. Simultaneous measurement of the¹²⁵I, used as a tracer forCl, and[³H]taurine effluxshowed that the time courses for the two effluxes differed. InCl-rich medium the increasein I efflux was transient,whereas that for taurine was sustained. OsmosensitiveCl conductance, assessed bymeasuring changes in cell volume, increased rapidly after hypotonicshock. The influx of taurine was able to counteractCl conductance-dependentcell shrinkage but only ~4 min after triggering cell swelling. Thistaurine-induced effect was blocked by DIDS. Differences in anionsensitivity, the time course of activation, and sensitivity to DIDSsuggest that the main cell swelling-activated permeability pathways fortaurine and Cl are separate.

     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Osmotic pressure regulates alpha beta gamma -rENaC expressed in Xenopus oocytes

The hypothesisthat amiloride-sensitive Na⁺channels (ENaC) are involved in cell volume regulation was tested.Anisosmotic ND-20 media (ranging from 70 to 450 mosM) were used tosuperfuse Xenopus oocytes expressing-rat ENaC (-rENaC). Whole cell currents werereversibly dependent on external osmolarity. Under conditions ofswelling (70 mosM) or shrinkage (450 mosM), current amplitude decreasedand increased, respectively. In contrast, there was no change incurrent amplitude of H₂O-injectedoocytes to the above osmotic insults. Currents recorded from-rENaC-injected oocytes were not sensitive to externalCl concentration or to theK⁺ channel inhibitorBaCl₂. They were sensitive toamiloride. The concentration of amiloride necessary to inhibit one-halfof the maximal rENaC current expressed in oocytes(K_i; apparentdissociation constant) decreased in swollen cells and increased inshrunken oocytes. The osmotic pressure-inducedNa⁺ currents showed propertiessimilar to those of stretch-activated channels, including inhibition byGd³⁺ andLa³⁺, and decreased selectivityfor Na⁺.-rENaC-expressing oocytes maintained a nearly constant cell volume in hypertonic ND-20. The present study is the firstdemonstration that -rENaC heterologously expressed inXenopus oocytes may contribute tooocyte volume regulation following shrinkage.

     

Human trabecular meshwork cell volume regulation

The volume ofcertain subpopulations of trabecular meshwork (TM) cells may modifyoutflow resistance of aqueous humor, thereby altering intraocularpressure. This study examines the contribution thatNa⁺/H⁺, Cl/HCOexchange, and K⁺-Cl efflux mechanisms have onthe volume of TM cells. Volume, Cl currents, andintracellular Ca²⁺ activity of cultured human TM cells werestudied with calcein fluorescence, whole cell patch clamping, and fura2 fluorescence, respectively. At physiological bicarbonateconcentration, the selective Na⁺/H⁺ antiportinhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicitytriggered a regulatory volume decrease (RVD), which could be inhibitedby the Cl channel blocker5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K⁺channel blockers Ba²⁺ and tetraethylammonium, and theK⁺-Cl symport blocker[(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism inisotonic conditions was dependent on bicarbonate; at physiologicallevels, the Na⁺/H⁺ exchange inhibitordimethylamiloride reduced cell volume, whereas at low levels theNa⁺-K⁺-2Cl symport inhibitorbumetanide had the predominant effect. Patch-clamp measurements showedthat hypotonicity activated an outwardly rectifying, NPPB-sensitiveCl channel displaying the permeability rankingCl > methylsulfonate > aspartate.2,3-Butanedione 2-monoxime antagonized actomyosin activity and bothincreased baseline [Ca²⁺] and abolishedswelling-activated increase in [Ca²⁺], but it did notaffect RVD. Results indicate that human TM cells display aCa²⁺-independent RVD and that volume is regulated byswelling-activated K⁺ and Cl channels,Na⁺/H⁺ antiports, and possiblyK⁺-Cl symports in addition toNa⁺-K⁺-2Cl symports.

     

Effect of glutamine supplementation on exercise-induced changes in lymphocyte function

The purpose of this study was toinvestigate the possible role of glutamine in exercise-inducedimpairment of lymphocyte function. Ten male athletesparticipated in a randomized, placebo-controlled, double-blindcrossover study. Each athlete performed bicycle exercise for 2 hat 75% of maximum O₂ consumption on 2 separate days.Glutamine or placebo supplements were given orally during and up to2 h postexercise. The trial induced postexercise neutrocytosisthat lasted at least 2 h. The total lymphocyte count increased bythe end of exercise due to increase of bothCD3⁺TCR⁺ andCD3⁺TCR⁺ T cells as well asCD3CD16⁺CD56⁺ naturalkiller (NK) cells. Concentrations of CD8⁺ andCD4⁺ T cells lacking CD28 and CD95 on their surfaceincreased more than those of cells expressing these receptors. Withinthe CD4⁺ cells, only CD45RA memory cells, butnot CD45RA⁺ naive cells, increased in response to exercise.Most lymphocyte subpopulations decreased 2 h after exercise.Glutamine supplementation abolished the postexercise decline in plasmaglutamine concentration but had no effect on lymphocyte trafficking, NKand lymphokine-activated killer cell activities, T cell proliferation,catecholamines, growth hormone, insulin, or glucose. Neutrocytosis wasless pronounced in the glutamine-supplemented group, but it is unlikelythat this finding is of any clinical significance. This study does notsupport the idea that glutamine plays a mechanistic role inexercise-induced immune changes.

     

PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-stimulated anion secretion and expresses numerous HCO3(-) transporters

Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ·cm²), confluent monolayers that responded to forskolin with an increase in short-circuit current (I_sc; 8 ± 1 µA/cm²) that required Cl^–, HCO₃^–, and Na⁺, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na⁺-HCO₃^– cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl^–/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I_sc. Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I_sc. RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the - and -subunits of the epithelia Na⁺ channels (- and -ENaC), but not -ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function. male reproductive tract; cystic fibrosis; epithelial Na⁺ channel expression; glucocorticoid receptor; adrenergic; vasopressin     

Contribution of chloride channels to volume regulation of cortical astrocytes

The objective of this study was todetermine the relative contribution of Cl channels tovolume regulation of cultured rat cortical astrocytes after hypotoniccell swelling. Using a Coulter counter, we showed that corticalastrocytes regulate their cell volume by ~60% within 45 min afterhypotonic challenge. This volume regulation was supported whenCl was replaced with Br,NO, methanesulfonate, oracetate but was inhibited when Cl wasreplaced with isethionate or gluconate.Additionally, substitution of Cl with Icompletely blocked volume regulation. Volume regulation was unaffected by furosemide or bumetanide, blockers of KCl transport, but was inhibited by Cl channel blockers, including5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB),4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumicacid. Surprisingly, the combination of Cd²⁺ with NPPB,DIDS, or niflumic acid inhibited regulation to a greater extent thanany of these drugs alone. Volume regulation did not differ amongastrocytes cultured from different brain regions, as cerebellar andhippocampal astrocytes exhibited behavior identical to that of corticalastrocytes. These data suggest that Cl flux through ionchannels rather than transporters is essential for volume regulation ofcultured astrocytes in response to hypotonic challenge.

     

Evidence for an Na+-K+-Cl- cotransporter in mammalian type I vestibular hair cells

In amniotes,there are two types of hair cells, designated I and II, that differ intheir morphology, innervation pattern, and ionic membrane properties.Type I cells are unique among hair cells in that their basolateralsurfaces are almost completely enclosed by an afferent calyceal nerveterminal. Recently, several lines of evidence have ascribed a motilefunction to type I hair cells. To investigate this, elevated externalK⁺, which had been used previouslyto induce hair cell shortening, was used to induce shape changes indissociated mammalian type I vestibular hair cells. Morphologicallyidentified type I cells shortened and widened when the externalK⁺ concentration was raisedisotonically from 2 to 125 mM. The shortening did not require externalCa²⁺ but was abolished whenexternal Cl was replacedwith gluconate or sulfate and when externalNa⁺ was replaced withN-methyl-D-glucamine.Bumetanide (10-100 µM), a specific blocker of theNa⁺-K⁺-Cl cotransporter,significantly reduced K⁺-inducedshortening. Hyposmotic solution resulted in type I cell shape changessimilar to those seen with highK⁺, i.e., shortening and widening.Type I cells became more spherical in hyposmotic solution, presumablyas a result of a volume increase due to water influx. In hypertonicsolution, cells became narrower and increased in length. These resultssuggest that shape changes in type I hair cells induced by highK⁺ are due, at least in part, toion and solute entry via anNa⁺-K⁺-Cl cotransporter, whichresults in cell swelling. A scheme is proposed whereby the type I haircell depolarizes and K⁺ leaves thecell via voltage-dependent K⁺channels and accumulates in the synaptic space between the type I haircell and calyx. Excess K⁺ couldthen be removed from the intercellular space by uptake via thecotransporter.

     

Red blood cells do not contribute to removal of K+ released from exhaustively working forearm muscle

K⁺ released from exercisingmuscle via K⁺ channels needs to beremoved from the interstitium into the blood to maintain high musclecell membrane potential and allow normal muscle contractility. Uptakeby red blood cells has been discussed as one mechanism that would alsoserve to regulate red blood cell volume, which was found to be constantdespite increased plasma osmolality and K⁺ concentration([K⁺_pl]). We evaluatedexercise-related changes in[K⁺_pl], pH, osmolality, meancellular Hb concentration, cell water, and red blood cellK⁺ concentration during exhaustivehandgrip exercise. Unidirectional ⁸⁶Rb⁺(K⁺) uptake by red blood cellswas measured in media with elevated extracellularK⁺, osmolarity, andcatecholamines to simulate particularly those exercise-related changesin plasma composition that are known to stimulateK⁺ uptake. During exercise[K⁺_pl] increased from 4.4 ± 0.7 to 7.1 ± 0.5 mmol/l plasma water and red blood cell K⁺ concentration increased from137.2 ± 6.0 to 144.6 ± 4.6 mmol/l cell water(P  0.05), but the intracellularK⁺-to-mean cellularHb concentration ratio did not change.⁸⁶Rb⁺uptake by red blood cells was increased by ~20% on stimulation, caused by activation of theNa⁺-K⁺pump andNa⁺-K⁺-2Clcotransport. Results indicate theK⁺ content of red blood cells didnot change as cells passed the exhaustively exercising forearm muscledespite the elevated [K⁺_pl]. The tendency for an increase in intracellularK⁺ concentration was due to aslight, although statistically not significant, decrease in red bloodcell volume. K⁺ uptake, althoughelevated, was too small to move significant amounts ofK⁺ into red blood cells. Ourresults suggest that red blood cells do not contribute to the removalof K⁺ released from muscle and donot regulate their volume by K⁺uptake during exhaustive forearm exercise.

     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Expression and function of the rat vesicular monoamine transporter 2

The vesicular monoamine transporters (VMATs) are essential proteins, involved in the storage of monoamines in the central nervous system and in endocrine cells, in a process that involves exchange of 2H⁺ with one substrate molecule. The VMATs interact with various native substrates and clinically relevant drugs and display the pharmacological profile of multidrug transporters. Vesicular transporters suffer from a lack of biochemical and structural data due to the difficulties in their expression. In this work we present the high-level expression of rat VMAT2 (rVMAT2) in a stable a human embryonic kidney cell line (HEK293), generated using the resistance to the neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) conferred by the protein. In addition, we describe novel procedures for the solubilization and purification of active protein, and its reconstitution into proteoliposomes. The partially purified protein in detergent binds the inhibitor tetrabenazine and, after reconstitution, displays high levels of µ_H+-driven electrogenic transport of serotonin. The reconstituted purified rVMAT2 has wild-type affinity for serotonin, and its turnover rate is 0.4 substrate molecule/s. membrane protein; ion-coupled transporters; neurotransmitter storage; monoamines     

Transforming growth factor-beta inhibition of proteasomal activity: a potential mechanism of growth arrest

Although the proteasome plays a critical role in the controlled degradation of proteins involved in cell cycle control, the direct modulation of proteasomal function by growth regulatory signaling has not yet been demonstrated. We assessed the effect of transforming growth factor (TGF)-, a potent inhibitor of cell growth, on proteasomal function. TGF- selectively decreased hydrolysis of the proteasomal substrate Cbz-Leu-Leu-Leu-7-amido-4-methyl-coumarin (z-LLL-AMC) in a concentration-dependent manner but did not inhibit hydrolysis of other substrates Suc-Leu-Leu-Val-Tyr-AMC (suc-LLVY-AMC) or Cbz-Leu-Leu-Glu-AMC (z-LLE-AMC). An increase in intracellular oxidative injury occurred during incubation with TGF-. Furthermore, in vitro hydrolysis of z-LLL-AMC, but not suc-LLVY-AMC, was decreased by hydrogen peroxide. TGF- did not increase cellular expression of heat shock protein (HSP)90, a potent inhibitor of z-LLL-AMC hydrolysis in vitro. The physiological relevance of TGF- inhibition of proteasomal activity was studied by assessing the role of z-LLL-AMC hydrolysis on cyclin-dependent kinase inhibitor expression and cell growth. TGF- increased expression of p27^KIP1 but did not alter expression of p21^WAF1 or p16^INK4A. The peptide aldehyde Cbz-Leu-Leu-leucinal (LLL-CHO or MG132) potently inhibited z-LLL-AMC hydrolysis in cell extracts as well as increasing p27^KIP1 and decreasing cell proliferation. Thus growth inhibition by TGF- decreases a specific proteasomal activity via an HSP90-independent mechanism that may involve oxidative inactivation or modulation of proteasomal subunit composition and results in altered cellular expression of key cell cycle regulatory proteins such as p27^KIP1. oxidative stress; cytokine; heat shock protein; cell cycle regulation     

Modulation of vascular smooth muscle cell migration by calcium/ calmodulin-dependent protein kinase II-delta 2

Previous studies demonstrated a requirement for multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (₂ or _C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII₂ were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr¹⁷, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII₂ inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII₂ autophosphorylation on Thr²⁸⁷ after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII₂ phosphorylated substrate in vitro without added Ca²⁺/calmodulin and in the intact cell without added Ca²⁺-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII₂ on Thr²⁸⁷. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII₂, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII₂ mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII₂ isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII₂ autophosphorylation may play an important role in PDGF-stimulated VSM cell migration. calcium/calmodulin-dependent protein kinase II; cell migration; adenovirus; autophosphorylation; chemotaxis; platelet-derived growth factor     

Genetic and biochemical determinants of abnormal monovalent ion transport in primary hypertension

Data obtainedduring the last two decades show that spontaneously hypertensive rats,an acceptable experimental model of primary human hypertension, possessincreased activity of both ubiquitous and renal cell-specific isoformsof theNa⁺/H⁺exchanger (NHE) andNa⁺-K⁺-2Clcotransporter. Abnormalities of these ion transporters have been foundin patients suffering from essential hypertension. Recent geneticstudies demonstrate that genes encoding the - and -subunits ofENaC, a renal cell-specific isoform of theNa⁺-K⁺-2Clcotransporter, and 3-, 1-, and 2-subunits of theNa⁺-K⁺pump are localized within quantitative trait loci (QTL) for elevated blood pressure as well as for enhanced heart-to-body weight ratio, proteinuria, phosphate excretion, and stroke latency. On the basis ofthe homology of genome maps, several other genes encoding these transporters, as well as theNa⁺/H⁺exchanger andNa⁺-K⁺-2Clcotransporter, can be predicted in QTL related to the pathogenesis ofhypertension. However, despite their location within QTL, analysis ofcDNA structure did not reveal any mutation in the coding region of theabove-listed transporters in primary hypertension, with the exceptionof G276L substitution in the1-Na⁺-K⁺pump from Dahl salt-sensitive rats and a higher occurrence of T594Mmutation of -ENaC in the black population with essential hypertension. These results suggest that, in contrast to Mendelian forms of hypertension, the altered activity of monovalent ion transporters in primary hypertension is caused by abnormalities ofsystems involved in the regulation of their expression and/or function.Further analysis of QTL in F₂hybrids of normotensive and hypertensive rats and in affected siblingpairs will allow mapping of genes causing abnormalities ofthese regulatory pathways.     

Inhibition of Ca2+-dependent Cl- secretion in T84 cells: membrane target(s) of inhibition is agonist specific

Previous studies have indicated thatCa²⁺-dependentCl secretion acrossmonolayers of T84 epithelial cells is subject to a variety of negativeinfluences that serve to limit the overall extent of secretion.However, the downstream membrane target(s) of these inhibitoryinfluences had not been elucidated. In this study, nuclide effluxtechniques were used to determine whether inhibition ofCa²⁺-dependentCl secretion induced bycarbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor,or insulin reflected actions at an apical Cl conductance, abasolateral K⁺ conductance, orboth. Pretreatment of T84 cell monolayers with carbachol or acell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced theability of subsequently added thapsigargin to stimulate apical¹²⁵I,but not basolateral⁸⁶Rb⁺,efflux. These data suggested an effect on an apicalCl channel. Conversely,epidermal growth factor reducedCa²⁺-stimulated⁸⁶Rb⁺but not¹²⁵Iefflux, suggesting an effect of the growth factor on aK⁺ channel. Finally, insulininhibited¹²⁵Iand⁸⁶Rb⁺effluxes. Thus effects of agents that inhibit transepithelial Cl secretion are alsomanifest at the level of transmembrane transport pathways. However, theprecise nature of the membrane conductances targeted are agonistspecific.

     

Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin alpha1/beta1/alphav and PDGF receptor pathways

Embryonic stem (ES) cells can differentiate into smooth muscle cells (SMCs) that can be used for tissue engineering and repair of damaged organs. However, little is known about the molecular mechanisms of differentiation in these cells. In the present study, we found collagen IV can promote ES cells to differentiate into stem cell antigen-1-positive (Sca-1⁺) progenitor cells and SMCs. Pretreatment of ES cells with antibodies against collagen IV significantly inhibited SMC marker expression. To further elucidate the effect of collagen IV on the induction and maintenance of SMC differentiation, Sca-1⁺ progenitor cells were isolated with magnetic beads, placed in collagen-IV-coated flasks, and cultured in differentiation medium with or without platelet-derived growth factor (PDGF)-BB for 6–90 days. Both immunostaining and fluorescence-activated cell sorter analyses revealed that the majority of these cells were positive for SMC-specific markers. Pretreatment of Sca-1⁺ progenitors with antibodies against integrin ₁, _v, and ₁, but not ₃, inhibited focal adhesion kinase (FAK) and paxillin phosphorylation and resulted in a marked inhibition of SMC differentiation. Various tyrosine kinase inhibitors, and specific siRNA for phosphatidylinositol 3-kinase (PI 3-kinase) and PDGF receptor- significantly inhibited SMC marker expression. Taken together, we demonstrate for the first time that collagen IV plays a crucial role in the early stage of SMC differentiation and that integrin (₁, ₁, and _v)-FAK-PI 3-kinase-mitogen-activated protein kinase and PDGF receptor- signaling pathways are involved in SMC differentiation. progenitor cells; extracellular matrix; growth factor receptors; platelet-derived growth factor     

Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate

Na⁺-K⁺-2Clcotransporters are important in renal salt reabsorption and in saltsecretion by epithelia. They are also essential in maintenance andregulation of ion gradients and cell volume in both epithelial andnonepithelial cells. Expression ofNa⁺-K⁺-2Clcotransporters in brain tissues is high; however, little is known abouttheir function and regulation in neurons. In this study, we examinedregulation of theNa⁺-K⁺-2Clcotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitiveK⁺ influx, and protein expressionwas evaluated by Western blot analysis. Glutamate was found to induce adose- and time-dependent stimulation ofNa⁺-K⁺-2Clcotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonistN-methyl-D-asparticacid (NMDA) and the metabotropic receptor agonist(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantlystimulated the cotransport activity in these cells.NMDA-mediated stimulation of theNa⁺-K⁺-2Clcotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate.trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)--methyl-(4-carboxyphenyl)glycine. The results demonstrate thatNa⁺-K⁺-2Clcotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.