Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat

Time-dependent changes in myosin heavy chain(MHC) isoform expression were investigated in rat soleus muscleunloaded by hindlimb suspension. Changes at the mRNA level weremeasured by RT-PCR and correlated with changes in the pattern of MHCprotein isoforms. Protein analyses of whole muscle revealed that MHCIdecreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showedthat MHCIId(x) displayed the steepest increase after 4 days andcontinued to rise until 28 days, the longest time period investigated.MHCIIb mRNA followed a similar time course, although at lower levels.MHCI mRNA, present at extremely low levels in control soleus, peakedafter 4 days, stayed elevated until 15 days, and then decayed.Immunohistochemistry of 15-day unloaded muscles revealed that MHCIwas present in muscle spindles but at low amounts also in extrafusalfibers. The slow-to-fast transitions thus seem to proceed in the orderMHCI  MHCIIa  MHCIId(x)  MHCIIb. Ourfindings indicate that MHCI is transiently upregulated in somefibers as an intermediate step during the transition from MHCI to MHCIIa.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging

The fluorescence of quinolinium-basedCl indicators such as6-methoxy-N-(3-sulfopropyl)quinolinium(SPQ) is quenched by Cl bya collisional mechanism without change in spectral shape. A series of"chimeric" dual-wavelengthCl indicators weresynthesized by conjugatingCl-sensitive and-insensitive chromophores with spacers. The SPQ chromophore(N-substituted 6-methoxyquinolinium; MQ) was selected as theCl-sensitive moiety[excitation wavelength(_ex) 350 nm, emission wavelength (_em) 450 nm]. N-substituted 6-aminoquinolinium (AQ) waschosen as theCl-insensitive moietybecause of its different spectral characteristics (_ex 380 nm,_em 546 nm), insensitivity toCl, positive charge (tominimize quenching by chromophore stacking/electron transfer), andreducibility (for noninvasive cell loading). The dual-wavelengthindicators were stable and nontoxic in cells and were distributeduniformly in cytoplasm, with occasional staining of the nucleus. Thebrightest and mostCl-sensitive indicatorswere -MQ-'-dimethyl-AQ-xylene dichloride andtrans-1,2-bis(4-[1-'-MQ-1'-'-dimethyl-AQ-xylyl]-pyridinium)ethylene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm(Cl sensitive; Stern-Volmerconstants 82 and 98 M¹)and 565 nm (Clinsensitive). Cystic fibrosis transmembrane conductanceregulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQby hypotonic shock or were labeled with its uncharged reduced form(octahydro-bis-DMXPQ) by brief incubation (20 µM, 10 min). Changes inCl concentration inresponse to Cl/nitrateexchange were recorded by emission ratio imaging (450/565 nm) at 365-nmexcitation wavelength. These results establish a first-generation setof chimeric bisquinoliniumCl indicators forratiometric measurement ofCl concentration.     

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.

     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio

We proposed andtested the use of nontraditional excitation wavelengths(₁ and ₂) and an emission wavelength(_em) to define conditions under which free calciumconcentration and a fluorescence ratio are linearly related.Fluorescence spectra were determined for aqueous solutions thatcontained 25 µM fura 2, 125 mM K⁺, and either 0 mM or 0.1 mM Ca²⁺. Effectively linear relationships between[Ca²⁺] and a fluorescence ratio, i.e., <5% bias when[Ca²⁺]  5 × dissociation constant, were apparentwhen ₁  400 nm, ₂  370 nm, and_em  510 nm. Combinations with longer ₁and _em and/or with shorter ₂ reduced thisbias further. Although the method described does not obviate thecomplications that surround the correction for fluorescence background,choosing a nontraditional combination of excitation and emissionwavelengths offers several practical advantages over more traditionalfura 2 fluorescence methodologies in a variety of experimental settings.

     

TGF-alpha reduces bradykinin-stimulated ion transport and prostaglandin release in human colonic epithelial cells

The effect of chronic exposure to transforming growth factor-(TGF-) on bradykinin-stimulated acute prostanoid production and ionsecretion in monolayers of HCA-7 colony 29 colonic epithelial cells hasbeen studied. Monolayers synthesized prostaglandinE₂ (PGE₂) at a basal rate of 2.10 ± 0.31 pg · monolayer¹ · min¹over 24 h. Bradykinin(10⁸-10⁵M) dose dependently increased acutePGE₂ release by three orders ofmagnitude. This was associated with a rise in cAMP from 1.60 ± 0.14 to 2.90 ± 0.1 pmol/monolayer (P < 0.02) and a dose-dependent increase in short-circuit current (SCC).When monolayers were primed by a 24-h exposure to TGF-, basalPGE₂ release rose to 6.31 ± 0.38 pg · monolayer¹ · min¹(TGF- concn 10 ng/ml; P = 0.001).However, the stimulation of acute prostaglandin release, intracellularcAMP, and increased SCC by bradykinin was significantly reduced bypreincubation with TGF-. Priming withPGE₂(10⁸-10⁶M) over 24 h mimicked the effect of TGF- on bradykinin-induced changes in cAMP and SCC. These data suggest that enhanced chronic release of prostaglandins in response to stimulation with TGF- maydownregulate acute responses to bradykinin. In vivo, TGF- could havean important modulatory function in regulating secretion underinflammatory conditions.     

CFTR induces the expression of DRA along with Cl(-)/HCO(3)(-) exchange activity in tracheal epithelial cells

Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO₃across the tracheal epithelium. The mechanism of the defect in HCO₃ secretion is ill defined; however, a lack ofapical Cl/HCO₃ exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO₃ exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO₃exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO₃ exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO₃ exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO₃ exchanger activity intracheal cells. We propose that the tracheal HCO₃secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO₃exchange activity mediated by DRA.

     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

Carbachol-induced desensitization of PLC-beta pathway in rat myometrium: downregulation of Gqalpha /G11alpha

In the estrogen-treated rat myometrium, carbachol increased thegeneration of inositol phosphates by stimulating the muscarinic receptor-G_q/G₁₁-phospholipaseC-3 (PLC-3) cascade. Exposure to carbachol resulted in a rapidand specific (homologous) attenuation of the subsequent muscarinicresponses in terms of inositol phosphate production, PLC-3translocation to membrane, and contraction. Refractoriness wasaccompanied by a reduction of membrane muscarinic binding sites and anuncoupled state of residual receptors. Protein kinase C (PKC) alteredthe functionality of muscarinic receptors and contributed to theinitial period of desensitization. A delayed phase of the muscarinicrefractoriness was PKC independent and was associated with adownregulation ofG_q/G₁₁.Atropine failed to induce desensitization as well asG_q/G₁₁downregulation, indicating that both events involve active occupancy ofthe receptor. Prolonged exposure toAlF₄ reduced subsequent AlF₄ as well as carbachol-mediatedinositol phosphate responses and similarly induced downregulation ofG_q/G₁₁. Data suggest that a decrease in the level ofG_q/G₁₁is subsequent to its activation and may account forheterologous desensitization.

     

Inhibition of cell differentiation by Galpha q in the renal epithelial cell line LLC-PK1

LLC-PK₁, an epithelial cellline derived from the kidney proximal tubule, was used to study theability of the G protein -subunit, G_q, to regulate celldifferentiation. A constitutively active mutant protein,_qQ209L, was expressed using theLacSwitch-inducible mammalian expression system. Induction of_qQ209L expression with isopropyl--D-thiogalactopyranoside(IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold.Increasing concentrations of IPTG progressively inhibited the activityof two differentiation markers,Na⁺-dependent hexose transport andalkaline phosphatase activity. Induction of_qQ209L expression also caused achange from an epithelial to a spindle-shaped morphology. The effectsof _qQ209L expression on celldifferentiation were similar to those observed with12-O-tetradecanoylphorbol 13-acetate(TPA) treatment. However, protein kinase C (PKC) levels weredownregulated in TPA-treated cells but not in_qQ209L-expressing cells,suggesting that the regulation of PKC byG_q may be different fromregulation by TPA. Interestingly, the PKC inhibitor GF-109203X did notinhibit the effect of IPTG on the development ofNa⁺-dependent hexose transport in_qQ209L-expressing cells. These data implicate PKC and PKC in the pathway used byG_q to block the development ofNa⁺-dependent hexose transport inIPTG-treated cells.

     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Calcium channels are composed of a pore-forming subunit,₁, and at least two auxiliarysubunits, - and₂-subunits. It is well knownthat -subunits regulate most of the properties of the channel. Thefunction of ₂-subunit isless understood. In this study, the effects of the calcium channel₂-subunit on the neuronal_1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe _1b- or the_2a-subunit. Most aspects of_1E function were affected by₂. Thus₂ caused a shift in thecurrent-voltage and conductance-voltage curves toward more positivepotentials and accelerated activation, deactivation, and theinstallation of the inactivation process. In addition, the efficiencywith which charge movement is coupled to pore opening assessed bydetermining ratios of limiting conductance to limiting charge movementwas decreased by ₂ byfactors that ranged from 1.6 (P < 0.01) for _1E-channels to 3.0 (P < 0.005) for_1E1b-channels. These results indicate that₂ facilitates the expressionand the maturation of_1E-channels and converts thesechannels into molecules responding more rapidly to voltage.

     

Apical and basolateral CO2-HCO-3 permeability in cultured bovine corneal endothelial cells

Corneal endothelial function is dependent onHCO₃ transport. However, the relativeHCO₃ permeabilities of the apical andbasolateral membranes are unknown. Using changes in intracellular pHsecondary to removingCO₂-HCO₃ (at constant pH) or removing HCO₃alone (at constant CO₂) fromapical or basolateral compartments, we determined the relative apicaland basolateral HCO₃ permeabilities and their dependencies on Na⁺ andCl. Removal ofCO₂-HCO₃from the apical side caused a steady-state alkalinization (+0.08 pHunits), and removal from the basolateral side caused an acidification(0.05 pH units). Removal ofHCO₃ at constantCO₂ indicated that the basolateralHCO₃ fluxes were about three to fourtimes the apical fluxes. Reducing perfusateNa⁺ concentration to 10 mM had noeffect on apical flux but slowed basolateralHCO₃ flux by one-half. In the absence of Cl, there was anapparent increase in apical HCO₃ fluxunder constant-pH conditions; however, no net change could be measuredunder constant-CO₂ conditions.Basolateral flux was slowed ~30% in the absence ofCl, but the net flux wasunchanged. The steady-state alkalinization after removal ofCO₂-HCO₃apically suggests that CO₂diffusion may contribute to apicalHCO₃ flux through the action of amembrane-associated carbonic anhydrase. Indeed, apicalCO₂ fluxes were inhibited by theextracellular carbonic anhydrase inhibitor benzolamide and partiallyrestored by exogenous carbonic anhydrase. The presence ofmembrane-bound carbonic anhydrase (CAIV) was confirmed byimmunoblotting. We conclude that theNa⁺-dependent basolateralHCO₃ permeability is consistent withNa⁺-nHCO₃cotransport. Changes inHCO₃ flux in the absence ofCl are most likely due toNa⁺-nHCO₃cotransport-induced membrane potential changes that cannot bedissipated. Apical HCO₃ permeabilityis relatively low, but may be augmented byCO₂ diffusion in conjunction witha CAIV.

     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport

A role for protein kinase C (PKC)- and -isotypes in ₁-adrenergicregulation of human tracheal epithelial Na-K-2Cl cotransport wasstudied with the use of isotype-specific PKC inhibitors and antisenseoligodeoxynucleotides to PKC- or - mRNA. Rottlerin, a PKC-inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive ³⁶Cl flux innystatin-permeabilized cell monolayers stimulated with methoxamine, an₁-adrenergic agonist, with a50% inhibitory concentration of 2.3 µM. Methoxamine increased PKCactivity in cytosol and a particulate fraction; the response wasinsensitive to PKC- and -_IIisotype-specific inhibitors, but was blocked by general PKC inhibitorsand rottlerin. Rottlerin also inhibited methoxamine-induced PKCactivity in immune complexes of PKC-, but not PKC-. At the subcellular level, methoxamine selectively elevated cytosolic PKC-activity and particulate PKC- activity. Pretreatment of cellmonolayers with antisense oligodeoxynucleotide to PKC- for 48 hreduced the amount of whole cell and cytosolic PKC-, diminished whole cell and cytosolic PKC- activity, and blockedmethoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotideto PKC- and antisense oligodeoxynucleotide to PKC- did not altermethoxamine-induced cotransport activity. These results demonstrate theselective activation of Na-K-2Cl cotransport by cytosolic PKC-.

     

AVP V1 receptor-mediated decrease in Cl- efflux and increase in dark cell number in choroid plexus epithelium

The cerebrospinalfluid (CSF)-generating choroid plexus (CP) has manyV₁ binding sites for argininevasopressin (AVP). AVP decreases CSF formation rate and choroidal bloodflow, but little is known about how AVP alters ion transport across theblood-CSF barrier. Adult rat lateral ventricle CP was loaded with³⁶Cl,exposed to AVP for 20 min, and then placed in isotope-free artificial CSF to measure release of³⁶Cl.Effect of AVP at 10¹² to10⁷ M on theCl efflux rate coefficient(in s¹) was quantified.Maximal inhibition (by 20%) ofCl extrusion at10⁹ M AVP was prevented bythe V₁ receptor antagonist[-mercapto-,-cyclopentamethyleneproprionyl¹,O-Me-Tyr²,Arg⁸]vasopressin.AVP also increased by more than twofold the number of dark and possiblydehydrated but otherwise morphologically normal choroid epithelialcells in adult CP. The V₁ receptorantagonist prevented this AVP-induced increment in dark cell frequency.In infant rats (1 wk) with incomplete CSF secretory ability,10⁹ M AVP altered neitherCl efflux nor dark cellfrequency. The ability of AVP to elicit functional and structuralchanges in adult, but not infant, CP epithelium is discussed in regardto ion transport, CSF secretion, intracranial pressure, and hydrocephalus.

     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.

     

Role of the alpha -subunit 326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

The platelet GPIIb-GPIIIa heterodimer (integrin_IIb3)binds fibrinogen with high affinity in response to activation byagonists, leading to platelet aggregation and formation of a hemostatic plug. The ³²⁶GRV motif in GPIIb ishighly conserved in the -subunit of other integrins, suggesting thatit might play an important functional role. Moreover,Arg³²⁷His substitution inGPIIb has been associated with defective platelet surface expression ofGPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidatingwhether the absence of Arg³²⁷ orits substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala³²⁷]GPIIb,[Gln³²⁷]GPIIb, or[Phe³²⁷]GPIIb intoChinese hamster ovary cells inherently expressing GPIIIa permittedsurface exposure of GPIIb-IIIa complexes, whereas [Glu³²⁷]GPIIb did not.These observations indicate that it is not the loss of[Arg³²⁷]GPIIb but thepresence of His³²⁷ or a negativelycharged residue like Glu at position 327 of GPIIb that prevents thesurface exposure of GPIIb-IIIa heterodimers. In contrast, changingGln³⁴⁴, the homologue toArg³²⁷ in the -subunit of thevitronectin receptor, to His did not prevent the surface expression of_v-GPIIIacomplexes. Thus the conformational constraint imposed byHis³²⁷ seems to be rather specificfor the heterodimerization and/or processing of GPIIb-IIIacomplexes.