PRODUCTION OF YEAST VARIANTS BY AUXIN AND EFFECTS OF PLANT GROWTH REGULATORS ON THESE VARIANTS

Using diploid strains of Saccharomyces cerevisiae and S. ellipsoideus,the following facts were found:

1. Indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid and -naphthaleneaceticacid produced stable variants differing in the cell form andin the response to the actions of auxin to elongate cells, toinduce respiration- deficient mutation and to promote sporulation.
2. The auxins also produced stable variants differing in theabilityto form spores.
3. Acetic acid had no above-menthionedactions of auxin.
4. Spore-formation and cell elongation of someof auxin-inducedvariants were controlled by auxin.

Biological significance of the auxin-induced variation is discussedand the usefulness of some of these variants as experimentalmaterial for auxin physiology in general is pointed out. (Received November 1, 1966; )     

Correlation between Lack of Receptacle Growth in Response to Auxin and Accumulation of a Specific Polypeptide in a Strawberry (Fragaria ananassa Duch.) Variant Genotype

Receptacle growth in strawberry (Fragaria ananassa Duch. cv.Ozark Beauty) occurred after either pollination or auxin treatment.In a strawberry variant genotype (Washington State UniversitySelection No. 12/13), pollination did not lead to receptaclegrowth but application of -naphthaleneacetic acid (NAA) at anthesisresulted in normal receptacle growth. The receptacles of OzarkBeauty retained their ability to respond to auxin at least upto 36 days after anthesis. However, delay of auxin applicationto the receptacles of the variant genotype resulted in decreasedauxin-responsive growth and auxin application after the 10thday of anthesis led to very little growth. The loss of auxin-responsivegrowth of the receptacle of the variant genotype was not associatedwith any loss of auxin binding activity of receptacle membranes.If auxin was not supplied to the receptacles of the variantgenotype at anthesis, the receptacles did not grow and a polypeptideof 52,000 M_r accumulated. Application of NAA to the receptaclesof the variant genotype at anthesis or on the fifth day afteranthesis resulted in the growth of the receptacle and the 52,000M_r polypeptide did not accumulate. Application of NAA to thereceptacles of the variant genotype on the 10th or the 15thday after anthesis led to very little growth of the receptacleand the 52,000 M_r polypeptide accumulated to high levels. Theseresults suggested a correlation between the lack of receptaclegrowth in response to auxin and accumulation of the 52,000 M_rpolypeptide. ¹ Current adress: The Institute of Applied Research, Ben GurionUniversity of the Negev, Beer-Sheva, Israel. (Received August 6, 1984; Accepted December 11, 1984)     

Molecular dynamics simulations of high-mannose oligosaccharides

Conformations of several high-mannose-type oligosaccharidesthat are generated during the biosynthetic degradation of Man₉GlcNAc₂to Man₅GlcNAc₂ have been studied by molecular dynamics (MD).Simulations were performed on NCI-FCRDC's Cray Y-MP 8D/8128supercomputer using Biosym's CVFF force field for 1000 Ps withdifferent initial conformations. The conformations of the two1,3- and the two 1,6-linkages in each oligomannose were different,suggesting that deriving oligosaccharide conformations basedon the conformational preferences of the constituent disaccharidefragments will not always yield correct results. Unlike otheroligomannoses, Man₉GlcNAc₂ appears to take more than one distinctconformation around the core 1,6-linkage. These various conformationsmay play an important role in determining the processing pathways.Using the data on the preferred conformations of these oligomannosesand the available experimental results, possible pathways forprocessing Man₉GlcNAc₂ to Man₅GlcNAc₂ by 1,2-linkage-specificmannosidases have been proposed. Conformational analysis ofMan₅GlcNAc₂ indicates that the addition of ß1,2-GlcNActo the 1,3-linked core mannose, besides serving as a prerequisitefor mannosidase II action as suggested earlier, may also preventthe removal of 1,3-mannose. The MD simulations also suggestthat the processing of the precursor oligosaccharide duringAsn-linked complex and hybrid glycan biosynthesis proceeds ina well-defined pathway involving more than one 1,2-linkage-specificmannosidase. Knowledge of the conformation of the processingintermediates obtained from the present study can be used todesign highly specific substrate analogues to inhibit a particularmannosidase, thereby blocking one processing pathway withoutinterfering with the others. carbohydrates conformation glycosidase inhibitors mannosidase oligosaccharide processing     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

The Effects of Auxin on the Mechanical Properties in Vivo of Cell Wall in Hypocotyl Segments from Gibberellin-Deficient Cowpea Seedlings

Auxin-induced changes in the mechanical properties of cell wallwere examined by both positive and negative pressure jump methodsusing hypocotyl segments excised from the 3-day-old seedlingsof cowpea that has been treated with uniconazole, a potent inhibitorof the biosynthesis of gibberellins. In such segments (U-segments)that were deficient in endogenous gibberellin, auxin increasedonly the effective turgor (Pⁱ–Y) and did not change theextensibility () of cell wall. As a result, the extent of theauxin-induced promotion of growth was halved. However, auxinwas able to increase of U-segments that has been pretreatedfor two hours with GA₃ prior to the application of IAA. Measurementof intracellular pressure (P_i) with a pressure probe revealedthat auxin did not change Pⁱ in either U-segments or GA₃-pretreatedsegments. The results suggest that auxin can decrease the yieldthreshold of the cell wall (Y) independently of gibberellinbut can increase only in the presence of gibberellin. The differencebetween and Y in terms of their requirement for gibberellinto respond to auxin suggests that they are mutually separablemechanical properties that originate from different molecularprocesses that occur in the architecture of yielding cell walls. ³Present address: Ohishi, Enden, Mori-machi, Shuchi-gun, Shizuoka,437-02 Japan     

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

We evaluated theeffects of acute hyperoxic exposure on alveolar epithelial cell (AEC)active ion transport and on expression ofNa⁺ pump(Na⁺-K⁺-ATPase)and rat epithelial Na⁺ channelsubunits. Rat AEC were cultivated in minimal defined serum-free medium(MDSF) on polycarbonate filters. Beginning on day5, confluent monolayers were exposedto either 95% air-5% CO₂(normoxia) or 95% O₂-5%CO₂ (hyperoxia) for 48 h.Transepithelial resistance(R_t) andshort-circuit current(I_sc) weredetermined before and after exposure.Na⁺ channel -, -, and-subunit andNa⁺-K⁺-ATPase₁- and₁-subunit mRNA levels werequantified by Northern analysis.Na⁺ pump₁- and₁-subunit protein abundance wasquantified by Western blotting. After hyperoxic exposure,I_sc across AECmonolayers decreased by ~60% at 48 h relative to monolayersmaintained under normoxic conditions.Na⁺ channel -subunit mRNAexpression was reduced by hyperoxia, whereas - and -subunit mRNAexpression was unchanged. Na⁺ pump₁-subunit mRNA was unchanged,whereas ₁-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in₁-subunit protein. Becausekeratinocyte growth factor (KGF) has recently been shown to upregulateAEC active ion transport and expression ofNa⁺-K⁺-ATPaseunder normoxic conditions, we assessed the ability of KGF to preventhyperoxia-induced changes in active ion transport by supplementingmedium with KGF (10 ng/ml) from day2. The presence of KGF prevented theeffects of hyperoxia on ion transport (as measured byI_sc) relativeto normoxic controls. Levels of₁ mRNA and protein wererelatively preserved in monolayers maintained in MDSF and KGF comparedwith those cultivated in MDSF alone. These results indicate that AECnet active ion transport is decreased after 48 h of hyperoxia, likelyas a result of a decrease in the number of functionalNa⁺ pumps per cell. KGF largelyprevents this decrease in active ion transport, at least in part, bypreserving Na⁺ pump expression.

     

Uptake and Accumulation of {alpha}-Naphthalene Acetic Acid by Cell Suspensions of Galium mollugo L.

Fidgeon, C. and Wilson, G. 1988. Uptake and accumulation ofa-naphthalene acetic acid by cell suspensions of Galium mollugoL.—J. exp. Bot. 39: 241-249. Galium mollugo cell suspensions require -NAA for continued growthand cell division. The kinetics of -NAA uptake from the medium(B₅) by Galium cells was assessed using 1-¹⁴C -NAA in a standardratio of cells to medium (0.25 g: 2.5 cm³). It was found thatthe uptake of -NAA was rapid, over 90% being taken up within4 h. Cells which had accumulated -NAA for 4 h or more did notrelease it back into the medium. It was found that Galium cellsaccumulated -NAA against a significant concentration gradient;suggesting the participation of an active component in the uptakemechanism. The effect of free-space and surface adsorption onthe uptake of -NAA was determined by means of a repeated washtechnique. These two factors were found to be of importanceonly during the first hour of uptake. Neither dead cells norplasmolysed cells absorbed -NAA. It is clear that, in the normal growth cycle, Galium cells cantake up the available -NAA within 3 or 4 h of inoculation andthat this can stimulate a cell division response of 3-4 generationsover the subsequent 14 d. Key words: Galium, cell suspension, -naphthalene acetic acid     

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.

     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium

In vitro mammary epithelial cell models typically fail to form a consistently tight barrier that can effectively separate blood from milk. Our hypothesis was that mammary epithelial barrier function would be affected by changes in luminal ion concentration and inflammatory cytokines. Bovine mammary epithelial (BME-UV cell line) cells were grown to confluence on permeable supports with a standard basolateral medium and either high-electrolyte (H-elec) or low-electrolyte (L-elec) apical medium for 14 days. Apical media were changed to/from H-elec medium at predetermined times prior to assay. Transepithelial electrical resistance (R_te) was highest in monolayers continuously exposed to apical L-elec. A time-dependent decline in R_te began within 24 h of H-elec medium exposure. Change from H-elec medium to L-elec medium time-dependently increased R_te. Permeation by FITC-conjugated dextran was elevated across monolayers exposed to H-elec, suggesting compromise of a paracellular pathway. Significant alteration in occludin distribution was evident, concomitant with the changes in R_te, although total occludin was unchanged. Neither substitution of Na⁺ with N-methyl-D-glucosamine (NMDG⁺) nor pharmacological inhibition of transcellular Na⁺ transport pathways abrogated the effects of apical H-elec medium on R_te. Tumor necrosis factor alpha, but not interleukin-1 nor interleukin-6, in the apical compartment caused a significant decrease in R_te within 8 h. These results indicate that mammary epithelium is a dynamic barrier whose cell-cell contacts are acutely modulated by cytokines and luminal electrolyte environment. Results not only demonstrate that BME-UV cells are a model system representative of mammary epithelium but also provide critical information that can be applied to other mammary model systems to improve their physiological relevance. transepithelial electrical resistance; apical cation concentration; paracellular permeability; mastitis; inflammatory cytokines; occludin     

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells

To clarifyinteractions between the cytoskeleton and activity of L-typeCa²⁺ (Ca_L) channels in vascular smooth muscle(VSM) cells, we investigated the effect of disruption of actinfilaments and microtubules on the L-type Ca²⁺ current[I_Ba(L)] of cultured VSM cells (A7r5 cellline) using whole cell voltage clamp. The cells were exposed to eachdisrupter for 1 h and then examined electrophysiologically andmorphologically. Results of immunostaining using anti--actin andanti--tubulin antibodies showed that colchicine disrupted both actinfilaments and microtubules, cytochalasin D disrupted only actinfilaments, and nocodazole disrupted only microtubules.I_Ba(L) was greatly reduced in cells that wereexposed to colchicine or cytochalasin D but not to nocodazole.Colchicine even inhibited I_Ba(L) by about 40%when the actin filaments were stabilized by phalloidin or when thecells were treated with phalloidin plus taxol to stabilize bothcytoskeletal components. These results suggest that colchicine mustalso cause some inhibition of I_Ba(L) due toanother unknown mechanism, e.g., a direct block of Ca_Lchannels. In summary, actin filament disruption of VSM cells inhibitsCa_L channel activity, whereas disrupting the microtubulesdoes not.

     

A Transient Stimulation of Net Photosynthesis of Rye Leaves by {alpha}-Hydroxy-2-pyridinemethanesulphonic Acid ({alpha}-HPMS) due to Inhibition of Photorespiratory CO2 Release

The effects of -hydroxy-2-pyridinemethanesulphonic acid (-HPMS)upon net photosynthesis (P_n, the CO₂ compensation point (),post-lower illumination burst of CO₂ (PLIB) and post-lower temperatureburst of CO₂ (PLTB) in detached rye (Secale cereale L.) leaveswere investigated. At low concentrations ( 0.5 mol m^–3),-HPMS initially stimulated P_n and decreased the magnitude ofboth PLIB and PLTB. The decreased at all concentrations of-HPMS (0.05–5.0 mol m^–3. The effects of -HPMS onP_n and were time-dependent and, after a few minutes, the P_nwas inhibited while values increased considerably. At a higherconcentration (5.0 mol m ^–3), the transient effects of-HPMS were shorter () or not observed at all (P_n. Both PLIBand PLTB, when expressed in relation to P_n, increased at higherlevels of this compound. Similar data with respect to the effectsof -HPMS on PLIB and PLTB were found for leaves of dandelion(Taraxacum officinale L.). The results suggest that -HPMS may stimulate P_n by inhibitingphotorespiration, as originally suggested by Zelitch (1966),but only at low concentrations and over a short time span. Thedecrease of PLIB and PLTB values at low -HPMS levels is consistentwith these processes being a residual activity of the glycolatepathway. Key words: CO₂ compensation point, -hydroxy-2-pyridinemethanesulphonic acid, photorespiration, photosynthesis     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

Photoinhibition and the diurnal variation of phytoplankton photosynthesis--I. Development of a photosynthesis--irradiance model from studies of in situ responses

Diurnal series of fluorescence and photosynthesis assays wereconducted in high altitude (3803 m), tropical (16°), LakeTiticaca (Peru/Bolivia). Near-surface diurnal thermoclines formedon typical days of high photon flux density (PFD, {small tilde}2000 µE m^–2 s^–1). In the depth range of diurnalstratification profiles of in vivo fluorescence, both without(F_a and with (F_b DCMU, exhibited a mean decrease of 64% frommorning to mid-day, but little change (mean increase of 1.5%)through the afternoon. Three times during the day surface, mid-depth(3–5 m) and deep (15–20 m) phytoplankton sampleswere incubated with H¹⁴CO₃^– under short (<2 h) exposuresto a range of in situ PFDs. Comparison of phytoplankton in differentsamples (ANOVA) showed identical photosynthetic response insunrise (isothermal) samples but a significant drop in surfaceand mid-depth photosynthesis at all PFDs during times of diurnalstratification. Similarly, both low-light () and light-saturated(P² _max photosynthetic parameters were lower in mid-day surfacesamples compared to deep samples. In addition, previously photoinhibitedsamples had a higher threshold intensity for photoinhibition,I_T. These results, together with diurnal time series of fluorescencefrom in situ incubations, demonstrate that recovery from extendedepisodes of photoinhibition during diurnal stratification isslower than suggested by previous observations in vitro. Photosynthesisby near-surface phytoplankton is different in light increasingup to I_T than light decreasing from I_T. This effect can be modeledby reducing and P_max as a function of the maximum photoinhibitingPFD in the diurnal light history. ¹Present address: Division of Molecular Plant Biology, Universityof California, Berkeley, Berkeley, CA 94720, USA     

Inhibition of Gibberellic Acid-induced Starch Mobilization by Salicylhydroxamic acid in Avena fatua L. Seed

Inhibition of GA₃-induced endosperm mobilization in Avena fatuaL. by salicylhydroxamic acid (SHAM), a widely used alternativerespiration inhibitor, was studied. SHAM strongly inhibitedthe GA₃-induced release of reducing sugars in the incubationmedium by 3 mm de-embryonated endosperm segments; at 4 mM SHAM,GA₃-induced sugar release was inhibited by 66–79 per cent.Extracts prepared from segments incubated in 0.05 mM GA₃ with2, 5 and 10 mM SHAM showed 30, 53 and 71 per cent lower -amylaseactivity, respectively, compared to the GA₃-alone treatment.Addition of SHAM (0.5–5 mM) during the enzyme assay hadno effect on the activity of -amylase. Thus, the inhibitionof starch mobilization in endosperm by SHAM is due to inhibitionof the production and not the activity of -amylase. The inhibitionof Avena fatua seedling growth by SHAM reported earlier may,in part, be due to its effect on endosperm mobilization. Since (1) Avena fatua seeds have been shown to have little orno SHAM-sensitive respiration, and (2) concentrations of SHAMnecessary for inhibiting endosperm mobilization were significantlyhigher than those generally necessary for inhibiting alternativerespiration, the inhibition of endosperm mobilization by thiscompound does not appear to involve its effect on alternativerespiration. Avena fatua L., wild oat, -amylase, endosperm, gibberellic acid, salicylhydroxamic acid, seed     

Fluid pressure in human dermal fibroblast aggregates measured with micropipettes

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (P_if). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (P_f) over a time course of a few minutes. P_f was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-µm diameter) and age (days 1-4) kept at different temperatures (15, 25, and 35°C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E₁ isopropyl ester (PGE₁), latanoprost (PGF₂), and ouabain. P_f was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. P_f decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 µl of 1 mM PGE₁ (P < 0.001). P_f increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 µl of 1.4 µM ouabain and from 3.1 to 4.4 mmHg after addition of 5 µl of 1.42 mM PGF₂ (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify P_f and studying in more detail specific cell types involved in control of P_f. This study also provides evidence that fibroblasts in the connective tissue can actively modulate P_f. micropuncture; prostaglandin E₁; prostaglandin F₂; ouabain; integrins     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

Brassinosteroid-Induced Bending of the Leaf Lamina of Dwarf Rice Seedlings: An Auxin-Mediated Phenomenon

A synthetic brassinosteroid (BR), 2,3,22ß,23ß-tetrahydroxy-24ß-methyl-B-homo7-oxa-5-cholestan-6-one, an isomer of the growth promoter brassinolide,when applied to seedlings of dwarf rice Oryza sativa var. Tan-ginbozuand Waito-C, induced a significant bending of the second leaflamina at 100 ng/plant and higher dosages. Promotion of thesecond leaf sheath elongation, the characteristic response ofdwarf rice varieties to gibberellins, was significantly butmodestly enhanced by BR at a dosage of 10,000 ng/plant, fiveorders of magnitude higher than the minimal dosage responseto GA₃. Gibberellin A₃ had no significant effect on the bendingof the second leaf lamina, nor did any synergism exist betweenBR and GA₃ in leaf lamina bending or leaf sheath elongation.Neither ethylene nor (2-chloroethyl)phosphonic acid (ethephon)caused the bending of the second leaf lamina, and neither synergizedthe BR effect. However, IAA and -naphthaleneacetic acid causedsignificant bending at 5,000 ng/plant, and both auxins significantlysynergized the effect of BR on the bending, IAA being effectiveat 500 ng/ plant in this regard. The antiauxins, 2,3,5-triiodobenzoicacid (TIBA) and -(p-chlorophenoxy)isobutyric acid (PCIB) completelynullified both the BR-induced bending and the BR$IAA-synergizedbending. The BR-induced bending response may thus be mediatedthrough endogenous auxin. (Received May 11, 1982; Accepted August 25, 1982)     

Modulation of alpha7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells

We showed previously that the expression of ₇-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on ₇-integrin expression and VSMC adhesion and migration. Expression of the ₇-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased ₇ cell surface protein expression (12 and 24 h: 3.3 ± 0.8- and 3.6 ± 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in ₇-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 ± 6% increase, P < 0.01), and this increase was partially inhibited by anti-₇-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-₇-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in ₇-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances ₇-integrin expression in VSMC and promotes ₇-integrin-mediated adhesion to laminin. vascular injury; laminin; mitogen-activated protein kinase