The Interactions of Auxins and Anti-auxins in the Stimulation of Root Growth

Experiments on the interactions of auxins and anti-auxins inroots have been extended to studies with concentrations givinggrowth stimulations using 2-mm. sections excised from the extensionzone of roots of Pisum sativum. The curves relating growth responsesto log₁₀ concentration for ß-indolylacetic acid (IAA)and three anti-auxins, -(I-naphthylmethylsulphide) propionicacid (NMSP), I-naphthylmethylsulphide acetic acid (NMSA), and4-chloro-3-nitrobenzoic acid (CNB), are of very similar shape.A fourth anti-auxin, p-chlorophenoxy-iso butyric acid (PCIB),shows negligible stimulation of growth in any concentration.In multifactorial experiments involving stimulatory concentrationsof IAA (10^–4 and 10^–5 p.p.m.) with several stimulatoryconcentrations of the first three anti-auxins (NMSP, NMSA, andCNB), consistent mutual antagonisms were clearly demonstrated.PCIB in non-inhibitory concentrations markedly antagonized stimulationby IAA Similar mutual antagonisms were shown in various mixturesof two anti-auxins. Both the similarities of the concentration-response curves andthe consistent mutual antagonisms suggest that both auxins andanti-auxins in stimulating root growth are exerting identicalphysiological actions in the same growth system. On the assumptionthat these substances are active when adsorbed at some enzymeor other protoplasmic surface, it has been demonstrated thatthese experimental results are more easily explained by a directaction at those growth centres than by a competitive antagonismof a natural endogenous growth inhibitor, as suggested in aprevious paper. The bearing of these results on current theories relating auxinand anti-auxin activities to molecular structure is discussed.     

Studies on the Geotropism of Roots:II.THE EFFECTS OF THE AUXIN ANTAGONIST {alpha}-(I-NAPHTHYLMETHYLSULPHIDE)PROPIONIC ACID (NMSP) AND ITS INTERACTIONS WITH APPLIED AUXINS

Previous experiments on the effects of auxins on the geotropicresponses of seedling pea roots (Audus and Brownbridge, 1957)have been extended using the ‘anti-auxin’ -(I-naphthylmethylsulphide)propionicacid (NMSP) alone and in combination with indole-3-acetic acid(IAA) and 2:4-dichlorophenoxyacetic acid (2:4-D). NMSP action differs from that of the auxins in that it reducesthe rate of curvature progressively as the concentration isincreased, irrespective of whether the overall extension growthof the roots is being stimulated (10 and 30 p.p.m.) or inhibited(100 p.p.m.). Correspondingly the reaction time is lengthenedby 25–50 per cent. in all concentrations. Studies of responsesin mixtures of growth-stimulating concentrations of NMSP (30p.p.m.) and growth-inhibiting concentrations of IAA (10^–8)and 2:4-D (3 x 10^–8) show that auxins and ‘antiauxins’are mutually antagonistic in most, if not all, their actionson growth and curvature. The results suggest that the anti-auxin NMSP may stimulate rootgrowth and inhibit curvature by interfering with the synthesisor distribution of a natural endogenous inhibitor, which isnot IAA. NMSP inhibition of root growth in high concentrationsmust, however, be exerted independently of this natural inhibitor.The mutual antagonisms shown between the auxins and NMSP arebest explained in terms of an interference with access to thegrowth centres; competitive action at the growth centres themselvesseems not to be involved.     

INTERACTION BETWEEN HELIANGINE AND PYRIMIDINES IN ADVENTITIOUS ROOT FORMATION OF PHASEOLUS CUTTINGS

1. Heliangine at 110^–4 M promoted the adventitious rootformation in hypocotyls of cuttings taken from light-grown (1,900lux) Phaseolus mungo seedlings. The promotion was almost completelyreversed by 310^–4 M uracil, uridine, cytidine, oroticacid or 610^–4 M carbamoyl DL-aspartic acid, and partlyby 310^–4 M thymine or thymidine. Neither 310^–4M cytosine, adenine, adenosine, guanine, guanosine nor a combinationof 310^–4 M carbamoyl phosphate and 310^–4 M L-asparticacid reduced the promotion by heliangine.
2. Uracil did not reducethe inhibiting effect of heliangine onthe indoleacetic acidinduced elongation of etiolated Avenacoleoptile sections.
3. Helianginein an aqueous uracil solution was recovered unchangedafter24-hr incubation at room temperature.
4. The root formation ofPhaseolus cuttings was promoted also by2-thiouracil and 5-fluorouracil.The effect was reversed byorotic acid or carbamoyl asparticacid, but not by carbamoylphosphate plus aspartic acid.
5. Ribonucleaseat 100 µg/ml increased the number of rootsprotruded fromhypocotyls of cuttings by about 260%.
6. A possible interpretationfor the promotion of root formationby heliangine is offered.

¹ Contribution No. 15 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. ² Dedicated to Prof. Dr. H. SODING in commemoration of the 70thbirthday.     

Auxin Induced Lateral Root Formation in Chicory

The supply of auxins [2,4-dichlorophenoxy acetic acid (2,4D),indole-3 acetic acid (1AA) and -naphthaleneacetic acid (NAA)]to excised chicory roots induced the formation of lateral rootmeristems mainly located close to the pre-existing apical rootmeristem. Lateral root growth induced in non-excised roots requiredhigher auxin concentrations. Inhibition of root elongation andconcomittant enlargement of the apices was also observed. SupplyingIAA induced the formation of lateral meristems earlier thanNAA, but subsequently favoured root elongation. Conversely,in the presence of 2,4D, reactivation of pericycle cells wasvery intense, but conversion of primordia to laterals was inhibited.Regardless of the auxin used, the responsive area in which lateralmeristems appeared was located a maximum of 4 mm away from theapical meristem. This region remained devoid of any lateralroot formation under control conditions. Pericycle cells oppositethe xylem poles in the diarch stele regained meristematic activityand divided transversally, giving rise to shorter cells. Thesecells subsequently divided periclinally, forming pairs of cellson the same transverse level. The root primordium extruded throughcortical cells and was surrounded by a lacuna formed to thedetriment of cortical cells.Copyright 1998 Annals of BotanyCompany Auxins,Cichorium intybus, chicory, lateral root, root elongation.     

The Inducible Formation and Stability of Nitrate Reductase in Higher Plants: II. EFFECTS OF ENVIRONMENTAL FACTORS, ANTIMETABOLITES, AND AMINO-ACIDS ON INDUCTION

The induction of nitrate reductase by molybdenum or nitratein excised tissues of cauliflower leaf was dependent on temperature;for the range 2^? to 12^? C, Q₁₀ was about 2; for the range 12^?to 22^? C, Q₁₀ was greater than 3. Enzyme formation was initiallymost rapid at 32^? C but did not continue for as long as it didat 22^? or 24^? C. Decreased oxygen supply lessened the rate ofenzyme formation. The effects on enzyme formation of a widerange of natural and synthetic antimetabolites were tested withrespect to induction by either nitrate or molybdenum, when introducedat the same time by infiltration. Actidione (cycloheximide),patulin, cycloserine, polymyxin B, L-2-thiolhistidine D-methionine,L-dihydroxyphenylalanine, D,L--methylglutamic acid, sarcosineand 1 ,2-dichloro-4-(p-nitrobenzenesulphonylamido)-5-nitrobenzene(DCDNS) were the most inhibitory compounds tested. Serine stimulatedproduction of enzyme activity; kinetin, benzimidazole, and p-fluorophenylalanine,3--methyltryptophane and the 4- isomer, chloramphenicol, gramicidin,and several thio- and^aza- derivatives of purines or pyrimidineswere practically without effect. Differential effects of inhibitorson enzyme formation in response to nitrate or molybdenum wererarely observed, and no deductions regarding the possible sequencein which the substrate and prosthetic metal induce activitycould be inferred from the results.     

Morphogenetic Responses of Browallia Leaf Sections and Callus

Leaf sections of Browallia viscosa and B. speciosa were placedon Murashige and Skoog (1962) salts and vitamins medium (MS)containing auxins and cytokinins, singly or in combination,to elicit morphogenetic responses. B. viscosa developed extensiveroots in 4 weeks on media supplemented with indolebutyric acid(IBA), indol-3-yl acetic acid (IAA) or naphthalene acetic acid(NAA) (0·01, 0·1, 1·0, 5·0 and 10·0mg^–1), but with 2, 4-D (0·1 mg^–1) only lightyellow friable callus was obtained. Shoot initiation and elongationoccurred consistently in 4–6 weeks on leaf sections inthe presence of 6---dimethylallyl amino purine (2iP). Similarly,shoot regeneration from leaf-derived callus, initiated and sub-culturedon MS + benzyladenine (BA) + NAA only induced callus on leafexplants of both species. B. speciosa did not respond exceptfor moderate and prolific callus formation on MS + BA + NAAand Uchimiya and Murashige (1974) media respectively. Browallia viscosa, Browallia speciosa, tissue culture, regeneration, morphogenetic potential     

Phytohormone-induced GABA production in transformed root cultures of Datura stramonium: an in vivo 15N NMR study

Primary nitrogen metabolism in transformed root cultures ofDatura stramonium was observed by in vivo ¹⁵N NMR. Treatmentof the root cultures with the plant growth regulators -naphthaleneaceticacid (NAA) and kinetin caused a de-differentiation of the roottissue, together with perturbation of primary and secondarynitrogen metabolism. The levels of newly-synthesized glutamineand glutamate during ammonium assimilation were depleted relativeto control cultures, whereas GABA biosynthesis was enhanced.Although GABA production could be stimulated by a decrease incytoplasmic pH (whether imposed artificially or induced by hypoxia),observation of the roots during phytohormone treatment by ³¹PNMR showed that the cytoplasmic pH remained stable, indicatingthat the perturbation of nitrogen metabolism in the de-differentiatedroots must be due to other causes. Key words: Datura, -aminobutyric acid, nitrogen metabolism, NMR, root cultures     

Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

Auxin and Ethylene-stimulated Adventitious Rooting in Relation to Tissue 9

We have previously shown that both endogenous auxin and ethylenepromote adventitious root formation in the hypocotyls of derootedsunflower (Helianthus annuus) seedlings. Experiments here showedthat promotive effects on rooting of the ethylene precursor,1-aminocyclopropane-l-carboxylic acid (ACC) and the ethylene-releasingcompound, ethephon (2-chloro-ethylphosphonic acid), dependedon the existence of cotyledons and apical bud (major sourcesof auxin) or the presence of exogenously applied indole-3-aceticacid (IAA). Ethephon, ACC, aminoethoxyvinylglycine (an inhibitorof ethylene biosynthesis), and silver thiosulphate (STS, aninhibitor of ethylene action), applied for a length of timethat significantly influenced adventitious rooting, showed noinhibitory effect on the basipetal transport of [³H]IAA. Theseregulators also had no effect on the metabolism of [³H]IAA andendogenous IAA levels measured by gas chromatography-mass spectrometry.ACC enhanced the rooting response of hypocotyls to exogenousIAA and decreased the inhibition of rooting by IAA transportinhibitor, N-1-naphthylphthalamic acid (NPA). STS reduced therooting response of hypocotyls to exogenous IAA and increasedthe inhibition of rooting by NPA. Exogenous auxins promotedethylene production in the rooting zone of the hypocotyls. Decapitationof the cuttings or application of NPA to the hypocotyl belowthe cotyledons did not alter ethylene production in the rootingzone, but greatly reduced the number of root primordia. We concludethat auxin is a primary controller of adventitious root formationin sunflower hypocotyls, while the effect of ethylene is mediatedby auxin. Key words: Auxin, ethylene, adventitious rooting, sunflower     

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsinwere analysed by sequential exoglycosidase digestion and gelfiltration chromatography, following reductive tritiation. Inaddition, selected tryptic glycopeptides obtained from frogretinal rod outer segment membranes were examined by electrospraymass spectrometry (ES-MS), fast atom bombardment mass spectrometry(FAB-MS), amino acid sequence and composition analysis, andcarbohydrate composition analysis. The amino acid sequence datademonstrated that the glycopeptides were derived from rhodopsinand confirmed the presence of twoN-glycosylation sites, at residuesAsn² and Asn¹⁵. The predominant glycan (60% of total) had thestructure GlcNAcß1–2Man1–3(Man1–6)Manß1–4GlcNAcß1–4GlcNAc-(Asn),with the remaining structures containing 1–3 additionalhexose residues, as reported previously for bovine rhodopsin.Unlike bovine rhodopsin, however, a sizable fraction of thetotal giycans of frog rhodopsin also contained sialic acid (NeuAc),with the sialylated oligosaccharides being present exclusivelyat the Asn² site. FAB-MS analysis of oligosaccharides releasedfrom the Asn² site gave, among other signals, an abundant quasimolecularion corresponding to a glycan of composition NeuAc₁Hex₆HexNAc₃(where Hex is hexose and HexNAc is N-acetylhexosamine), consistentwith a hybrid structure. The potential biological implicationsof these results are discussed in the context of rod outer segmentmembrane renewal. glycoforms oligosaccharide structure rhodopsin     

The effect of ammonium ions on uptake of glutamine and other amino compounds by cultured cells of rapeseed

Rooting responses and ethylene production by hypocotyl cuttings from etiolated mung-bean seedlings treated with the auxins -naphthaleneacetic acid, -(indole-3)-n-butyric acid (IBA) and 2,4,5-trichloro-phenoxypropionic acid were determined. There was no relationship between the abilities of the auxins to induce root formation and their capacities for inducing ethylene production. Studies with mixtures of 3-indoleacetic acid, a poor stimulator of rooting but an effective inducer of ethylene production, and IBA, an effective rooting stimulator but a poor inducer of ethylene production, exposure of cuttings to ethylene or (2-chloroethyl) phosphonic acid (Ethephon), hypobaric storage (150 mb) of treated cuttings, and exposure of auxin-treated cuttings to 7% CO₂ also indicated that ethylene is not directly involved in initiation of adventitious roots in this plant material.Abbreviations IAA indole-3-acetic acid - IBA -(indole-3)-n-butyric acid - NAA -naphthaleneacetic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid     

Adventitious Root Formation in Young Shoots of Cedrus Deodara

The effect of auxins [indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA)], phenolics (phloroglucinol and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin, containing 50% carbendazim) on adventitious root formation in stem cuttings (current season's growth) of Cedrus deodara L. during winter and monsoon (rainy) seasons has been examined. Significant stimulation of rooting due to treatments was observed in cuttings planted in winter in the following order: 0.25 mM IBA (87.5% rooting) > 5 mM coumarin (70.8%) > IBA (0.5 mM) + coumarin (5 mM) (50.0%). In cuttings planted in monsoon only 0.05% Bavistin was found to be effective in inducing rooting (83.3%). Other treatments were ineffective and in some treatments drying of cuttings was noticed.     

PROMOTING ACTIVITY OF TERPENIC LACTONES IN PHASEOLUS ROOTING AND THEIR REACTIVITY TOWARD CYSTEINE

Sesquiterpenoids which contain exomethylenes conjugated to -lactonecarbonyl-heliangine, helianginol, pyrethrosin and cyclopyrethrosinacetate—promoted the adventitious root formation on hypocotylsof cuttings taken from light-grown (1900 lux) 6-day old Phaseolusmungo seedlings, but their derivatives in which the methylene-lactone systems are reduced-to the saturated lactones—dihydroheliangine,hexahydro-heliangine, dihydrohelianginol, dihydrocyclopyrethrosinacetate and tetrahydrocyclopyrethrosin acetate—showedno effect on the root formation. As far as the present experiment is concerned, every substancewhich showed promoting activity in Phaseolus rooting reactedwith cysteine and formed an adduct, but any of the substancesincapable of promoting root formation did not react with cysteine.The correlation of promoting activity of terpenic lactones inPhaseolus rooting with their reactivity toward SH groups maythus be demonstrated. ¹ Contribution No. 14 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo.     

Interaction of abscisic acid and auxins in rooting of cuttings

Abscisic acid (ABA) at optimum concentrations promoted rootingof Phaseolus aureus ROXB. and Lycopersicon esculentum MILL,stem cuttings. In combination with IAA (indole-3-acetic acid)ABA has mostly given additive effects. Synergistic effect ofABA was noted on IBA (-indolebutyric acid)-induced rooting ofLycopersicon cuttings. Rooting of Phaseolus vulgaris L. cuttingscompletely failed when ABA (50 mg/liter) was applied in combinationwith IBA or NAA (-naphthaleneacetic acid). The results suggestthat abscisic acid may be an important natural regulator ofrooting in cuttings. (Received March 19, 1970; )     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

Acclimation to low light intensity in photosynthesis and growth of Pseudo-nitzschia multiseris Hasle, a neurotoxigenic diatom

Pseudo-nitzschia multiseries, a neurotoxigenic diatom, was grownin batch culture at light intensities between 53 and 1100 µmolm^–2 s^–1. Cellular contents of carbon. nitrogen andchlorophyll a, and the relationship between photosynthesis andlight levels, were studied during exponential (day 4) and stationaryphases (day 12). In the stationary phase at low light, therewas an increase in cellular chlorophyll a and the initial slopeof P-I curves (^B), which permitted a photosynthetic assimilationof energy equivalent to that of cells grown at high light. Inpast incidents of domoic acid poisoning, this may have facilitateddomoic acid production at low light intensities.     

Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current season's growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.     

Biosynthetic mechanism of glycolate in Chromatium IV. Glycolate-glycine transformation

The metabolic transformation of glycolate to glycine occurringin photosynthesizing cells of Chromatium was investigated bythe radioisotopic technique and by amino acid analysis. By analyzingthe distribution of radiocarbon upon feeding [1-¹⁴C] glycolate,[2-¹⁴C] glyoxylate and [1-¹⁴C] glycine to bacterial cells, itwas demonstrated that glycolate is converted to glycinc viaglyoxylate, and both glycolate and glycine are excreted extracellularly.Although the formation of serine was barely detected by theabove two techniques in both N₂ and O₂ atmospheres, it was foundthat ¹⁴CO₂ is evolved quite markedly from both [1-¹⁴C] glycolateand [1-¹⁴C] glycine fed to the Chromatium cells. Analyticalresults of transient changes in amino acid compositions underatmospheric changes of N₂O₂ and by the addition of exogenousglycolate in N₂ confirm the notion that glycolate is convertedto glycine. Acidic amino acids (glutamic acid and aspartic acid)appear to take part in glycine formation as amino donors. Theformation of glycine from glycolate in a N₂ atmosphere suggeststhat an unknown glycolate dehydrogenation reaction may operatein the overall process. ¹ This is paper XXXVII in the series ‘Structure and Functionof Chloroplast Proteins’. Paper XXXVI is ref. (5). Theresearch was supported in part by grants from the Ministry ofEducation of Japan (No. 111912), the Toray Science Foundation(Tokyo) and the Naito Science Foundation (Tokyo). (Received July 14, 1976; )     

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