Control of carbohydrate metabolism by ethylene in latex vessels ofHevea brasiliensis MUEL. ARC in relation to rubber production

The treatment of the bark of regularly tapped trees with 2-ohloroethylphosphonio acid (CEPA) in the long run brings down sucrose level in the latex even when latex production is not increased. The sucrose deficit thus caused persists for a number of months after the treatment probably due to the depletion of carbohydrate reserves by the stimulation. The loss of carbohydrates not related to an increase of rubber produotion grows as the concentrations of applied CEPA are raised. The treatment increases the activity of phosphoenolpyruvate (PEP) carboxylase and decreases pyruvate deoarboxylation in mitochondria-free oytoplasmic latex serum. The pyruvate deoarboxylase has a sharp optimum at pH 6.5 and is almost inactive at pH 7.5 while PEP oarboxylase, like invertase, has the optimum at weakly alcaline pH and is only slightly active at pH 6.5. The CEPA-induced changes in the activity of these enzymes are related to an increase of latex pH after the treatment. Consistently with this the evolution of CO2 from sugars catabolised in the serum was found to decrease with increasing pH. It is concluded that the observed ethylene-induced changes in enzyme aotivities bring about in latex vessels a deviation of the glycolysis-fed metabolio flux from synthetic pathways to mitochondrial oxidations and consequently a decrease in the efficiency of sucrose conversion to rubber.     

Stimulatory effects of 2,4-dichlorophenoxyacetic acid and of 1-naphthylacetic acid on sucrose level,invertase activity and sucrose utilization in the latex ofHevea brasiliensis

Summary Treatment of the bark ofHevea brasiliensis with 2,4-dichlorophenoxyacetic acid (2,4-D) or l-naphthylacetic acid (NAA) greatly increases sucrose level, invertase activity and sucrose utilization in the latex; the efficacy of 2,4-D is considerably greater than that of NAA. The greater sucrose utilization is the consequence of increased invertase activity. The changes occur as soon as the first tapping following bark treatment. It is suggested that the rise in both sucrose level and utilization in the latex serum mediate the effect of auxins on latex production. This is most likely related to a faciliation of latex outflow resulting from an increase in the osmotic and turgor pressure in the laticiferous tissue, as well as to enhanced regeneration of latex.The latex invertase has been found to be of a weakly alkaline type, with a sharp pH optimum at 7.15–7.20 in citrate-phosphate buffer. Its activity falls of rapidly on the acid side, being almost zero at pH 6.4. Since the natural pH of latex generally varies between pH 6.5 and 7.0, it is suggested that pH is an important factor in the regulation of invertase activity in the latex, and that the limiting nature of invertase-mediated sucrose hydrolysis in latex serum is caused by unfavourable conditions for invertase activity rather than by a scarcity of this enzyme.Expert of the International Atomic Energy Agency.     

Sucrose Synthetase in the Latex of Hevea brasiliensis Muell. Arg

Sucrose synthetase (EC 2.4.1.13 [EC] ) was found in the latex of therubber tree but the activity of sucrose phosphate synthetase(EC 2.4.1.14 [EC] ) was not detected. The enzyme was purified andsome properties have been investigated. Examination of the kineticsof sucrose synthesis revealed K_m of 0.56 mM for uridine diphosphoglucoseand 3.85 mM for fructose. Mg²⁺ and cyanide activated sucrosesynthesis but reduced the cleavage reaction. Increased pH hadthe same effect, the synthetic activity being higher than theactivity of sucrose breakdown within the physiological levelsof latex pH. In the latex of regularly tapped trees, the total enzyme activityin the direction of synthesis was about 10% or less of the totalinvertase activity at pH 7.0. Because of the strong limitationof invertase under natural conditions, the proportion of actualsynthetase activity is, however, much higher and evidence ispresented that in the latex of regularly tapped trees this activitysignificantly reduces carbohydrate breakdown. Some indications have been obtained that this involvement ofsucrose synthetase is weakened by application of Ethrel to thebark. A reduction of its synthetic activity, accompanied byan acceleration of sucrose utilization in latex cytoplasm andby an increase of latex yield, could be observed before thetreatment-induced rise of pH enhancing inver.     

Effects of Tapping, Wounding, and Growth Regulators on Turgor Pressure in Hevea brasiliensis Muell. Arg.

Various forms of wounding result in increases in hydrostaticpressure in the laticiferous phloem tissue of Hevea brasiliensis;regular tapping, a form of controlled wounding, probably causesa similar response. When a tree is ring-barked, there is a transientfall in turgor immediately above and below the ring, presumablyowing to loss of latex during cutting. This is followed by apressure increase which is particularly marked above the ring,suggesting the accumulation of metabolites. Isolation of an‘island’ of tissue, by cuts down to the wood, resultsin a steep fall in turgor within it, although a relatively slowrecovery may follow. Partially isolated panels show smallerfalls and a more rapid rise. It appears that a functional phloemconnexion permits a more rapid recovery of osmotic and turgorpressures following latex losses, both in the isolation experimentsand in normal tapping. Growth regulators such as 2,4,5-trichlorophenoxyaceticacid (2,4,5-T) increase latex yields by prolonging latex flowafter tapping. In untapped trees treatment with 2,4,5-T resultsin a slow and fairly small increase in turgor pressure, butthis effect may not be apparent if trees are regularly tapped.When the tapping cut is opened, there is a rapid fall in pressureimmediately under the cut. This loss in turgor spreads throughthe latex-vessel system as latex flows towards the cut, butrecovery is apparent near the cut even before flow ceases. Pressuregradients indicate a rapid formation of a localized resistanceto flow at or near the cut surface. This process appears toplay a major part in restricting flow. The increased yieldswhich result both from regular tapping and after 2,4,5-T treatmentappear to result from a delay in this sealing process. The mechanismsby which the barrier to flow is built up and delayed by 2,4,5-Ttreatment are not clearly understood.     

Does resin tapping affect the tree-ring growth and climate sensitivity of the Chinese pine (Pinus tabuliformis) in the Loess Plateau,China?

Resin tapping could affect water and nutrient transport processes in Chinese pine trees, rendering them more vulnerable to extreme climatic events, such as drought, and affecting the ecological function of forests in semi-arid regions. This study evaluated how resin tapping affects the tree-ring growth and climate sensitivity of Chinese pine in the Loess Plateau. We compared tree-ring growth patterns between the tapped and untapped faces of tapped trees, and investigated tree-ring growth and its response to climate between tapped and untapped trees in a forest stand during the 1967–2017 period. Tapped trees showed asymmetrical growth patterns after resin tapping, with narrower rings near the tapped face and wider ones near the untapped face. Furthermore, tapped trees had inter-annual variations consistent with those of untapped trees except for the years 2000 and 2001, with significantly lower values following resin tapping, and tree-ring growth then returning to normal. The climate response analysis indicated that the tree-ring growth of both tapped and untapped trees was negatively affected by monthly mean temperatures during the early growing season (May to July) in the post-resin-tapping period. Furthermore, tree-ring growth in tapped trees also revealed significant correlation with water vapour deficit and the Palmer drought index, which indicates that tapped trees are more vulnerable to drought. Further studies based on stable isotopes (i.e. δ¹³C, δ¹⁸O, and δ¹⁵N) could improve our understanding of the physiological mechanisms that regulate the effects of resin tapping on tree-ring growth.     

Photosynthate allocation and productivity of latex vessels inHevea brasiliensis

Manipulations of production systems in rubber tree which were intended to improve sucrose translocation in tapped bark resulted in an increase of latex sucrose and of latex production and reduced the incidence of nonyielding laticiferous tissue. This was achieved by shortening the tapping cut from full to half spiral, by changing the descending direction into an ascending mode of tapping or by annual change-over of tapping panel allowing for a longer time the regeneration of bark removed above the location of the cut. The increase of latex yield did not result in a significant decrease in the growth of trees over a period of three years. Clonal differences in nonyielding bark appeared to be related to differences in sucrose depletion by tapping. In clone PB 235 which exhibited low latex sucrose, a reduction of tapping frequency resulted in an increase in sucrose level and in a decrease of bark “dryness” tending to an increase in total yield. The tapping manipulations examined did not affect latex flow characteristics such as the plugging index of latex vessels and the bursting index of lutoids. The results stress the importance of photosynthate allocation for the physiology of laticiferous system productivity and indicate the possibilities of improving assimilate economy in rubber trees. On mission as export of the International Atomic Energy Agency.     

Short-term resin tapping activities had a minor influence on physiological responses recorded in the tree-ring isotopes of Chinese pine (Pinus tabuliformis)

Resin tapping might affect tree-ring growth, but details on the physiological responses of trees to resin tapping are still lacking, particularly for long-term responses. This study aimed to explore the physiological processes underlying resin-tapping of Chinese pine (Pinus tabuliformis) by using tree-ring stable isotopes. We compared tree-ring earlywood and latewood stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotopes in the pre-resin tapping and post-resin tapping period for tapped trees and compared their values between tapped and untapped trees and their responses to climate variables in a forest stand from 1984 to 2017. Furthermore, we used a dual isotope model to distinguish between the effects of the photosynthetic assimilation rate and stomatal conductance. Results indicated that tapped and untapped trees showed similar inter-annual variation for two isotopes, while the absolute values of tapped trees were slightly (P > 0.05) lower than tapped trees in the two years following resin tapping. Climate response analysis indicated that resin tapping had no significant effect on climatic sensitivity for either stable isotope. Earlywood stable isotopes were mainly influenced by temperature, relative humidity, and Palmer Drought Severity Index (PDSI) from May to July, while latewood isotopes were mainly influence by relative humidity form July to August and PDSI from July to September. The conceptual model results indicated that resin tapping lead to a slight, but not significant, decrease in the intrinsic water-use efficiency caused by increased stomatal conductance for the first two to three years following resin tapping. We conclude that tree-ring physiological responses could be less affected by short-term resin tapping activities.     

Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production

Background and Aims

The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel^®) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described.

Methods

Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel.

Key Results

Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application.

Conclusion

The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.Key words: Hevea brasiliensis, laticifers, latex production, ethylene, sucrose transporters     

Trunk contraction ofHevea brasiliensis due to tapping

Determination of bark contraction due to tapping made simultaneously at different positions on the trunk ofHevea trees (young PR 107, S/2 ↓) provided information about the flow area and the speed of contration spreading. The relation between truck contraction at the given distance from the cut and the amount of latex tapped was experessed in mathematical terms. A quantitative relation was also established between the yields of latex and the linear dimension of flow areas in longitudinal direction below the middle of tapping cut. From the contraction curves it is deduced that the lateral flow of latex becomes relatively more important with increasing yields.     

Purification of an Acid Invertase from Washed Discs of Storage Roots of Red Beet (Beta vulgaris L)

The purification of an acid invertase from washed discs of storageroots of red beet (Beta vulgaris L.) is described. An overallpurification of 1210-fold was obtained using a combination of(NH₄)₂SO₄ precipitation, size-exclusion chromatography, ion-exchangechromatography, conA-sepharose chromatography and two roundsof FPLC on Mono Q HR 5/5, the first at pH 7·5, the secondat pH 6·5. The purified enzyme had a specific activityof 206     

Morphogenetic Effects on Vegetative Plants of Poa pratensis L. of 6-Azauracil, (2-Chloroethyl)- phosphonic acid, and (2-Chloroethyl) trimethyl-ammonium chloride and their Interaction with Gibberellic Acid

Fresh and dry weights and leaf size of Poa pratensis were reducedwhen treated with 6-azauracil (AzU), (2-chloroethyl)phosphonicacid (CEPA), or (2-chloroethyl)trimethylammonium chloride (CCC).AzU and CEPA inhibited epidermal cell division without inhibitingcell elongation, while CCC inhibited mainly cell elongationand cell division to a small extent. The ratio of blade lengthto sheath length and the blade length/width ratio were reduced,but leaf emergence and tillering were increased by AzU and CEPA.CCC affected only the latter three features. Like GA₃, CEPAinduced stem formation, but internodes were shorter. GA₃ was ineffective in preventing leaf-growth inhibition byAzU, which inhibited Ga₃-induced cell elongation. The inhibitoryeffect of CEPA on leaf growth was apparently reversed by GA₃,but this was due solely to increased cell elongation, the reductionin cell number being unaffected. Ga₃ reversed the effect ofCCC on leaf length, as well as on cell size and number. Simultaneousapplication of the inhibitors produced a complex interactionin reducing leaf length and number and size of epidermis cells.It is postulated that AzU, CEPA, and CCC have different modesof action because they have specific effects on plant growthand different effects on GA₃-induced cell elongation.     

Ethrel (Ethylene Releaser)-Induced Increases in the Adenylate Pool and Transtonoplast DeltapH within Hevea Latex Cells

The treatment of rubber tree (Hevea brasiliensis) bark with chloro-2-ethyl phosphonic acid (ethrel), an ethylene-releasing chemical, induced, after a lag period of 13 to 21 hours, a marked increase in the total adenine nucleotides (essentially ATP and ADP) of latex cells. This rise in the latex adenylate pool was concomitant with a marked decrease in the [ATP]/[ADP] ratio without significant changes in the adenylate energy charge. The apparent equilibrium constant for the adenylate kinase, which appeared to behave as a key enzyme in maintaining the adenylate energy charge in the latex, was considerably reduced, probably as a consequence of the alkalinization of the latex cytosol induced by the treatment with ethrel. To reduce the “sink effect” and activation of the metabolism induced in Hevea bark by regular tapping, the latex was collected by micropuncture (few drops) at increasing distance (5-50 centimeters) above and below an ethrel-treated area on the virgin bark of resting trees. The effect of ethrel was shown to spread progressively along the trunk. The increase in the adenylate pool (essentially ATP) was detectable as early as 24 hours after the bark treatment and was maximum after 6 or 8 days, 5 centimeters as well as 50 centimeters above and below the stimulated bark ring. The correlative vacuolar acidification and cytosolic alkalinization, i.e. the increase in the transtonoplast ΔpH, induced in the latex cells by ethrel were shown to be concomitant with the rise in ATP content of the latex. This suggests that the tonoplast H⁺-pumping ATPase, which catalyzes vacuolar acidification in the latex, is directly and essentially under the control of the availability of its substrate (i.e. ATP) in the latex. The results are discussed in relation to energy-dependent activation of metabolism, and increased rubber production, as induced by the stimulation of rubber trees with ethrel.     

Control of RNA level and of RNA ratios in the latex ofHevea brasiliensis Müll. Arg. effect of latex tapping and of growth regulators

The association between latex RNA and latex production was examined using MAK column chromatography techniques. In young untapped trees the introduction of tapping or the treatment of bark with growth regulators resulted in an increase of RNA level and of rRNA/tRNA ratio in the latex. In regularly tapped trees an increase in rRNA but not in tRNA was brought about by increasing the tapping frequency. Treatment with growth regulators had the same effect but essentially only through the related enhancement of latex export from latex vessels. During latex flow, the highest RNA level was registered in latex fractions originating from the most heavily drained areas of bark. Using³²P labeling, evidence was obtained that the export of latex results in an enhancement of rRNA migration into the inner latex containing space of the vessels. This is considered as the reason of the generally observed association of high RNA level and of high rRNA/tRNA ratio with high latex yield. It is proposed that in controlling the RNA level and RNA proportions in the latex an important role is played by changes in turgor pressure associated with the loss of latex which may influence the export of RNA from the nucleus through related induction of pressure disequilibrium between the nucleoplasm and the latex cytoplasm.     

Some aspects of sucrose transport and utilization in latex producing bark ofHevea brasiliensis Muel. Arg

The radiotracer technique is described for transport and metabolic studies in latex producing bark. This technique provided further evidence that carbohydrates are supplied to latex vessels as sucrose and that sucrose is readily used for rubber synthesis. Bark treatment with ethylene resulting in latex flow stimulation is shown to activate within a few hours sucrose and water transport into the latex vessels. The available knowledge suggests an implication of an energy-dependent process of proton excretion which could explain the earlier observed ethylene induced alkalization of latex cytosol enhancing invertase and sucrose utilization. An increase of tapping intensity decreases the level of latex sucrose and changes its distribution pattern in drained bark. There is a clonal difference in the early response to exploitation. Bark excision by conventional tapping inhibits downward sucrose translocation, the inhibition being proportional to the surface of consumed bark above the tapping cut. Seasonal variation in the level of latex sucrose is related to variations in sunshine duration and sucrose concentration rises during the process of refoliation. The results stress the importance of sucrose supply to latex vessels for latex producing potentialities and indicate an association of low sucrose availability in latex vessels with premature latex vessel degeneration and bark dryness.     

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.     

Immunological Reactions of Single Pollen Grains, Electrophoresis and Enzymology of Pollen Protein Exudates

Single intact pollen grains of Oenothera organensis, when placedupon a thin layer of agar containing pollen antiserum, producecircular areas of precipitate. Pollen grains from an S₂S₂ plantdo not produce precipitate in S₆ antiserum. Pollen grains froman S₆S₆ plant and an S₂'S₄' self-compatible plant produce precipitatesin S₆ antiserum. Fifty per cent of the pollen grains from anS₂S₆ plant produce precipitate in S₆ antiserum. Protein diffusesinto buffer solutions from intact pollen grains within 212 min.As much as 40 per cent of the total protein diffuses out inan hour. Amylase and invertase were detected in the diffusatefrom pollen grains. Alkaline and acid phosphatases were confinedto the pollen grains and did not diffuse out. The serologicalprecipitates are specific to the incompatibility system.     

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50

The effect of CO₂-induced acidification on transjunctional voltage (V_j) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO₂ application greatly decreased the V_j sensitivity of Cx50 channels, and increased that of Cx50-D3N channels. CO₂ also affected the kinetics of V_j dependent inactivation of junctional current (I_j), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO₂ sensitivity of chemical gating such that even CO₂ concentrations as low as 2.5% significantly lowered junctional conductance (G_j). With Cx50 channels G_j dropped by 78% with a drop in intracellular pH (pH_i) to 6.83, whereas with Cx50-D3N channels G_j dropped by 95% with a drop in pH_i to just 7.19. We have previously hypothesized that the way in which V_j gating reacts to CO₂ might be related to connexin’s gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO₂ on V_j gating sensitivity and speed. cell communication; lens; gap junctions; chemical gating; channel gating; Xenopus oocytes     

The Purification and Characterization of Soluble Acid Invertase from Coleoptiles of Wheat (Triticum aestivum L. cv. Avalon)

Soluble acid invertase from wheat coleoptiles was purified toelectrophoretic homogeneity. A comparison of molecular weightby SDS-PAGE and gel filtration suggested that the enzyme wasa monomer of M_r50 000. The enzyme was a glycoprotein and, afterchemical deglycosylation, possessed a M_rof 48000. A polyclonalantiserum was raised against the deglycosylated protein. Thiscross-reacted specifically with acid invertase. A putative precursorof invertase synthesized in a cell-free translation system wasdetected by SDS-PAGE and fluorography of the immunoprecipitatedpolypeptides. The distribution of acid invertase in wheat seedlingshoots was investigated both by visualizing invertase activityafter starch gel electrophoresis and by immunoblotting. Bothtechniques identified two forms of invertase in extracts ofthe primary leaf and only one form in extracts of coleoptiles.The low pH optimum and the glycoprotein nature of wheat coleoptileinvertase are consistent with a vacuolar location. Fructoseinhibited its activity, suggesting that enzyme activity couldbe modulated by end-product inhibition. Key words: Acid invertase, purification, antiserum, glycoprotein, Triticum aestivum, wheat, coleoptiles     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

Expression, regulation, and function of P2X4 purinergic receptor in human cervical epithelial cells

Micromolarconcentrations of ATP stimulate biphasic change in transepithelialconductance across CaSki cultures on filters, an acute transientincrease (phase I response; triggered by P2Y₂ receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated byaugmented calcium influx and protein kinase C-dependent increase intight junctional resistance. The objective of the study was todetermine the role of P2X₄ receptor as a mediator ofphase II response. Human cervical epithelial cells expressP2X₄ receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms byNorthern blot analysis) and P2X₄ protein. Depletion ofvitamin A reversibly downregulated P2X₄ receptor mRNA andprotein and ATP-induced calcium influx. Depletion of vitamin Aabrogated phase II response, and the effect could bepartially reversed only with retinoic acid receptor (RAR)-selectiveretinoids but not retinoid X receptor (RXR) agonists. Depletion ofvitamin A also abrogated protein kinase C increase in tight junctionalresistance, and the effect could not be reversed with retinoids.Depletion of vitamin A also abrogated phase I increase inpermeability and reversibly downregulated P2Y₂ receptormRNA and ATP-induced calcium mobilization. However, in contrast tophase II response, both RAR and RXR agonists could fullyreverse those effects. These results suggest that phase IIresponse is mediated by a P2X₄ receptor mechanism.