The Apparent Induction of Nitrate Uptake by Chara corallina Cells following Pretreatment with or without Nitrate and Chlorate

Nitrate provision has been found to regulate the capacity forChara corallina cells to take up nitrate. When nitrate was suppliedto N sufficient cells maximum nitrate uptake was reached after8 h. Prolonged treatment of the cells in the absence of N alsoresulted in the apparent ability of these cells to take up nitrate.Chlorate was found to substitute partially for nitrate in the‘induction’ step. The effects on nitrate reductionwere separated from those on nitrate uptake by experiments usingtungstate. Tungstate pretreatment had no effect on NO^–₃uptake ‘induced’ by N starvation, but inhibitedNO^–₃ uptake associated with NO^–₃ pretreatment. Chloridepretreatment similarly had no effect on NO^–₃ uptake ‘induced’by N deprivation, but inhibited NO^–₃ uptake followingNO^–₃ pretreatment. The data suggest that there are atleast two mechanisms responsible for the ‘induction’of nitrate uptake by Chara cells, one associated with NO^–₃reduction and ‘induced’ by CIO^–₃ or NO^–₃and one associated with N deprivation. Key words: Nitrate, Chlorate, Chara corallina, Induction     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

Characterization of the Ca2+-Dependent Cl- Efflux in Perfused Chara Cells

The mechanism of the Ca²⁺-dependent Cl^– efflux was studiedin tonoplast-free cells, in which the intracellular chemicalcomposition can be freely controlled. Tonoplast-free cells wereprepared by perfusing the cell interior of internodal cellsof Chara corallina with a medium that contained EGTA. The Ca²⁺-inducedCl^– efflux was measured together with the membrane potentialduring continuous intracellular perfusion. The dependenciesof Cl^– efflux and the membrane potential on the intracellularCa²⁺ or Cl^– concentrations were analyzed. When perfusionwas started with medium that contained Ca²⁺ ions, Cl^–efflux and membrane depolarization were induced. The amountof Cl^– efflux varied considerably among individual cells.The rate of efflux decreased exponentially but a residual effluxremained detectable. The Cl^– efflux was induced at concentrationsof Ca²⁺ ions above 1 µM and reached a maximum at 1 mM.By contrast, the membrane depolarization reached a maximum atabout 10 µM Ca²⁺. The rate of Cl^– efflux increasedlinearly with logarithmic increases in the intracellular Cl^–concentrations. These findings suggest that more than two kindsof Ca²⁺-dependent Cl^– channel might be present in theplasma membrane. Addition of ATP or its removal from the perfusion medium didnot affect the Ca²⁺-dependent Cl^– efflux. Calmodulin antagonistsslightly inhibited the Ca²⁺-dependent Cl^– efflux. ¹Present address: Biological Laboratory, Hitotsubashi University,Naka 2-1, Kunitachi, Tokyo, 186 Japan.     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

Internal Factors Regulating Nitrate and Chloride Influx in Plant Cells

The primary factor determining the observed decrease in activeC1^– influx during salt accumulation in carrot and barleyroot cells has been shown to be the concentration of C1^–+ NO₃^– in the vacuole. The relationship between C1^– influx and the vacuolar concentrationsof various substances was examined after the tissues had accumulatedions from various salt solutions. After accumulating K⁺ malate,C1^– influx was not reduced, but after accumulating C1^–or NO₃^– salts, C1^– influx was reduced by up to 90per cent. Considering all treatments, C1^– influx was notcorrelated with the vacuolar concentration of K⁺, Na⁺, (K⁺+Na⁺),reducing sugars, malate, C1^–, or NO₃^–, nor withthe cellular osmotic pressure. The correlation coefficient betweenCl^– influx and log (C1^– + NO₃^– concentrationin the vacuole) was highly significant, and accounted for allthe variation in C1^– influx in this experiment. Net NO₃^– influx is similarly reduced by a high C1^–concentration in the vacuole. External Cl^– and NO₃^–have quantitatively different, apparently competitive, effectson C1^– influx. These differ from the apparently negative-feedbackeffects of C1^– and NO₃^– in the vacuole, which arequantitatively similar. Decreasing the internal hydrostatic pressure by raising theexternal osmotic pressure increased active K⁺ influx in Valoniaventricosa, but had no effect on C1^– or K⁺ influx in carrotor maize root cells. Cl^– influx is not related to thereducing sugar concentration during ageing drifts in excisedcarrot root tissue. Acetazolamide did not inhibit C1^– influx to carrot tissue. The implications of this type of negative feedback regulation,and the relationship between C1^– and NO₃^– transportare discussed.     

Rates of Photosynthesis in Characean Cells: II. PHOTOSYNTHETIC 14CO2 FIXATION AND 14C-BICARBONATE UPTAKE BY CHARACEAN CELLS

Photosynthetic ¹⁴C fixation by Characean cells in solutionsof high pH containing NaH¹⁴CO₃ gave a measure of the abilityof these cells to take up bicarbonate (H¹⁴CO₃^–). Whereascells of Nitella translucens from plants collected and thenstored in the laboratory absorbed bicarbonate at 1–1.5µµmoles cm^–2 sec^–1, rates of 3–8µµmoles cm^–2 sec^–1 were obtained withN. translucens cells from plants grown in the laboratory. Influxesof 5–6 µµmoles cm^–2 sec^–1 wereobtained with Chara australis, 3–8 µµmolescm^–2 sec^–1 with Nitellopsis obtusa, and 1–5µµmoles cm^–2 sec^–1 with Tolypella intricata.It is considered that these influxes represent the activityof a bicarbonate pump, which may be an electrogenic process. In solutions of lower pH, H¹⁴CO₃^– uptake would be maskedby rapid diffusion of ¹⁴CO₂ into the cells: the four Characeanspecies fixed ¹⁴CO₂ at maximum rates of 30–40 µµmolescm^–2 sec^–1 (at 21° C).     

Ion Content of the Halotolerant Alga Dunaliella salina

The intracellular concentration of the major ions in Dunaliellasalina cells were determined, following the removal of extracellularions by ion-exchange minicolumns. Log phase cells, grown inmedia containing 1–4 molar NaCl, contained 30–50mM chloride and 200–350 mM magnesium (5 mM in medium).Phosphorus, which is present intracellularly mostly as polyphosphate,was present in amounts of 60–100 fmoles per cell, equivalentto a concentration of 600–1,000 mM (0.2 mM in medium).Previous data indicated that such cells contained 20–40mM Na⁺, 150–300mM K⁺, 20mM SO^2–₄, and very low concentrationsof Ca2⁺ and charged nitrogenous compounds. Mg²⁺ and K⁺ seemto serve as the major counter ions for the intracellular negativecharge present in the massively accumulated polyphosphates.The former accounts for about 2/3 of the required positive charge.This is supported by the observation that limitation in thephosphate or K⁺ supply in the medium lead to a parallel decreasein the accumulation of intracellular phosphorus, Mg²⁺ or K⁺. ¹Present address: Department of Vegetables, The Volcani Center,Bet-Dagan 50250, Israel. (Received June 13, 1988; Accepted August 25, 1988)     

Roles of complex gangliosides in the development of experimental autoimmune encephalomyelitis

We induced experimental autoimmune encephalomyelitis (EAE) inGM2/GD2 synthase knockout mice (GM2/GD2^–/–), whichcannot synthesize complex gangliosides, such as GM1, GD1a, GD1b,GT1b, and GQ1b, to investigate the roles of complex gangliosidesin the pathogenesis of this disease. We used myelin-oligodendrocyteglycoprotein (MOG) as an immunogen. In active immunization EAE,the severity of clinical score was not different but the diseaseonset was significantly delayed in GM2/GD2^–/– comparedwith those in wild-type mice. When we transferred MOG-reactiveT cells from GM2/GD2^–/– or wild-type mice to wild-typemice, no significant differences were observed between the twogroups. In contrast, when we transferred MOG-reactive T cellsfrom wild-type mice to GM2/GD2^–/– or to wild-typemice, the onset of EAE in GM2/GD2^–/– mice was delayed.The recall response of MOG-specific T cells, the function ofantigen presenting cells, or the expression of several adhesionmolecules in the endothelium were not significantly differentbetween GM2/GD2^–/– and wild-type mice. On the otherhand, quantitative analysis of cellular infiltration in thecentral nervous system (CNS) on day 9 of active immunizationEAE showed that the CD4⁺ cell number in the CNS isolated fromGM2/GD2^–/– mice was significantly less than thatfrom wild-type mice. It indicated that the delayed onset ofEAE in GM2/GD2^–/– mice was due to the delay of themigration of pathogenic T cells into the CNS. Thus, the complexgangliosides may be involved in the T cell–endothelialcell interaction in the pathogenetic process of EAE.     

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells

The eye’s aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl^– enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl^– channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl^– channels with a unitary conductance of 272 ± 2 pS (n = 80). Voltage steps from 0 to ±80 mV, but not to ±40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl^– channels. cAMP also activated the maxi-Cl^– channels in outside-out patches. In both cases, maxi-Cl^– channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl^– concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl^– concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at ±80 but not at ±40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl^– channels of native PE cells that contribute to Cl^– release particularly from Cl^–-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl^– reabsorption into the ciliary stroma. cell volume; chloride secretion; aqueous humor formation     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

Synthesis of an S-(2-Aminoethyl)-L-Cysteine Conjugate in S-(2-Aminoethyl)-L-Cysteine- Resistant Adenine-Auxotrophic Cells of Datura innoxia Mill

A line of S-(2-aminoethyl)-L-cysteine-resistant adenine-auxotrophiccells (Ad^–AEC^r strain) was isolated from adenine-auxotrophiccells (Ad^– strain) of Datura innoxia Mill by a stepwiseselection method. Ad^–AEC^r and Bl cells, which were clonedfrom the original Ad^–AEC^r cells, were able to grow activelyon medium that contained 10 mM S-(2-aminoethyl)-L-cysteine (AEC),whereas the growth of Ad^– cells ceased completely in thepresence of 0.5 mM AEC. The resistant phenotype has been maintainedfor at least 10 months in culture on medium without AEC. Levels of free lysine in Ad^–AEC^r and Bl cells were similarto that in Ad^– cells. By contrast, the level of free AECin Ad^–AEC cells was 10-fold lower than in Ad^– cellsand no free AEC was detectable in Bl cells. However, acid hydrolysisof extracts from Ad^–AEC^r and Bl cells resulted in a remarkableincrease in levels of detectable AEC. This result indicatesthat conjugated AEC is synthesized and accumulated in the AEC-resistantcells. The level of the AEC conjugate in Bl cells increasedwith increases in the concentration of AEC in the culture medium,while intracellular levels of AEC were so low as not to be detectablein the case of cells grown on medium supplemented with AEC atless than 1 mM. The AEC conjugate was also detected in Ad^–cells, but at lower levels than in the AEC-resistant cells.In addition, AEC was found to be incorporated into soluble proteinsin Ad^– cells. These results suggest that the resistance of AEC-resistant cellsof Datura innoxia is accomplished via acceleration of the synthesisof the AEC conjugate which prevents any increase in intracellularlevels of free AEC. ¹Present address: Institute for Biology and Chemistry, TsumuraCo.Ltd., Inashiki, Ibaraki, 300-03 Japan. ²Present address: North Kanto Shop, Sakata Seed Co. Ltd.,Saitama,347 Japan.     

Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry

The aim of the present study was to investigate the properties and role of capacitative Ca²⁺ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca²⁺ entry in IC was larger in cells with depleted intracellular Ca²⁺ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca²⁺ entry blockers Gd³⁺ (10 µM), La³⁺ (10 µM), and Ni²⁺ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca²⁺ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at –60 mV. These events were not significantly affected by Gd³⁺ (10 µM) or La³⁺ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni²⁺. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents     

Anchoring protein is required for cAMP-dependent stimulation of L-type Ca2+ channels in rabbit portal vein

Stimulation of cardiac L-typeCa²⁺ channels by cAMP-dependentprotein kinase (PKA) requires anchoring of PKA to a specificsubcellular environment by A-kinase anchoring proteins (AKAP). Thisstudy evaluated the possible requirement of AKAP in PKA-dependentregulation of L-type Ca²⁺ channelsin vascular smooth muscle cells using the conventional whole cellpatch-clamp technique. Peak Ba²⁺current in freshly isolated rabbit portal vein myocytes wassignificantly increased by superfusion with either 0.5 µM isoproterenol (131 ± 3% of the control value,n = 11) or 10 µM 8-bromoadenosine3',5'-cyclic monophosphate (8-BrcAMP; 114 ± 1%,n = 8). The PKA-induced stimulatory effects ofboth isoproterenol and 8-BrcAMP were completely abolished by a specificPKA inhibitor KT-5720 (0.2 µM) or by dialyzing cells with Ht 31 (100 µM), a peptide that inhibits the binding of PKA to AKAP. In contrast,Ht 31 did not block the excitatory effect of the catalytic subunit ofPKA when dialyzed into the cells. These data suggest that stimulationof Ca²⁺ channels in vascularmyocytes by endogenous PKA requires localization of PKA through bindingto AKAP.

     

Distribution of Growth in the Apical Region of the Shoot of Lupinus albus

The purpose of the investigation is the determination of thevolumes and numbers of cells of the meristematic dome and ofeach of the first 7 primordia and internodes at the apex ofthe shoot of Lupinui albus. This system occupies a zone whichis about 0·4 mm. in length. Techniques are describedfor dissecting the region in which the observations are made,for determining the numbers of cells and the volumes of theseveral fragments. From the number of cells and the volume ofeach fragment an average cell volume it calculated. It is shown that in the midphase of the plastochron the domecontains 3,500 cells and has a volume of 1·6 x10^–3mm.³,the first primordium contains 1,630 cells and has a volume of0·38 x10^–3 mm.³, and the first intemode containsabout 700 cells and has a volume of about 1·4 x10^–3mm³The number of cells and the volume of the primordium increaseexponentially with increasing plastochron age, and the seventhprimordium contains 26,000 cells and has a volume of 20·9x 10^–3mm³ The seventh intemode contains about 5,000 cellsand has a volume of 8·6x10^–3mm³ The average cell volume in the dome is 4·7 x 10^–7mm.³in the first primorndium it is 2·3 x 10^–7mm.³ andin the first internode it is 20·9x 10^–7mm.³ Inthe seventh primordium the average cell volume increases to7·9 x 10^–7mm.³ In the internodes there is little,if. any, change in cell volume from the first to the seventhof the series. The significance of these changes is discussed.     

Ultrastructural Localization of Ions: IV. LOCALIZATION OF CHLORIDE AND BROMIDE IN NITELLA TRANSLUCENS AND X-RAY ENERGY SPECTROSCOPY OF SILVER PRECIPITATION PRODUCTS

The cytoplasmic distribution of Cl^– in Nitella translucenswas assessed by means of two contrasting approaches. The firstinvolved the histochemical precipitation of Cl^– with Ag⁺followed by X-ray analytical verification of the silver precipitationproducts, and the second, quench-freezing of whole Nitella cellsfollowed by freeze-substitution in the presence of silver underanhydrous conditions. Both methods produced identical evidencefor Cl^– distribution, showing that a large proportionof the Cl^– is present in the stationary cortical gel layerwhich includes the chloroplasts. However, the chloroplasts appearedto be low in Cl^– content while the bulk of the Cl^–appeared to be situated between the chloroplasts and plasmalemma. Experiments were carried out in other to detect the pathwayof the ‘fast component’ of halide ion transfer tothe vacuole. Br^– was supplied for various time intervalsto low Cl^– Nitella cells, followed by attempts to differentiatebetween AgCl and AgBr deposits. Solutions of various strengthof NH₄OH or ammonium carbonate were used to remove AgCl (butnot AgBr) deposits by formation of a Ag(NH₃)₂⁺ complex. AlthoughX-ray analytical verification showed that the method had somepotential usefulness it could not be carried out successfullybecause of loss of structural detail caused by NH⁺₄. The distribution and density of deposits near the plasmalemmasuggested the occurrence of a process in which cytoplasmic loadingis achieved by a sequential rupture and repair of the plasmalemmamembrane. Vesicles and reticulate structures in the streamingcytoplasmic phase generally showed very little deposit, butthese structures, together with the tonoplast, became greatlyenriched with deposits when cells had been given a brief exposers(3 min) to a Cl^– or Br^– solution. These rapid changesmay possibly be related to the ‘fast component’of halide ion transfer to the vacuole.