Pumpkin (Cucurbita sp.) seed globulin VI. Proteolytic activities appearing in germinating cotyledons

N--benzoyl D,L-arginine p-nitroanilide (BAPA), leucine p-nitroanilide(LPA) and casein hydrolytic activities were assayed in germinatingcotyledons. BAPA hydrolytic activity was not detected in dryseeds, but increased rapidly from 1 to 4 days of germinationand then decreased. LPA and casein hydrolytic activities weredetected in dry seeds and increased from 2 to 4 days. Caseinhydrolytic activity decreased faster than the other two activities. BAPA hydrolytic enzyme was partially purified. It was inhibitedby p-chloromercuribenzoate and activated by ß-mercaptoethanoland dithiothreitol, but was not affected by EDTA, phenylmethylsulfonylfluoride, pumpkin trypsin inhibitor and several divalent cations.It had no ability to hydrolyze globulin or the chain to produce F_ß or smaller polypeptides,respectively, which was referred to as proteolytic activityI in the preceding paper (14), but released small peptides andamino acids from the chain and F_ß. However, it wasdifferent from proteolytic enzyme II which was present in dryseeds and inhibited by EDTA (14). Pumpkin trypsin inhibitor was purified. Its molecular weightwas estimated to be 10,500 by gel filtration. It did not inhibitthe BAPA hydrolytic enzyme. Both proteolytic activities I andII were also not reduced by the inhibitor (14). The inhibitoryactivity decreased gradually during germination. (Received November 9, 1979; )     

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.

     

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II

The effect ofCa²⁺/calmodulin-dependent protein kinase II (CaMKII)stimulation on unitary low voltage-activated (LVA) T-type Ca²⁺ channel currents in isolated bovine adrenalglomerulosa (AG) cells was measured using the patch-clamp technique. Incell-attached and inside-out patches, LVA channel activity wasidentified by voltage-dependent inactivation and a single-channelconductance of ~9 pS in 110 mM BaCl₂ orCaCl₂. In the cell-attached patch, elevation of bathCa²⁺ from 150 nM to 1 µM raised intracellularCa²⁺ in K⁺-depolarized (140 mM) cells andevoked an increase in the LVA Ca²⁺ channel probability ofopening (NP_o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblanksweeps, a rise in the frequency of channel opening in nonblank sweeps,and a 30% reduction in first latency. No apparent changes in thesingle-channel open-time distribution, burst lengths, or openings/burstwere apparent. Preincubation of AG cells with lipophilic or peptideinhibitors of CaMKII in the cell-attached or excised (inside-out)configurations prevented the rise in NP_o elicited by elevated Ca²⁺ concentration.Furthermore, administration of a mutant recombinant CaMKIIexhibiting cofactor-independent activity in the absence of elevatedCa²⁺ produced a threefold elevation in LVA channelNP_o. These data indicate that CaMKII activity isboth necessary and sufficient for LVA channel activation byCa²⁺.

     

ATP stimulation of Na+/Ca2+ exchange in cardiac sarcolemmal vesicles

In cardiacsarcolemmal vesicles, MgATP stimulatesNa⁺/Ca²⁺exchange with the following characteristics:1) increases 10-fold the apparentaffinity for cytosolic Ca²⁺;2) a Michaelis constant for ATP of~500 µM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory;4) not observed in the presence of20 µM eosin alone but reinstated when vanadate is added;5) mimicked by adenosine5'-O-(3-thiotriphosphate), without the need for vanadate, but not by ,-methyleneadenosine 5'-triphosphate; and 6) notaffected by unspecific protein alkaline phosphatase but abolished by aphosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLCeffect is counteracted by phosphatidylinositol. In addition, in theabsence of ATP,L--phosphatidylinositol4,5-bisphosphate (PIP₂) was ableto stimulate the exchanger activity in vesicles pretreated with PI-PLC.This MgATP stimulation is not related to phosphorylation of thecarrier, whereas phosphorylation appeared in the phosphoinositides,mainly PIP₂, thatcoimmunoprecipitate with the exchanger. Vesicles incubated with MgATPand no Ca²⁺ show a markedsynthesis ofL--phosphatidylinositol4-monophosphate (PIP) with little production ofPIP₂; in the presence of 1 µM Ca²⁺, the net synthesis of PIP issmaller, whereas that of PIP₂increases ninefold. These results indicate thatPIP₂ is involved in the MgATPstimulation of the cardiacNa⁺/Ca²⁺exchanger through a fast phosphorylation chain: aCa²⁺-independent PIP formationfollowed by a Ca²⁺-dependentsynthesis of PIP₂.

     

PGE1, dexamethasone, U-74389G, or Bt2-cAMP as an additive to promote protection by UW solution in I/R injury

Chiang, Chi-Huei, Kang Hsu, Horng-Chin Yan, Horng-Jyh Harn,and Deh-Ming Chang.PGE₁, dexamethasone,U-74389G, or Bt₂-cAMP as anadditive to promote protection by UW solution in I/R injury. J. Appl. Physiol. 83(2): 583-590, 1997.A method to reduce ischemia-reperfusion (I/R) injury can be animportant criterion to improve the preservation solution. AlthoughUniversity of Wisconsin solution (UW) works as a lung preservationsolution, its attenuation effect on I/R injury has not beeninvestigated. We attempted to determine whether, by adding variousprotective agents, modified UW solutions will enhance the I/Rattenuation by UW. We examined the I/R injury in an isolated rat lungmodel. Various solutions, e.g., physiological salt solution (PSS), UW,and modified UW solutions containing various protective agents such asprostaglandin E₁, dexamethasone, U-74389G, or dibutyryl adenosine 3,5-cyclic monophosphatewere perfused individually to evaluate the I/R injury. Isolated rat lung experiments, with ischemia for 45 min, then reperfusion for 60 min, were conducted in a closed circulating system.Hemodynamic changes, lung weight gain (LWG), capillary filtrationcoefficient (K_fc), proteincontent of lavage fluid, concentration of cytokines, and lunghistopathology were analyzed. Results showed that the acute I/R lunginjury with immediate permeability pulmonary edema was associated withan increase in tumor necrosis factor- (TNF-) production. A significant correlation existed betweenTNF- and K_fc(r = 0.8, P < 0.0001) and TNF- and LWG(r = 0.9, P < 0.0001), indicatingthat TNF- is an important cytokine modulating early I/R injury.Significantly lower levels ofK_fc, LWG,TNF-, and protein concentration of lung lavage(P < 0.05) were found in theUW-perfused group than in the control group perfused with PSS. ModifiedUW promoted the protective effect of UW to further decreaseK_fc, LWG, andTNF- (P < 0.05).Histopathological observations also substantiated this evidence. In theUW+U-74389G group, bronchial alveolar lavage fluid contained lowestprotein concentration. We conclude that the UW solution attenuates I/Rinjury of rat lung and that the modified UW solutions further enhancethe effect of UW in reducing I/R injury. Among modified solutions,UW+U-74389G is the best. Further investigation of the improved effectsof the modified UW solutions would be beneficial in lungtransplantation.

     

Studies on cutin-esterase II. Characteristics of cutin-esterase from Botrytis cinerea and its activity on tomato-cutin

The activity of cutin-esterase, cutinase, was detected in themycelial homogenate of Botrytis cinerea cultured in a peptone-sucrosemedium at 25°C for 7 days. The crude enzyme solution wasprepared from the homogenate by centrifugation at 106,600xg,treatment with (NH₄)₂SO₄ at 70% saturation, and dialysis against0.01 M phosphate buffer. The optimum pH, temperature and assayduration for enzyme activity were 5.0, 25°C and 18 hr, respectively.Specific activity was 255 mµmoles/mg protein/18 hr aspalmitate under optimum conditions. 83% of the activity waslost by heating the enzyme solution (pH 7.6) for 4 min at 95°C.Palmitic, stearic, oleic, 9, 10-dihydroxystearic or linoleic,dihydroxyeicosanoic and octadecanedioic acids were recognizedin the enzymic hydrolysate of tomato-cutin using gas-liquidchromatography. Among these fatty acids, palmitic, oleic andoctadecanedioic acids were readily liberated by the enzyme,but dihydroxyeicosanoic acid, the major component of tomato-cutin,was isolated only in small amounts. The enzyme is, therefore,an exo-type cutinase which hydrolyses minor side chains of fattyacids bound to the major structure of cutin. Cutinesterase mayfacilitate cuticular invasion by fungi as a result of reductionin mechanical strength of the cuticle by the enzyme ¹Biological Laboratory, Research Department, Nihon Noyaku Co.,Ltd., Kawachinagano, Osaka, Japan (Received June 16, 1970; )     

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     

Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration

Previous studies have shown that inhibition of L-type Ca²⁺ current (I_Ca) by cytosolic free Mg²⁺ concentration ([Mg²⁺]_i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of I_Ca, rat cardiac myocytes and tsA201 cells expressing L-type Ca²⁺ channels were whole cell voltage-clamped with patch pipettes in which [Mg²⁺] ([Mg²⁺]_p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca²⁺ channels (_1C/_2A/₂), increasing [Mg²⁺]_p from 0.2 mM to 1.8 mM decreased peak I_Ca by 76 ± 4.5% (n = 7). Mg²⁺-dependent modulation of I_Ca was also observed in cells loaded with ATP--S. With 0.2 mM [Mg²⁺]_p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP_2A) produced large changes in I_Ca amplitude; however, with 1.8 mM [Mg²⁺]_p, these same manipulations had no significant effect on I_Ca. With mutant channels lacking principal PKA phosphorylation sites (_1C/S1928A/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p had only small effects on I_Ca. However, when channel open probability was increased by _1C-subunit truncation (_1C1905/_{2A/S478A/S479A}/₂), increasing [Mg²⁺]_p greatly reduced peak I_Ca. Correspondingly, in myocytes voltage-clamped with pipette PP_2A to minimize channel phosphorylation, increasing [Mg²⁺]_p produced a much larger reduction in I_Ca when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg²⁺ modulates the extent to which channel phosphorylation regulates I_Ca. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca²⁺ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A     

Thyroid hormone inhibits biliary growth in bile duct-ligated rats by PLC/IP(3)/Ca(2+)-dependent downregulation of SRC/ERK1/2

The role of the thyroid hormone agonist 3,3',5 L-tri-iodothyronine (T3) on cholangiocytes is unknown. We evaluated the in vivo and in vitro effects of T3 on cholangiocyte proliferation of bile duct-ligated (BDL) rats. We assessed the expression of ₁-, ₂-, ₁-, and ₂-thyroid hormone receptors (THRs) by immunohistochemistry in liver sections from normal and BDL rats. BDL rats were treated with T3 (38.4 µg/day) or vehicle for 1 wk. We evaluated 1) biliary mass and apoptosis in liver sections and 2) proliferation in cholangiocytes. Serum-free T3 levels were measured by chemiluminescence. Purified BDL cholangiocytes were treated with 0.2% BSA or T3 (1 µM) in the absence/presence of U-73122 (PLC inhibitor) or BAPTA/AM (intracellular Ca²⁺ chelator) before measurement of PCNA protein expression by immunoblots. The in vitro effects of T3 (1 µM) on 1) cAMP, IP₃, and Ca²⁺ levels and 2) the phosphorylation of Src Tyr139 and Tyr530 (that, together, regulate Src activity) and ERK1/2 of BDL cholangiocytes were also evaluated. ₁-, ₂-, ₁-, and ₂-THRs were expressed by bile ducts of normal and BDL rats. In vivo, T3 decreased cholangiocyte proliferation of BDL rats. In vitro, T3 inhibition of PCNA protein expression was blocked by U-73122 and BAPTA/AM. Furthermore, T3 1) increased IP₃ and Ca²⁺ levels and 2) decreased Src and ERK1/2 phosphorylation of BDL cholangiocytes. T3 inhibits cholangiocyte proliferation of BDL rats by PLC/IP₃/Ca²⁺-dependent decreased phosphorylation of Src/ERK1/2. Activation of the intracellular signals triggered by T3 may modulate the excess of cholangiocyte proliferation in liver diseases. cholestasis; cholangiopathies; hyperplasia; intrahepatic biliary epithelium; mitosis     

Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action

To determinethe mechanism of fatty acid modulation of rabbit pulmonary arterylarge-conductance Ca²⁺-activated K⁺(BK_Ca) channel activity, we studied effects of fatty acidsand other lipids on channel activity in excised patches withpatch-clamp techniques. The structural features of the fatty acidrequired to increase BK_Ca channel activity (or averagenumber of open channels, NP_o) were identified tobe the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which havea sufficiently long alkyl chain (C  8), produced a decrease inNP_o. Neutral and short-chain lipids did notalter NP_o. Screening of membrane surface chargewith high-ionic-strength bathing solutions (330 mM K⁺ or130 mM K⁺, 300 mM Na⁺) did not alter themodulation of the BK_Ca channel NP_oby fatty acids and other charged lipids, indicating that channelmodulation is unlikely to be due to an alteration of the membraneelectric field or the attraction of local counterions to the channel.Fatty acids and other negatively charged lipids were able to modulate BK_Ca channel activity in bathing solutions containing 0 mMCa²⁺, 20 mM EGTA, suggesting that calcium is not requiredfor this modulation. Together, these results indicate that modulationof BK_Ca channels by fatty acids and other charged lipidsmost likely occurs by their direct interaction with the channel proteinitself or with some other channel-associated component.

     

Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells

We used theCa²⁺-sensitive fluorescent dyefura 2, together with measurements of intracellularD-myo-inositol1,4,5-trisphosphate [Ins(1,4,5)P₃],to assess the inhibitory effects of caffeine on signal transduction viaG protein-coupled receptor pathways in isolated rat mandibular salivaryacinar cells. ACh, norepinephrine (NE), and substance P (SP) all evokedsubstantial increases in the intracellular freeCa²⁺ concentration([Ca²⁺]_i).Responses to ACh and NE were markedly inhibited by prior application of20 mM caffeine. The inhibitory effect of caffeine was not reproduced byphosphodiesterase inhibition with IBMX or addition of cell-permeantdibutyryl cAMP. In contrast to the ACh and NE responses, the[Ca²⁺]_iresponse to SP was unaffected by caffeine. Despite this, SP and AChappeared to mobilize Ca²⁺ from acommon intracellular pool. Measurements of agonist-induced changes inIns(1,4,5)P₃levels confirmed that caffeine inhibited the stimulus-response couplingpathway at a point beforeIns(1,4,5)P₃ generation. Caffeine did not, however, inhibit[Ca²⁺]_iresponses evoked by direct activation of G proteins with 40 mMF. These data show thatcaffeine inhibits G protein-coupled signal transduction in these cellsat some element that is common to the muscarinic and -adrenergicsignaling pathways but is not shared by the SP signaling pathway. Wesuggest that this element might be a specific structural motif on the Gprotein-coupled muscarinic and -adrenergic receptors.     

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     

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     

Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells

We have examined theinteraction between muscarinic and ₂-adrenergic receptoractivation on inositol phosphate (IP) formation in the nonpigmentedcells of the ciliary body epithelium (NPE cells) of the rabbit. We havecompared these changes with those previously observed in theintracellular free Ca²⁺ concentration. Whereas muscarinicreceptor activation causes an increase in intracellularCa²⁺ and IP formation, activation of₂-receptors does not significantly increase eitherintracellular Ca²⁺ or IPs over basal levels. However,simultaneous activation of muscarinic and ₂-adrenergicreceptors with the specific agonists carbachol and UK-14304 producesmassive Ca²⁺ increases and results in a synergisticincrease in IP formation. This synergistic IP formation is inhibited byboth muscarinic and ₂-adrenergic receptor antagonists aswell as by pertussis toxin and an inhibitor of phospholipase C. IPformation is predominantly independent of intracellularCa²⁺, because it is decreased but not prevented by blockingthe entry of Ca²⁺ with LaCl₃ or chelatingintracellular Ca²⁺ with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca²⁺ resulting fromsimultaneous activation of muscarinic and ₂-adrenergic receptors.

     

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

A voltage-gated, small, persistent Na⁺ current (I_Na) has been shown in mammalian cardiomyocytes. Hypoxia potentiates the persistent I_Na that may cause arrhythmias. In the present study, we investigated the effects of n-3 polyunsaturated fatty acids (PUFAs) on I_Na in HEK-293t cells transfected with an inactivation-deficient mutant (L409C/A410W) of the -subunit (hH1) of human cardiac Na⁺ channels (hNav1.5) plus ₁-subunits. Extracellular application of 5 µM eicosapentaenoic acid (EPA; C20:5n-3) significantly inhibited I_Na. The late portion of I_Na (I_{Na late}, measured near the end of each pulse) was almost completely suppressed. I_Na returned to the pretreated level after washout of EPA. The inhibitory effect of EPA on I_Na was concentration dependent, with IC₅₀ values of 4.0 ± 0.4 µM for I_Na peak (I_{Na peak}) and 0.9 ± 0.1 µM for I_{Na late}. EPA shifted the steady-state inactivation of I_{Na peak} by –19 mV in the hyperpolarizing direction. EPA accelerated the process of resting inactivation of the mutant channel and delayed the recovery of the mutated Na⁺ channel from resting inactivation. Other polyunsaturated fatty acids, docosahexaenoic acid, linolenic acid, arachidonic acid, and linoleic acid, all at 5 µM concentration, also significantly inhibited I_Na. In contrast, the monounsaturated fatty acid oleic acid or the saturated fatty acids stearic acid and palmitic acid at 5 µM concentration had no effect on I_Na. Our data demonstrate that the double mutations at the 409 and 410 sites in the D1–S6 region of hH1 induce inactivation-deficient I_Na and that n-3 PUFAs inhibit mutant I_Na. human cardiac sodium channel     

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     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Transcaltachia (the Rapid Hormonal Stimulation of Intestinal Calcium Transport): A Component of Adaptation to Calcium Needs and Calcium Availability

Calcium is one of the key elements required for maintenanceand survival of life in animals with both endo- and exoskeletons.Because there is a wide variation in dietary calcium (dependentupon the local habitat) and dietary practices, and because thereis a changing physiological need throughout life (e.g., formammals, during growth, puberty, pregnancy, lactation, and menopause),it is essential that the process of intestinal calcium absorptionbe adaptable and responsive to both the dietary and physiologicalcircumstances. This article reviews the evidence that transcaltachia,or the rapid stimulation of intestinal Ca²⁺ transport by thesteroid hormone, l, 25(OH)₂-vitamin D₃, as studied in the chicken,meets many of the objectives of an adaptive intestinal calciumtransport process. Transcaltachia is studied in a perfused chickduodenum, where ⁴⁵Ca²⁺ is placed in the lumen and potentialagonists are perfused into the celiac artery; the transcaltachicreponse represents the stimulation within 4–8 min of thetransfer of ⁴⁵Ca²⁺ from the lumen to the vascular perfusate.l,25(OH)₂-vitamin D₃ stimulation of transcaltachia occurs vianongenomic mechanisms which involve a plasma membrane receptorfor the secosteroid and the coupled opening of voltage-gatedCa²⁺ channels on the basal lateral membrane of the intestinalepithelial cell and the activation of the second messengers,protein kinase C and cAMP.     

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.

     

Acute hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K⁺ (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [I_K(V)] is selective to PASMC; hypoxia has little effect on I_K(V) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming -subunits and regulatory -subunits. KCNA5 is a Kv channel -subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits I_K(V) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased I_K(V) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect I_K(V) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of PO₂ from 145 to 35 mmHg reduced I_K(V) by 40% in rat PASMC transfected with human KCNA5 but had no effect on I_K(V) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on I_K(V) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels. membrane potential; potassium channels; vascular smooth muscle