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.

     

Contractile responsiveness of coronary arteries from exercise-trained rats

Parker, Janet L., Mildred L. Mattox, and M. Harold Laughlin.Contractile responsiveness of coronary arteries from exercise trained rats. J. Appl. Physiol. 83(2):434-443, 1997.The purpose of this study was to determine whetherexercise training alters vasomotor reactivity of rat coronary arteries.In vitro isometric microvessel techniques were used to evaluatevasomotor properties of proximal left anterior artery rings (1 ring peranimal) from exercise-trained rats (ET;n = 10) subjected to a 12-wk treadmill training protocol (32 m/min, 15% incline, 1 h/day, 5 days/wk) andcontrol rats (C; n = 6) restricted tocage activity. No differences in passive length-tension characteristicsor internal diameter (158 ± 9 and 166 ± 9 µm) were observedbetween vessesls of C and ET rats. Concentration-response curves toK⁺ (5-100 mM), prostaglandinF₂(10⁸-10⁴M), and norepinephrine(10⁸-10⁴)were unaltered (P > 0.05) incoronary rings from ET rats compared with C rats; however, lower valuesof the concentration producing 50% of the maximal contractile responsein rings from ET rats (P = 0.05)suggest that contractile sensitivity to norepinephrine wasenhanced. Vasorelaxation responses to sodium nitroprusside (10⁹-10⁴M) and adenosine(10⁹-10⁴M) were not different (P > 0.05)between vessels of C and ET rats. However, relaxation responses to theendothelium-dependent vasodilator acetylcholine (ACh;10¹⁰-10⁴M) were significantly blunted (P < 0.001) in coronary rings from ET animals; maximal ACh relaxationaveraged 90 ± 5 and 46 ± 12%, respectively, in vessels of Cand ET groups. In additional experiments, two coronary rings (proximaland distal) were isolated from each C(n = 7) and ET(n = 7) animal. Proximal coronaryartery rings from ET animals demonstrated decreased relaxationresponses to ACh; however, ACh-mediated relaxation of distal coronaryrings was not different between C and ET groups.N^G-monomethyl-L-arginine(inhibitor of nitric oxide synthase) blocked ACh relaxation of allrings. L-Arginine (substrate fornitric oxide synthase) did not improve the blunted ACh relaxation in proximal coronary artery rings from ET rats. These studies suggest thatexercise-training selectively decreases endothelium-dependent (ACh) butnot endothelium-independent (sodium nitroprusside) relaxation responsesof rat proximal coronary arteries; endothelium-dependent relaxation ofdistal coronary arteries is unaltered by training.

     

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitroresponses of pulmonary arteries (6-mm OD) from the dorsocaudal (top)and cranioventral (bottom) lung regions to endothelium-dependentvasodilators (methacholine, bradykinin, and calcium ionophore A-23187).Methacholine relaxed endothelium-intact top vessels; however, in bottomvessels, a small relaxation preceded a profound contraction. In topvessels, removal of endothelial cells converted relaxation tocontraction, and in bottom vessels it abolished relaxation and enhancedcontraction. Bradykinin and A-23187 were more potent and caused greaterendothelium-mediated relaxation in top than in bottom arteries. Theendothelium-independent vasodilator sodium nitroprusside caused similarrelaxations in all rings.N-nitro-L-arginine andN^G-monomethyl-L-arginine andmethylene blue abolished relaxation of top and bottom arteries tomethacholine; meclofenamate had little effect. We conclude thatregional differences in endothelium-mediated relaxation are caused bydifferences in the magnitude of the endothelial release of nitricoxide. Similar differences in endothelium-dependent flow-mediatedvasodilation and endothelial nitric oxide release may result inpreferential perfusion of caudodorsal lung regions.

     

Colchicine-Induced Effects in the Scaly Green Alga, Mesostigma viride: Loss of Microtubules and Paracrystal Formation

The effects of colchicine treatment upon the scale-covered,disc-shaped prasinophyte, Mesostigma viride, are profound. 2–4mg ml^–1 of the alkaloid induces the loss of pit and othercytoskeletal microtubules within 6 h of treatment. Subsequently,the organism loses its distinctive shape and becomes ellipsoidal.During treatment, prominent, intracellular crystalline bandsconsisting of 25 nm hexagonal subunits form in close proximityto the former pit region. The Golgi apparatus remains intactduring colchicine application but scale ontogenesis is distinctlyaltered. All effects are reversible upon removal of colchicine.A discussion of the cytoskeletal role of parallel series ofmicrotubules in unicellular algae is presented. Key words: Mesostigma, Colchicine, Cytoskeletal     

Effect of Lead and Colchicine on Morphogenesis in Protonemata of the Moss Funaria hygrometrica

Protonemata of Funaria hygrometrica grown in artificial mediacontaining different lead concentrations grow more slowly thancontrols and show a disturbance of polar growth, changed arrangementof chloroplasts, alterations of nucleus and septa position.Morphological effects are dose-dependent. At the lowest leadconcentration (10^-6 M), only a delay in development was observed,but no cellular alterations, At 10^-5 M Pb nuclear migration,cellular shape, size and position of plastids, were alteredand a variety of aberrant forms were present. At 10^-4 M, besidesthese alterations, a drastic reduction of the protonemal system,high vacuolation and the growth of protonemal filaments fromleaves were evident. The highest concentration, (10^-3 M), causeddeath. Patterns of protonemal development and cellular arrangementin lead-treated samples showed similarities as well as differences,if compared to alterations induced by colchicine. Indirect immunofluorescencedemonstrated a correlation between lead concentration and alterationof cytoskeletal organization (alterations similar to those inducedby colchicine). Hypotheses are raised to account for effects of lead on microtubulestructure, arrangement and cytoplasm organization.Copyright1995, 1999 Academic Press Funaria hygrometrica, lead, protonemal development, cytomorphogenesis, microtubules     

Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release. intracellular calcium stores; oscillations; pulmonary hypertension     

Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle

We investigatedthe role of the integrin-associated proteins focal adhesion kinase(FAK) and paxillin as mediators of mechanosensitive signal transductionin tracheal smooth muscle. In muscle strips contracted isometricallywith ACh, we observed higher levels of tyrosine phosphorylation of FAKand paxillin at the optimal muscle length(L_o) than atshorter muscle lengths of 0.5 or 0.75 L_o. Paxillinphosphorylation was also length sensitive in muscles activated byK⁺ depolarization and adjustedrapidly to changes in muscle length imposed after contractileactivation by either ACh or K⁺depolarization. Ca²⁺ depletion didnot affect the length sensitivity of paxillin and FAK phosphorylationin muscles activated with ACh, indicating that the mechanotransductionprocess can be mediated by aCa²⁺-independent pathway. SinceCa²⁺-depleted muscles do notgenerate significant active tension, this suggests that themechanotransduction mechanism is sensitive to muscle length rather thantension. We conclude that FAK and paxillin participate in anintegrin-mediated mechanotransduction process in tracheal smoothmuscle. We propose that this pathway may initiate alterations in smoothmuscle cell structure and contractility via the remodeling of actinfilaments and/or via the mechanosensitive regulation ofsignaling molecules involved in contractile protein activation.

     

Progression of coronary and mesenteric vascular dysfunction in Zucker obese and Zucker diabetic fatty rats

We investigated the progression of vascular dysfunction associated with the metabolic syndrome with and without hyperglycemia in lean, Zucker obese, and Zucker diabetic fatty (ZDF) rats. Responses of aorta and small coronary and mesenteric arteries were measured to endothelium-dependent and -independent vasodilators. Indices of oxidative stress were increased in serum from ZDF rats throughout the study, whereas values were increased in Zucker obese rats later in the study [thiobarbituric acid reactive substances: 0.45 +/- 0.02, 0.59 +/- 0.03 (P < 0.05), and 0.58 +/- 0.03 (P < 0.05) mug/ml in serum from 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively]. Acetylcholine (ACh)-induced relaxation was not altered in vessels from lean animals from 8-40 wk. ACh-induced relaxation was nearly abolished in coronary arteries from 28- to 36-wk-old Zucker obese rats and by 16-36 wk in ZDF rats and was attenuated in aorta and mesenteric vessels from ZDF rats [%relaxation to 10 muM ACh: 72.2 +/- 7.1, 17.9 +/- 5.9 (P < 0.05), and 23.0 +/- 4.5 (P < 0.05) in coronary vessels; and 67.9 +/- 9.2, 50.1 +/- 5.5, and 42.3 +/- 4.7 (P < 0.05) in mesenteric vessels from 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively]. The attenuated ACh-induced relaxation was improved when vessels were incubated with tiron, suggesting superoxide as a mechanism of endothelial dysfunction. Sodium nitroprusside-induced relaxation was not altered in aorta or coronary arteries and was potentiated in mesenteric arteries from Zucker obese rats. Our data suggest that diabetes enhances the progression of vascular dysfunction. Increases in indices of oxidative stress precede the development of dysfunction and may serve as a marker of endothelial damage.     

Effects of microtubules and microfilaments on [Ca(2+)](i) and contractility in a reconstituted fibroblast fiber

We used a reconstituted fiber formed when 3T3fibroblasts are grown in collagen to characterize nonmusclecontractility and Ca²⁺ signaling. Calf serum (CS) andthrombin elicited reversible contractures repeatable for >8 h. CSelicited dose-dependent increases in isometric force; 30% produced thelargest forces of 106 ± 12 µN (n = 30), whichis estimated to be 0.5 mN/mm² cell cross-sectionalarea. Half times for contraction and relaxation were 4.7 ± 0.3 and 3.1 ± 0.3 min at 37°C. With imposition of constant shortening velocities, force declined with time, yieldingtime-dependent force-velocity relations. Forces at 5 s fit thehyperbolic Hill equation; maximum velocity(V_max) was 0.035 ± 0.002 L_o/s.Compliance averaged 0.0076 ± 0.0006 L_o/F_o. Disruption of microtubules with nocodazole in a CS-contracted fiber had no net effects on force, V_max, or stiffness; force increased in 8, butdecreased in 13, fibers. Nocodazole did not affect baselineintracellular Ca²⁺ concentration([Ca²⁺]_i) but reduced (~30%) the[Ca²⁺]_i response to CS. The force afternocodazole treatment was the primary determinant of stiffness andV_max, suggesting that microtubules were not amajor component of fiber internal mechanical resistance. Cytochalasin Dhad major inhibitory effects on all contractile parameters measured butlittle effect on [Ca²⁺]_i.

     

Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca²⁺-activated K⁺ (K_Ca) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI₂). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI₂ is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-mediated vascular relaxation. The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway. Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells. Consequently, activation of TRPV4 channels induces smaller changes in whole-cell current density. Thus, ovariectomy leads to a reduction in endothelial SK3 channel activity thereby reducing the SK3 contribution to EDH vasorelaxation.     

Modification in Cell Shape Unrelated to Cellulose Microfibril Orientation in Growing Thallus Cells of Chaetomorpha moniligera

Cellulose microfibril orientation patterns in thallus cellsof Chaetomorpha moniligera were studied, and the relationshipbetween the microfibril and the peripheral microtubule arrangementsduring cell-shape modification by colchicine was examined. Inthe cuttings from growing thalli, linearly arranged cylindricalcells developed into cask-shaped cells during 4–6 daysof culture at 27?C. In the cylindrical cells, microfibrils formingthe innermost portion of the wall were arranged alternatelyin longitudinal and transverse directions, but peripheral microtubuleswere always arranged only in a longitudinal direction. Thesefeatures were also noted in the cask-shaped cells. Colchicineat 10^–3M and 3?10^–3M accelerated both cell expansionand wall thickening with matrix deposition, but the directionsin which both microfibrils and microtubules were arranged werethe same as those of the cylindrical cells. These results indicatethat (1) the microfibril and microtubule arrangements of Chaetomorphaare not necessarily correlated, (2) changes in cell shape ofChaetomorpha are not necessarily accompanied by changes in thearrangement of cell-wall microfibrils, and (3) colchicine playsa role in the loosening and thickening of cell walls by enhancingmatrix deposition. (Received June 2, 1986; Accepted February 13, 1987)     

Involvement of Microtubules on Nuclear Positioning during Apical Growth in Adiantum protonemata

The position of the nucleus during apical growth of a single-celledprotonema in Adiantum capillus-veneris under continuous redlight was observed to find whether any cytoskeletons were involvedin determining its location. The nucleus migrated through thefilamentous cell keeping a constant distance of ca. 55 µmfrom the tip, but was not able to maintain this position inthe presence of colchicine. The nuclei in most cells could bedisplaced by centrifugation at 110?g for 15 min in the presenceof anti-micro-tubule drugs such as colchicine, ethyl N-phenylcarbamateand griseofulvin, but not when these drugs were absent. Similartreatment with cytochalasin B did not cause the displacing effect.These results suggest that microtubules have a role determiningthe position of the nucleus when it migrates during apical growth. ¹ Present address: Department of Developmental Biology, ResearchSchool of Biological Sciences, The Australian National University,Canberra City, A.C.T. 2601, Australia. (Received November 27, 1984; Accepted February 18, 1985)     

Differential relaxant responses of pulmonary arteries and veins in lung explants of guinea pigs

Shi, Weibin, David H. Eidelman, and René P. Michel.Differential relaxant responses of pulmonary arteries and veins inlung explants of guinea pigs. J. Appl.Physiol. 83(5): 1476-1481, 1997.The endotheliumregulates vascular tone through release of relaxing or contractingfactors, with nitric oxide (NO) being a major endothelium-derivedrelaxing factor. In the present study, we used a lung explant techniqueto determine the differential abilities and mechanisms of pulmonaryarteries and veins of normal guinea pigs to relax after precontraction.Excised lungs of 15 guinea pigs were filled through the airways with1% agarose, cut into 1-mm-thick slices, and cultured overnight.Luminal areas of vascular cross sections were measured with animage-analysis system. Vessels were precontracted with U-46619, andresponses to histamine, acetylcholine (ACh), sodium nitroprusside, andpapaverine were examined. We also determined the effects ofN-nitro-L-arginineand of indomethacin on ACh-induced responses. We found that histaminerelaxed arteries more than veins and that ACh relaxed only arteries.N-nitro-L-arginine pretreatmentabolished ACh-induced relaxation of arteries and caused ACh-inducedcontraction of veins, whereas indomethacin markedly augmentedACh-induced relaxation of arteries (maximal relaxation: 48.5 ± 4.7 vs. 19.2 ± 5.1% without it) and induced a dose-dependentrelaxation of veins (maximal relaxation: 17.0 ± 4.1%). Sodiumnitroprusside induced a significantly greater relaxation of arteriesthan veins, whereas papaverine relaxed them equally. We conclude thatin guinea pigs endothelial NO-mediated relaxation is greater inpulmonary arteries than in veins and that ACh-induced NO-mediatedrelaxation is reduced by the simultaneous production ofcyclooxygenase-derived vasoconstrictors.

     

Superoxide dismutase restores endothelium-dependent arteriolar dilatation during acute infusion of nicotine

We previouslyshowed [Am. J. Physiol. 272 (Heart Circ. Physiol. 41):H2337-H2342, 1997] that nicotine impairsendothelium-dependent arteriolar dilatation. However, mechanisms thataccounted for the effect of nicotine on endothelium-dependentvasodilatation were not examined. Thus the goal of this study was toexamine the role of oxygen radicals in nicotine-induced impairment of arteriolar reactivity. We measured diameter of cheek pouch resistance arterioles (~50 µm diameter) in response to endothelium-dependent (ACh and ADP) and -independent (nitroglycerin) agonists before andafter infusion of vehicle or nicotine in the absence or presence ofsuperoxide dismutase. ACh, ADP, and nitroglycerin produced dose-relateddilatation of cheek pouch arterioles before infusion of vehicle ornicotine. Infusion of vehicle, in the absence or presence of superoxidedismutase (150 U/ml), did not alter endothelium-dependent or-independent arteriolar dilatation. In contrast, infusion of nicotine(2 µg · kg¹ · min¹)impaired endothelium-dependent, but not -independent, arteriolar dilatation. In addition, the effect of nicotine onendothelium-dependent vasodilatation was reversed by topicalapplication of superoxide dismutase. We suggest that nicotine impairsendothelium-dependent arteriolar dilatation via an increase in thesynthesis/release of oxygen-derived free radicals.

     

Overexpression of human KCNA5 increases IK V and enhances apoptosis

Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K⁺ efflux and K⁺ channel activity. Inhibited apoptosis and downregulated K⁺ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K⁺ (Kv) channel, increases K⁺ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (I_K(V)) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K⁺ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K⁺ currents (i.e., K⁺ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH). potassium ion channel; pulmonary hypertension     

Disruption of endothelial caveolae is associated with impairment of both NO- as well as EDHF in acetylcholine-induced relaxation depending on their relative contribution in different vascular beds

Caveolae represent an important structural element involved in endothelial signal-transduction. The present study was designed to investigate the role of caveolae in endothelium-dependent relaxation of different vascular beds. Caveolae were disrupted by cholesterol depletion with filipin (4x10(-6) g L(-1)) or methyl-beta-cyclodextrin (MCD; 1x10(-3) mol L(-1)) and the effect on endothelium-dependent relaxation was studied in rat aorta, small renal arteries and mesenteric arteries in the absence and presence of L-NMMA. The contribution of NO and EDHF, respectively, to total relaxation in response to acetylcholine (ACh) gradually changed from aorta (71.2+/-6.1% and 28.8+/-6.1%), to renal arteries (48.6+/-6.4% and 51.4+/-6.4%) and to mesenteric arteries (9.1+/-4.0% and 90.9+/-4.1%). Electron microscopy confirmed filipin to decrease the number of endothelial caveolae in all vessels studied. Incubation with filipin inhibited endothelium-dependent relaxation induced by cumulative doses of ACh (3x10(-9)-10(-4) mol L(-1)) in all three vascular beds. In aorta, treatment with either filipin or MCD only inhibited the NO component, whereas in renal artery both NO and EDHF formation were affected. In contrast, in mesenteric arteries, filipin treatment only reduced EDHF formation. Disruption of endothelial caveolae is associated with the impairment of both NO and EDHF in acetylcholine-induced relaxation.     

Permeation of Ca2+ and monovalent cations through an outwardly rectifying channel in maize root stelar cells

A depolarization-activated outwardly-rectifying channel (OR),most likely involved in the passive release of K⁺ from the rootsymplasm into the stelar apoplast (for subsequent transportto the shoot via the xylem vessels), has been characterizedin the plasma membrane of maize root stelar cells (Roberts andTester, 1995). In the present study, the selectivity of thischannel was further characterized using single channel current-voltagecurves generated using a voltage ramp protocol. This protocolpermitted the accurate and unambiguous measurement of the reversalpotentials of currents resulting from single channel openings.Using the voltage ramp protocol, it was shown that the OR allowsboth K⁺ efflux and Ca²⁺ influx at potentials positive of E_Kand negative of E_Ca. The OR had a P_Ca/P_K of 1.72–0.21decreasing as extracellular Ca²⁺ was increased. The permeabilityof the OR for monovalent cations other than K⁺ was also investigated.In biionic conditions, a relative permeability sequence of was determined (i.e. Eisenman sequenceIV). The physiological implications of the selectivity of theOR are discussed. Key words: Maize roots, K⁺ channel selectivity, Ca²⁺ permeation     

Effects of Colchicine, Vinblastine, Cytochalasin B and Phalloidin on the Seismonastic Movement of Mimosa pudica Leaf and on Motor Cell Ultrastructure

Fleurat-Lessard, P., Roblin, G., Bonmort, J. and Besse, C. 1988.Effects of colchicine, vinblastine, cytochalasin B and phalloidinon the seismonastic movement of Mimosa pudica leaf and on motorcell ultrastructure.—J. exp. Bot. 39: 209–221. Colchicine at 1 x 10^–3 mol dm^–3 does not affectthe seismonastic movement of Mimosa pudica leaves but disruptsmicrotubules in motor cells. Vinblastine at 5 x 10^–3 moldm^–3 does not affect this movement and partly disruptsmicrotubules. Vinblastine at 1 x 10^–4 mol dm^–3 alwaysdisrupts microtubules, even after a 12 h reversibility whenthe movement is restored. These drugs, applied at the same respectiveconcentrations, do not alter cytoplasmic and vacuolar fibrils.Cytochalasin B and phalloidin alter the seismonastic movementof Mimosa leaves when applied at concentrations of 1.25 x 10^–3and 2.4 x 10^–4 mol dm^–3 respectively. These drugs,used at the same respective concentrations, also affect themotor cell structure and, in particular, modify the arrangementand the structure of the fibrils but they do not destroy themicrotubules. These data suggest that microtubules are not directly involvedin the seismonastic reaction whereas fibrils, formed by thin(3.0 nm wide) filaments, may be implicated in this reaction. Key words: Colchicine, cytochalasin B, phalloidin, Mimosa pudica, motor cells, vinblastine     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium     

Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids

O'Donnell, Denise C., Mary L. Tod, and John B. Gordon.Developmental changes in endothelium-dependent relaxation of pulmonary arteries: role of EDNO and prostanoids. J. Appl. Physiol. 81(5): 2013-2019, 1996.Wehypothesized that maturational changes in both prostaglandin andendothelium-derived nitric oxide (EDNO) activity contribute todevelopmental changes in endothelium-dependent relaxation of newbornpulmonary arteries. Responses to endothelium-dependent vasodilatorsacetylcholine, bradykinin, and calcium ionophore A-23187 weredetermined in phenylephrine-constricted third- and fourth-generation(1- to 2-mm-diameter) pulmonary artery rings from 2-day (2d)- and 1-mo(1m)-old lambs under control conditions (Con), after inhibition of EDNOsynthesis withN-nitro-L-arginine(L-NNA), after inhibition ofprostanoid synthesis with meclofenamate (Mec), or both modulators withboth inhibitors. Endothelium-independent responses to sodiumnitroprusside (SNP) were also measured in Con rings.Endothelium-dependent relaxation was greater in 2d than 1m Con rings,particularly at high concentrations when an increase in tensionoccurred in 1m rings. L-NNAattenuated endothelium-dependent relaxation more in 2d rings, and SNPcaused greater relaxation in 2d rings. However, Mec abolished allage-related differences by attenuating relaxation in 2d rings andconstriction in 1m rings. These data suggest that developmental changesin endothelium-dependent responses of ovine pulmonary artery rings reflect both a decrease in EDNO activity and maturational differences in the relative influence of dilator and constrictor prostanoids.