Mechanism of protein kinase c potentiation of airway β-adrenergic relaxation

To evaluate the regulatory action of protein kinase C (PKC) on airway β-adrenergic function, the relaxant effects of isoproterenol (ISO) and 8 bromo-cyclic AMP (BrcAMP) were examined in tracheal smooth muscle (TSM) segments half-maximally contracted with acetylcholine in the absence (control) and presence of PKC activation with the phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPS). Relative to control tissues, TSM treated with 0.1μM DPB depicted significantly enhanced maximal relaxation and sensitivity to ISO but not to BrcAMP. The enhancing effect of DPB on ISO responsiveness was completely inhibited in the presence of the PKC antagonist H-7. Inhibition of the Na+-K+ pump with either ouabain or K+-free buffer diminished the TSM relaxant response to ISO but not to BrcAMP. Inhibition of the Na+-K+ pump also ablated the DPB-induced potentiation of β-adrenoceptor responsiveness. Collectively, these data demonstrate that: 1) PKC activation enhances TSM relaxant responsiveness to β-adrenoceptor stimulation; 2) inhibition of the airway Na+-K+ pump markedly blunts the relaxant response to β-adrenoceptor stimulation; and 3) inhibition of the Na+-K+ pump abolishes the above potentiating effect of DPB on β-adrenoceptor-mediated relaxation of rabbit TSM. Thus, the above findings provide new evidence that PKC activation enhances the airway relaxant response to β-adrenoceptor stimulation, and that the latter effect is dependent on potentiated stimulation of the airway electrogenic Na+-K+ pump.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Cyclosporin A inhibits proteolytic cleavage and degradation of membrane-bound protein kinase C in hepatocytes after stimulation by phorbol ester

Stimulation of hepatocytes by the tumor promoter phorbol 12-myristate 13-acetate (PMA) caused translocation of cytosolic Ca2+/phospholipid-dependent protein kinase C (PK-C). The major part of PK-C activity (greater than 80%) was associated with the membrane fraction after 30 min. During the following 6 h protein kinase C activity decreased to less than 10%. Minor amounts of Ca2+/phospholipid-independent PK-C activity were found in the cytosol fraction at all times; they temporarily increased 2.5-fold with PMA and decreased after 1 h. Cyclosporin A did not affect the translocation of PK-C from the cytoplasm to the membrane fraction, but the decrease of PK-C activity following translocation was blocked. No marked increase of Ca2+/phospholipid-independent PK-C activity was observed in the cytosol in the presence of cyclosporin A. Leupeptin, which is known to inhibit Ca2+-requiring non-lysosomal proteinases (e.g. calpain), showed an effect similar to cyclosporin A. Both agents reduced proteolytic degradation of cellular proteins observed in isolated hepatocytes after PMA treatment. Ca2+-ionophore A23187 in high doses (greater than 10(5) M) partly reversed cyclosporin A and leupeptin action.     

Inhibitory action of opioid peptides on ouabain-sensitive Na+-K+ and Ca2+-dependent ATPase activities in bovine cardiac sarcolemma

The present study demonstrates that morphine (10(-6) and 10(-5) M), methionine-enkephalin or leucine-enkephalin (10(-10), 10(-8), and 10(-6) M) were able to inhibit significantly, in a dose-dependent manner, both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. The inhibitory action of these opioids on the two ATPases was not antagonized by preincubation with naloxone (10(-6) M). Naloxone alone (10(-8), 10(-6) and 10(-5) M) did not affect both the sarcolemmal Ca2+-dependent ATPase and the ouabain-sensitive Na+-K+ ATPase activities. Heat-denatured methionine-enkephalin (10(-6) M) or leucine-enkephalin (10(-6) M) also unaffected both the ATPases. The possibility is also discussed that opioid peptides may regulate myocardial contractility by modulating the movement of ions across the heart sarcolemma.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

Tunicamycin reduces Na(+)-K(+)-pump expression in cultured skeletal muscle.

The purpose of this study was to examine effects of tunicamycin (TM), which inhibits core glycosylation of the beta-subunit, on functional expression of the Na(+)-K+ pump in primary cultures of embryonic chick skeletal muscle. Measurements were made of specific-[3H]-ouabain binding, ouabain-sensitive 86Rb uptake, resting membrane potential (Em), and electrogenic pump contribution to Em (Ep) of single myotubes with intracellular microelectrodes. Growth of 4-6-day-old skeletal myotubes in the presence of TM (1 microgram/ml) for 21-24 hr reduced the number of Na(+)-K+ pumps to 60-90% of control. Na(+)-K+ pump activity, the level of resting Em and Ep were also reduced significantly by TM. In addition, TM completely blocked the hyperpolarization of Em induced in single myotubes by cooling to 10 degrees C and then re-warming to 37 degrees C. Effects of tunicamycin were compared with those of tetrodotoxin (TTX; 2 x 10(-7) M for 24 hr), which blocks voltage-dependent Na+ channels. TM produced significantly greater decreases in ouabain-binding and Em than did TTX, findings that indicate that reduced Na(+)-K+ pump expression was not exclusively secondary to decreased intracellular Na+, the primary regulator of pump synthesis in cultured muscle. Similarly, effects of TM were significantly greater than those of cycloheximide, which inhibits protein synthesis by 95%. These findings demonstrate that effects were not due to inhibition of protein synthesis. We conclude that glycosylation of the Na(+)-K+ pump beta-subunit is required for full physiological expression of pump activity in skeletal muscle.     

The effect of endothelin-1 on Na+/H+ metabolism in vascular smooth muscle cells]

The effects of endothelin on intracellular pH (pHi) were examined in cultured rat vascular smooth muscle cells (VSMC) using the fluorescent probe BCECF. Endothelin induced biphasic changes in pHi: initial decrease followed by a subsequent increase above the basal level due to activation of the Na+/H+ exchange. The elevation of pHi was slow and sustained, but depended on the dose of endothelin: IC50 was about 3 x 10(-8) M. Na+/H+ exchange inhibition by EIPA (10(-7) M) or by equimolar replacement of external Na+ by choline abolished the pHi increase by enhancing the first phase of cytoplasm acidification. Effects of endothelin were compared with the action of protein kinase C (PK-C) activator phorbol 12-13 myristate ester (PMA). PMA induced a monophasic slow and sustained increase in pHi. The treatments of VSMC with H-7 and staurosporine (PK-C) inhibitors prevented the pHi response to endothelin and PMA. These results suggest that protein kinase C may play an important role in mediating the effects of endothelin on Na+/H+ exchange in VSMC.     

Ca(2+)-dependent K(+) channels and Na(+)-K(+)-ATPase mediate H(2)O(2)- and superoxide-induced relaxations in canine trachealis.

We examined the ionic mechanisms underlying the responses of canine trachealis to superoxide (generated in vitro by using xanthine oxidase or added exogenously) and peroxide (generated spontaneously in vitro by the dismutation of superoxide or added exogenously). Although neither had any effect on resting tone, both triggered relaxations in carbachol-precontracted tissues. These relaxations were eliminated by catalase but were much less sensitive to the hydroxyl radical scavenger dimethylthiourea, indicating they were mediated primarily by peroxide. These relaxations were decreased in magnitude and/or slowed by nifedipine (10(-6) M), ouabain (10(-6) M), or tetraethylammonium (25 mM), but not by 4-aminopyridine (5 mM), and were small or absent in tissues precontracted with 30 mM KCl. Finally, peroxide triggered membrane hyperpolarization and elevated cytosolic concentration of Ca(2+) (primarily via release from the internal store). Thus peroxide-mediated relaxations seem to involve Ca(2+) release, opening of Ca(2+)-dependent K(+) channels, hyperpolarization, closure of Ca(2+) channels, and relaxation. In addition, some other free radical (hydroxyl radical?) may activate the Na(+)-K(+) pump, also hyperpolarizing the membrane and causing relaxation.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

Effects of temperature on cholinergic contractility of rabbit airway muscle

To elucidate the mechanism underlying temperature-induced changes in airway cholinergic contractility, the effects of organ bath cooling were evaluated in isolated rabbit airway smooth muscle (ASM) segments isometrically contracted with methacholine (METH) (10(-8)-10(-3) M) and electrical field stimulation (ES), wherein the ES stimulus frequency was varied between 1 and 100 Hz. Cooling from 37 to 25 degrees C produced systematic increases (P less than 0.01) in isometric tension at various administered doses of METH and at different levels of ES. Since the potentiated contractions to ES significantly exceeded (P less than 0.001) the corresponding increases in METH-induced contractility, we evaluated whether the latter was attributed to temperature-mediated changes in intrinsic airway neuronal acetylcholine (ACh) release. Accordingly, the effects of ASM cooling were independently determined before and after inhibition of the Na+-K+ electrogenic pump with ouabain (10(-5) M), and depletion of intrinsic neuronal ACh stores with hemicholinium-3 (HC-3) (10(-3) M). In the presence of either ouabain or HC-3 the above responses to temperature reduction were reversed, and airway cooling was associated with abrupt relaxation of ASM segments precontracted with METH. In contrast, neither inhibition of cyclooxygenase products with indomethacin (10(-6) M) nor cholinesterase inhibition with neostigmine (10(-3) M) notably influenced the ASM responses to organ bath cooling. Thus these findings demonstrate that 1) both METH-induced and neurally mediated cholinergic contractility are augmented during airway cooling; 2) the potentiated cholinergic responses are attributed to enhanced presynaptic release of ACh at the airway neuromuscular junction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bleomycin stimulates both membrane (Na+-K+) ATPase and electrogenic (Na+-K+) pump and partially removes the inhibition by vanadium ions

Bleomycin 2 X 10(-6) and 6 X 10(-6) mol.1(-1) increased the activity of specific (Na+-K+) ATPase of the rat brain microsomes. It also stimulated the electrogenic (Na+-K+) pump in intact skeletal muscle cells. The blocking effect of vanadyl (+4V) on membrane (Na+-K+) ATPase was eliminated completely by the drug, but the action of vanadate (+5V) was counteracted only partially. Electron paramagnetic resonance spectra revealed the formation of a +4V - bleomycin complex which is still able to activate the (Na+-K+) ATPase.     

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+-K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.     

The effect of vanadate on the electrogenic Na+/K+ pump, intracellular Na+ concentration and electrophysiological characteristics of mouse skeletal muscle fibre

The present study reports a discrepancy between the effects of vanadate on the membrane Na+-K+-ATPase and the Na+/K+ pump of the skeletal muscle. Vanadate in concentration 4 X 10(-6) mol/l which is necessary to block the enzyme Na+-K+-ATPase activity of membrane fractions failed to inhibit the electrogenic Na+/K+ pump of intact muscle cells. The effect of vanadate on the electrophysiological parameters of the muscle fibre membrane required much higher vanadate levels, but again, Na+/K+ pump was still active. Vanadate in concentrations 4 X 10(-4) and 4 X 10(-5) mol/l depolarized the membrane potential and decreased the membrane resistance [apparently in consequence of enhanced passive membrane permeability for Na+ ions]. Action potentials and the electrical excitability of the muscle fibre membrane were reduced by these vanadate concentrations.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Dual effect of dihydropyridines on 45Ca uptake induced by the K+ -channel blocker, 4-aminopyridin on mast cells

The 1,4-dihydropyridines (DHP) are calcium antagonists and represent a new class of drugs which act by a selective inhibition of Ca++ influx through voltage-operated calcium channels. We report the effect of nifedipine (Bay A 1040), nisoldipine (Bay K 5552) and nitrendipine (Bay E 5009) on the histamine release and on the 45Ca uptake promoted by 4-aminopyridin in mast cells. These cells treated with DHP (10(-12)-10(-3) M) activated the secretory response in a dose-dependent manner in the range of concentrations 10(-6)-10(-3) M, whereas concentrations of 10(-12)-10(-6) M did not significantly inhibit the secretion. 4-Aminopyridin, a known K+ -channel blocker, induced 45Ca uptake. Pretreatment of mast cells with DHP prior to 4-aminopyridin stimulation inhibited or stimulated 45Ca uptake depending on concentration; thus, concentrations of DHP below 10(-12) of nitrendipine and 10(-9) for nisoldipine and nifedipine were inhibitory, while higher doses potentiated 45Ca uptake. These results demonstrate a diversity of pharmacological effects of DHP on mediator secretion and 45Ca uptake in mast cells and throw into question their only properties as Ca++ antagonists.     

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.     

Ca2+-induced down-regulation of Na+ channels in toad bladder epithelium

Regulation of epithelial Na+ channels was investigated by measuring the amiloride-blockable 22Na+ fluxes in apical membrane vesicles, derived from cells exposed to various treatments. Maximal amiloride-blockable 22Na+ uptake into vesicles was obtained if the cells were preincubated at 25 degrees C in a Ca2+-free [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) solution. Including 10(-5) M Ca2+ in the cell incubating medium blocked nearly all of the amiloride-sensitive flux in vesicles, even though the Ca2+ was removed before homogenization of the cells. This Ca2+-dependent inhibition of Na+ channels could be induced in whole cells only; incubating cell homogenates with Ca2+ had no effect on the transport in vesicles. The dose-response relationships of this effect were measured by equilibrating cell aliquots with various Ca2+-EGTA buffers, preparing membrane vesicles (in the absence of Ca2+ ions), and assaying them for amiloride-sensitive Na+ permeability. It was found that the Ca2+ blockage is highly cooperative (Hill coefficient of nearly 4) and is characterized by an inhibition constant which varies between 6.4 X 10(-8) to 8.15 X 10(-6)M Ca2+. Thus, it is likely that the above process is involved in the physiological control of Na+ transport. The Ca2+-dependent transport changes were not affected by the calmodulin inhibitor trifluoperasine, vanadate (VO3-), phorbol ester, colchicine, cytochalasin B, 3-deazaadenosine, and 8-bromo-cAMP. Vanadyl (VO2+) ions, on the other hand, produced a "Ca2+-like" inhibition of transport.     

beta-Adrenergic effect on Na+-K+ transport in rat skeletal muscle.

1. Intact rat extensor digitorum longus muscles soaked in L-isoproterenol plus 10(-5) M ouabain gained less sarcoplasmic Na+ than did muscles soaked in ouabain alone. Half maximal effect was produced by 10(-8) M L-isoproterenol. 2. D-Isoproterenol and oxidized L-isoproterenol were only 3 and 1%, respectively, as potent as L-isoproterenol. Other catechols tested had no effect. 3. The effect of L-isoproterenol on sarcoplasmic Na+ content appears to be a beta-adrenergic function in that it was blocked by propranolol, but not by phentolamine, and could be mimicked by dibutyryl cyclic AMP or by caffeine. 4. Reduced gain in sarcoplasmic Na+ was accompanied by reduced loss of sarcoplasmic K+. 5. L-Isoproterenol increased loss of sarcoplasmic Na+ in the absence of ouabain, in muscles recovering from cold treatment. 6. Results suggest that the beta-adrenergic system stimulates a coupled Na-K+ pump. 7. A model is proposed in which stimulation of the Na+-K+ pump in response to beta-adrenergic agents involves a number of intermediate steps, identified tentatively.     

Extracellular Ca2+ mobilization in potential-dependent contraction of trachealis of maturing swine.

We studied the effect of maturation on potassium-induced parasympathetic activation and Ca2+ entry in tracheal smooth muscle (TSM) from fifteen 2-wk-old (2ws) and sixteen 10-wk-old (10ws) male domestic farm swine. Atropine (10(-7) M) caused inhibition of the maximal contraction elicited by potassium to 50.3 +/- 2.6% maximum of control response (P less than 0.001) in TSM from 2ws but had no significant effect in TSM from 10ws (94.6 +/- 4.2% maximum; P = NS vs. control). Verapamil (10(-7) M) plus 10(-7) M atropine reduced contraction elicited by potassium in both 2ws (23.7 +/- 5.8% maximum; P less than 0.001 vs. control) and 10ws (50.6 +/- 6.3% maximum; P less than 0.001 vs. control, P less than 0.05 vs. 2ws); 10(-6)M verapamil caused greater than 95% blockade of contraction caused by potassium in both 2ws and 10ws. In separate studies, atropine-treated strips were equilibrated with extracellular Ca2+ concentrations ([Ca2+]o) ranging from normal (1X [Ca2+]o) to four times normal (4x [Ca2+]o). Increasing [Ca2+]o increased maximal contractile response in atropine-treated TSM strips from 68.7 +/- 3.8% maximum for 1x [Ca2+]o to 100.8 +/- 4.8% maximum for 4x [Ca2+]o (P less than 0.001) in 2ws. Neither atropine nor [Ca2+]o affected maximal responses of TSM in 10ws (103.5 +/- 3.0% maximum for 1x [Ca2+]o; P = NS vs. control). However, in the presence of atropine and verapamil, 4x [Ca2+]o augmented KCl-elicited contraction of TSM from both 2ws (46.9 +/- 6.3% maximum; P less than 0.01 vs. control) and 10ws (78.6 +/- 2.3% maximum; P less than 0.005 vs. control).(ABSTRACT TRUNCATED AT 250 WORDS)     

Time- and dose-dependent influence of ouabain on the ultrastructure of optic neurones

The cardiac glycoside ouabain was injected into the eye-bulb of the teleost fish, Carassius carassius. Three doses of ouabain were used: 10(-4) M, 10(-5) M, 10(-6) M. The final concentrations in the vitreous body of the eye were approximately 3-10(-5) M, 3-10-6 M and 3-10-7 M, respectively. After 8 hrs, 1, 2, 4, 6 and 8 days the ultrastructural alterations of retinal ganglion cells, the optic axons near the bulb and the terminal segments in the optic tectum were studied. The high doses of ouabain induced an early necrobiosis of the cell bodies in the retina followed by degeneration in the nerve. This is characterized as a protracted form of Wallerian degeneration. The significance of the inhibition of Na+ -K+-activated ATPase at the perikaryal level for both the integrity of axonal morphology and the axonal flow is discussed.