Rhodamine B, a fluorescent probe for acidic organelles in denervated skeletal muscle.

We describe a very efficient method for fluorescent labeling of acidic structures in denervated skeletal muscle with rhodamine B. Rhodamine B at 50 ng/ml gave selective and distinct segmental labeling of denervated muscle fibers after 5-min incubation at room temperature. Labeling was also achieved at 4 degrees C. The labeling was disrupted by the ionophores monensin and nigericin, suggesting a labeling confined to acidic structures. Rhodamine B co-localized with the lysosomotropic dye Lyso Tracker Green and a marker for endocytosis (fluorescein isothiocyanate-labeled dextran). Rhodamine B, which is highly lipophilic, showed pH-dependent fluorescence emission in saturated aqueous N-octanol. Tetramethylrhodamine showed similar characteristics for labeling of denervated muscle fibers and pH-dependent fluorescence in N-octanol. The carboxyl group present in these two compounds appears important, because structurally related compounds that either lack this group or have it esterified failed to label denervated muscle fibers and showed no pH-dependent fluorescence in N-octanol. The results suggest that rhodamine B labels acidic organelles belonging to the endosomal/lyosomal system of denervated skeletal muscle fibers. Nevertheless, it failed to label such organelles in a number of mammalian cell types other than denervated skeletal muscle fibers.

Secretion of plasminogen activator and lysosomal enzymes from mouse skeletal muscle: effect of denervation.

Levels of hydrolytic enzymes increase in skeletal muscle after denervation and their activities in the extracellular matrix appear to be important for interaction between muscle and nerve. Using enzymatic assays for beta-glucuronidase, beta-galactosidase, and plasminogen activator, we show that secretion of these enzymes from mouse skeletal muscle increases after denervation and that drugs interfering with the secretory pathway or the reuptake of enzymes modulate this release. Thus, brefeldin A inhibited secretion of plasminogen activator activity and mannan increased secreted amounts of beta-glucuronidase, but not of beta-galactosidase, in denervated muscle. In innervated muscle, brefeldin A decreased secreted activity of plasminogen activator, but mannan had no effect on secretion of either beta-glucuronidase or beta-galactosidase. Furthermore, secretion of plasminogen activator was temperature dependent. These observations, together with previous studies, suggest that secretion of hydrolytic enzymes from adult skeletal muscle may be of physiological significance in nerve-muscle communication.

Reversal by concanavalin A of the inhibitory effects of extracellular Ca2+ on pinocytosis in Amoeba proteus.

When Concanavalin A, 1-20 micrograms ml-1 binds to the surface of Amoeba proteus the cell's response to Ca2+ and to cationic inducers of pinocytosis is strikingly altered. Separately, Concanavalin A and Ca2+ are weak inducers but their combined effects are intense pinocytosis and suppression of the normal inhibitory effect of Ca2+ on cation-induced pinocytosis. At high concentrations (greater than 25 micrograms ml-1) the lectin increases cellular uptake and binding of 45Ca in the cell surface but the ionic permeability and the potential of the cell membrane are little changed by treatment with Concanavalin A. Subsequent addition of Ca2+ starts the pinocytic cycle and causes a fivefold increase of the membrane resistance without depolarizing the cell. Neither a rise of free intracellular calcium nor an influx of 45Ca precedes formation of pinocytotic channels when Con A is applied to cells in the presence of calcium. All effects of Concanavalin A are blocked by alpha-methylmannoside while succinyl-Concanavalin A is without effects on pinocytosis. These findings suggest that crosslinking of cell surface carbohydrates reverses calcium inhibition of pinocytosis and increases the cell's pinocytotic response to cations without modifying either the bioelectrical effects or the intracellular free concentration of calcium.

Possible regulation of cation-induced pinocytosis in Amoeba proteus by phospholipase A.

We have studied the effects of exogenous phospholipids and compounds which are known to alter the activity of phospholipase A (PLA) on Ca2+-dependent, Na+-induced pinocytosis in Amoeba proteus. The PLA-inhibitors mepacrine, p-bromophenacyl bromide (pBPB) and Rosenthal's inhibitor depressed pinocytosis. Normal pinocytotic intensity was restored by the addition of Ca2+ or picomolar concentrations of lysolecithin. Very low concentrations of lysophospholipids and different molecular species of lecithins increased the capacity for pinocytosis in starved amoebae. The effect of the lecithins but not of the corresponding lysolecithins was abolished by PLA-inhibitors. Also, the restoration of the pinocytotic capacity of starved amoebae by melittin and mastoparan, which are known to stimulate PLA, was inhibited by mepacrine and pBPB. Isolated amoeba plasma membranes contain phospholipase A1 and A2 activity and the amoebae secrete a lipid (PRF, pinocytosis regulating factor) which has lysolecithin-like effects on pinocytosis. The enzyme activities and the release of PRF were markedly decreased by the PLA-inhibitors. Our observations support the hypothesis that PRF is a lysophospholipid that may constitute a signal for the formation of pinocytotic channels in the initial stages of pinocytosis. The phospholipase A activity of the amoeba must therefore be assigned an important role in the regulation of the Ca2+-dependent, cation-induced pinocytosis.

Peptides as modifiers of Na+-induced pinocytosis in starved Amoeba proteus.

Low concentrations of six peptide hormones; glucagon, vasoactive intestinal peptide, substance P, angiotensin II, lysine-vasopressin, arginine-vasopressin, and the chemotactic peptide fMet-Leu-Phe, activated the capacity for pinocytosis in starved Amoeba proteus. Competitive inhibitors of the chemotactic peptide in leucocytes inhibited activation by fMet-Leu-Phe, suggesting that its action in the amoeba is mediated by specific receptors. The opioid peptides, beta-endorphin, dynorphin (1-13) and leu-enkephalin abolished through a naloxone-sensitive mechanism activation by hormones and several other activating agents. Also, low concentrations of beef and pork insulin inhibited activation by peptide hormones. An insulin analogue of low potency in mammalian cells was inactive in the amoeba. These results support the hypothesis that besides opioid receptors, there may be insulin receptors and possibly receptors for several other peptide hormones in Amoeba proteus.

Induction and inhibition of pinocytosis by aminoglycoside antibiotics.

We investigated whether differences in induction or stimulation of pinocytosis by six amino-glycosides reflected reported differences in their nephrotoxicity. Pinocytosis induced by antibiotics, Na+, K+ or Ca2+ was quantified by the number of pinocytotic channels in Amoeba proteus, a cell suitable for the study of the pinocytotic process. The aminoglycosides were potent inducers of pinocytosis. They were effective in the order of their cationic charge: neomycin greater than gentamicin greater than netilmicin = tobramycin greater than kanamycin greater than streptomycin. Factors which reduced the charge of the molecules, i.e. alkaline pH and combination with carbenicillin or heparin, diminished pinocytosis. Like La3+ the antibiotics inhibited Na+ -induced pinocytosis. The order of efficacy was netilmicin greater than gentamicin greater than neomycin. A similar rank order, which is the reverse of the order of nephrotoxicity, was observed for inhibition of Ca2+ -stimulated, Na+ -induced pinocytosis. Netilmicin was also the most potent inhibitor of the Ca2+-induced pinocytosis in cells treated with concanavalin A. Inhibition of Ca2+ -stimulated pinocytosis by netilmicin was reversed by Ca2+, the calcium ionophore A 23187, or 4-aminopyridine. We have shown that several nephrotoxic cations are strong inducers of pinocytosis in the amoeba, that aminoglycosides in Ringer solution induce pinocytosis in the approximate order of their nephrotoxicity and that factors which are known to diminish toxicity reduce pinocytosis. It, therefore, appears that the mechanism of aminoglycoside nephrotoxicity is related to their ability to induce pinocytosis in the amoeba. Low inducing potency and strong Ca2+ -antagonism, as for netilmicin, are qualities which may reduce the tendency of polycationic compounds to damage proximal tubular cells.

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus.

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.

Activation of the capacity for sodium-induced pinocytosis in Amoeba proteus.

Amoebae treated with cycloheximide or starved for 8-10 days lose their pinocytotic response to Na+. Their capacity for Na+-induced pinocytosis was activated after application of various physical or chemical stimuli (electrical stimulation, mechanical shearing forces, osmotic pressure, UV-light, alkali metal ions, capsaicin, and indole). The degree of activation was related to the intensity and duration of the stimulus and lasted several hours after the stimulus had been withdrawn. The dose-response curves of activating stimuli were always biphasic. Strong activating agents reduced the sensitivity of the amoeba to the inducer. At concentrations lower than those which induced pinocytosis, but in the same order of efficacy, inorganic cations were potent activating agents. Like induction of pinocytosis, activation by cations required minute amounts of Ca2+ and was inhibited by high concentrations of this ion. Activation may therefore be an early event during the induction of pinocytosis. Capsaicin and indole were potent activators, indicating that specific chemical stimuli may increase the capacity for pinocytosis. The activation may be the result of a secretory process adding area and structures to the old membrane which are necessary for the induction of pinocytosis.

Cold enhanced secretion of a pinocytosis-regulating factor in Amoeba proteus.

Low temperature inhibited preferentially pinocytosis induced by Na+ and Na+-like inducers, a category of inducers which are particularly dependent on the concentration of Ca++ that means susceptible to blockade by EGTA, La+++, or high Ca++. Coldness inhibited Na+-induced pinocytosis more effectively in cells from a calcium deficient medium than in normal cells and the inhibition was abolished by Ca++. In normally fed cells, a period of coldness prevented the subsequent induction of pinocytosis by Na+. This block was of the same type as that observed when the concentration of Ca++ was increased in the medium of normal cells kept at room temperature. In contrast, starved cells responded to coldness by an increased capacity for pinocytosis. These effects lasted for several hours after the cells had been rewarmed and were mediated by a cell-derived soluble factor, PRF, pinocytosis regulating factor. Solubility characteristics indicated that PRF is a lipid. Ca++, low pH, and low temperature stimulated accumulation of the factor in the medium. Preincubation with PRF or addition of PRF to the inducer stimulated pinocytosis in starved or Ca++-deficient cells and reduced the sensitivity to Na+-like inducers in normal cells. The amount of PRF secreted from cold cells was sufficient to cause the Ca++-like blockade of pinocytosis in the cold, the diminished pinocytotic activity after a cold period and the activating effect of coldness on starved cells. PRF appears to be an important physiological regulator of the capacity for pinocytosis in the amoeba.

Lysolecithin as a modifier of induced pinocytosis in Amoeba proteus.

Lysolecithin was found to modify cation-induced pinocytosis in Amoeba proteus. It is shown here that lysolecithin (LPC) in the concentration range of 10(-15) to 10(-10) g/ml has the same effect on Na+ -induced pinocytosis as cAMP and a pinocytosis regulating factor (PRF) which is secreted by the amoeba. Thus, LPC activated Na+-induced pinocytosis in starved amoebae and decreased the sensitivity to the inducer in normal cells. Pinocytosis depressed by treatment with EGTA or dibucaine became normal upon addition of LPC to the inducer. These effects were also obtained with lysolecithin isolated from the amoeba. It is suggested that PRF and amoeba LPC may be closely related and that phospholipase activity of the amoeba may regulate its capacity for pinocytosis.

Naloxone-reversible effect of opioids on pinocytosis in Amoeba proteus.

A characteristic feature of induced pinocytosis in Amoeba proteus is the formation of broad channels by invagination of the cell membrane. This process, which requires Ca2+, occurs in response to depolarising cations. High Ca2+ levels reduce pinocytosis induced by cations such as Na+ and Tris+, whereas pinocytosis induced by K+ is less affected by Ca2+ (ref. 4). Agents which interfere with the calcium metabolism of the amoeba will therefore either stimulate or inhibit pinocytosis induced by Na+ (ref. 5). Among the agents which are supposed to reduce Ca2+ influx across cell membranes or otherwise decrease cellular availability of Ca2+ are the opiates and opioid peptides, high doses of which have been reported to affect the amoeba. Accordingly, Met-enkephalin, morphine and codeine potentiate the inhibition of pinocytosis caused by Ca2+-binding agents and reverse the calcium blockade of pinocytosis mediated by caffeine. In this report we show that pinocytosis induced by Na+ or Tris+ is suppressed by beta-endorphin, Metenkephalin and morphine. These effects were abolished or diminished by an opiate receptor antagonist, (-)naloxone, by increasing the Na+ concentration, or by addition of Ca2+.

Effect of lanthanum on pinocytosis induced by cations in Amoeba proteus.

Lanthanum chloride (greater than or equal to 10(-5) M) induced pinocytosis in normal and at greater than or equal to 10(-4) M in Ca++-deficient amoeba. With respect to the Ca++-requirement of the pinocytotic response low and high concentrations of La+++ had effects like Na+ and K+, respectively. The concentration of La+++ stimulated or inhibited other types of pinocytosis. Thus all concentrations of La+++ inhibited sodium induced pinocytosis while high concentrations (greater than 10(-3) M) stimulated and low concentrations diminished potassium induced pinocytosis. Only the latter effect required the presence of Ca++. In the presence of La+++ other inducers acted either like K+ or Na+. Inducers may cause channel formation by opening a pore for Ca++ in the plasma membrane, Na+ like inducers being less effective than K+-like inducers, and by releasing Ca++ into the cytoplasm from the glycocalyx (Na+-like inducers) or from the entire cell membrane (K+-like inducers). La+++ may diminish the effect of Na+-like inducers and vice versa by direct competition for sites in the glycocalyx and the effect of a K+-like inducer by redistribution of Ca++ in the cell surface. At high concentrations or in the presence of a K+-like inducer La+++ may enter the Ca++ pore, release Ca++ from the interior of the membrane and so induce or stimulate pinocytosis.

Effect of ultraviolet radiation on pinocytosis in Amoeba proteua.

Ultraviolet (UV) irradiation (4 000-10 000 erg X mm(-2) decreased membrane potential and input resistance of Amoeba proteus and induced formation of pinocytotic channels. Submaximal pinocytosis induced by UV light was additive to pinocytosis induced by K+ or Na+ and stimulated in the presence of EGTA. It was not inhibited by the presence of La+++ or by pretreatment with dibucaine. In these respects and with respect to optimum pH and pCa, UV induced pinocytosis. Accumulation of K+ in the amoeba membrane after a dose of radiation may explain the similarity between pinocytosis induced by UV light and potassium salts. Ca++ present during the period of irradiation inhibited the effect of UV light. Instead Ca++ applied after irradiation (1-20 mM) increased channel formation. This effect was stimulated the presence of local anesthetic drugs. It is suggested that high doses of UV light may induce channel formation by releasing Ca++ from the cell membrane into the cell (UV induced pinocytosis). Ca++ may be released at the moment of absorption of UV light in the membrane as well as during the period of depolarization which follows irradiation. Low doses of UV light may permit extracellular Ca++ to enter the cell and stimulate channel formation (calcium induced pinocytosis). Dithiotreitol (1 mM) applied after irradiation depressed both UV and calcium induced pinocytosis so these may be the result of the same structural change which involves the formation of disulphide bonds in the membrane.

Inhibition of induced pinocytosis in Amoeba proteus by membrane stabilizing drugs.

The effect of membrane stabilizing drugs on cation induced pinocytosis was studied in Amoeba proteus. Initially the presence of local anesthetic drugs during a pinocytosis cycle had a stimulating effect on channel formation, however, the capacity to develop pinocytotic channels was reversibly inhibited after a period of treatment with these drugs. Imipramine, vinblastine and the phenothiazines had effects similar to local anaesthetics. The local anesthetics inhibited pinocytosis in the following order: dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine greater than procaine, and the phenothiazines: thioridazine greater than prochlorperazine greater chlorpromazine greater than prometazine. Pinocytosis, when induced by Na+ or tris, was more affected by the drugs and by calcium binding agents than pinocytosis induced by K+. After pretreatment with inhibitory concentration of dibucaine (3 x 10(-4) M) the depolarization of the membrane and the conductance increase during pinocytosis were normal, while the increase of oxygen uptake during the pincoytosis cycle was abolished. Addition of Ca++ before, during or after dibucaine treatment decreased the effect of the drug. Conversely, in dibucaine-treated cells, cation induced pinocytosis was less inhibited by Ca++ than pinocytosis in normal cells. Addition of EGTA to the inducing solutions potentiated the inhibitory effect of the drug. It is suggested that these drugs release Ca++ from the cell surface and at higher concentration or after prolonged incubation time interfere with a Ca++ mechanism which couples the membrane and contractile systems in the cytoplasm.

Membrane potential and conductance during pinocytosis induced in Amoeba proteus with alkali metal ions.

An investigation of the relationship between the polarized state of the membrane and the onset and the intensity of pinocytosis was made in Amoeba proteus. Membrane potential and input resistance was in all instances found to decrease in approximate proportion to the number of channels when pinocytosis was induced by a variety of alkali metal ions at varying pH. Channels began to appear when the membrane was depolarized to -30 mV by the inducer of pinocytosis. With all inducers the maximum pinocytosis was encountered at membrane potentials close to zero. No positive potentials were recorded when the chloride salts of the inducing cations were used. At high concentrations of alkali ions a transient increase of the chloride permeability caused short-lasting hyperolarizations of the membrane. Inhibition of pinocytosis by Ca++ was accompanied by an increase of input resistance and membrane potential. The selectivity of the membrane to different alkali metal ions observed as changes in pinocytosis intensity, membrane potential and input resistance was found to vary with the concentration of the inducer and with the Ca++ concentration of the extracellular solution. Displacement of membrane bound Ca++ appeared to decrease the field strength of charged groups in the membrane altering its selectivity among alkali cations. The formation of pinocytotic channels is suggested to require translocation of Ca++ from the membrane into the cell and would therefore be closely related to the electrical properties of the amoeba.