Antitumor activity of ethanol extract from Hippophae rhamnoides L. leaves towards human acute myeloid leukemia cells in vitro.

We studied the effects of ethanol extract from Hippophae rhamnoides L. leaves on the growth and differentiation of human acute myeloid leukemia cells (KG-1a, HL60, and U937). The extract of Hippophae rhamnoides L. leaves inhibited cell growth depending on the cell strain and extract dose. In a high concentration (100 µg/ml), the extract also exhibited a cytotoxic effect on HL60 cells. Hippophae rhamnoides L. leaves extract did not affect cell differentiation and did not modify the differentiating effect of calcitriol, active vitamin D metabolite. Inhibition of cell proliferation was paralleled by paradoxical accumulation of phase S cells (synthetic phase) with a reciprocal decrease in the count of G1 cells (presynthetic phase). The extract in a concentration of 100 µg/ml induced the appearance of cells with a subdiploid DNA content (sub-G1 phase cells), which indicated induction of apoptosis. The antiproliferative effect of Hippophae rhamnoides L. extract on acute myeloid leukemia cells was at least partially determined by activation of the S phase checkpoint, which probably led to deceleration of the cell cycle and apoptosis induction.

[The role of a GTP-binding protein in coupling of a muscarinic cholinergic receptor and Na,K-ATPase in myocardial sarcolemma]. / Rol' GTP-sviazyvaiushchego belka v sopriazhenii muskarinovogo kholinoretseptora i Na,K-ATPase v sarkolemme miokarda.

The effects of the cholinergic agonist carbachol (Cch) and guanine nucleotides on the Na,K-ATPase and K-dependent p-nitrophenylphosphatase (K-p-NPPase) activities in rabbit and dog myocardial sarcolemma vesicles in the presence of the pore-forming antibiotic alamethicin (20 micrograms/ml), was studied. Cch (0.01-100 microM) inhibited the both enzymatic activities by 40-45% (IC50 = 0.3-0.5 microM) only after addition of GTP (50 microM) or its analogs: GTP gamma S (0.1-1.0 microM) and Gpp(NH)p (10 microM). The muscarinic acetylcholine receptor (mAchR) antagonist atropine (10 microM) blocked the effect of Cch. GTP gamma S alone produced a concentration-dependent decrease in the both Na,K-ATPase and K-p-NPPase activities by 40-45% (IC50 = 1-2 microM) with a lag period of about 3 minutes; this lag disappeared in the presence of the agonist. The GDP analog GDP beta S (0.01-100 microM) neither affected these activities nor promoted the inhibiting effect of Cch. Pretreatment of sarcolemmal vesicles with 20 micrograms/ml of pertussis toxin in the presence of 100 microM NAD abolished the inhibiting effect of Cch on the Na,K-ATPase and phosphatase activities. Under these conditions pertussis toxin catalyzed the ADP-ribosylation of alpha-subunits of the inhibitory GTP-binding protein (G1) which were identified immunochemically as alpha i2, alpha i3 and, possibly, alpha i1. The data obtained testify to the involvement of G1 in the mAchR-mediated inhibition of myocardial sarcolemmal Na,K-ATPase as well as in the signal transduction from the receptor to the enzyme.

Na(+)-K(+)-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution.

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 microM) decrease in the Na(+)-K(+)-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[gamma-thio]triphosphate (0.1 microM GTP gamma S) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 microM) and is abolished in sarcolemma treated with pertussis toxin (20 micrograms/ml) in the presence of 100 microM NAD. GTP gamma S alone reduces Na(+)-K(+)-ATPase activity by 45% (half-maximum inhibitory = 1 microM). The apparent affinity of the enzyme for GTP gamma S is increased approximately 10-fold in the presence of 1 microM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTP gamma S-dependent inhibition of the Na(+)-K(+)-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na(+)-K(+)-ATPase and pertussis toxin-sensitive Gi activities. Na(+)-K(+)-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20-30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTP gamma S, or Gpp(CH2)p] at micromolar concentrations were restored when the Na(+)-K(+)-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive Gi protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5'-[beta-thio]diphosphate, or App(NH)p were unable to induce the Gi protein-mediated attenuation of Na(+)-K(+)-ATPase activity in the reconstitution system.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of carbacholine and serotonin on the ATPase activity in synaptosomes of the projection and association areas of the feline cerebral cortex]. / Vliianie karbakholina i serotonina na ATP-aznuiu aktivnost' sinaptosom proektsionnoi i assotsiativnoi zon kory golovnogo mozga koshki.

Na, K-ATPase and Mg-ATPase activities were measured in the synaptosomes of the temporal auditory projection area and the frontal association area. Moreover, the effects of carbacholine and serotonin on those activities were investigated. Na, K-ATPase activity in the synaptosomes of the association area was shown to be reliably higher that in the synaptosomes of the projection area (11.02 +/- 0.45 vs 8.40 +/- 0.55 microM Pi/mg of protein hr; P less than 0.05). Mg-ATPase activity was higher in the second case as compared to the first one (11.40 +/- 0.38 vs 9.04 +/- 0.35; p less than 0.05). Carbacholine and serotonin (10(-8)-10(-3) M) were found to induce equal inhibition of Na, K-ATPase activity in the synaptosomes of both cortices (1 max = 25-30%, 1C50 = 0.2-0.3 microM) which is blocked respectively with atropine (10(-6) M) and methysergide (10(-6) M) and enhanced in presence of GTP (5.10(-5) M). The enzyme activity is also inhibited by the non-hydrolysable guanine nucleotide, GTP gamma S (10(-8)-10(-4) M), in the absence of the antagonists (1 max = 35-40%, 1 C50 = 0.02 microM). In the methysergide-containing medium serotonin exerts a dose-dependent stimulatory effect on Na, K-ATPase which is more pronounced in the synaptosomes of the association area (A max = 25%, A C50 = 0.05 microM). Mg-ATPase activity of membrane preparations is liable to be stimulated by both serotonin and carbacholine, stimulation being more pronounced in the synaptosomes of the association cortex as well (A max = 35%, A C50 = 0.2-0.3 microM). This effect is insensitive either to the antagonists of the corresponding receptors or to GTP. GTP gamma S does not cause alterations in the enzymatic activity. Na, K-ATPase is suggested to be coupled to muscarine and serotonin receptors in the synaptic membranes of both projection and association cortical areas via a GTP-binding protein. At the same time, the agonists of receptors mentioned above are presumably also capable to effect Mg-ATPase activity by the receptor-independent way.

Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma.

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.

[Characteristics of ATPase activity in the sarcolemma of longitudinal and circular muscles of the canine ileum]. / Kharakteristika ATFaznoi aktivnosti sarkolemmy prodol'noi i tsirkuliarnoi muskulatury podvzdoshnoi kishki sobaki.

The subcellular fraction enriched in sarcolemmal vesicles was isolated from the longitudinal muscle (LM) and the circular muscle (CM) of the canine ileum by sucrose density gradient centrifugation. Treatment of the LM and CM membranes with sodium dodecylsulfate (0.2 mg/kg protein) led to a 3-fold increase in Na,K-ATPase activity (up to 24 and 39 mumol Pi/mg protein/h, respectively) and to a 90-95% inactivation of Mg-ATPase which was 2 and 8 times (for the CM and the LM, respectively) more active than Na,K-ATPase in the untreated sarcolemma. A specific inhibition of Na,K-ATPase activity by acetylcholine (Ach) and serotonin (ST) was observed which could de blocked in the presence of muscarinic and serotonin receptor antagonists. Sensitivity of the enzyme to ST was more than one order of magnitude higher than to Ach (IC50 = 10(-8) vs 1.2 x 10(-7) M). The inhibition of Na,K-ATPase activity by the neurotransmitters was more pronounced in the LM membranes (30-40%) than in the CM ones (10-20%). These data indicate that cell membranes of the LM and CM differ both in specific ATPase activities and the responsiveness of Na,K-ATPase to the receptor-mediated effects of Ach and ST.

The effect of neomycin on contractile activity of the canine cervical lymphatic vessel induced by various agents.

The effects of neomycin on isometric contractions induced by norepinephrine (NE), alpha 1-adrenoceptor agonist phenylephrine (PE), KCl, and an activator of GTP-binding proteins (G-proteins) NaF were studied in the isolated canine cervical lymphatic vessel (CLV). Incubation of the CLV with 0.3 or 1.3 mmol/l neomycin for 30-180 min did not affect significantly either the basal vascular tone or the response to NE, and potentiated the response to KCl by 24 +/- 6% (p less than 0.05). On the other hand, neomycin (1.3 mmol/l) treatment reduced by 22 +/- 6% (p less than 0.05) the contractions induced by PE and completely (by 96 +/- 3%, p less than 0.001) suppressed the effects of NaF. Upon the combined action of NaF and NE, neomycin reduced only NaF-component of the total response. Verapamil (100 mumol/l) had no effect on the magnitude of NaF-induced tension and partially inhibited NE- and PE-induced contractions (by 20 +/- 4% (p less than 0.05) and 53 +/- 5% (p less than 0.01), respectively). Indomethacin (10 mumol/l) did not influence significantly the contractions evoked by NE, KCl, and NaF either under control conditions or in the presence of neomycin. These data suggest that the phosphoinositides may considerably contribute to the CLV contractions evoked by NaF, but do not play a substantial role in the responses of the vessel to alpha-adrenergic stimulation and KCl.

[The effect of neomycin on the contractile activity of the skeletal muscles in the frog]. / Vliianie neomitsina na sokratitel'nuiu aktivnost' skeletnykh myshts liagushki.

Neomycin, a selective inhibitor of the phosphoinositide metabolism, depending on its concentration and the incubation time, depressed the K+-contractures and contractions of m. sartorius R. ridibunda caused by a single or a tetanic electrostimulation. In the experiments on m. rectus abdominis, neomycin inhibited the Ach- and oubaine contractures but not the K+ ones. The contractures of both muscles induced with caffeine did not change in presence of the above antibiotic. The data obtained suggest a participation of phosphoinositides contractions of skeletal muscles induced by excitation of the plasma membrane.

[Na,K-ATPase activity in the myocardial sarcolemma in ischemia]. / Na,K-ATP-aznaia aktivnost' v sarkolemme miokarda pri ishemii.

The total time-controlled ischemia (up to 45 min) was studied for its effect on the Na,K-ATPase activity, content of nonesterified fatty acids (NEFA) and intensity of lipid peroxidation (LP) in sarcolemmal (SL) preparations and soluble fractions (SF) from the rat and guinea-pig left ventricles. A strong correlation between Na, K-ATPase inhibition and NEFA accumulation was revealed in the SF. On the contrary, changes in the NEFA content and LP level both in SL and SF did not correlate with a decrease in the enzymic activity. Pretreatment with albumin (0.5 mg/ml) induced equally small increase both in the control and in the ischemic SL preparations. It is suggested that the Na,K-ATPase activity during a short period of myocardial ischemia (up to 45 min) may be due to the NEFA accumulation in the cytosolic and/or extracellular space, but not in SL.

[M-cholinergic regulation of Na+,K+-ATPase activity of vesicular preparations of sarcolemma from the myocardium and intestinal smooth muscles]. / M-kholinergicheskaia reguliatsiia aktivnosti Na+,K+-ATP-azy v vezikuliarnykh preparatakh sarkolemmy miokarda i gladkikh myshts kishechnika.

Acetylcholine (10(-7)-10(-2) M) enhanced the Na+, K+-ATPase activity in sarcolemmal vesicles from myocardium and intestinal smooth muscle. The stimulation of the enzyme from canine ventricles reaches 150% and was less pronounced (10-20%) in the case of frog myocardium and canine ileal muscles. The activating action of the neurotransmitter was simulated by gramicidin D (1-5 microM), but not by valinomycin 1-5 microM), blocked both by ouabain (200 microM) and atropine (0.1 microM), a muscarinic cholinergic antagonist. The activating action disappeared after treatment of membranes with alamethicin, a pore-producing antibiotic (0.8 mg/mg of protein). It is suggested that an increase in the Na+, K+-ATPase activity caused by acetylcholine is induced by Na+ which permeate the sarcolemmal vesicles through the ionic channel coupled with muscarinic acetylcholine receptor.

[Changes in the activity and regulatory properties of Na, K-ATPase from the myocardial sarcolemma in total graded ischemia]. / Izmenenie aktivnosti i reguliatornykh svoistv Na, K-ATFazy sarkolemmy miokarda pri total'noi dozirovannoi ishemii.

Na, K-ATPase activity of the rat and guinea-pig myocardial sarcolemma and its sensitivity to digoxin (DG) and carbamylcholine (CCh) were investigated during experimental ischemia. Ischemia was induced by the incubation of hearts in the air at 37 degrees C. This 15-, 30- and 45-min treatment led to a decrease in enzymatic activity which was similar in both animal species. Dose-related dependence of DG effect (10(-8)-10(-2) M) on sarcolemmal Na, K-ATPase activity of guinea-pig ischemic hearts did not differ from the control, whereas the rat enzyme sensitivity to glycosides rose with the progress of ischemia. CCh (10(-7)-10(-3) M) produced an inhibition of Na, K-ATPase activity which had reached 40% both in the rat and guinea-pig myocardial preparations. This effect was blocked by atropine (10(-6) M). The magnitude of enzyme responses to CCh declined depending on the duration of ischemia, with it being greater in guinea-pig sarcolemma than in rat membrane. The increased sensitivity of the rat Na, K-ATPase to CCh was also observed.

[Cholinergic regulation of the Na, K-ATPase activity of the pig kidney]. / Issledovanie kholinergicheskoi reguliatsii aktivnosti Na, K-ATFazy iz pochki svin'i.

Acetylcholine does not change the activity of Na, K-ATPase isolated from pig kidney. The enzyme is shown to have insignificant acetylcholinesterase activity. It is suggested that Na, K-ATPase sensitivity to acetylcholine disappears in the course of enzyme purification and that acetylcholinesterase activity is extrinsic.

[M-cholinoreceptor-mediated inhibition of Na, K-ATPase activity in the myocardial sarcolemma and intestinal smooth muscles by acetylcholine]. / Oposredovannoe M-kholinoretseptorom ingibirovanie Na, K-ATFaznoi aktivnosti sarkolemmy miokarda i gladkikh myshts kishechnika atsetilkholinom.

Acetylcholine (ACH) produced specific inhibition of Na, K-ATP-ase activity in sarcolemmic preparations of the frog heart (K0.5 = 1 microM), dog atria (K0,5 = 5 microM) and ventricles (K0.5 = 1 microM), and dog small intestinal smooth muscles (K0,5 = 0.5 microM). K0.5 is the concentration causing a half-maximal effect. Atropine (10(-7) = 10(-6) M) blocked the inhibitory effect of ACH. The preparations contained a considerable number of 3H-quinuclidinyl benzilate (3H-QNB) binding sites. Treatment of atrial sarcolemma with a mixture of digitonin and sodium cholate resulted in a substantial decrease in the number of 3H-QNB binding sites in the membrane, while Na,K-ATPase lost responsiveness to ACH. In the presence of 10 microM GTP there was a noticeable decrease in sensitivity of the enzyme to ACH. It is assumed that inhibition of Na, K-ATPase activity by acetylcholine is mediated by muscarinic receptor activation with the involvement in this process of GTP-binding proteins.

[Effect of acetylcholine on the Na+,K+-ATPase activity of different preparations of myocardial sarcolemma]. / Deistvie atsetilkholina na Na, K-ATF-aznuiu aktivnost' paznykh preparatov sarkolemmy miokarda.

Dose-dependent inhibition of Na+, K+-ATPase by acetylcholine was found in dog heart sarcolemma obtained after treatment with NaI. The enzyme was completely inhibited at 1 X 10(-2) M concentration of acetylcholine (Ki = 14 +/- 2 mM). Low concentrations of acetylcholine (1 X 10(-6)--1 X 10(-5) M) increased the Na+, K+-ATPase activity, the intermediate (5 X 10(-5)--5 X 10(-4) M) and high (1 X 10(-3)--1 X 10(-2) M) concentrations decreased the activity in the preparations enriched with sarcoplasmic vesicles. Sodium dodecylsulfate enhanced the activating effect but did not affect the inhibition. Atropine (1 X 10(-6)--1 X 10(-4) M) decreased the Na+, K+-ATPase activity and protected the enzyme against the inhibitory effect of acetylcholine. The data obtained suggest that interaction of acetylcholine with integral membrane proteins (Na+, K+-ATPase and/or muscarinic acetylcholine receptors) is apparently responsible for the neurotransmitter inhibitory effect.

[Sensitivity of brain Na, K-ATPase to acetylcholine following heat treatment of membrane preparations]. / Poiavlenie chuvstvitel'nosti Na, K-ATFazy mozga k atsetilkholinu posle teplovoi obrabotki membrannykh preparatov.

Microsomal preparations of Na, K-ATPase, isolated from dog and bovine brains, were completely insensitive to acetylcholine (10(-6)-10(-2) M) under standard experimental conditions. At the same time, heat pretreatment of membranes induced alterations of the enzymatic activity in presence of low concentrations (10(-6)-10(-5) M) of the neurotransmitter. Acetylcholine stimulated the Na, K-ATPase activity after storage of the preparations at 22 degrees within 30 or 60 min inhibited the activity at 37 degrees. Addition of the transmitter during the preincubation increased its effect. The maximal values of Na, K-ATPase activation and inhibition constituted 30% and 24%, respectively. D-tubocurarine (10(-6)-10(-5) M) and atropine (10(-4) M) did not eliminate and eserine (10(-4) M) did not alter the induced effect of acetylcholine. The effects of acetylcholine were changed to the opposite ones within 70-90 days of storage of the preparations. The phenomenon might be simulated by "ageing" of microsomes at 37 degrees within 3-6 hrs.