Additive feeding inhibitory and aversive effects of naltrexone and exendin-4 combinations.

OBJECTIVE: One developing strategy for obesity treatment has been to use combinations of differently acting pharmacotherapies to improve weight loss with fewer adverse effects. The purpose of this study was to determine whether the combination of naltrexone (Nal), an opioid antagonist acting on the reward system, and exendin-4 (Ex-4), a glucagon-like peptide 1 agonist acting on satiety signaling, would produce larger reductions in food intake than either alone in rats. Because the anorectic potencies of both compounds have been associated with nausea and malaise, the influence of these drug combinations on the acquisition of a conditioned taste aversion (CTA) was also determined. METHODS: In Experiment 1, the acute anorectic effects of Nal (0.32-3.2 mg kg(-1); intraperitoneally (i.p.)) and Ex-4 (1-10 µg kg(-1); i.p.) were assessed alone or in combination. Combinational doses were further investigated by the repeated daily administration of 1 mg kg(-1) Nal+3.2 µg kg(-1) Ex-4 for 4 days. In Experiment 2, both compounds alone or in combination were used as unconditioned stimuli in a series of CTA tests. RESULTS: Nal and Ex-4, alone or in combination, suppressed food intake in a dose-dependent manner, and the interaction on food intake between Nal and Ex-4 was additive. In the CTA paradigm, Nal (1 mg kg(-1)) alone did not support acquisition, whereas a CTA was evident with doses of Ex-4 (1 or 3.2 µg kg(-1)). Combinations of Nal and Ex-4 also resulted in a more rapid and robust acquisition of a CTA. CONCLUSION: Given that the Nal and Ex-4 combination produces additive effects on not only food intake reduction but also food aversion learning, this specific drug combination does not have the benefit of minimizing the adverse effects associated with each individual drug. These data suggest that it is necessary to evaluate both the positive and adverse effects at early stages of combinational drug development.

Risedronate pretreatment does not hamper the anabolic effects of prostaglandin E2 in ovx rats.

Pretreatment of an anti-resorptive agent on the anabolic effects of prostaglandin E2 (PGE2) was studied on the proximal tibia and tibial shaft of ovariectomy (ovx) rats. Two days after ovx, rats were treated with either risedronate (Ris, 5 micrograms/kg twice weekly) or vehicle (V) for 60 days and then switched to 3 or 6 mg/kg/d PGE2 for 21 or 90 days. Bone area of both proximal tibial metaphysis (PTM) and tibial shaft (TX) were measured. Pretreatment with Ris increased the bone mass in PTM but not in TX of ovx rats. In the PTM, PGE2 produced the same percentage of new bone mass in both V- and Ris-pretreated ovx rats. The amount of new bone was almost the same after 3 weeks and 12 weeks of PGE2 treatment. There was no difference in the anabolic effects of 3 and 6 mg PGE2/kg/d in V-pretreated rats; however, the effects in Ris-pretreated groups were greater with 6 mg PGE2/kg/d than with 3 mg PGE2/kg/d. In TX, only the 6mg PGE2/kg/d administration added new bone on endocortical surfaces of both V- or Ris-pretreatment rats which leads to thickening the minimal cortical width, decreasing the marrow cavity and increasing total bone area. Both doses of PGE2 created new trabecular bone in the marrow cavity of tibial shaft in both vehicle- and Ris-pretreated ovx rats. These results suggest that Ris-pretreatment did not hamper the anabolic effects of PGE2 on either PTM or TX in ovx rats.

Studies on the inhibitory effects of quercetin on the growth of HL-60 leukemia cells.

Quercetin, a naturally occurring flavonoid, has been shown to exert multiple pharmacological effects and to be an anticancer agent or a supplementary anticancer agent. In this report, the human HL-60 promyelocytic leukemia cell line was used to study the effects of quercetin on the growth, cell cycle, activities of cytosolic and membrane protein kinase C (PKC) and tyrosine protein kinase (TPK), and phosphoinositide production of the tumor cells. The results showed that quercetin inhibited the growth of HL-60 cells in a concentration-dependent manner, with an IC50 value of about 7.7 microM after 96 hr of treatment; when the concentration of quercetin was 10 microM, the percent inhibition on the growth of HL-60 cells was 17.1, 27.3, 40.1, and 52.7% after 24, 48, 72, and 96 hr of treatment, respectively. Flow cytometric analyses showed that quercetin caused an increase in cells in the G2/M phase and a decrease in cells in the G0/G1 phase of the cell cycle in a concentration-dependent manner; these effects were reversed when quercetin was removed from the culture medium. Quercetin strongly inhibited the activities of cytosolic PKC and membrane TPK from HL-60 cells in vitro, with IC50 values of about 30.9 and 20.1 microM, respectively, but did not affect membrane PKC or cytosolic TPK activity from HL-60 cells in vitro. Quercetin markedly inhibited in a concentration-dependent manner the production of phosphoinositides in intact HL-60 cells. The results provide evidence that the inhibitory effect of quercetin on the growth of HL-60 cells may be related to its inhibitory effects on PKC and/or TPK in vitro and/or on the production of phosphoinositides.

[Quantitative study on the effect of osthole on proximal tibiae in ovariectomized (OVX) rats].

Thirty-one 3-month-old Female Sprague-Dawley rats were randomly divided into 5 groups, basal control (group 1, killed at the begining), aging control (group 2), ovariectomized (OVX, group 3), OVX with nilestriol treatment group (group 4) and OVX with osthole treatment group (group 5). Group 2 and group 3 ig with water 5 ml.kg-1 and group 5 ig with osthole 6.7 mg.kg-1, all once a day for 6 d; group 4 ig with nilestriol 1 mg.kg-1, once a week. After 12 weeks, all rats were killed. The proximal tibiae of rats were processed to undecalcified sections at 20 microns thickness for histomorphometric analysis. OVX was shown to reduce markedly the trabecular bone mass (%Tb. Ar-59%) due to increase of bone turnover with the result that bone resorption exceeded bone formation, as compared with aging controls. In contrast, treatment of OVX rats with Osthole and nilestriol increased significantly the trabecular area (increased 68% and 27.1% compared with that of OVX respectively). Our results indicate that osthole and nilestriol treatment provides protection against osteoporosis in OVX rats. The protective mechanism of osthole and nilestriol involves supression of bone turnover, but the effects of osthole is lower than that of nilestriol (trabecular area decreased 55% more in osthole group than that with nilestriol treatment). Our finding may provide theoretical evidence for the clinical use of osthole or nilestriol for treatment and prevention of osteoporosis.

Palatable food avoidance and acceptance learning with different stressors in female rats.

Stress activates the hypothalamus-pituitary-adrenal (HPA) axis leading to the release of glucocorticoids (GC). Increased activity of the HPA axis and GC exposure has been suggested to facilitate the development of obesity and metabolic syndrome. Nonetheless, different stressors can produce distinct effects on food intake and may support different directions of food learning e.g. avoidance or acceptance. This study examined whether interoceptive (LiCl and exendin-4) and restraint stress (RS) support similar or distinct food learning. Female rats were exposed to different stressors after their consumption of a palatable food (butter icing). After four palatable food-stress pairings, distinct intakes of the butter icing were observed in rats treated with different stressors. Rats that received butter icing followed by intraperitoneal injections of LiCl (42.3mg/kg) and exendin-4 (10µg/kg) completely avoided the palatable food with subsequent presentations. In contrast, rats experiencing RS paired with the palatable food increased their consumption of butter icing across trials and did so to a greater degree than rats receiving saline injections. These data indicate that interoceptive and psychosocial stressors support conditioned food avoidance and acceptance, respectively. Examination of c-Fos immunoreactivity revealed distinct neural activation by interoceptive and psychosocial stressors that could provide the neural basis underlying opposite direction of food acceptance learning.

Cell death induced by ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid in anaplastic thyroid carcinoma cells is via a mitochondrial-mediated pathway.

The chemical compound ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), isolated from the Chinese herbal medicine plant Pteris semipinnata L, has been known to exert antitumor activity. However, the molecular mechanism of the action is not understood. In this study we demonstrated that apoptotic cell death induced by 5F in FRO cells was concentration- and time-dependent. The rapid increase in intracellular reactive oxygen species (ROS) levels was involved in the mechanism of cell death. c-Jun N-terminal kinase (JNK) activation and G2 block were related to cell death induced by 5F. Extracellular signal-related kinase (ERK) and p38 were also activated, but as survival signals in response to 5F treatment to counteract the induction of cell death. In the process of the induction of apoptotic cell death, Bax translocated into mitochondria, a reduction in Delta psi(m) was observed and a release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria into the cytosol occurred, indicating that cell death induced by 5F was through a mitochondrial-mediated pathway.

Increase in cytoskeletal actin induced by inositol 1,4-bisphosphate in saponin-permeated pig platelets.

Inositol 1,4-bisphosphate (IP2) which rapidly accumulates during cell activation, strongly stimulates an increase in cytoskeletal actin in saponin-permeated platelets, and the effect is insensitive to 5'-Chloro-5'-deoxyadenosine. Within 10 s, the amount of cytoskeletal actin in platelets rapidly increases by 41%, and then slowly increases further. IP2 induces the increase in cytoskeletal actin in a dose-dependent manner. The half-maximal effect requires approximately 2 microM of IP2. Inositol 1,4,5- triphosphate, the messenger for Ca2+ release, causes the increase in cytoskeletal actin, but is less effective than IP2. Inositol 1-monophosphate and inositol 2-monophosphate have no effect on cytoskeletal actin. Phorbol 12-myristate 13-acetate, which has been shown to activate IP3 5'-phosphatase through protein kinase C, stimulates the increase in cytoskeletal actin. Spermine, an inhibitor of IP3 5'-phosphatase, inhibits the thrombin stimulated increase in cytoskeletal actin. These results suggest that IP2 may be a messenger that controls the organization of actin filaments during cell activation. This study presents the first evidence for IP2 as a messenger during cell activation.

Inhibition of 5'-chloro-5'-deoxyadenosine on phosphoinositide and protein phosphorylation in swine platelets.

AIM: To study the effects of adenosine on the phosphorylation of phosphoinositides and proteins in platelets. METHODS: In the presence of Mg2+ and/or Ca2+, swine thrombocytic membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min and the incorporations of 32P into phospholipids or proteins were measured. RESULTS: 5'-Chloro-5'-deoxyadenosine decreased the formation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate [IC50 71 and 75 (95% confidence limits 60-85 and 62-90) mumol.L-1, respectively], behaving as a competitive inhibitor to ATP, and inhibited the phosphorylation of pleckstrin (the major protein kinase C substrate) and myosin light chain [IC50 75 and 82 (95% confidence limits 62-90 and 66-102) mumol.L-1, respectively]. CONCLUSION: Adenosine affects the phosphoinositide signaling pathway in platelets, which helps to clarify the inhibition of adenosine on platelet activation.

[Inhibition effects of adenosine and its analogues on actin polymerization in pig platelets and the possible mechanism].

The effects of adenosine and its analogues on the polymerization of actin in pig platelets and the possible mechanism were investigated. The results show that: Thrombin (0.5 U/ml) and ADP (50 mumol/L) stimulate actin polymerization in pig platelets: Adenosine, 5'-chloro-5'-deoxyadenosine, 2'-deoxyadenosine strongly inhibit thrombin- and/or ADP-induced actin polymerization. Adenosine and 5'-chloro-5'-deoxyadenosine strongly inhibit the phosphorylation of phosphatidylinositol in dose-dependent manner, and adenosine reverses the formation of thrombin-stimulated inositol bisphosphate, which has proved to promote the polymerization of actin in saponin-permeated platelets. These suggest that the inhibition of adenosine and its analogues on phosphatidylinositol turnover might involve in their inhibition on actin polymerization in platelets, and phosphatidylinositol turnover might play an important role in actin polymerization during cell activation.

[Effects of spermine on phosphorylation of phosphoinositide and 47 K protein in platelet membranes of pig].

In the presence of Mg2+ and exogenous phosphatidylinositol-4-phosphate, swine thrombocytic membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min and the incorporations of 32P into phospholipids and proteins were measured. Spermine stimulated the formation of phosphatidylinositol-4,5-bisphosphate (optimal concentration 0.25 mmol.L-1) and inhibited the phosphorylation of 47 K protein (IC50 1 mmol.L-1). This helps to clarify the effect of spermine on cell growth and proliferation.

Inhibitory effect of disodium quercetin-7,4'-disulfate on aggregation of pig platelets induced by thrombin and its mechanism.

AIM: To study the inhibitory effect of semi-synthesized quercetin derivatives--disodium quercetin-7,4'-disulfate (DQD) on the platelet aggregation induced by thrombin and its mechanism. METHODS: Platelet aggregation was analysed by turbidimetry. Cytosolic free calcium concentration ([Ca2+]i) was determined by Fura-2 fluorescence technique. Activity of Ca2+/PL dependent protein kinase C (PKC) was assayed by incubating PKC with histone III S and [gamma-32P]ATP. The cytoskeletal proteins were precipitated by Triton and separated by SDS-PAGE. RESULTS: DQD inhibited the platelet aggregation induced by thrombin (500 U/L), when DQD concentrations were 100, 200, and 400 mumol/L, the inhibition rates were 77%, 86%, and 82% respectively. DQD inhibited Ca2+ influx in platelets induced by thrombin (500 U/L) in the presence of extracellular Ca2+ 1 mmol/L in a concentration-dependent manner (10-80 mumol/L); DQD also had inhibitory effect on intracellular Ca2+ mobilization in the absence of extracellular Ca2+. DQD (10-160 mumol/L) inhibited the cytosolic Ca2+/PL dependent PKC from platelets in a concentration-dependent manner, but had no effect on membrane PKC. DQD (20-200 mumol/L) inhibited the actin polymerization induced by thrombin (500 U/L) in platelets in a concentration-dependent manner. CONCLUSION: DQD inhibited pig platelet aggregation induced by thrombin and its molecular mechanism was due to its inhibition of Ca2+ influx, intracellular Ca2+ mobilization, Ca2+/PL dependent PKC activity, and actin polymerization.

Effect of quercetin on activities of protein kinase C and tyrosine protein kinase from HL-60 cells.

AIM: To study the effect of quercetin (Que) on the activities of cytosol and membrane protein kinase C (PKC) and tyrosine protein kinase (TPK) from HL-60 cells in vitro. METHODS: The number of viable cells was counted by a trypan blue dye exclusion test. PKC activity was assayed by incubating PKC with histone III S and [gamma-32P]ATP. TPK activity was assayed by incubating TPK with poly glutamate.tyrosine (4:1). RESULTS: Que inhibited the proliferation of HL-60 cells in a concentration-dependent manner, its IC50 was 29 (22-37) mumol.L-1 after 48-h treatment; Que strongly inhibited the activity of cytosol PKC and membrane TPK with IC50 31 (20-48) mumol.L-1, 24 (13-45) mumol.L-1, respectively, but did not affect membrane PKC and cytosol TPK from HL-60 cells in vitro. CONCLUSION: The inhibitory effect of Que on the growth of tumor cells is related to its inhibitory effects on PKC and/or TPK.

[Effects of heparin and other chemicals on phosphorylation of phosphatidylinositol in pig erythrocyte membrane].

In the presence of Mg2+, the pig erythrocyte membranes were incubated with [gamma-32P]ATP at 30 degrees C for 3 min to study the effects of some chemicals on the phosphorylation of phosphatidylinositol. The incubations were stopped by the addition of chloroform/methanol (2:1, vol/vol) and the phospholipids were extracted with acid chloroform/methanol and separated on silica gel TLC plates. [gamma-32P] phosphatidyl-inositol-4-phosphate was quantitated by scintillation counting and autoradiography. The results indicated that heparin and neomycin inhibited the phosphorylation of phosphatidylinositol in a concentration-dependent manner, while dimethylsulfoxide stimulated the phosphorylation at low concentration (less than 10%), but inhibited at high concentration (greater than 10%). Hexachlorocyclohexane stimulated the phosphorylation within a certain limit of concentration up to 6.4 micrograms/ml. Li2SO4 (10 mmol/L), EGTA (100 mumol/L) and theophylline (100 mumol/L) had no significant effects.

Effects of genistein on aggregation and cytosolic free calcium in pig platelets.

AIM: To study the effects of genistein on aggregation and cytosolic free calcium concentration in platelets. METHODS: Using turbidimetry to analyse aggregation and using Fura-2 fluorescence technique to determine Ca2+ level. RESULTS: Genistein strongly inhibited the pig platelet aggregation induced by thrombin (250 U.L-1). When genistein concentrations were 5 and 20 mumol.L-1, the inhibition rates on the aggregation were 52% and 73%, respectively. Genistein inhibited the rise of cytosolic free calcium concentration in platelets stimulated by thrombin (500 U.L-1) in the presence of extracellular Ca2+ 1 mmol.L-1. When genistein concentrations were 10, 20, 40, and 80 mumol.L-1, the inhibition rates were 24%, 40%, 63%, and 65%, respectively, but no effect on thrombin-induced internal Ca2+ release from dense tubular system. CONCLUSION: Genistein is a potential anti-platelet agent, mainly due to an inhibition of Ca2+ influx.

Action of 3 tyrphostin derivatives on casein kinase II from rat liver.

AIM: To study the action of tyrphostin on casein kinase (CK) II. METHODS: CK II was partially purified from rat livers by sequential DE52 and heparin-Sepharose chromatography. CK II activity was assayed by incubating CK II with dephosphorylated casein and [gamma-32P]ATP. RESULTS: AG34 inhibited the activity of CK II with IC50 33 (27-41) mumol.L-1. Both AG372 (121 mumol.L-1) and AG1112 (150 mumol.L-1) displayed inhibitory effects on the activity of CK II. Kinetic studies of AG34 on CK II showed that it was noncompetitive with casein and ATP. CONCLUSION: AG34, AG372, and AG1112 were potent inhibitors of CK II, and the inhibitory action of AG34 was noncompetitive with casein and ATP.

Effects of tyrphostins on activity of casein kinase II from rat liver.

AIM: To investigate the effects of tyrphostins, (AG213, AG1394, AG114, AG1109, AG555) on the activity of casein kinase (CK) II. METHODS: CK II was partially purified from rat livers by sequential DE52 and heparin-Sepharose chromatography. CK II activity was assayed by incubating CK II with dephosphorylated casein and [gamma-32 P]ATP. RESULTS: AG213 inhibited the activity of CK II with IC50 44.7 mumol.L-1 (41.5-47.9 mumol.L-1), and AG1394 (144 mumol.L-1) strongly inhibited the activity of CK II with an inhibitory ratio of 89%. AG114 (174 mumol.L-1) and AG1109 (126 mumol.L-1) had inhibitory effects on the activity of CK II (p < 0.01). AG555 (136 mumol.L-1) had little effect on CK II activity. CONCLUSION: Some tyrphostins are potent inhibitors of CK II.

Effects of aprotinin in platelet aggregation and cytosolic free calcium in swine platelets.

Aprotinin inhibited platelet aggregation induced by thrombin (0.25 U.ml-1) with IC50 200 kIU.ml-1, and inhibited the rise of cytosolic free calcium concentration in platelets stimulated by thrombin (0.1 U.ml-1) in the absence and in the presence of Ca2+ 0.5 mmol.L-1 (IC50 117 and 50 kIU.ml-1, respectively), but had no effect on the amounts of actin and myosin heavy chain associated with cytoskeletons. These suggest that aprotinin is an anti-platelet agent and may exert its action through inhibiting the Ca2+ flux.

Dimethylamiloride-induced differentiation of HL-60 cells.

AIM: To study the effect of 5-(N,N-dimethyl) amiloride (DMA) on the proliferation and differentiation of HL-60 cells in vitro. METHODS: MTT assay to test cytotoxicity; cell staining and NBT reduction to test cell differentiation. RESULTS: DMA inhibited HL-60 cells growth in a concentration-dependent manner, and IC50 value for 96 h was 31.7 (95% confidence limits: 6.3-57.1) mumol.L-1. DMA also induced granulocytic differentiation in HL-60 cells. The percentage of differentiating cells increased from 6.5% to 70% after DMA 100 mumol.L-1 treatment for 3 d. The differentiating effect of DMA was distinguishable from amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), and (5-(N-ethyl-N-isopropyl)amiloride (MIA). None among the amiloride, EIPA, and MIA were capable of triggering the differentiation of HL-60 cells. CONCLUSION: DMA inhibited the proliferation of HL-60 cells and induced differentiation of HL-60 cells.

Inhibitory effects of sodium quercetin monosulfate on pig platelet aggregation induced by thrombin.

AIM: To study the inhibitory effects of sodium quercetin monosulfate (SQMS) on pig platelet aggregation induced by thrombin. METHODS: Platelet aggregation was analyzed by turbidimetry. Cytosolic free calcium concentration ([Ca2+]i) was determined by Fura-2 fluorescence. Activity of protein kinase C (PKC) was assayed by incubating PKC with histone III S and [gamma-32 P] ATP. The cytoskeletal proteins were precipitated by Triton X-100 and separated by SDS-PAGE. RESULTS: SQMS inhibited the platelet aggregation induced by thrombin 500 U.L-1 with IC50 132 (50-347) mumol.L-1. SQMS inhibited Ca2+ influx in blood platelets induced by thrombin 500 U.L-1 in the presence of extracellular Ca2+ 1 mmol.L-1 with IC50 20 (9-46) mumol.L-1; SQMS inhibited the internal Ca2+ release in the absence of extracellular Ca2+. SQMS also decreased [Ca2+]i level in quiescent blood platelets. SQMS (10-160 mumol.L-1) inhibited the activity of cytosolic PKC from blood platelets in a concentration-dependent manner, but had no effect on membrane PKC. SQMS (20-80 mumol.L-1) inhibited the actin polymerization induced by thrombin 500 U.L-1 inblood platelets in a concentration-dependent manner. CONCLUSION: SQMS inhibited pig platelet aggregation induced by thrombin and its mechanism might be due to its inhibitions of Ca2+ influx, internal Ca2- release, PKC activity, and actin polymerization.

Effects of antitumor compounds isolated from Pteris semipinnata L on DNA topoisomerases and cell cycle of HL-60 cells.

AIM: To study the effect of the antitumor compounds 5F, 6F, and A from Pteris semipinnata L on the activities of DNA topoisomerases and cell cycle of HL-60 cells, and the synergism of compound 6F in combination with genistein in vitro. METHODS: DNA topoisomerases were isolated from HL-60 cell lines, and supercoiled pBR322 DNA was used as substrate to determine the activities of DNA topoisomerase I and II. Cell cycle was analyzed by flow cytometry (FCM). Cytotoxicity assay was tested by MTT method. RESULTS: Compounds 5F, 6F, and A inhibited the activities of DNA topoisomerase I and II. After exposure of the cells to compound 6F, an increase in cells in the S and G2/M phases and a decrease in cells in the G0/G1 phase of the cell cycle were observed. At low concentrations (57.8 and 115.6 nmol.L-1), compound 6F enhanced the cytotoxicity against HL-60 cell line in combination with genistein, q values were > 1.15. The enhancement times of 57.8 and 115.6 nmol.L-1 of 6F by genistein were 2.60 and 4.65, respectively. CONCLUSION: Compounds 5F, 6F, and A inhibited the activities of DNA topoisomerases of HL-60 cells. Compound 6F increased the number of cells in S and G2/M phases, decreased the population of G0/G1 phase cells, and enhanced the cytotoxicity of genistein, which had synergism with 6F in antitumor action.