Adenylate cyclase system is essential for long-term facilitation at the crayfish neuromuscular junction. (1/5)

Long-term facilitation (LTF), a form of synaptic plasticity demonstrated at the crayfish neuromuscular junction, is induced by tetanic stimulation and persists for hours. LTF can be divided into 2 phases: a tetanic phase, which occurs during stimulation, and a long-lasting phase, which persists after stimulation. Activators and potentiators of cAMP (forskolin and 3-isobutyl-methyl-xanthine) produce facilitation of excitatory postsynaptic potentials, which attain approximately the amplitude of the long-lasting phase of LTF but last for a shorter time. Localized presynaptic injection of a protein inhibitor ("Walsh inhibitor") specific for the cAMP-dependent protein kinase blocks the long-lasting phase of LTF at synapses near the injection site with no apparent effect on the tetanic phase. Normal LTF develops and persists at synapses of the same axon distant from the injection site. Localization of the injected inhibitor was confirmed by fluorescent tagging. Localized injection of SQ22,536, an adenylate cyclase inhibitor, also blocks the second phase of LTF near the injection site, but not at distant synapses. These experiments establish a role for adenylate cyclase activation in the long-lasting phase of LTF. The phosphatidylinositol second-messenger system is not important in LTF as inhibition of phospholipase C by injection of RA233, which blocks facilitatory effects of serotonin, does not affect any aspect of LTF.  (+info)

Drug treatments for metastasis of the Lewis lung carcinoma: lack of correlation between inhibition of lung metastasis and survival. (2/5)

The abilities of the Eli Lilly compounds LY150310, LY189332, and LY135305 to inhibit spontaneous metastasis and to increase animal survival were evaluated. These compounds represent widely varied structures and were evaluated because they have been found to inhibit thromboxane synthetase, cyclooxygenase, and thrombin activation, respectively. These biochemical processes have been proposed in the literature as targets for antimetastatic drugs. The purpose of this investigation was twofold: (a) to compare the antimetastatic activities of the Eli Lilly compounds to those of the reference antimetastatic compounds nafazatrom and RA233, and (b) to examine the correlation between inhibition of spontaneous lung metastasis and survival. Spontaneous metastasis of the Lewis lung carcinoma was used to evaluate the antimetastatic activity of the compounds. In this model 5 x 10(5) tumor cells were implanted into the gastrocnemius muscle, the primary tumor was resected on Day 14, and metastatic lung lesions were counted on Day 25. Compounds were administered every 12 h on Days 5 through 19. Nafazatrom, LY150310, LY189332, and LY135305 were found to inhibit spontaneous lung metastasis in a dose-dependent manner. The ED50 values for the respective inhibitions with these compounds were 50, 0.5, 2, and 0.35 mg/kg/day; the respective therapeutic indexes (LD50/ED50) were 7, 180, 255, and 511. To evaluate the effect of nafazatrom, LY150310, LY189332, and LY135305 on animal survival, the compounds were given at maximally antimetastatic doses of 200, 60, 20, and 6 mg/kg/day, respectively. Two dosing schedules were used: (a) on Days 5 through 19 and (b) on Day 5 until death. Neither the median survival times nor the numbers of long-term survivors were significantly changed with any of the compounds at any dosing schedule. RA233, given to a maximally tolerated dose of 200 mg/kg/day on Day 5 until death, did not inhibit lung metastasis and did not increase median survival time. Postmortem examination of animals dosed with nafazatrom, LY150310, LY189332, and LY135305 showed complete inhibition in lung lesions and the appearance of lesions in the liver, kidney, spleen, and brain. The results of this investigation show that the effect a compound has on the number of metastatic lesions in a target organ may not be predictive of its effect on survival. To successfully translate laboratory data into the clinic, survival should be considered as a predictor of a compound's potential clinical utility.  (+info)

Direct effects of the pyrimido-pyrimidine derivative RA 233 (Rapenton) on rat 13762NF mammary tumor cell clones in vitro. (3/5)

Studies with the pyrimido-pyrimidine analogue RA 233 (Rapenton) suggest that its antimetastatic action may not be mediated entirely by inhibition of platelet function. Little is known about its direct effects on tumor cells. We investigated the in vitro effects of RA 233 on clones MTLn3 and MTC of differing metastatic potentials, isolated from the 13762NF rat mammary adenocarcinoma. The results indicated that RA 233 is cytostatic (EC50 of approximately 140 microM and approximately 180 microM for MTLn3 and MTC cells, respectively) rather than cytotoxic by determining changes in viable cell number, thymidine uptake, and incorporation of thymidine and methionine. In both clones RA 233 inhibited cAMP-dependent phosphodiesterase activity and affected cAMP accumulation in intact cells. In contrast, clonal heterogeneity in drug-induced morphological changes, such as vacuole formation and altered organization of cytoskeletal structures, as well as increased tumor cell growth at 50 microM RA 233 was observed between clones MTLn3 and MTC. These data could explain the conflicting results obtained with RA 233 when evaluated as an antimetastatic agent.  (+info)

Inhibitors of cyclic nucleotide phosphodiesterases inhibit protein carboxyl methylation in intact blood platelets. (4/5)

The cycle of protein-carboxyl methylation and demethylation was studied in intact blood platelets. Platelets rapidly incorporated L-[methyl-3H]methionine and after a delay of about 20 min, they evolved [3H]methanol. This evolution, and the amount of [3H] methanol liberated by treatment with base, was inhibited in a dose-dependent fashion by the cyclic nucleotide phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine, papaverine, dipyridamole, and RA233 (2,6-bis(diethanolamino)-4-piperidinopyrimido[5,4-d] pyrimidine). Each of these compounds increased the incorporation of [3H]methionine into platelets. The effects of RA233 were studied in more detail. Inhibition of [3H]methanol production was not potentiated by stimulators of the adenylate cyclase or the guanylate cyclase. The majority of the base-labile radioactivity was trichloroacetic acid precipitable. Thin layer chromatography of extracts of platelets incubated with L-[35S]methionine showed that RA233 did not induce a cellular accumulation of [35S]S-adenosylhomocysteine, and that it actually increased the amount of cellular [35S]S-adenosylmethionine. Discontinuous polyacrylamide gel electrophoresis at acid pH using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride of platelets incubated with [3H]methionine showed incorporation of radioactivity into more than 30 protein bands, including one which co-migrates with calmodulin. The incorporation into the majority of these bands was inhibited by RA233 in a dose-dependent fashion. It is suggested that caution should be used in ascribing the pharmacological effects of known phosphodiesterase inhibitors to increases in cyclic nucleotides, because some of these effects could be due to inhibition of protein carboxyl methylation.  (+info)

Interruption of tumor-associated platelet consumption with platelet enzyme inhibitors. (5/5)

Twenty dogs with naturally occurring metastatic tumors were treated with anticoagulants (Warfarin) or platelet enzyme inhibitor drugs (dipyridamole, dipyridamole plus aspirin, RA233, sulfinpyrazone, or a combination of RA233 and sulfinpyrazone) to determine if tumor-related reductions in platelet survival and concentration could be reversed. Anticoagulation was ineffective, while platelet enzyme inhibitors were able to produce improvements in platelet survival. Of the 18 dogs with metastatic tumor treated with platelet enzyme inhibitors, only 5 (28%) showed a reduction in platelet survival during the first week of observation on therapy compared to their baseline survivals. This is significantly different than the decreases in platelet survivals observed in 8 of 10 untreated dogs (80%) with metastatic tumor observed for the same interval. Furthermore, 8 of the 18 treated dogs (44%) had platelet survivals within 2 standard deviations of normal, compared to only 1 of 10 untreated dogs. Of the 8 dogs with normal platelet survivals, 6 were treated with a combination of a phosphodiesterase inhibitor (RA233 or dipyridamole) and a cyclooxygenase inhibitor (sulfinpyrazone or aspirin). The combination of RA233 and sulfinpyrazone was the best drug program tested and resulted in normal platelet survivals in 63% and improved platelet counts in 75% of the animals treated. Thus, platelet enzyme inhibitors with different mechanisms of action may have a synergistic effect in reversing the abnormal platelet hemostasis found in a variety of spontaneously occurring canine neoplasms.  (+info)