A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing “tethered-ligand” then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by α-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of γ-thrombin, and the α- and ζ- thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of α-thrombin involved in human platelet activation.

1-(Cyclohexylmethyl)-4-[4-[(2,3-dihydro-2-oxo-1H-imidazo[4,5-b] quinolin-7-yl)oxy]-1-oxobutyl]piperazine (2) was previously identified as a potent, water-soluble inhibitor of human blood platelet cAMP phosphodiesterase and of induced aggregation in vitro that demonstrated effective antithrombotic activity in animal models of thrombosis. Although 2 exhibited 25% oral bioavailability in rats, pharmacokinetic studies conducted in monkeys revealed that the parent compound was less than 5% bioavailable, the result of extensive first-pass biotransformation in the liver. In an effort to identify potent platelet aggregation inhibitors with enhanced metabolic stability, the side-chain amide moiety of 2 was replaced with chemically more stable urea (6a-s), sulfonamide (13a-m), sulfone (19a-r), and tetrazole (23a-s) moieties. Many representatives from each of these structural types effectively combined potent inhibition of ADP-induced human platelet aggregation in vitro with excellent aqueous solubility, and several are superior to 2. Within each series, the N-(cyclohexylmethyl)-, N-(2-ethylbutyl)-, N-benzyl-, and N-(4-fluorobenzyl)-substituted derivatives were evaluated for in vitro metabolic stability by incubating with the S-9 fraction of monkey liver for 2 h, and the extent of biotransformation was compared with that of the prototype 2. The sulfone 19e and the tetrazoles 23e, 23g, 23j, and 23q were significantly more stable than 2 under these conditions, and 19e and 23e were selected for evaluation in vivo. Tetrazole 23e exhibited 72% bioavailability following ip administration to rats compared with 35% bioavailability for 2 and 19e under the same conditions. However, the oral bioavailability of 19e and 23e in the rat was estimated to be only 3%, suggesting that 19e and 23e are less readily absorbed from the gastrointestinal tract than 2.

Synthesis and in vitro pharmacological profile of several 1,3-dioxane and 1,3-dioxolane analogs are described. Compounds 1c and 1f are the two most potent thromboxane receptor antagonists with Kd values of 8.0± 0.4 nM and 8.2 ±0.4 nM, respectively at the TxA2PGH2 receptor in human platelet membrane.

Kidney renin concentrations were significantly lower in spontaneously hypertensive rats (SHR) than in normotensive Wistar Kyoto (WKY) rats. After treatment of both SHR and WKY rats with captopril (100 mg/kg p.o. for 3 months), kidney renin concentration increased dramatically in SHR and slightly, but significantly, in WKY. After captopril treatment, kidney renin content of SHR was still significantly lower than WKY. Because of the lower content of kidney renin in SHR and the proportionately greater increase in kidney renin content in SHR after captopril treatment than in WKY, it is proposed that a fundamental difference(s) in the control of the renin-angiotensin system exists in SHR, an effect which may or may not be related to SHR hypertension.

A series of cyclic 2′,3′-nucleotides, cyclic 3′,5′-nucleotides and derivatives of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) with a substituent at the C-8 position were investigated as inhibitors of partially purified cyclic AMP phosphodiesterases (PDE) of cat heart and rat brain. The assays were carried out at a substrate concentration (0.06 μM) where the contribution to the total enzyme activity by phosphodiesterases with Km values for cyclic AMP above 100 μM was insignificant; consequently the activity measured was that of low Km enzymes.

Mammary adenocarcinomas of the rat induced by gastric intubation of 7,12-dimethylbenz(a)anthracene will regress after ovariectomy. The activities of enzymes and the levels of nucleic acids and lipids were measured in tumors of animals sacrificed 1, 5, and 14 days after ovariectomy. Palpation of the tumors showed that no significant decreases in tumor size or volume occurred until 14 days after ovariectomy, at which time tumor size and volume had decreased 27 and 62%, respectively. Significant changes in biochemical parameters were first observed as follows: Day 1, glutamate dehydrogenase, hexokinase, aspartate aminotransferase, and cholesterol; Day 5, pyruvate kinase, glucose-6-phosphate dehydrogenase, NADP-malate dehydrogenase, phosphoglucomutase, RNA, and RNA/DNA ratios; and Day 14, NADP-isocitrate dehydrogenase and glucosephosphate isomerase. The activities of α-glycerolphosphate dehydrogenase and glucokinase, as well as the levels of DNA, free fatty cids, and triglycerides, were not significantly altered at any time. Similar results were obtained in animals ovariectomized when the first tumor that appeared approximated 2 × 2 cm and in others ovariectomized 35 days after the first tumor had reached 0.5 × 0.5 cm, and the first three tumors were analyzed. These data indicate that certain specific biochemical changes precede measurable changes in tumor size and are implicated as essential components for continued neoplastic growth.

Summary The administration of a phenothiazine tranquilizer, fluphenazine HCl, caused a decrease in the growth of the R3230AC mammary adenocarcinoma of the Fischer rat. Examination of various biochemical parameters in the cancers of fluphenazine HCl-treated animals showed that drug treatment caused a decrease in DNA levels, an increase in free fatty acids and triglycerides, and significant elevations in the activities of glucose-6-phosphate dehydrogenase, NADP-malate dehydrogenase, phosphoglucomutase, and aspartate aminotransferase. Mammary glands from the drug-treated tumor-bearing animals were stimulated and demonstrated striking increases (2 to 5 times per unit of tumor weight or 10 to 20 times per mg DNA) in enzyme activities compared with those found in control animals. The alterations reported were ascribed to the known ability of phenothiazines to stimulate endogenous secretion of prolactin. Thus, creating a hormonal milieu that stimulates normal mammary glands does not necessarily stimulate growth of mammary tumors.

In order to establish valid experimental systems for the study of breast cancer, an examination of the biochemical and morphologic characteristics of transplantable and carcinogen-induced mammary tumors in rats and human infiltrating ductal carcinomas of the breast was undertaken. A comparison of measurements of several selected enzymes, nucleic acids and lipids has demonstrated that the biochemical characteristics of the rodent carcinomas induced by a single dose of 7,12-dimethylbenz (a) anthracene are similar to those of human breast cancers studied. The importance of validating experimental models, in order to achieve a logical translation of information to the human disease, is discussed.

Selected enzyme activities and nucleic acid content of the transplantable, dimethylbenz(a)anthracene-induced 13762 mammary adenocarcinoma and 3 sublines were measured as influenced either by the sex of the tumor-bearing host or by treatment of the host with androgen or estrogen. The normal female line tumor had the highest enzyme activities and the normal male line tumor had the lowest enzyme activities when growing in their respective hosts. Transplantation of these neoplasms into animals of the opposite sex produced either increased or decreased enzyme activities in direction of the normal tumor lines of the respective hosts. The tumor, conditioned by testosterone propionate, grew more rapidly in the presence of exogenous androgen and showed marked elevations in enzyme activities in comparison with those of the conditioned tumor line in the absence of hormone treatment. In general, the alterations in enzyme activities reflected the increased or decreased growth rate resulting from hormonal treatment. The exception to this was the response of the androgen-conditioned line to treatment with estrogen; this treatment produced no increase in tumor weight, but it increased enzyme activities and induced gross lactation. Continuous treatment with testosterone propionate for 5, 6, and 7 weeks in animals bearing the testosterone propionate-conditioned tumor resulted in a progressive increase in the activity of glucose-6-phosphate dehydrogenase, malate dehydrogenase, and α-glycerolphosphate dehydrogenase.

Summary The R3230AC mammary adenocarcinoma responds to estrogen treatment by increased glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarboxylating), and phosphoglucomutase activities. Various doses of actidione (cycloheximide), an antibiotic reported to inhibit protein synthesis in mammalian systems without inhibiting RNA synthesis, were employed to determine their effect on the estrogen-induced alterations in enzyme activities in this neoplasm as well as in the uteri and mammary glands of the tumor-bearing animal. Actidione caused a decrease in the activities of glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarboxylating), and phosphoglucomutase in the carcinoma. The antibiotic, at certain doses, completely prevented the estrogen-induced elevations in the activities of these enzymes in the neoplasm and prevented the estrogen-induced decrease in isocitrate dehydrogenase (decarboxylating) activity in the tumor. Although actidione did not prevent the uterotropic response to estrogen, the antibiotic did block the estrogen-induced elevations of glucose-6-phosphate dehydrogenase, malate dehydrogenase (decarboxylating), glucosephosphate isomerase, and glutamate dehydrogenase activities in the uterus. Actidione, administered alone or with estrogen, produced an increase in the RNA concentration and RNA/DNA ratios in the tumors and uteri and had no significant effect on the concentration of DNA in these tissues. The activities of most of these enzymes in the mammary gland were not notably elevated by estrogen treatment under these experimental conditions, and both malate dehydrogenase (decarboxylating) and α-glycerolphosphate dehydrogenase activities were significantly reduced. Actidione treatment, either alone or with estrogen, produced a dose-related decrease in the activities of these enzymes in the mammary gland. The estrogen-induced decrease in α-glycerolphosphate dehydrogenase activity was prevented at only the lowest dose of actidione employed. Protein and RNA synthesis were estimated by determining the amount of leucine- 14 C and uridine-5- 3 H incorporated into these substances in the neoplasm and uteri. Estrogen treatment produced a 2- to 3-fold increased in both the total activity and specific activity of both radioactive substrates in the carcinoma in vivo and actidione treatment prevented this estrogen-induced response. Similar results were obtained in the uteri of these tumor-bearing animals. These data, together with results of our earlier experiments employing actinomycin D, suggest that the estrogen-induced elevations of enzyme activities in the R3230AC mammary adenocarcinoma are dependent on protein synthesis.