A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice.

Constitutively active Ras proteins, their regulatory components, and overexpressed tyrosine kinase receptors that activate Ras, are frequently associated with cell transformation in human tumors. This suggests that functional Ras antagonists may have anti-tumor activity. Studies in rodent fibroblasts have shown that S-trans, transfarnesylthiosalicylic acid (FTS) acts as a rather specific nontoxic Ras antagonist, dislodging Ras from its membrane anchorage domains and accelerating its degradation. FTS is not a farnesyltransferase inhibitor, and does not affect Ras maturation. Here we demonstrate that FTS also acts as a functional Ras antagonist in human pancreatic cell lines that express activated K-Ras (Panc-1 and MiaPaCa-2). In Panc-1 cells, FTS at a concentration of 25-100 microM reduced the amount of Ras in a dose-dependent manner and interfered with serum-dependent and epidermal growth factor-stimulated ERK activation, thus inhibiting both anchorage-dependent and anchorage-independent growth of Panc-1 cells in vitro. FTS also inhibited tumor growth in Panc-1 xenografted nude mice, apparently without systemic toxicity. Daily FTS treatment (5 mg/kg intraperitoneally) in mice with tumors (mean volume 0.07 cm3) markedly decreased tumor growth (after treatment for 18 days, tumor volume had increased by only 23+/-30-fold in the FTS-treated group and by 127+/-66-fold in controls). These findings suggest that FTS represents a new class of functional Ras antagonists with potential therapeutic value.

Altered ontogenesis of muscarinic cholinergic receptor in mouse brain: effect of L-thyroxine and betamethasone.

The effects of L-T4 and betamethasone treatment of newborn mice on the development of the cholinergic muscarinic receptor in certain brain regions was studied using the potent labeled muscarinic antagonist [3H]4-N-methyl-piperidyl benzilate. Treatment with both L-T4 and betamethasone caused an accelerated accumulation of muscarinic receptors in the cortex 16 days post partum, with a subsequent reduction in level at 30 days. In the cerebellum and caudate putamen, only betamethasone caused a similar early accumulation of muscarinic receptors, while the later effect, namely a reduction in the level at 30 days, was seen with both hormones in these two regions as well as in the hippocampus. The results can explain some behavioral effects observed in other studies after treatment with these hormones.

Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging.

The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.

Identification of two distinct protein carboxyl methyltransferases in eucaryotic cells.

Two distinct protein carboxyl methyltransferases (PCM) were identified in the electric organ of Torpedo ocellata. They were separated from each other in the active form by means of nondenaturing gel electrophoresis and by p-(chloromercuri)benzoate-agarose chromatography, and were individually identified by specific polyclonal antibodies. The existence of at least two distinct PCMs in eucaryotic cells raises the possibility that these enzymes are involved in distinct transmethylation reactions.

Prenylated protein methyltransferase of rat cerebellum is developmentally co-expressed with its substrates.

High levels of prenylated protein methyltransferase are expressed in the developing rat cerebellum and are responsible for methylation of endogenous G-proteins and 50-52 kDa synaptosomal proteins. Enzyme activity in cerebellar synaptosomes of 3 week postnatal rats is 2-fold higher than that found in adult rat cerebellum. A 10-fold rise in activity occurs at the end of the second and during the third postnatal weeks, followed by a subsequent decline. Expression of the enzymes' substrates follows the same pattern. The high methyltransferase activity in 3-week-old cerebellum coincides with the period of granule cell migration and synaptogenesis, suggesting a regulatory role for the enzyme and its substrates in cerebellar ontogenesis.

Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth.

Inhibitors of the enzyme that methylates ras proteins, the prenylated protein methyltransferase (PPMTase), are described. They are farnesyl derivatives of rigid carboxylic acids that recognize the farnesylcysteine recognition domain of the enzyme but do not serve as substrates. They also inhibit ras-dependent cell growth by a mechanism that is probably unrelated to inhibition of ras methylation, even though their potencies as PPMTase inhibitors and cell-growth inhibitors correlate well. The most potent inhibitor is S-trans,trans-farnesylthiosalicylic acid (FTS) (2). FTS (2) selectively inhibits the growth of human Ha-ras-transformed Rat1 cells in vitro (EC50 = 7.5 microM).

Muscarinic involvement in the regulation of gonadotropin-releasing hormone in the cyclic rat.

The release of gonadotropin-releasing hormone (GnRH) from the median eminence (ME) in cyclic rats was stimulated to a significant extent by the selective muscarinic antagonists 11[(2)(diethylamino)methyl][-1-piperidinyl]-acetyl-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX-116) and methoctramine, and to a lesser extent also by other ligands selectively antagonistic to m1 and m3 receptors. Such stimulation was estrous-cycle-dependent and was not achieved by muscarinic agonists. We suggest that the effect is induced via the m4 receptor subtype. Attempts to block the muscarinic-antagonist-induced stimulation of GnRH release with a variety of drugs were successful only in the presence of prazosin, an antagonist to alpha 1-adrenergic receptors. One possible explanation for this muscarinically mediated stimulation of GnRH release is that it results from cross-talk between the muscarinic and the alpha 1-adrenergic receptors, i.e., muscarinic agonists might inhibit the release induced by alpha 1-agonists, and muscarinic antagonists, by cancelling this inhibitory effect, might thus allow the endogenous alpha 1-agent, norepinephrine, to induce the release of GnRH.

On the interaction of drugs with the cholinergic nervous system. II. Cross-tolerance between phencyclidine derivatives and cholinergic drugs.

A symmetrical cross-tolerance was found between two phencyclidine derivatives--phencyclidine and cyclohexamine--and also between two cholinergic drugs--physostigmine and oxotremorine. On the other hand, mice rendered tolerant to the phencyclidine derivatives showed cross-tolerance to these cholinergic drugs, but no cross-tolerance was observed in the opposite direction. The applicability of such experiments to the elucidation of neurochemical interactions of centrally acting drugs is discussed.

Inhibition of dopamine release by melatonin: regional distribution in the rat brain.

Dopamine release evoked by electrical field stimulation of slices from various regions of rat brain was assessed in the presence of 10-10-10-5 M melatonin. Inhibition of dopamine release by melatonin was observed in the ventral hippocampus, medulla pons, preoptic area and median and posterior hypothalamus. No inhibitory effect of melatonin on dopamine release was observed in the cerebral cortex, cerebellum, dorsal hippocampus and striatum. Equal concentrations of melatonin were needed to produce half-maximal inhibition in all the regions affected. The results indicate that the brain sites for inhibitory effect of melatonin on dopamine neurosecretion overlap the sites reportedly involved in its modulation of neuroendocrine functions.

Acetylcholine rhythm in the preoptic area of the rat hypothalamus is synchronized with the estrous cycle.

Acetylcholine levels in the preoptic area of rat hypothalamus were found to change rhythmically, in synchronization with the estrous cycle. During the proestrous critical period, these levels demonstrated a drop followed by a sharp rise. The absence of this phenomena in males indicates sexual dimorphism. The close association between this newly detected hypothalamic acetylcholine rhythm and the estrous cycle suggests the possible involvement of acetylcholine in regulation of the estrous cycle.

Acetylcholine in the rat pituitary: a possible humoral factor.

Significant amounts of acetylcholine (ACh) were detected in each of the 3 lobes of the rat pituitary (3-6 pmol/anterior lobe, 3 pmol/intermediate lobe and 1.8 pmol/posterior lobe). In the anterior lobes of cyclic rats the levels of ACh varied with the estrous cycle, with daily peaks being observed on the days of proestrus and estrus. The occurrence of ACh, apparently as a humoral factor, appears to be unique to the anterior pituitary.

Abortifacient potential for the epigeal parts of Peganum harmala.

The effect of methanol and acetone extracts of the epigeal parts of Peganum harmala, a common medicinal plant among Bedouins in Israel, was studied on several parameters of reproduction in female rats. The methanol extract at a dose of 2.5 g/kg/day, offered in food or in drinking suspension for 30 days, significantly prolonged diestrus by 1.0 day. The methanol extracts at doses of 2.0, 2.5 and 3.5 g/kg/day appeared to produce a dose-dependent significant decrease in litter size. No change in the physical and nutritional status of the animals and no adverse toxicological effects were observed.

Muscarinic receptors in the preoptic area are sensitive to 17 beta-estradiol during the critical period.

The relationship between the steroid hormone 17 beta-estradiol and the muscarinic cholinergic receptors present in the preoptic area (POA), median hypothalamus and posterior hypothalamus of female rats was examined in vitro at various stages of the estrous cycle. Muscarinic receptors varied in a cyclic manner, specifically in the POA, as shown by an increase in the proportion of high-affinity agonist binding sites (RH) to 60% during the proestrus, as compared to RH proportion observed during diestrus-2 and during the afternoon of proestrus (35%). Exposure of POA homogenates to 17 beta-estradiol resulted in conversion of RH to low-affinity agonist binding sites (RL). This effect of the hormone was also restricted to the POA taken from rats during the morning of proestrus. It was blocked by the antiestrogenic drug, clomiphene, and could be prevented by preoccupation of the muscarinic receptors by their own ligands prior to the addition of hormone. It follows that significant changes in POA muscarinic receptors in situ exactly coincided with the known critical time period characterized by high estrogen levels and high levels of estrogen receptors in the POA. These changes in muscarinic receptors might thus conceivably reflect variations in cholinergic activity in the POA during the estrous cycle.

Studies on muscarinic receptors in mouse and rat hypothalamus: a comparison of sex and cyclical differences.

The ligand binding properties of muscarinic receptors present in whole hypothalamus as well as in its three regions (preoptic area, median and posterior regions) were studied in male rats and mice, as well as in female rats during various stages of the estrous cycle, using the tritiated antagonist N-methyl-4-piperidyl benzilate. Kinetic and equilibrium analysis of antagonist binding as well as competition experiments with agonists, were used to probe the nature of the binding sites and possible differences between specific muscarinic sites in the various preparations. We could detect differences in agonist binding parameters between male and female rats in the preoptic area. In female rats binding of agonist to high affinity state is characterized by a lower affinity (27 nM) than in males (3.7 nM). Secondly, the population of agonist high affinity sites at the proestrous stage is much higher than that at other stages of cycle (66 vs. 38%). In addition, the binding properties of muscarinic receptors following intracisternal 6-hydroxydopamine lesion were investigated. This treatment resulted in a decrease of about one sixth of the total muscarinic receptors in the hypothalamus. The data are best interpreted as a degeneration of existing presynaptic muscarinic receptors located on catecholamine terminals in the hypothalamus. The pre- and postsynaptic nature of the muscarinic receptors, their localization in view of their binding properties, and their possible physiological role are discussed.