Transcriptional repression of the rat osteocalcin gene: role of two intronic CCTCCT motifs.

The regulation of osteocalcin gene transcription is complex, involving multiple positive and negative regulators. Previous studies have demonstrated that an intronic sequence, TTTCTTT (+118 to +124) is capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in cells of the osteoblast lineage, by interacting with a specific nuclear protein. Further analyses of intronic sequences have identified a second silencer motif in this region. Two copies of a CCTCCT motif are present within the first intron of the rat osteocalcin gene (+106 to +111 and +135 to +140) and are capable of mediating transcriptional repression of osteocalcin-CAT fusion genes in rat osteosarcoma cells. Transient gene expression assays of wild-type and mutant osteocalcin-CAT fusion genes into ROS 17/2.8 cells demonstrate that mutagenesis of either of these CCTCCT motifs in isolation results in a 1.6-fold increase in CAT activity relative to the parent fusion gene. Moreover, a 5-fold increase in reporter gene activity is observed when both motifs are mutated together. These sequences are also capable of suppressing osteocalcin promoter activity when placed upstream to the osteocalcin promoter. Gel retardation and southwestern analyses demonstrate that the CCTCCT motifs interact with specific proteins present in nuclear extracts from ROS 17/2.8 and UMR 106 osteosarcoma cells but not COS-7 kidney cells. Mutations that abolish suppressor function of this motif markedly impair interactions with this specific nuclear protein. These data demonstrate that at least two different silencer motifs (TTTCTTT and CCTCCT) in the first intron of the rat osteocalcin gene contribute to its transcriptional repression.

A simple non-radioactive method for the simultaneous quantitative determination of stomach empyting and intestinal propulsion in the intact conscious rat.

A simple non-radioactive method for the simultaneous assessment of stomach emptying and intestinal propulsion in intact fasted conscious rats was developed employing Amberlite pellets. The Amberlite pellets were administered by gastric gavage and the rats were killed 20 or 120 min later. The number and percent of the pellets in the stomach and intestines and the distance travelled by each pellet in the small intestine were determined. The distance travelled by the leading pellet in the small intestine was employed as a parameter to determine effects on intestinal propulsion independent of the stomach emptying activity. Chlorisondamine (s.c.), atropine (s.c.), pentobarbital (i.p.) and sesame seed oil (p.o.) inhibited both stomach emptying and intestinal propulsion in a dose-related manner. All these agents also caused a dose-related displacement of the pellets in the small intestine which resulted in a more cephalad-oriented distribution of the pellets. Propantheline (s.c.) exerted a dose-related inhibition on the stomach emptying but not on intestinal propulsion. Carbachol (s.c.) increased both the rate of stomach emptying and that of propulsion in the small intestine.

Troponoids. 3. Synthesis and antiallergy activity of N-troponyloxamic acid esters.

A number of oxamic acid derivatives of tropones and tropolones were synthesized and their antianaphylactic activity was determined in passive paw anaphylaxis (PPA). Several of these esters possessed oral activity. A comparison of the effect on the biological activity of the esters and the corresponding acid and its salt is reported. The experiments suggesting a relationship between the activity and the bioavailability of the ester 19 are also described. A study of the fate of ester 19 in serum on oral or intravenous administration to rats and dogs is reported. In vitro results of the effect of the compounds 19, 45, and 45a on the activity of the guinea pig lung and beef heart phosphodiesterase are presented. The various factors that may contribute to the antiallergy activity of compounds of this series are discussed.

Effect of acute and chronic treatment of tandamine, a new heterocyclic antidepressant, on biogenic amine metabolism and related activities.

The effects of tandamine, a clinically effective heterocyclic antidepressant, administered either acutely (10 mg/kg i.p.) or chronically (10 mg/kg i.p. daily for 21 days) on biogenic amine uptake and metabolism in the rat were determined and a comparison with desipramine was made. Tandamine, similarly to desipramine, blocked norepinephrine (NE) uptake in rat brain and heart following both acute and chronic administration. No effect of tandamine on dopamine (DA) or serotonin (5-HT) uptake was observed. Both drugs lowered endogenous brain NE when given chronically but not acutely. In contrast, no such effect on brain DA and 5-HT or heart NE was observed. Tandamine, like desipramine, administered chronically prior to an intraventricular injection of 3H-NE, produced increases in the decline of 3H-NE as indicated by decreased 3H-NE with increased levels of 3H-normetanephrine in brain stem of rats, suggesting an increased turnover of NE. No such effect was observed following acute treatment. Both drugs increased the behavioural effects of L-Dopa following and acute oral administration, with tandamine appearing superior to desipramine at the lower dose examined (10 mg/kg). Tandamine was 57--833 times less effective in binding to rat brain muscarinic receptors than desipramine, imipramine, butriptyline and amitriptyline, respectively. Thus, tandamine affects biogenic amine mechanism following either acute or chronic administration in a fashion similar to desipramine, but unlike desipramine, it exhibits relatively little anticholinergic properties, a further indication of the potential use of tandamine in the treatment of human depression, particularly where an increase in drive is desired.

Sucralfate: antipeptic, antiulcer activities and antagonism of gastric emptying.

The antiulcerogenic and antipeptic activities of sucralfate (Ulcerlmin), a basic aluminum sucrose sulfate complex, were investigated. In rats, sucralfate inhibited the formation of ulcers induced by pyloric ligation, indometacin and cysteamine. In doses antagonizing forestomach ulcer formation, sucralfate increased the pH of the gastric juice in a dose-related manner. In vitro, at pH 1.9, sucralfate inhibited rat, dog and hog pepsin in a concentration-related manner and also the peptic activity in human gastric juice. Sucralfate may act as an inhibitor of pepsin by precipitating the enzyme or by binding with it reversibly. The antipeptic activity appears to be directly related to the amount of sucralfate in suspension rather than that in solution. In the gastrointestinal tract, the basic nature of sucralfate may enhance the antipeptic activity of the sucralfate molecule. In rats, sucralfate decreases the rate of gastric emptying.

Effects of [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) on cyclic 3',5'-nucleotide formation and phosphodiesterase activity.

The orally-effective antiallergic compound [N-(2-oxo-3,5,7-cycloheptatrien-1-yl)] aminooxoacetic acid ethyl ester (AY-25,674) exhibited a potency equivalent to or 3 times less than theophylline in inhibiting guinea-pig lung and beef heart PDE, respectively, AY-25,674 did not affect the basal activity of guinea-pig lung adenyl cyclase. Although part of the antiallergic activity of AY-25,674 may be due to the ability to elevate cyclic AMP levels by PDE inhibition, other modes of action appear to be of greater relevance.

Effect of alrestatin sodium on glucose-stimulated insulin secretion in the fasted anaesthetized rat.

In the anaesthetized fasted non-diabetic male intact rat, alrestatin sodium injected as a bolus (0.75 mmol/kg, i.v.) did not affect basal plasma insulin or glucose levels. However, in response to an intravenous glucose tolerance test, plasma insulin levels were significantly increased above the values observed in the animal during a control test. The decreases in plasma glucose levels after alrestatin were significantly greater than in the control study. In rat pancreatic preparations in vitro, alrestatin lowered the basal release of 3H-norepinephrine and also the release obtained with the catecholamine-releasing agent tyramine. A modulation of catecholamine release appears to be of importance in the mode of action of alrestatin with respect to the insulin secretion and plasma glucose levels. It is suggested that alrestatin may play a useful role in the therapy of diabetes mellitus since it can augment insulin secretion when glucose is administered to a fasted animal in which the acute insulin response has been shown to be like that of the human diabetic, and in addition, can lower arginine-stimulated glucagon secretion in the animal, the latter being a model of an action that is observed in the human diabetic. The net effect of these hormonal changes has been predicted previously to be a lowering of the blood glucose levels in the human diabetic patient.

A relatively specific and quantitative assay for histamine H2-receptor blocking activity by determination of inhibition of histamine-induced gastric acid secretion in the rat.

A relatively specific method for the quantitative assay of histamine H2-receptor antagonists has been developed. The method is based on the antagonism of the histamine-induced, and the spontaneous, gastric acid secretion in the stomach-perfused, urethane-anesthetized acute rat. For the induced gastric acid secretion, the animal received two consecutive equal injections of histamine (2 mg/kg, intrajugularly), the second 1.7 h after the termination of the first histamine-induced acid hypersecretion. Atropine (5 mg/kg), chlorisondamine (2 mg/kg), imipramine (10 mg/kg) and tripelennamine (5 mg/kg), administered i.p., failed to inhibit the histamine-induced, or the spontaneous, gastric acid secretion. Metiamide inhibited the histamine-induced acid secretion (ED50 1.85 mg/kg, i.p.) and the spontaneous acid secretion (10 mg/kg, i.p.). These results suggest that in the rat model employed in this study, histamine and H2-receptors play a very important role in the regulation of the gastric acid secretion. Other mechanisms involving cholinergic tone, biogenic amine uptake and histamine H1-receptors do not seem to intervene.

Effect of alrestatin on arginine-induced secretion of glucagon and insulin in the rat.

Alrestatin, a lens aldose reductase inhibitor, decreased i.v. arginine-induced glucagon levels and augmented arginine-stimulated insulin release in the ether anesthetized rat. Alrestatin may then be useful in the treatment of diabetes mellitus, due to its actions on insulin and glucagon, and its capacity to delay the onset of sugar-induced cataracts in the rat.

Effect of somatostatin analogues and 17-alpha-dihydroequilin on rat brain opiate receptors.

Two somatostatin analogues (SA) and 17-alpha-dihydroequilin (E) inhibited the stereospecific binding of 3H-naloxone in vitro to rat brain opiate receptors in the absence of sodium. The addition of sodium indicated the SA to be greater or similar in potency to somatostatin as opiate agonists and E to be an opiate antagonist. The results indicate that SA and certain steroids may affect endorphin containing neurons.

Inhibition of prostaglandin-induced cyclic AMP accumulation in the rat anterior pituitary by alrestatin.

Alrestatin at 25-1 X 10(-4) M inhibited the accumulation of cyclic AMP induced by prostaglandin E2, but not theophylline, in the rat anterior pituitary in vitro. Somatostatin, at lower concentrations, inhibited both; maximal inhibition of the prostaglandin effect was greater with alrestatin. As cyclic AMP is considered to be a mediator in induced-hormonal release, it appears from the present findings that alrestatin may be of potential use in altering hormonal release.

Alrestatin: gastric acid antisecretory-antiulcer activity in the rat.

Alrestatin sodium (AY-22,284-A) inhibited gastric acid secretion and decreased the volume of gastric juice produced in the pylorus-ligated rat when administered intraperitoneally or perorally. Pentagastrin-induced gastric acid output in the unanesthetized rat was antagonized. The pyloric ligation-induced ulcer formation in the rat was inhibited. Alrestatin sodium did not exhibit an anticholinergic profile. The drug did not block the norepinephrine neuronal uptake mechanism in rat brain or heart; it did not alter the endogenous norepinephrine concentration in the heart and decreased endogeneous brain norepinephrine concentration. Alrestatin sodium is an effective inhibitor of gastric acid secretion and ulcer formation in the rat.

Effect of butaclamol, a new neuroleptic, on serotoninergic mechanisms.

Butaclamol (1.0-0.1 mg kg-1, i.p.) and spiroperidol (1-0-0-5 mg kg-1, i.p.) but not (-)-butaclamol (15 mg kg-1, i.p.), blocked the hyperactivity induced in rats by tranylcypromine-L-tryptophan pretreatment. Neither butaclamol nor spiroperidol altered the accumulation of brain 5-HT following parglyine or the decline of brain 5-HT following inhibition with the tryptophan hydroxylase inhibitor alpha-propyldopacetamide thus indicating that butaclamol and spiroperidol do not affect either the synthesis or the turnover of brain 5-HT. It is concluded that the antagonism of the tranylcypromise-L-tryptophan-induced hyperactivity by butaclamol and spiroperidol is due to their blockade of dopaminergic receptors rather than an action on neuronal serotoninergic mechanisms.