HERP, a new primary target of Notch regulated by ligand binding.

Notch signaling dictates cell fate and critically influences cell proliferation, differentiation, and apoptosis in metazoans. Ligand binding initiates the signal through regulated intramembrane proteolysis of a transmembrane Notch receptor which releases the signal-transducing Notch intracellular domain (NICD). The HES/E(spl) gene family is a primary target of Notch and thus far the only known Notch effector. A newly isolated HERP family, a HES-related basic helix-loop-helix protein family, has been proposed as a potential target of Notch, based on its induction following NICD overexpression. However, NICD is physiologically maintained at an extremely low level that typically escapes detection, and therefore, nonregulated overexpression of NICD-as in transient transfection-has the potential of generating cellular responses of little physiological relevance. Indeed, a constitutively active NICD indiscriminately up-regulates expression of both HERP1 and HERP2 mRNAs. However, physiological Notch stimulation through ligand binding results in the selective induction of HERP2 but not HERP1 mRNA and causes only marginal up-regulation of HES1 mRNA. Importantly, HERP2 is an immediate target gene of Notch signaling since HERP2 mRNA expression is induced even in the absence of de novo protein synthesis. HERP2 mRNA induction is accompanied by specific expression of HERP2 protein in the nucleus. Furthermore, using RBP-Jk-deficient cells, we show that an RBP-Jk protein, a transcription factor that directly activates HES/E(spl) transcription, also is essential for HERP2 mRNA expression and that expression of exogenous RBP-Jk is sufficient to rescue HERP2 mRNA expression. These data establish that HERP2 is a novel primary target gene of Notch that, together with HES, may effect diverse biological activities of Notch.

HERP, a novel heterodimer partner of HES/E(spl) in Notch signaling.

HERP1 and -2 are members of a new basic helix-loop-helix (bHLH) protein family closely related to HES/E(spl), the only previously known Notch effector. Like that of HES, HERP mRNA expression is directly up-regulated by Notch ligand binding without de novo protein synthesis. HES and HERP are individually expressed in certain cells, but they are also coexpressed within single cells after Notch stimulation. Here, we show that HERP has intrinsic transcriptional repression activity. Transcriptional repression by HES/E(spl) entails the recruitment of the corepressor TLE/Groucho via a conserved WRPW motif, whereas unexpectedly the corresponding-but modified-tetrapeptide motif in HERP confers marginal repression. Rather, HERP uses its bHLH domain to recruit the mSin3 complex containing histone deacetylase HDAC1 and an additional corepressor, N-CoR, to mediate repression. HES and HERP homodimers bind similar DNA sequences, but with distinct sequence preferences, and they repress transcription from specific DNA binding sites. Importantly, HES and HERP associate with each other in solution and form a stable HES-HERP heterodimer upon DNA binding. HES-HERP heterodimers have both a greater DNA binding activity and a stronger repression activity than do the respective homodimers. Thus, Notch signaling relies on cooperation between HES and HERP, two transcriptional repressors with distinctive repression mechanisms which, either as homo- or as heterodimers, regulate target gene expression.

Novel calcium antagonists. Synthesis and structure-activity relationship studies of benzothiazoline derivatives.

A series of novel compounds having a benzothiazoline skeleton was studied for their structure-activity relationship (SAR) with respect to Ca2+ antagonistic activity. As test compounds, analogues of 3-acyl-2-arylbenzothiazolines (3) were synthesized. Benzothiazoline derivatives (3) exerted higher Ca2+ antagonistic activity than the corresponding thiazolidine derivatives (2). Effects of substituents R1-R4, the substitution position of the aminoalkoxy group and R2, and the length of the methylene chain on biological activities were examined. Compound 4 [3-acetyl-2-[5-methoxy-2-[4-[N-methyl-N-(3,4,5-trimethoxyphenethyl ) amino]butoxy]phenyl]benzothiazoline hydrochloride] showed a potent Ca2+ antagonistic activity in vitro and dual inhibition on the fast Na+ inward channel and the slow Ca2+ inward channel in Langendorff perfused rabbit hearts. Compound 4 also showed a long-acting hypotensive effect in spontaneously hypertensive rats and prevented acute pulmonary thrombotic death in mice.

Antianaphylactic effects of dipivalyl epinephrine and related compounds in rat conjunctiva.

Topically applied dipivalyl epinephrine (DPE) and related compounds have been found to inhibit passive anaphylactic reaction in rat conjunctiva. The order of activity is as follows: isoproterenol greater than DPE greater than epinephrine greater than norepinephrine. The antianaphylactic effect of DPE was antagonized by propranolol but was not affected by phentolamine. The effects of epinephrine, norepinephrine, and isoproterenol were also antagonized by propranolol but potentiated by pentolamine. From these findings, it was suggested that DPE not only exerts its antianaphylactic action through activation of beta-adrenergic receptor but also itself has a little different action from epinephrine.

Effects of sodium loading on antihypertensive responses to an angiotensin-converting enzyme inhibitor in spontaneously hypertensive and renal hypertensive rats.

Sodium loading significantly attenuated the antihypertensive potency of an angiotensin-converting enzyme inhibitor, SA446, in 2-kidney, 1-clip renal hypertensive rats, but the potency of SA446 remained unchanged in spontaneously hypertensive rats. Basal levels of plasma renin activity of both types of hypertensive rats were suppressed by sodium loading. From these results, it appeared that a different mechanism was involved in the maintenance of hypertension in 2-kidney, 1-clip renal hypertensive rats and in spontaneously hypertensive rats. It also appeared that the mechanism of the antihypertensive action of angiotensin-converting enzyme inhibitors was not explainable only by the suppression of the plasma renin-angiotensin system.

Potentiating mechanism of bradykinin action on smooth muscle by sulfhydryl compounds.

(4R)-3-[(2S)-3-Mercapto-2-methylpropanoyl]-4-thiazolidinecarboxylic acid (YS-980), a potent angiotensin I (AI) converting enzyme inhibitor, inhibited the contractile response of isolated guinea-pig ileum to AI but not to AII, while it potentiated the response to bradykinin. Other sulfhydryl compuonds also produced inhibition of AI action, and the effects were closely correlated with their potentiating activities against bradykinin action. These results suggest that it is mainly inhibition of the enzyme in the tissue which participates in the mechanism of potentiation of kinin action by YS-980.

Renal excretion of an angiotensin I converting enzyme inhibitor (SA-446) in dogs.

The renal excretory mechanism of an orally active inhibitor of angiotensin converting enzyme (SA-446) was examined in anesthetized dogs. Parenteral administration of this compound resulted in production of constant levels of about 2 mg/l in the plasma (PSA) and the urine concentration was 726 +/- 200 mg/l, a level significantly higher than that in the plasma. Renal clearance of SA-446 (CSA) was 2.24 +/- 0.34 ml/g X min and was significantly higher than GFR. The clearance ratio (CSA/GFR) of over 1.0 was indicative of a net tubular secretion. Administration of probenecid resulted in a significant rise in PSA and in a significant decrease in urinary excretion but with no change in the plasma protein binding ratio. CSA decreased significantly from 2.24 +/- 0.34 to 0.71 +/- 0.14 ml/g X min. The inhibitory action of SA-446 (0.02 mg/kg, i.v.) on the pressor response to angiotensin I disappeared at about 50 min, this action being maintained for about 2 h in the probenecid pretreated dog. Since probenecid is a competitive inhibitor of organic anion secretory transport, our results show the net tubular secretion of SA-446, via organic anion transport systems. Prolongation of the action of SA-446, as induced by probenecid may be due to the increase of plasma concentration, by the inhibition of tubular secretion.

Cardiovascular characterization of SD-3211, a novel benzothiazine calcium channel blocker, in isolated rabbit hearts.

The cardiovascular effects of SD-3211, a novel benzothiazine Ca++ channel blocker, were compared with those of diltiazem and nicardipine in Langendorff-perfused rabbit hearts. SD-3211 was more potent in increasing coronary artery flow than in depressing cardiac function (i.e., contractile force, heart rate and conduction time). The relative specificity of SD-3211 for coronary vasodilation to cardiodepression was clearly greater than that of diltiazem, but less than that of nicardipine. Thus, the present study demonstrates that SD-3211, despite a non-dihydropyridine type of Ca++ channel blocker, can be characterized as a potent coronary vasodilator with a little effect on cardiac function.

Effect of tiopronin on prostaglandin synthesis in rabbit kidney medulla slices.

The effect of 2-mercaptopropionylglycine (tiopronin), which is widely used for the treatment of various hepatic disorders, on the generation of medullary prostaglandins (PG) E2 and F2 alpha has been examined. Tiopronin had a potent inhibitory effect on PG E2 formation. Simultaneously, PG F2 alpha production was increased. In the presence of tiopronin the net increased amount of PG F2 alpha was much smaller than the net decreased amount of PG E2 (6-20%). These results suggest that tiopronin has the potential to modulate PG E2 and F2 alpha synthesis by affecting endoperoxide E2 isomerase or endoperoxide reductase and that this effect may represent some pharmacological action of the drug.

pH dependency of effect of topically applied dipivefrine hydrochloride on intraocular pressure and pupil size in rabbits: comparative studies with epinephrine.

The pH and dose dependencies of topically applied dipivefrine hydrochloride (DPE) on intraocular pressure (IOP) reduction and mydriasis were compared with those of epinephrine bitartrate (EPI) in normal rabbits. Statistically significantly greater IOP lowering and mydriatic effects of DPE solutions were achieved by increasing their pH from 3 to 6 or their concentrations from 0.02% to 0.1%. The ocular hypotensive and mydriatic effects of EPI also depended on their concentrations, but not on their pH. On the other hand, blink rates of rabbits following topical application of DPE tended to be reduced by the increase of pH, while they were not influenced by the concentrations. From these results, it was indicated that the pharmacological effects of DPE were augmented about 3 times by increasing the pH of its solutions from 3 to 5, which also was associated with reduction of ocular irritation, while the effects of EPI were not altered by the changes of pH. As the result of the increase of pH from 3 to 5, DPE became about 50 times more potent than EPI as a ocular hypotensive agent.

Potentiative effect of angiotensin converting enzyme inhibitor on carrageenan edema in rats and the role of tissue kininogen.

Roles of kininogens in the development of potentiation of carrageenan edema in rats by angiotensin converting enzyme (ACE, = kininase II) inhibitor were studied. Carrageenan-induced edema was potentiated by oral administration of YS980, an ACE inhibitor, at a dose of 1 mg/kg 0.5 h before carrageenan injection. Intravenous injection of bromelain, a kininogen (KGN) depletor, at 3 mg/kg produced reduction of plasma KGN (total and high molecular weight KGN), which resulted in suppression of carrageenan edema and suppression of edema potentiation induced by YS980. Even after the plasma KGN level and inflammatory response to carrageenan returned to normal 24 h after the administration of bromelain, the potentiative effect of YS980 on the carrageenan edema remained suppressed. Thus, some factor other than plasma KGN is thought to be involved in the potentiation mechanisms on the carrageenan edema by YS980. Partially purified KGN from rat plasma (0.1 mg/site: liberated 4.4 X 10(-8) g bradykinin eq by trypsin digestion) completely restored the suppressed potentiation to normal by local preinjection to the inflamed site. In addition, such restoration was not observed in the animal in which plasma KGN was reduced 3 h after administration of bromelain. These results suggest that KGN, not only in plasma but also in tissue, play an active role in the development of potentiation of carrageenan edema by ACE inhibitor.