Nelson W. Chong

l-NG-nitro arginine (l-NOARG), a novel, l-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro

1 The effect of l‐NG‐nitro arginine (l‐NOARG) was compared with that of l‐NG‐monomethyl arginine (l‐NMMA) on vasodilatation of the isolated aorta of the rabbit and perfused mesentery of the rat in response to acetylcholine (ACh) and sodium nitroprusside (NP). 2 l‐NOARG (1.5–100 μm) and l‐NMMA (3–100 μm) produced concentration‐related contraction of the rabbit aorta precontracted with phenylephrine (700–900 nm). Similarly, l‐NOARG (10–200 μm) and l‐NMMA (30–100 μm) elevated perfusion pressure of the noradrenaline (NA, 0.6–2.5 mm)‐preconstricted rat mesentery preparation. 3 l‐NOARG (1.5–100 μm) and l‐NMMA (3–100 μm) caused concentration‐related inhibition of the vasodilator effect of ACh (0.01–1.0 μm) on the rabbit aorta without influencing responses to NP (0.03–0.5 μm). l‐NOARG methyl ester (30 μm) also inhibited ACh‐induced vasorelaxation with similar potency to NOARG. l‐arginine (30–150 μm) but not d‐arginine (100 μm) caused graded reversal of the inhibitory effect of both l‐NOARG (15 μm) and l‐NMMA (30 μm). Complete reversal of the effect of both inhibitors was achieved with 150 μm l‐arginine. l‐Alanine (50 μm), l‐arginosuccinic acid (5 μm), l‐citrulline (50 μm), l‐methionine (50 μm) and l‐ornithine (50 μm) failed to reverse the inhibitory effect of l‐NOARG (15 μm). 4 l‐NOARG (10–200 μm) and l‐NMMA (30–100 μm) inhibited the vasodilator effect of ACh (0.006–18.0 nmol) in the rat mesentery without affecting vasodilatation due to NP (1.1–11.1 nmol). l‐Arginine (100 μm) but not d‐arginine (100 μm) produced partial reversal of the effect of l‐NOARG (30 μm) and l‐NMMA (30 μm). 5 l‐ and d‐Nα‐butyloxycarbonyl NG‐nitro arginine (100 μm) produced modest (approximately 20%) inhibition of the effect of ACh on the rabbit aorta; this effect was not reversible with l‐arginine (100 μm). l‐Nα‐monocarbobenzoxy arginine (l‐NMCA, 50 μm), l‐Nα‐NG‐dicarbobenzoxy arginine (l‐NDCA, 5 μm) and l‐NG‐tosyl arginine (50 μm) were inactive. 6 These results identify l‐NOARG as a potent, l‐arginine reversible inhibitor of endothelium‐dependent vasodilatation. The available data suggests that l‐NOARG, like l‐NMMA, inhibits endothelial nitric oxide (NO) biosynthesis.

Characterization of the chicken serotonin N-acetyltransferase gene: activation via clock gene heterodimer/E-box interaction

The abundance of serotoninN-acetyltransferase (arylalkylamineN-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5′-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT genein vitro.

Chickens' Cry2: molecular analysis of an avian cryptochrome in retinal and pineal photoreceptors

We have identified and characterized an ortholog of the putative mammalian clock gene cryptochrome 2 (Cry2) in the chicken, Gallus domesticus. Northern blot analysis of gCry2 mRNA indicates widespread distribution in central nervous and peripheral tissues, with very high expression in pineal and retina. In situ hybridization of chick brain and retina reveals expression in photoreceptors and in visual and circadian system structures. Expression is rhythmic; mRNA levels predominate in late subjective night. The present data suggests that gCry2 is a candidate avian clock gene and/or photopigment and set the stage for functional studies of gCry2.

Reduction of gaba and glutamate transporter messenger rnas in the severe-seizure genetically epilepsy-prone rat

The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-1 and EAAC-1, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immunoblotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype-specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (-8 to -24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 (-8 to -12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (-20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-1, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins.

Temporal-spatial characterization of chicken clock genes: circadian expression in retina, pineal gland, and peripheral tissues.

The molecular core of the vertebrate circadian clock is a set of clock genes, whose products interact to control circadian changes in physiology. These clock genes are expressed in all tissues known to possess an endogenous self‐sustaining clock, and many are also found in peripheral tissues. In the present study, the expression patterns of two clock genes, cBmal1 and cMOP4, were examined in the chicken, a useful model for analysis of the avian circadian system. In two tissues which contain endogenous clocks – the pineal gland and retina – circadian fluctuations of both cBmal1 and cMOP4 mRNAs were observed to be synchronous; highest levels occurred at Zeitgeber time 12. Expression of these genes is also rhythmic in several peripheral tissues; however, the phases of these rhythms differ from those in the pineal gland and retina: in the liver the peaks of cMOP4 and cBmal1 mRNAs are delayed 4–8 h and in the heart they are advanced by 4 h, relative to those in the pineal gland and retina. These results provide the first temporal characterization of cBmal1 and cMOP4 mRNAs in avian tissues: their presence in avian peripheral tissues indicates they may influence temporal features of daily rhythms in biochemical, physiological, and behavioral functions at these sites.

Structural requirements at the melatonin receptor

1 High affinity, specific binding sites for the pineal hormone, melatonin (5‐methoxy N‐acetyltryptamine) can be detected in chick brain membranes by use of the radiolabeled agonist, 2‐[125I]‐iodomelatonin (2‐[125I]‐aMT). 2 The affinity of a number of analogues of melatonin at the 2‐[125I]‐aMT binding site was determined and compared with an analysis of their electronic structure and significant quantitative relationships obtained. 3 The best correlations indicated that binding affinity was correlated with ΔE, the difference between the frontier orbital energies, and QNH, the electron density in the highest occupied molecular orbital of the side‐chain nitrogen atom. 4 These findings suggest that ligand binding may involve hydrogen bonding between the 5‐methoxy and amide moieties of melatonin and complementary amino acid residues, and charge transfer interactions between the indole ring of melatonin and an aromatic amino acid in the receptor binding site. 5 A molecular model of a putative binding site is proposed based on the predicted amino acid sequence of the cloned Xenopus laevis melanophore melatonin receptor and the quantitative structure‐affinity relationships observed in the present study.

Pharmacological identity of 2-[125I]iodomelatonin binding sites in chicken brain and sheep pars tuberalis

2-[125I]Iodomelatonin was used to compare the binding and pharmacological characteristics of the melatonin receptor sites found in chicken brain and sheep pars tuberalis. Scatchard analysis and kinetic experiments showed that 2-[125I]iodomelatonin binds to a single class of site in both tissues with high affinity (Kd 20-34 pM). Competition experiments, using 21 analogues of melatonin, gave inhibition constants (Ki) for the two sites which were significantly correlated (r = 0.985, n = 21, P less than 0.0001). We conclude that the 2-[125I]iodomelatonin binding sites in sheep pars tuberalis and chicken brain have identical binding and pharmacological characteristics.

Circadian expression of tryptophan hydroxylase mRNA in the chicken retina

Many aspects of retinal physiology are controlled by a circadian clock located within the eye. This clock controls the rhythmic synthesis of melatonin, which results in elevated levels during the night and low levels during the day. The rate-limiting enzyme in melatonin biosynthesis in retina appears to be tryptophan hydroxylase (TPH)[G.M. Cahill and J.C. Besharse, Circadian regulation of melatonin in the retina of Xenopus laevis: Limitation by serotonin availability, J. Neurochem. 54 (1990) 716-719]. In this report, we found that TPH mRNA is strongly expressed in the photoreceptor layer and the vitread portion of the inner nuclear layer; the message is also expressed, but to a lesser extent, in the ganglion cell layer. The abundance of retinal TPH mRNA exhibits a circadian rhythm which persists in constant light or constant darkness. The phase of the rhythm can be reversed by reversing the light:dark cycle. In parallel experiments we found a similar pattern of expression in the chicken pineal gland. However, whereas a pulse of light at midnight suppressed retinal TPH mRNA by 25%, it did not alter pineal TPH mRNA, suggesting that there are tissue-specific differences in photic regulation of TPH mRNA. In retinas treated with kainic acid to destroy serotonin-containing amacrine and bipolar cells, a high amplitude rhythm of TPH mRNA was observed indicating that melatonin-synthesizing photoreceptors are the primary source of the rhythmic message. These observations provide the first evidence that chick retinal TPH mRNA is under control of a circadian clock.

ms1, a novel stress‐responsive, muscle‐specific gene that is up‐regulated in the early stages of pressure overload‐induced left ventricular hypertrophy

We have identified and characterised a cDNA encoding a novel gene, designated myocyte stress 1 (ms1), that is up‐regulated within 1 h in the left ventricle following the application of pressure overload by aortic banding in the rat. The deduced ms1 protein of 317 amino acids contains several putative functional motifs, including a region that is evolutionarily conserved. Distribution analysis indicates that rat ms1 mRNA expression is predominantly expressed in striated muscle and progressively increases in the left ventricle from embryo to adulthood. These findings suggest that ms1 may be important in striated muscle biology and the development of pressure‐induced left ventricular hypertrophy.

Circadian clock genes cause activation of the human PAI-1 gene promoter with 4G/5G allelic preference

Increased plasminogen activator inhibitor‐1 (PAI‐1) activity is associated with greater risk of myocardial infarction. PAI‐1 expression is regulated by a 4G/5G promoter polymorphism. The 4G allele is associated with higher PAI‐levels and greater circadian variation. Here we show that clock protein heterodimers BMAL/CLOCK cause greater activation (≈2‐fold, P < 0.05) of the 4G allele. Site‐directed mutagenesis studies suggest that clock genes act on two canonical E‐boxes to regulate PAI‐1 promoter activity. These results identify a potential novel mechanism whereby allele‐specific clock genes – mediated modulation of PAI‐1 expression may contribute to circadian variation in cardiac risk.