Circadian regulation of iodopsin gene expression in embryonic photoreceptors in retinal cell culture.

A circadian clock regulates a number of diverse physiological functions in the vertebrate eye. In this study, we show that mRNA for the red-sensitive cone pigment, iodopsin, fluctuates with a circadian rhythm in chicken retina. Transcript levels increase in the late afternoon just prior to the time of cone disc shedding. Furthermore, iodopsin mRNA levels are regulated similarly by a circadian oscillator in primary cultures of dispersed embryonic chick retina. Nuclear run-on experiments show that the circadian regulation of iodopsin transcript abundance occurs at the level of gene transcription. Our results provide a demonstration of clock-regulated gene expression in a vertebrate preparation maintained in cell culture.

Dopamine mediates circadian rhythms of rod-cone dominance in the Japanese quail retina.

A circadian clock modulates the functional organization of the Japanese quail retina. Under conditions of constant darkness, rods dominate electroretinogram (ERG) b-wave responses at night, and cones dominate them during the day, yielding a circadian rhythm in retinal sensitivity and rod-cone dominance. The activity of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, also exhibits a circadian rhythm in the retina with approximately threefold higher levels during the day than at night. The rhythm of tyrosine hydroxylase activity is opposite in phase to the circadian activity of tryptophan hydroxylase, the first enzyme in the melatonin biosynthetic pathway. We tested whether dopamine may be related to the physiological rhythms of the retina by examining the actions of pharmacological agents that effect dopamine receptors. We found that blocking dopamine D2 receptors in the retina during the day mimics the nighttime state by increasing the amplitude of the b-wave and shifting the retina to rod dominance. Conversely, activating D2 receptors at night mimics the daytime state by decreasing the amplitude of the b-wave and shifting the retina to cone dominance. A selective antagonist for D1 dopamine receptors has no effect on retinal sensitivity or rod-cone dominance. Reducing retinal dopamine partially abolishes rhythms in sensitivity and yields a rod-dominated retina regardless of the time of day. These results suggest that dopamine, under the control of a circadian oscillator, has a key role in modulating sensitivity and rod-cone dominance in the Japanese quail retina.

Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length.

In lower vertebrates, cone retinomotor movements occur in response to changes in lighting conditions and to an endogenous circadian clock. In the light, cone myoids contract, while in the dark, they elongate. In order to test the hypothesis that melatonin and dopamine may be involved in the regulation of cone movement, we have used an in vitro eyecup preparation from Xenopus laevis that sustains light- and dark-adaptive cone retinomotor movement. Melatonin mimics darkness by causing cone elongation. Dark- and melatonin-induced cone elongation are blocked by dopamine. Dopamine also stimulates cone contraction in dark-adapted eyecups. The effect of dopamine appears to be mediated specifically by a dopamine receptor, possibly of the D2 type. The dopamine agonist apomorphine and the putative D2 agonist LY171555 induced cone contraction. In contrast, the putative D1 agonist SKF38393-A and specific alpha 1-, alpha 2-, and beta-adrenergic receptor agonists were without effect. Furthermore, the dopamine antagonist spiroperidol not only blocked light-induced cone contraction, but also stimulated cone elongation in the light. These results suggest that dopamine is part of the light signal for cone contraction, and that its suppression is part of the dark signal for cone elongation. Melatonin may affect cone movement indirectly through its influence on the dopaminergic system.

Neuropeptide Y stimulates luteinizing hormone-releasing hormone release from superfused hypothalamic GT1-7 cells.

The effects of neuropeptide Y (NPY) on LHRH release from an immortalized cell line were investigated using a flow-through cell culture superfusion system. Immortalized hypothalamic GT1-7 cells were cultured for 72 h and superfused for a total of 180 min. In initial experiments, discrete 5-min pulses of NPY (10(-12)-10(-5) M) were administered to the cells. A clear dose-dependent stimulatory effect on NPY on LHRH release from the cells was observed with a calculated 50% effectiveness concentration of 33 nM. The stimulatory effects of brief NPY exposure were rapid and robust, e.g. reaching and maintaining levels of 173% over baseline for 20 min at the 10(-7) dose. The lowest dose of NPY that showed a significant effect was 10(-10) M; maximal responses were observed at 10(-6) M and reached a plateau thereafter. Control pulses of Dulbecco's modified Eagle's medium (DMEM) and 10(-6) M substance P or arg-vasopressin were also presented to the cells to serve as controls for our pulse protocol, and these challenges produced no significant LHRH responses. The NPY receptor antagonists, PYX1 and PYX2, at 10(-8) M, completely blocked the observed NPY responses in these cells. To assess the NPY receptor subtypes that mediate the NPY effects pharmacologically, GT1-7 cells were challenged with a Y1 receptor agonist, (Leu31Pro34)NPY, a Y2 receptor agonist, NPY(13-36), or peptide YY, at doses 10(-12)-10(-5) M. All four peptides stimulated LHRH release from GT1-7 cells with a rank-ordered potency of NPY = peptide YY > Y1 agonist = Y2 agonist. To examine possible signal transduction mechanism(s) involved in mediating this effect, pertussis toxin, RpcAMPs (cyclic adenosine-3'5'-monophosphothioate Rp diastereomer), Ca(2+)-free DMEM and TMB-8 (3, 4, 5-trimethoxybenzoic acid 8-(diethylamino) octylester) were used to treat the cells before and during superfusion with NPY. Treatment with pertussis toxin, RpcAMPs, and Ca(2+)-free DMEM did not significantly alter NPY-stimulated LHRH release responses to 10(-7) M NPY. However, the addition of 100 microM and 250 microM TMB-8 to Ca(2+)-free DMEM almost completely blocked this NPY effect, as did 10 microM ryanodine. Finally, the locus of action for this NPY effect was examined using tetrodotoxin to reduce action potential propagation in the GT1-7 cells. Tetrodotoxin treatment blocked the LHRH response to NPY by more than 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

Circadian regulation of retinomotor movements: II. The role of GABA in the regulation of cone position.

Cone photoreceptor movements in lower vertebrates are regulated by the interaction of the light-dark cycle and an endogenous circadian clock. We have suggested that melatonin and dopamine interact to regulate dark- and light-adaptive movements, respectively, and that melatonin affects cones indirectly by inhibiting dopamine release. In fact, any factor modulating dopaminergic neurons in the retina may have effects on either cone elongation or contraction. We have utilized an in vitro eyecup preparation from the African clawed frog, Xenopus laevis, to evaluate a possible role of the neurotransmitter GABA, which is thought to tonically suppress dopamine release. GABA agonists mimic the effects of darkness and induce cone elongation; the effects of GABA agonists are blocked by dopamine. Muscimol-induced cone elongation occurs at low light intensity but is inhibited by bright light in eyecups prepared from cyclic-light-maintained animals. Although neither melatonin nor muscimol stimulates cone elongation in bright light, simultaneous application of both drugs induces elongation. The GABA antagonist picrotoxin induces cone contraction which is blocked by the dopamine receptor antagonist spiroperidol, which suggests that GABA may affect cone movement in Xenopus by regulating dopamine neurons. Consistent with this, picrotoxin-induced cone contraction is Ca+2 dependent and is blocked by high Mg+2 or the Ca+2 antagonist nifedipine. Pharmacological analysis suggests that the effects of GABA may result from its action at more than one receptor subtype. Our results support the hypothesis that dopamine is part of the light signal for cone contraction and that its suppression is part of the signal for cone elongation.

Nonpeptide angiotensin II receptor antagonists: the discovery of a series of N-(biphenylylmethyl)imidazoles as potent, orally active antihypertensives.

A new series of nonpeptide angiotensin II (AII) receptor antagonists has been prepared. These N-(biphenylyl-methyl)imidazoles, e.g. 2-butyl-1-[(2'-carboxybiphenyl-4-yl)methyl]-4-chloro-5- (hydroxymethyl)imidazole, differ from the previously reported N-(benzamidobenzyl)imidazoles and related compounds in that they produce a potent antihypertensive effect upon oral administration; the earlier series generally were active only when administered intravenously. It has been found that the acidic group at the 2'-position of the biphenyl is essential. Only ortho-substituted acids possess both high affinity for the AII receptor and good oral antihypertensive potency. The carboxylic acid group has been replaced with a variety of acidic isosteres, and the tetrazole ring has been found to be the most effective. The tetrazole derivative, DuP 753, is currently in development for the treatment of hypertension.

RNA synthesis inhibitors increase melatonin production in Y79 human retinoblastoma cells.

Y79 human retinoblastoma cells synthesize melatonin in cell culture thus providing a unique preparation for studying the regulation of melatonin biosynthesis in mammalian retinas. We have previously demonstrated that Y79 cells express NAT and HIOMT activity and produce melatonin in a cAMP- and protein synthesis-dependent manner by increasing NAT, and not HIOMT activity, as has been demonstrated in other retinal and pineal melatonin synthesizing systems. We have extended these studies to investigate the role of RNA synthesis in melatonin regulation, and report here that RNA synthesis inhibitors do not suppress melatonin production in Y79 retinoblastoma cells. Rather, at intermediate concentrations, the inhibitors actinomycin D and camptothecin increase melatonin levels. Camptothecin, a topoisomerase I inhibitor, also increased NAT activity and accumulated cAMP levels in a calcium-dependent manner. This effect on cAMP did not appear to occur through phosphodiesterase, and other regulators of retinal melatonin such as melatonin degradation or components of the dopamine system were unaffected. These results are in contrast with the suppression of melatonin synthesis by RNA synthesis inhibitors observed in rat and chick pineal glands and in chick retinas.

Melatonin and rhythmic photoreceptor metabolism: melatonin-induced cone elongation is blocked at high light intensity.

We have proposed a model for circadian regulation of cone position in Xenopus laevis that involves interaction of melatonin and dopamine as signals for darkness and light respectively. One problem, however, is that the effects of melatonin have not been detected in eye cups prepared from animals maintained on a cyclic light schedule. Since melatonin's effect would be expected to occur in low light intensity at night, we have investigated the relationship among melatonin, light intensity, and cone length. We report that melatonin mimics the effects of darkness and stimulates cone elongation in eye cups from cyclic light animals incubated at low but not at high light intensities.

N-acetyltransferase and protein synthesis modulate melatonin production by Y79 human retinoblastoma cells.

Melatonin is synthesized by the vertebrate pineal gland in a circadian fashion and is involved in numerous circadian and seasonal processes in the organism. The vertebrate retina also produces melatonin rhythmically to regulate rhythmic physiological processes in the eye. In both organs, melatonin is synthesized from serotonin by the sequential action of the enzymes, N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), and can be stimulated by increases in cyclic AMP through a mechanism requiring protein synthesis. The regulation of ocular melatonin biosynthesis in mammals and particularly humans, has not been well studied. Recently, we have shown that Y79 human retinoblastoma cells produce melatonin and that cAMP can stimulate melatonin production. Y79 cells, therefore, provide a model system in which to study melatonin synthesis in human tissue. We report that cAMP stimulates NAT, but not HIOMT activity in Y79 cells, and that stimulation of NAT activity is linearly related to melatonin release. In addition, the stimulation of NAT and melatonin requires protein synthesis. The turnover of NAT is rather rapid, with a half-life of about 20 min. These results suggest that the regulation of melatonin in Y79 retinoblastoma cells is similar to that found in the retina and pineal of other vertebrates.

Circadian organization in quail retina: differential regulation of melatonin synthesis and iodopsin gene expression in vitro.

Adult Japanese quail have an endogenous circadian clock located in their eyes that has been shown to regulate melatonin biosynthesis. We investigated if a circadian oscillator is present in cultures of dispersed embryonic quail retina. Melatonin release in retinal cell culture is modulated by the light cycle, indicating that there are functional photoreceptors in culture. However, when cultures were placed in constant darkness no rhythm of melatonin was observed, indicating that at this period of development the circadian oscillator does not influence melatonin release. To explore further the question of whether a circadian oscillator is present in embryonic cell culture, we examined expression of iodopsin, the red visual pigment. Iodopsin mRNA is expressed in a circadian rhythm with peak levels occurring late in the afternoon (ZT 9). Analysis indicates that the clock influence is at the level of gene transcription. These results suggest that a clock is not "hooked up" to melatonin release embryonically or that a different oscillator regulates photopigment expression versus melatonin release.

Identification of hepatic cirrhosis by duplex doppler ultrasound value of the hepatic artery resistive index.

Pulsed Doppler Ultrasound was used to analyze hepatic artery wave forms near the porta hepatis. The Resistive Index (RI) = [peak systolic frequency shift (A)--minimum diastolic frequency shift (B)]/[peak systolic frequency shift (A)] has been calculated from this information. Two populations have been compared; 30 fit hospital staff members, 23 male, 7 female, age mean 37 years and range 19 to 73 years, and 33 cirrhotic potential liver transplant recipients, 16 male, 17 female, age mean 48 years and range 11 to 78 years. The RI was successfully obtained in 94% of the potential transplant patients. There is a significant difference between the RI of the controls (mean = 0.72, SE = 0.2, n = 27) and the cirrhotics (mean = 0.82, SE = 0.2, n = 31), P less than 0.0001. Using a cut off of greater than 0.77 this index has a sensitivity, specificity and overall accuracy of 68%, 70% and 69% respectively.

The falling anterior labrum.

Two cases of a falling anterior glenoid labrum are presented. The anterior labrum appears normal on supine double contrast computed tomographic arthrography (DCCTA). However, on prone DCCTA the anterior labrum falls away from the glenoid. The addition of prone DCCTA in these two cases reveals a labral tear, occult on routine supine DCCTA.

Forskolin and camptothecin induce a 30 kDa protein associated with melatonin production in Y79 human retinoblastoma cells.

The synthesis of melatonin in Xenopus retinas, chick and quail retinal cell cultures, and Y79 human retinoblastoma cells is stimulated by cAMP through a protein synthesis-dependent mechanism. In Y79 retinoblastoma cells, combined treatment with the RNA synthesis inhibitor camptothecin and agents that elevate cAMP, such as forskolin, causes a synergistic elevation of melatonin. Using two-dimensional gel analysis we have identified a 30 kDa cytosolic protein (p30) whose radiolabeling was consistently increased in parallel with increases in arylalkylamine N-acetyltransferase activity and melatonin production that were induced by forskolin and/or camptothecin. Pulse-chase experiments suggest that the elevation in radiolabeling of p30 is due to increased synthesis. Three candidate proteins found in the mammalian pineal, protein 14-3-3, malate dehydrogenase, and recoverin, do not comigrate with p30.

Cyclic AMP-dependent melatonin production in Y79 human retinoblastoma cells.

Melatonin is rhythmically synthesized in some vertebrate retinas and has been implicated in the regulation of key rhythmic events in the photoreceptor-pigment epithelial complex. In human retina, melatonin is present; however, no information exists on the cellular regulation of this hormone. We report here that the established human retinoblastoma cell line Y79 synthesizes and releases melatonin. Treatments that elevate cyclic AMP (cAMP) levels (forskolin, 8-Br-cAMP, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine) all stimulate melatonin release from static cultures of Y79 cells. Other 8-bromo nucleotide analogues (cyclic GMP, ATP, and AMP) are not effective. These results suggest that Y79 human retinoblastoma cells require a cAMP-dependent mechanism for melatonin biosynthesis similar to that described previously in other vertebrates. This is the first demonstration of melatonin release from a cultured human cell line. These results support the idea that human retinal cells share homologies with pineal cells, as suggested by the condition trilateral retinoblastoma.

Giant adrenal myelolipoma.

Adrenal myelolipomas are nonfunctioning benign tumors composed of hematopoietic elements and mature adipose tissue. These tumors usually remain small and asymptomatic; occasionally, however, they reach massive proportions and become symptomatic. To date, only two giant adrenal myelolipomas (>4,000 g) have been described in the literature. We describe the diagnostic evaluation and the operative management of the third largest adrenal myelolipoma reported.