Biomedical subjects

D S Gregory

Publications and source records attributed to D S Gregory.

At least 19 recordsLinked to original sources

PubMed2001

Circadian change of adenylate cyclase activity in rabbit ciliary processes.

PURPOSE: To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle. METHODS: After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin. RESULTS: The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a beta-adrenergic antagonist, or brimonidine, an alpha2-adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an alpha2-adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX. CONCLUSIONS: The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of beta-adrenergic receptors.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Hydroxyamphetamine increases intraocular pressure in rabbits.

OBJECTIVE: To determine the effect of norepinephrine (NE) released from endogenous ocular stores on intraocular pressure (IOP) and aqueous flow in rabbits. METHODS: The IOP was measured with a pneumatonometer, the aqueous flow with a scanning fluorophotometer, and the aqueous NE by methylation with catechol-O-methyltransferase in the presence of S-adenosyl-L-[methyl-(3)H]methionine. RESULTS: Hydroxyamphetamine increased IOP in a dose-dependent fashion. Surgical removal of the superior cervical sympathetic ganglion eliminated the increase in IOP and pupil diameter; preganglionic section of the cervical sympathetic trunk did not. Hydroxyamphetamine increased the concentration of NE in the aqueous. Increased IOP was not accompanied by increased aqueous flow and was eliminated by blockade of alpha(1)-adrenergic receptors but not beta- or alpha(2)-adrenergic receptors. CONCLUSIONS: Increased IOP after hydroxyamphetamine application is consistent with earlier suggestions that the nocturnal circadian increase in IOP in rabbits is mediated in part by NE released from ocular sympathetic nerves. However, failure of hydroxyamphetamine to increase aqueous flow and of beta-adrenergic blockade to blunt the increase in IOP does not support our suggestion that the nocturnal increase in IOP results in part from NE stimulation of ciliary process beta-adrenergic receptors and increased aqueous flow. CLINICAL RELEVANCE: In addition to increasing pupil diameter, hydroxyamphetamine increases IOP.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2000

Response of rabbit ciliary process cyclic nucleotides to specific phosphodiesterase inhibitors.

PURPOSE: To determine which cyclic nucleotide phosphodiesterase family activities can be identified in rabbit ciliary processes. METHODS: Freshly excised rabbit ciliary processes were incubated in vitro with family selective phosphodiesterase inhibitors in the absence or presence of activators of soluble or membrane bound guanylate cyclase. The resulting increases of cyclic AMP and cyclic GMP were measured by RIA. RESULTS: Rabbit ciliary process cyclic AMP levels were increased by the phosphodiesterase 1 and 4 selective inhibitors, 8-methoxymethyl-IBMX and rolipram, respectively. Cyclic GMP levels were increased by 8-methoxymethyl-IBMX; whereas the phosphodiesterase 5 and 6 selective inhibitor, zaprinast, increased cyclic GMP little. Nitric oxide donors increased cyclic AMP in addition to cyclic GMP, and inhibition of soluble guanylate cyclase eliminated the increase of cyclic AMP. The effects of sodium nitroprusside and the phosphodiesterase 3 selective inhibitor, cilostamide, on cyclic AMP were not additive suggesting that the sodium nitroprusside mediated increase of cyclic GMP, like cilostamide, inhibited phosphodiesterase 3. CONCLUSIONS: Cyclic AMP in rabbit ciliary processes is hydrolyzed primarily by phosphodiesterases 1 and 4, and, when cyclic GMP levels are low, by phosphodiesterase 3; cyclic GMP is hydrolyzed primarily by phosphodiesterase 1.

Animals↗

PubMed1999

Crystal structure of human DT-diaphorase: a model for interaction with the cytotoxic prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954).

The crystal structure of human DT-diaphorase (NAD(P)H oxidoreductase (quinone); EC 1.6.99.2) has been determined to 2.3 A resolution. There are only minor differences in shape and volume between the active sites of the rat and human enzymes and in the hydrophobic environment in the vicinity of the substrate. The isoalloxazine ring of the bound FAD is more buried in the human structure. Molecular modeling was used to examine optimal positions for the antitumor prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) in both the human and rat enzyme active sites. This suggests that the position of CB1954 in the active site of the human enzyme is very similar to that in the rat, although there are detailed differences in the predicted patterns of hydrogen bonding between side chains and the drug. Some of the differences are a consequence of the shift in position for the FAD molecule and may contribute to the observed differences in rate of the two-electron reduction of CB1954.

Amino Acid Sequence↗

PubMed1997

Response and level of beta-adrenergic, vasoactive intestinal peptide, and PACAP receptors during the circadian cycle.

PURPOSE: To determine whether a nocturnal increase of ciliary process beta-adrenergic receptor responsiveness can explain the observation that timolol decreased the aqueous flow rate and intraocular pressure (IOP) during the night but not during the day in rabbits. METHODS: Rabbits were housed in alternating 12-hour periods of light and dark. In vitro stimulation of tissue cyclic adenosine monophosphate (cAMP) levels by isoproterenol (ISO), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP), or a soluble derivative of forskolin (sFSK) was measured in ciliary processes harvested at mid-light phase and early and late dark phase. Inhibition of ISO and VIP stimulation of ciliary process cAMP by an alpha 2-adrenergic agonist and maximal binding of [125I]I-pindolol, [125I]I-VIP, and [125I]I-PACAP in ciliary process membranes were measured at the same three times. RESULTS: Although there may have been a nocturnal increase in the sensitivity of ciliary process cAMP levels to stimulation by ISO, this was not observed consistently, VIP, but not PACAP, stimulation increased at night, but there was no change in the response to sFSK. Inhibition by apraclonidine of elevated ciliary process cAMP levels was constant at all three times. Ligand-binding studies showed little change in ciliary process beta-adrenergic, VIP-, or PACAP-receptor levels at the three times. CONCLUSIONS: There is no convincing evidence for a nocturnal increase in beta-adrenergic receptor sensitivity in rabbit ciliary processes that could explain the difference between day and night effects of timolol on aqueous flow and IOP.

Adrenergic alpha-Agonists↗

PubMed1996

cAMP levels increased by activation of metabotropic glutamate receptors correlate with visual plasticity.

We have investigated the cAMP level increased by stimulation of metabotropic glutamate receptors (mGluRs) in cat visual cortex during development. The cAMP level increases activated by the general mGluR agonist (1S,3R)-1-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were closely correlated with the critical period for ocular dominance plasticity in both light- and dark-reared animals. Activation of either group I or group II mGluRs increased the cAMP level. Group II mGluR activation also reduced the forskolin-stimulated cAMP increase. The correlation was emulated by a mixture of groups I, II, and III mGluR agonists but not by agonists applied singly; therefore, the correlation is attributable to activation of multiple groups of mGluRs. The cAMP level increased by the mixture was greater than the sum of the increases produced by the agonists applied singly (super-additive effect), suggesting an interaction between the G-proteins and/or second messengers controlled by these mGluRs. The basal cAMP level also correlated closely with the critical period until shortly after the peak of the critical period. Therefore, the major factor that contributes to the correlation between the ACPD-stimulated cAMP increase and the peak of the critical period is the basal level of cAMP: the activation of multiple mGluRs amplifies the basal cAMP. We suggest that both basal activity of cAMP production and activation of mGluRs may be important in plasticity in the visual cortex.

Animal Husbandry↗

PubMed1996

Glutamate receptors and development of the visual cortex: effect of metabotropic agonists on cAMP.

Glutamate receptors are more active in several respects in young animals than in adults. Here we examine the effect of metabotropic glutamate agonists on rat cortical cAMP during and after the critical period for visual cortex plasticity. Quisqualate produced a substantial increase in cAMP, which was larger during the critical period than in the adult. The increase was not affected by CNQX or APV, showing that it was not due to the action of quisqualate on ionotropic glutamate receptors. Both Type I mGluRs (mGluRs 1 and/or 5) and Type II mGluRs (mGluRs 2 and/or 3) probably contributed to the cAMP increase because (i) ACPD and L-CCG-I, which are more active on Type II mGluRs, were more effective than DHPG, which is more active on Type I mGluRs; and (ii) there was a significant difference in the effect of ACPD on the increase in cAMP, comparing mGluR1 knockout mice with control mice. Agonists which produce large stimulation of cAMP production (ACPD, L-CCG-I), as well as L-AP4, also produced small attenuations of forskolin-stimulated cAMP, but only at high concentrations. Thus, we conclude that it is the stimulation and/or potentiation of cAMP production that is significant, rather than the attenuation of forskolin-stimulated cAMP. Since this stimulation and/or potentiation is higher during the critical period than in the adult, and the cAMP second messenger system has been implicated in hippocampal plasticity, it may also play a role in visual cortex plasticity.

Animals↗

PubMed1996

Neuropeptide Y levels in the aqueous humor of rabbits.

The concentration of neuropeptide Y in the aqueous humor (NPYaq) did not change during the circadian cycle in rabbits. However, NPYaq also did not decrease after superior cervical ganglionectomy or preganglionic section of the cervical sympathetic trunk (decentralization). Nor did NPYaq change after either stimulation, with para-aminoclonidine, or blockade, with rauwolscine, of prejunctional ocular alpha2-adrenergic receptors. Therefore, NPYaq does not reflect NPY release from ocular sympathetic nerves in rabbits.

Adrenergic alpha-Agonists↗

PubMed1995

Light exposure decreases IOP in rabbits during the night.

Elevated IOP observed in rabbits during the dark phase of the circadian cycle decreased rapidly and reversibly when rabbits were exposed to light during the dark phase. The decrease of IOP does not result from decreased aqueous flow and only part of the decrease requires intact ocular sympathetic innervation.

Animals↗

PubMed1993

The prediction and characterization of metal binding sites in proteins.

The rational engineering of novel functions into proteins can only be attempted when the underlying structural scaffold on which the new function is displayed and the structure of the target protein are both well understood. To introduce functions mediated by metals it is therefore necessary to identify the principal liganding residues for the chosen metal, the required architecture of the metal-ligand complex and sites within the target protein that could accommodate such sites. Here we present a method that applies structural information from the protein data bank to the ab initio design and characterization of novel metal binding sites. The prediction method has been tested on 28 metalloprotein structures from the Brookhaven Protein Data Bank. It successfully identified > 90% of the metal binding sites. In addition, we have used the method to design and characterize zinc binding sites in two antibody structures. Metal binding studies on one of these putative metalloantibodies showed metal binding, confirming the predictive power of the method.

Algorithms↗

PubMed1993

Melatonin does not increase IOP significantly in rabbits.

New Zealand white rabbits have a circadian rhythm of intraocular pressure; pressure is higher during the dark than during the light. We explored the possibility that the dark phase increase of serum and/or ocular melatonin plays a role in regulating the rhythm of intraocular pressure. Exogenous melatonin was delivered by four routes: topical application (0.1 and 10 micrograms), intravenous injection (1 microgram), intravitreal injection (9 micrograms) and intra-arterial infusion (50 ng and 1 microgram). Melatonin delivered by these routes did not increase intraocular pressure. We also measured the concentration of melatonin in the aqueous after unilateral intra-arterial infusion to confirm that melatonin delivered by this route reached the eye. The concentration was 419 +/- 99 and 109 +/- 17 pg/ml (n = 8, p < 0.025) in the ipsilateral and contralateral eyes, respectively. The concentration of melatonin in the aqueous after intra-arterial infusion of saline was less than the sensitivity of the assay (< 16 pg/ml, n = 6). The results of these experiments indicate that melatonin does not increase IOP significantly under the experimental conditions employed in this study.

Administration, Topical↗

PubMed1992

Rabbits have a circadian rhythm of aqueous humor cyclic AMP.

Rabbits have circadian rhythms of intraocular pressure (IOP) and aqueous flow. The dark phase increases of IOP and flow are associated with increased aqueous cyclic AMP. If the daily rhythm of aqueous cyclic AMP reflects changes of tissue levels of cyclic AMP which mediate one of the mechanisms which control the rhythms of IOP and aqueous flow, then it must be a circadian rhythm as are the rhythms of IOP and flow. A recent report showed no change in aqueous cyclic AMP from 2 hrs before to 2 hrs after the beginning of subjective dark in animals housed in constant dark. Because the increase of intraocular pressure at the same times did persist in constant dark, the authors concluded that a significant portion of the circadian rhythm of IOP is unrelated to cyclic AMP mediated ocular mechanisms. However, if an inhibitor of cyclic nucleotide phosphodiesterase is added to aqueous samples after harvesting by paracentesis, it is possible to show that most of the increase of aqueous cyclic AMP from mid-light phase to mid-dark phase persists in constant dark and is therefore likely to reflect a circadian rhythm of tissue cyclic AMP.

1-Methyl-3-isobutylxanthine↗

PubMed1992

Do alpha-adrenergic receptors participate in control of the circadian rhythm of IOP?

The alpha 2-adrenergic antagonists, yohimbine and rauwolscine, and the alpha 1-adrenergic antagonist, bunazosin, were used to explore the role of alpha-adrenergic receptors in the regulation of the circadian rhythms of intraocular pressure and aqueous flow in New Zealand white rabbits. Blockade of alpha 2-adrenergic receptors with yohimbine or rauwolscine produced small decreases in intraocular pressure during both light and dark phases. Rauwolscine had no effect on aqueous flow during the light or dark, but it increased the concentration of norepinephrine in the aqueous during both light and dark. These observations are difficult to reconcile with earlier suggestions that increased sympathetic input to the eye increases intraocular pressure and aqueous flow during the dark. The role of alpha 2-adrenergic receptors in the control of the circadian rhythm of intraocular pressure is unclear. Blockade of alpha 1-adrenergic receptors with bunazosin produced a dose-dependent reduction of IOP during the dark phase of the circadian cycle, a smaller reduction during the light phase, and no reduction during either light or dark in rabbits after superior cervical ganglionectomy or preganglionic section of the cervical sympathetic trunk (decentralization). Bunazosin decreased pupil diameter during the dark phase but had no effect on aqueous flow. Because it is unlikely that alpha 1-adrenergic blockade increased outflow facility or uveoscleral outflow, the mechanism for the role of alpha 1-adrenergic receptors in the control of the circadian rhythm of intraocular pressure in rabbits remains to be identified.

Adrenergic alpha-Antagonists↗

PubMed1991

Ocular adrenergic nerves contribute to control of the circadian rhythm of aqueous flow in rabbits.

Aqueous flow was measured fluorophotometrically in New Zealand white rabbits after unilateral decentralization of the cervical ganglion or cervical ganglionectomy to determine the role of ocular adrenergic input in regulating the circadian rhythm of aqueous flow. Both surgical procedures decreased the rate of aqueous flow during the dark phase when flow is high. During the light phase when flow is low, cervical ganglionectomy increased aqueous flow; decentralization may have increased flow also, but the increases were not statistically significant. Aqueous flow was also measured in normal rabbits after topical application of timolol during the light or dark to determine whether beta-adrenergic receptors play a role in controlling the circadian rhythm of flow. Timolol produced a small reduction of the rate of aqueous flow when applied topically during the dark but not during the light. These results suggest that part of the increase of aqueous flow during the dark phase is produced by adrenergic input to the ciliary processes and that beta-adrenergic receptors mediate part of this increase.

Administration, Topical↗

PubMed1991

Changes in aqueous norepinephrine and cyclic adenosine monophosphate during the circadian cycle in rabbits.

The concentrations of catecholamines and cyclic adenosine monophosphate (AMP) in the aqueous of rabbits entrained to 12-hr light:12-hr dark were measured at six times during the circadian cycle. Aqueous norepinephrine was higher in the dark than in the light; the minimum concentration at 1.5 hr after lights on, and maximum concentration at 6 hr after lights off, were 1.21 +/- 0.17 and 4.60 +/- 0.79 ng/ml, respectively. The concentrations of epinephrine and dopamine were less than the sensitivity of the assay (0.1 and 0.4 ng/ml, respectively) at all times. Aqueous cyclic AMP was also higher in the dark than in the light; the minimum concentration at 6 hr after lights on, and maximum concentration at 10.5 hr after lights off, were 11.5 +/- 1.2 and 29.4 +/- 2.7 pmol/ml, respectively. Superior cervical ganglionectomy reduced aqueous norepinephrine during light and dark to less than the sensitivity of the assay (0.1 ng/ml); preganglionic section of the cervical sympathetic trunk (decentralization) did not reduce aqueous norepinephrine as much as ganglionectomy but markedly reduced its concentration during the light and dark. Superior cervical ganglionectomy or pretreatment with 0.1% timolol virtually eliminated, and decentralization markedly reduced, the dark-phase increase of aqueous cyclic AMP. Most of the daily changes in norepinephrine and cyclic AMP persisted in animals housed in constant dark; this is consistent with the fraction of both changes which persisted in constant dark being circadian. These results support the idea that increased adrenergic activity during the dark increases the rate of aqueous flow and intraocular pressure in rabbits.

Animals↗

PubMed1990

Antibody-combining sites: prediction and design.

For maximum value, a predicted model of an antibody-combining site should have an accuracy approaching that of an X-ray structure (1.6-2.7 A). In addition, the method by which the combining site is modelled should make no demands on the user of a sort that require arbitrary or subjective decisions to be made during the process. We have made substantial progress towards this objective and some recent results are reviewed. In addition, we describe how the modelling protocols developed can aid in the design of novel features within the antibody-combining site. The particular design example reported here suggests an approach for the introduction of metal-binding sites to create metallo-antibodies. This type of modification may be useful in the design of immunobiosensors, the induction of catalytic activity or simply as an alternative to metal chelates in the preparation of antibodies for imaging.

Algorithms↗

PubMed1990

Timolol reduces IOP in normal NZW rabbits during the dark only.

The role of beta-adrenergic receptors in mediating the circadian rhythm of intraocular pressure was examined by determining the effect on intraocular pressure of timolol applied topically at different times during the circadian cycle to New Zealand White rabbits entrained to alternating 12 hr periods of light and dark. Timolol lowered intraocular pressure when applied during the dark but not during the light phase of the circadian cycle. Timolol did not lower intraocular pressure when applied during the dark to animals after cervical ganglionectomy. These results are consistent with the idea that beta-adrenergic receptors participate in mediating the circadian rhythm of intraocular pressure, and suggest that endogenous agonists released by adrenergic nerves in the eye during the dark stimulate ocular beta-adrenergic receptors to increase intraocular pressure in rabbits.

Administration, Topical↗

PubMed1989

Alpha 2-adrenergic and VIP receptors in rabbit ciliary processes interact.

The interaction between alpha 2-adrenergic and VIP receptors has been studied by examining inhibition of VIP-stimulated cyclic AMP production by adrenergic agonists in intact, excised rabbit ciliary processes. Epinephrine, norepinephrine, isoproterenol, dopamine, and the specific alpha 2-adrenergic agonists clonidine and p-aminoclonidine exhibit dose-dependent inhibition of VIP-stimulated cyclic AMP production. I50s, clonidine (0.05 microM) = p-aminoclonidine (0.05 microM) congruent to epinephrine (0.1 microM) less than norepinephrine (2.0 microM) less than isoproterenol (15 microM) = dopamine (15 microM), are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors. Inhibition of VIP-stimulated cyclic AMP production by clonidine, epinephrine, isoproterenol, and dopamine is blocked by yohimbine but not by prazosin. These data establish the alpha 2-adrenergic specificity of the inhibitory effects observed. We have previously shown that beta 2-adrenergic receptor-mediated stimulation of cyclic AMP production in rabbit ciliary processes is also inhibited by postjunctional alpha 2-adrenergic receptors. These studies support the idea that the catecholamines may regulate aqueous humor formation by inhibiting stimulation of cyclic AMP production via postjunctional alpha 2-adrenergic receptors in ciliary processes.

Adrenergic alpha-Agonists↗