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Biomedical subjects

D Sugden

Publications and source records attributed to D Sugden.

At least 91 records · Page 5Linked to original sources
PubMed1986

Purification of rat pineal hydroxyindole-O-methyltransferase using S-adenosyl-L-homocysteine agarose chromatography.

Rat pineal hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) was purified by affinity chromatography using an S-adenosyl-L-homocysteine agarose column. This single-step procedure, which is rapid, simple, and applicable to small quantities of tissue, gave a large enrichment of a protein (Mr approximately 38,000) identified by SDS-PAGE and silver staining. The amino acid composition of rat HIOMT was generally similar to that of the bovine enzyme, although some differences were apparent. This method will be valuable in isolating sufficient rat HIOMT to enable its primary amino acid sequence to be determined.

Acetylserotonin O-Methyltransferase↗
PubMed1985

Development of the rat pineal alpha 1-adrenoceptor.

Pineal alpha 1-adrenoceptors in rats from 19 days of gestation until 11 months of age were studied using [125I]iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ( [125I]HEAT). The number of specific [125I]HEAT binding sites increased markedly between 18 days of gestation (101.7 +/- 13.1 fmol/mg protein) and 10 days of age (336.2 +/- 34.3 fmol/mg protein). A significant decline occurred after 1 month of age. A saturation study showed similar changes in receptor density with age (Bmax; 20 days of gestation, 130.5 fmol/mg protein; 35 days old, 288.1 fmol/mg protein) but no difference in Kd (58.4 pM at both -1 and +35 days). The developmental appearance of the pineal alpha 1-adrenoceptor and the decline in its density with age are remarkably similar to changes reported for pineal beta-adrenoceptors.

Animals↗
PubMed1985

Serum melatonin and pineal indoleamine metabolism in a species with a small day/night N-acetyltransferase rhythm.

Ovine serum and pineal melatonin levels are low during the day, increase five to ten-fold at night, decrease during a light pulse at night, and rapidly increase to night levels following the light-dark transition. N-Acetyltransferase activity increases three-fold at night, falls significantly in response to the light pulse, but does not increase following the light pulse. No significant change in N-acetylserotonin occurs under these conditions. These results suggest that the biochemical mechanisms controlling pineal melatonin synthesis in the sheep pineal gland may be different from those in the rat.

Acetyltransferases↗
PubMed1985

Rapid nocturnal increase in ovine pineal N-acetyltransferase activity and melatonin synthesis: effects of cycloheximide.

Thirty minutes after the onset of darkness, ovine pineal arylalkylamine N-acetyltransferase, N-acetylserotonin, and melatonin increase 5- to 10-fold. No significant changes in hydroxyindole-O-methyltransferase, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxyindoleacetic acid, and 5-methoxytryptophol are detected at this time. Administration of cycloheximide inhibits the rise in N-acetyltransferase and N-acetylserotonin, but not melatonin. Unexpectedly, 5-methoxytryptophol increases after cycloheximide treatment. Taken together, these results, although consistent in part with a role for serotonin N-acetylation in the regulation of melatonin synthesis in sheep, indicate that an N-acetyltransferase-independent mechanism may also be involved.

Acetylserotonin O-Methyltransferase↗
PubMed1985

Regulation of rat pineal alpha 1-adrenoceptors.

Some aspects of the physiological regulation of the pineal alpha 1-adrenoceptor have been studied using the selective, high-affinity ligand [125I] iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([125I]HEAT). Pineal glands taken from rats housed in a diurnal lighting cycle showed no circadian rhythm in the number of specific [125I]HEAT binding sites, although a characteristic rhythm in pineal melatonin was seen. It was established that the pineal alpha 1-adrenoceptor is under neural control because interruption of neural stimulation of the pineal by bilateral superior cervical ganglionectomy (SCGX) or by exposing rats to constant light for 3 weeks doubled receptor density but did not change affinity for [125I]HEAT. Administration of various alpha 1-adrenoceptor agonists either acutely (i.p. injection) or chronically (s.c. infusion) did not alter the number of specific [125I]HEAT binding sites. Together these results indicate that the pineal alpha 1-adrenoceptor, like the pineal beta-adrenoceptor, is regulated by sympathetic nerve activity, probably through the physiological release of the neurotransmitter norepinephrine. However the absence of a circadian rhythm in alpha 1-adrenoceptor number and lack of down-regulation by adrenergic agonists imply different mechanisms of regulation.

Adrenergic alpha-Antagonists↗
PubMed1985

Ovine pineal indoles: effects of L-tryptophan or L-5-hydroxytryptophan administration.

L-5-Hydroxytryptophan (L-5-HTP) (20 or 200 mg/kg i.p.) but not L-tryptophan (500 mg/kg i.p.) loading substantially increases serum melatonin in sheep. In the present study we examined the effects of these compounds on pineal serotonin and six serotonin metabolites. L-Tryptophan failed to increase 5-hydroxytryptamine (5-HT; serotonin) or any of its metabolites despite a five-fold increase in pineal tryptophan. In contrast, L-5-HTP loading produced a marked increase in pineal 5-HT and its metabolites, including N-acetylserotonin (NAS) and melatonin, indicating that an increased synthesis of melatonin is responsible for the increased serum melatonin concentration after loading with this precursor. No change in pineal indoleamine N-acetyltransferase (NAT) activity was seen. These results are consistent with the suggestion that, during daytime in the sheep, 5-HT availability may limit the production of melatonin.

5-Hydroxytryptophan↗
PubMed1985

Ovine pineal alpha 1-adrenoceptors: characterization and evidence for a functional role in the regulation of serum melatonin.

Plasma melatonin in sheep increases to nocturnal levels rapidly (10-20 min) after dark onset. This increase is blocked by iv prazosin (1 mg), but not propranolol (6 mg). Prazosin also blocks the elevation in pineal melatonin content after dark onset, but does not significantly alter the rise in N-acetyltransferase activity or the elevation in pineal N-acetylserotonin content. Since the nocturnal elevation in N-acetyltransferase, a neurally regulated event, was unaltered, this suggests that prazosin does not significantly impair the transmission of neural signals from the eye to the gland, but does act on pineal alpha 1-adrenoceptors to block melatonin production. This is supported by binding studies in ovine pineal membranes using [125I] iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone, which revealed that binding is rapid, reversible, saturable, and stereo-specific. Saturation studies indicated the presence of a single class of binding sites, with an equilibrium binding constant (Kd) of 32 +/- 6 pM and a maximum binding of 139 +/- 19 fmol/mg protein. The relative potencies of several adrenergic agonists and antagonists in competition studies indicated that the receptor belongs to the alpha 1-subclass of adrenoceptors. Together, these data suggest that melatonin synthesis in the sheep pineal gland is controlled in part by an alpha 1-adrenoceptor mechanism at a step beyond N-acetylation.

Acetyltransferases↗
PubMed1985

Atypical synergistic alpha 1- and beta-adrenergic regulation of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in rat pinealocytes.

The adrenergic control of cAMP and 3',5'-cyclic GMP (cGMP) in dispersed adult rat pinealocytes was investigated. Norepinephrine treatment increased cAMP and cGMP content 60- and 400-fold, respectively; both alpha- and beta-adrenoceptors had to be activated for these responses to occur. Beta-Adrenergic stimulation alone produced only about 6- and 2-fold increase in cAMP and cGMP content, respectively. Alpha-Adrenergic stimulation, which alone had no effect on either cyclic nucleotide concentration, markedly amplified the beta-adrenergic stimulation of both cAMP and cGMP. The relative potency of alpha-adrenergic agonists and antagonists indicates the alpha 1-subclass of adrenoceptors is involved. A role of alpha 1-adrenoceptors in the control of pineal cAMP is consistent with published evidence of the presence of alpha 1-adrenoceptors on pinealocytes and their role in the regulation of N-acetyltransferase activity and melatonin production.

Animals↗
PubMed1984

Alpha-adrenergic potentiation of beta-adrenergic stimulation of rat pineal N-acetyltransferase. Studies using cirazoline and fluorine analogs of norepinephrine.

Recent evidence indicates that melatonin production is controlled by norepinephrine acting via alpha 1-and beta 1-adrenoceptors on pinealocytes; activation of alpha 1-adrenoceptors appears to potentiate the effects of beta 1-adrenoceptor activation. However, alpha-adrenergic potentiation of beta 1-adrenergic activation has been demonstrated with only one alpha-adrenergic agonist. For this reason, this issue was reinvestigated using two other alpha-adrenergic agonists, 6-fluoronorepinephrine and cirazoline. Both compounds, which were found to have a high affinity for pineal alpha 1-adrenoceptors, potentiated the stimulatory effects of isoproterenol on pineal N-acetyltransferase. 6-Fluoronorepinephrine also potentiated the stimulation of N-acetyltransferase activity produced by another beta-adrenergic agonist, 2-fluoronorepinephrine. These findings support the hypothesis that pineal N-acetyltransferase activity is regulated by norepinephrine acting through both alpha 1- and beta 1-adrenoceptors.

Acetyltransferases↗
PubMed1984

Rat pineal alpha 1-adrenoceptors: identification and characterization using [125I]iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone.

[125I]Iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone ([125I]HEAT), a selective, high affinity, high specific activity alpha 1-adrenoceptor ligand, is used to characterize alpha-adrenoceptors in the rat pineal gland. Binding of [125I]HEAT to membranes is rapid [association rate constant (Kon) = 3.1 nM-1 min-1] and readily reversible either by 100-fold dilution or by addition of excess unlabeled HEAT [apparent dissociation rate constant (Koff) = 0.153 min-1). Saturation experiments indicate a single class of noncooperative binding sites with an equilibrium binding constant (KD) of 41 +/- 9 pM and a Bmax of 399 +/- 63 fmol/mg protein. The relative potency of a number of adrenoceptor agonists and antagonists in competing with [125I] HEAT indicates the receptor is an alpha 1-subtype. In addition, inhibition of binding is stereospecific; (-)epinephrine and norepinephrine are more than 100-fold more potent than their (+)isomers. The identification of alpha 1-adrenoceptors in the pineal gland is consistent with evidence indicating a role for these receptors in the regulation of melatonin synthesis and phosphatidylinositol turnover.

Adrenergic beta-Agonists↗
PubMed1984

Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity.

Studies of [3H]diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot [Kd = 23 nM, maximum binding sites (Bmax) = 1.56 pmol/mg of protein for cells in monolayer culture; Kd = 7 nM, Bmax = 1.3 pmol/mg of protein for cells in suspension culture]. Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the "peripheral" type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the [3H]diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

Acetyltransferases↗
PubMed1983

5-hydroxytryptophan elevates serum melatonin.

Daytime administration of 5-hydroxytryptophan to sheep elevated serum melatonin more than sevenfold within 2 hours. This suggests that administration of 5-hydroxytryptophan could be used as the basis of a clinical test of pineal function and that melatonin might mediate some clinical effects of 5-hydroxytryptophan.

Animals↗
PubMed1983

Postsynaptic alpha-adrenergic receptors potentiate the beta-adrenergic stimulation of pineal serotonin N-acetyltransferase.

The role played by postsynaptic alpha-adrenergic receptors in the stimulation of pineal N-acetyltransferase (EC 2.3.1.5) and [3H]melatonin production was investigated in the rat. In vivo studies indicated that phenylephrine, an alpha-adrenergic agonist, potentiated and prolonged the effects of isoproterenol, a beta-adrenergic agonist. Similar observations were made in organ culture with glands devoid of functional nerve endings. In addition, a combination of 1 microM prazosin, an alpha 1-adrenergic blocking agent, and 1 microM propranolol, a beta-adrenergic blocking agent, was many times more potent then either agent alone in blocking the stimulatory effects of norepinephrine on N-acetyltransferase activity and [3H]melatonin production. These findings establish that norepinephrine acting through alpha- and beta-adrenergic receptors stimulates rat pineal N-acetyltransferase activity and, as a result, the production of melatonin. Apparently, beta-adrenergic activation is an absolute requirement, and an alpha-adrenergic receptor mechanism potentiates beta-adrenergic activation. These findings are significant because they demonstrate alpha-adrenergic potentiation of beta-adrenergic effects. In addition, they indicate that the widely held belief that melatonin production is regulated exclusively by a postsynaptic beta-adrenergic mechanism must be revised.

Acetyltransferases↗
PubMed1983

In vivo release from cerebral cortex of [14C]glutamate synthesized from [U-14C]glutamine.

Awake, unrestrained, and behaviourally normal animals with superfusion cannulae implanted over the sensorimotor cortex were used in a study of the capacity of infused [U-14C]glutamine for labelling glutamate and other amino acids released by depolarising stimuli. A spontaneous background release of [14C]glutamate was detected. This was increased by tityustoxin (1 microM). The specific radioactivity of glutamate increased eightfold during the evoked-release period. [14C]Aspartate was also detected and showed increased release, but not increased specific labelling, in response to depolarisation. Evoked gamma-aminobutyric acid (GABA) release occurred but only small amounts of [14C]GABA were detected. Glutamine showed increased rates of uptake to the sensorimotor cortex during stimulation periods, suggesting an accelerated breakdown via glutaminase.

Animals↗
PubMed1983

Regulation of rat pineal hydroxyindole-O-methyltransferase in neonatal and adult rats.

The relative importance of neural, and some nonneural, mechanisms in the control of pineal hydroxyindole-O-methyltransferase (HIOMT) activity during development and in the adult rat was studied. In neonatal rats, guanethidine-treatment, bilateral superior cervical ganglionectomy (SCGX), or exposure to constant light did not prevent the initial appearance of HIOMT activity, indicating that neural stimulation of the gland is not essential for the development of HIOMT activity. In adult rats, decentralization or removal of the SCG led to a slow fall in HIOMT activity, to about 30% of control activity, indicating that the enzyme is largely under neural control. Additionally, adrenalectomy or hypophysectomy had no effect on HIOMT activity, refuting the suggestion that adrenal and/or gonadal steroids are of major importance in the regulation of this enzyme. The fall in activity of the enzyme after SCGX or exposure to constant light probably does not represent a shift in the Km of the enzyme nor the selective disappearance of a distinct molecular species. Similar changes in HIOMT activity and cyclic GMP responsiveness occur in response to alterations in the length of the daily dark period, adding further evidence to our earlier speculation that there may be a functional relationship between these two.

Acetylserotonin O-Methyltransferase↗
PubMed1983

Beta-adrenergic receptor control of rat pineal hydroxyindole-O-methyltransferase.

The nature of the postsynaptic adrenergic receptor on rat pinealocytes which controls hydroxyindole-O-methyltransferase (HIOMT) activity was studied. This enzyme is involved in the synthesis of the pineal hormone melatonin. Adrenergic drugs were administered continuously for a 7-day period to rats in which neural stimulation of the pineal gland was blocked by either superior cervical ganglionectomy or exposure to constant light. l-Isoproterenol, a beta-adrenergic agonist, prevented the fall in enzyme activity that occurs when neural stimulation is interrupted; d-isoproterenol was ineffective. The potency order of different adrenergic agonists was d,l-isoproterenol greater than l-norepinephrine greater than l-epinephrine. Terbutaline, a selective beta 2-adrenergic agonist, was ineffective. The selective alpha 1-adrenergic agonists phenylephrine and methoxamine and the alpha 2-agonist clonidine were also ineffective. High doses of the beta-adrenergic blocker propranolol antagonized the effect of isoproterenol and caused a fall in HIOMT activity in normal rats housed under normal diurnal lighting. This in vivo evidence is consistent with the hypothesis that the neural control of pineal HIOMT is mediated via a beta-adrenergic receptor.

Acetylserotonin O-Methyltransferase↗
PubMed1983

Reciprocal day/night relationship between serotonin oxidation and N-acetylation products in the rat pineal gland.

Pineal tryptophan, serotonin [5-hydroxytryptamine (5-HT)], N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxytryptophol, and 5-methoxyindoleacetic acid were measured by high pressure liquid chromatography with electrochemical detection. A complete analysis required less than the equivalent of two rat pineal glands. Samples were obtained at eight time points. A reciprocal physiological relationship was found between oxidation and N-acetylation products of 5-HT. 5-HT and the oxidation products 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, 5-methoxytryptophol, and 5-methoxyindoleacetic acid decreased at night, when the N-acetylation products N-acetylserotonin and melatonin increased. These observations are consistent with the hypothesis that circadian changes in the N-acetylation of 5-HT by indoleamine N-acetyltransferase is the major factor controlling circadian changes in the amounts of 5-HT, and 5-HT oxidation and N-acetylation products in the rat pineal gland.

Acetylation↗