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D C Klein

Publications and source records attributed to D C Klein.

At least 127 records · Page 7Linked to original sources
PubMed1987

Multiple forms of arylalkylamine N-acetyltransferases in the rat pineal gland: purification of one molecular form.

Rat pineal serotonin N-acetyltransferase (EC 2.3.1.87) activity is isolated in two molecular forms (Mr approximately equal to 10,000 and 95,000) by high performance size exclusion liquid chromatography in the presence of ammonium acetate (0.1 M, pH 6.5). In the presence of sodium citrate (0.1 M, pH 6.5), however, it is eluted as a single peak of intermediate size (Mr approximately equal to 30,000). A highly enriched preparation of one of the molecular forms has been obtained by a two-step purification procedure involving disulfide-exchange and anion-exchange chromatography. The N-acetyltransferase in 250 pineal glands obtained from isoproterenol-treated rats can be purified about 80-fold in 1 day; recovery is about 3%. Polyacrylamide gel electrophoresis of the final preparation indicates that a single major band (Mr approximately equal to 11,000) is present; this appears to be serotonin N-acetyltransferase.

Acetyltransferases↗
PubMed1987

Characterization of the alpha +-like Na+,K+-ATPase which mediates ouabain inhibition of adrenergic induction of N-acetyltransferase (EC 2.3.1.87) activity: studies with isolated pinealocytes.

Ouabain inhibits (IC50 congruent to 200 nM) the congruent to 100-fold adrenergic cyclic AMP stimulation of rat pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87, serotonin N-acetyltransferase, NAT) activity in intact pineal glands. In the present study, ouabain binding sites in pineal membranes were characterized in detail and compared to sites in isolated pinealocytes, which mediate the inhibition of Na+,K+-ATPase, as indicated by 86Rb uptake and norepinephrine (NE) stimulation of NAT activity. High affinity ouabain-binding sites were identified in crude preparations of pineal membranes (Kd congruent to 14 nM; Bmax congruent to 4 pmol/mg of protein) and similar sites were also found in ovine and bovine pineal tissue. The ouabain Kd value for the rat pineal binding sites was similar to the estimated ouabain IC50 values for 86Rb uptake and the NE stimulation of NAT activity in intact rat pinealocytes. In addition, the relative orders of potency of four cardiac glycosides in displacing [3H]ouabain from high affinity binding sites and inhibiting both 86Rb uptake and NE stimulation of NAT activity were the same (acetyldigitoxin greater than ouabain greater than digitoxin greater than strophanthidin). The similarities in the characteristics of the high affinity [3H]ouabain-binding sites and the sites involved in the inhibition of 86Rb uptake and stimulation of NAT activity indicate that an alpha +-like Na+,K+-ATPase mediates the inhibitory effects of ouabain on the adrenergic induction of pineal NAT activity.

Acetyltransferases↗
PubMed1986

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes.

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.

Animals↗
PubMed1986

Subfornical organ: effects of salt loading and water deprivation on in vitro radioamino acid incorporation into individual proteins.

The subfornical organ of the brain has a role in the regulation of fluid balance in higher animals. In this study the effects of salt loading and water deprivation on specific proteins in this organ were investigated. For 4 days, 3 groups of rats were given an appropriate fluid diet (control, 2% NaCl and water deprived), with all groups having free access to food. Animals were killed by decapitation, and the subfornical organ was quickly dissected out and incubated for 6 h in a medium containing [35S]methionine and [35S]cysteine. Proteins from these organs were then separated by two-dimensional electrophoresis, and the resulting autofluorographs were analyzed by scanning densitometry. The results show that the incorporation of labeled amino acids into 8 proteins was changed due to the experimental manipulations.

Animals↗
PubMed1986

Pinealocyte projections into the mammalian brain revealed with S-antigen antiserum.

Neural processes from mammalian pinealocytes have been discovered in several brain areas. These processes were visualized immunocytochemically in the Djungarian hamster, Phodopus sungorus, with an antiserum against bovine retinal S-antigen and traced as far as the region of the posterior commissure and habenular nuclei. This result indicates that pineal-to-brain connections exist in the mammal, and that the mammalian pineal gland, currently thought of only as a neuroendocrine organ, may communicate directly with select brain regions by way of these projections. The existence of mammalian pinealocyte projections is consistent with the view that these cells are not of glial origin but are derivatives of photoreceptor cells of the pineal complex of lower vertebrates that transmit signals to the brain by neural projections.

Animals↗
PubMed1986

S-antigen-like immunoreactivity in a human pineocytoma.

A pineocytoma was investigated by means of immunocytochemistry with the use of a polyclonal antibody against bovine retinal S-antigen. Several cells of this tumor displayed strong S-antigen-like immunoreaction in analogy to certain pinealocytes in normal human pineal organs. This study indicates that S-antigen immunocytochemistry may be applied to characterize tumors of the pineal region.

Antigens↗
PubMed1986

Characterization of benzodiazepine receptors in the bovine pineal gland: evidence for the presence of an atypical binding site.

Bovine and rat pineal benzodiazepine receptors were characterized using ligands with high affinities for either 'central-type' (CBR) or 'peripheral-type' (PBR) benzodiazepine receptors. The characteristics (Bmax = 83 +/- 10 fmol/mg protein, Kd = 3.88 +/- 0.46 nM) of benzodiazepine receptors in bovine pineal membranes measured with [3H]flunitrazepam (using flunitrazepam to define non-specific binding) were consistent with previously reported values. However, if non-specific binding was defined using Ro 15-1788 (a selective CBR ligand), the Bmax and Kd of [3H]flunitrazepam decreased 51 and 58%, respectively. In addition, when using PK 11195 to determine non-specific binding, the Bmax of [3H]flunitrazepam binding to bovine pineal decreased further (approximately 80%, Kd decreased approximately 39%). Together, these observations strongly suggested the presence of PBR in the bovine pineal. Bovine pineal PBR characterized with [3H]PK 11195 revealed a high density (relative to CBR) of high affinity binding sites (Kd = 1.08 +/- 0.30, Bmax = 776 +/- 33.0 fmol/mg protein). In contrast, when [3H]Ro 5-4864 (1-20 nM) was used to define PBR, no binding was detectable. These observations are in sharp contrast to the rat pineal gland, in which both [3H]Ro 5-4864 and [3H]PK 11195 bind to a large number of PBR with high affinity (Kd approximately equal to 1.9 nM, Bmax approximately equal to 26 pmol/mg protein). Bovine pineal PBR were further characterized with compounds structurally related to either Ro 5-4864 or PK 11195.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

alpha-Transducin immunoreactivity in retinae and sensory pineal organs of adult vertebrates.

Antiserum against the alpha subunit of bovine rod-outer-segment transducin was used in an immunocytochemical study that identified the protein in retina (human, baboon, owl monkey, cow, rat, quail, newt, frog, salmon, eel, and lamprey), pineal organ (quail, newt, frog, salmon, eel, and lamprey), and parapineal organ (salmon and lamprey). No reaction was observed in the cow or rat pineal organ or the eel parapineal organ. The immunoreaction was very strong in outer segments but weak in perikarya. Immunoblots of crude tissue extracts of bovine rod-outer-segment membranes and frog and fish retina revealed a 39-kDa immunopositive band. The fish retina also contained two additional bands of mass 43 kDa and 25 kDa. Only the 43-kDa band was present in the fish pineal organ, which is photosensitive. This raises the possibility that the 43-kDa alpha transducin-immunopositive molecule present in the fish pineal organ and retina may be involved in phototransduction.

Animals↗
PubMed1986

Development and regulation of rhodopsin kinase in rat pineal and retina.

Rhodopsin kinase, once thought to be a retinal enzyme, was recently found at high levels in the pineal gland. In the present study the developmental pattern and the regulation by environmental lighting of this enzyme in both tissues was studied in the rat. Enzyme activity was present in the neonatal pineal gland several days earlier than in the retina, and increased gradually up to 20 days of age and remained at that level thereafter; the retinal enzyme appeared to increase until day 60. Pineal and retinal rhodopsin kinase activities showed a 25% increase in in the middle of the dark and the beginning of the light period, respectively. Exposure to constant light caused a 50% decrease in rhodopsin kinase levels in both tissues. However, only pineal rhodopsin kinase activity declined followed bilateral superior cervical ganglionectomy. This indicates pineal rhodopsin kinase activity is similar to other pineal enzymes in that it is controlled by light acting through the sympathetic nervous system. In contrast, the light-induced decrease in retinal rhodopsin kinase may be due to the direct destructive effect of light on the retina. The finding of neural control of pineal rhodopsin kinase in the pineal gland of adult rats is consistent with a function of the enzyme in the neural regulation of pineal function.

Animals↗
PubMed1986

See-saw signal processing in pinealocytes involves reciprocal changes in the alpha 1-adrenergic component of the cyclic GMP response and the beta-adrenergic component of the cyclic AMP response.

Pineal cyclic AMP and cyclic GMP are regulated by norepinephrine (NE) acting through alpha 1- and beta-adrenoceptors. beta-Adrenergic stimulation appears to be an absolute requirement and alpha 1-adrenergic activation amplifies beta-adrenergic stimulation of the cyclic AMP response 10-fold and the cyclic GMP response 100-fold, respectively. Chronic deprivation of adrenergic stimulation, due to exposure to constant light (LL) or by surgical denervation, enhances the cyclic AMP response and diminishes the cyclic GMP response as compared to control animals in a 10:14 light/dark (LD) cycle. This phenomenon is termed see-saw signal processing. In the current study we find these changes do not reflect shifts in the time course or Ka of these responses. Dose-response studies indicate the beta-adrenergic component of cyclic AMP stimulation is enhanced and the alpha 1-adrenergic component of cyclic GMP stimulation is diminished in LL pinealocytes. Several observations indicate these changes may reflect alterations in Ca2+-sensitive postreceptor mechanisms.

Animals↗
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↗
PubMed1986

Retinal S-antigen: immunocytochemical and immunochemical studies on distribution in animal photoreceptors and pineal organs.

Antiserum against bovine retinal S-antigen, a soluble protein (MW = 50 kDa) thought to be involved in phototransduction, was used in an immunohistochemical and immunochemical study of vertebrate eyes and pineal systems and invertebrate photoreceptor organs. Positive reactions, not seen with antiserum preabsorbed with highly purified S-antigen, were observed in planarian and starfish ocelli; scallop eyes; polychaete eye; crayfish compound eye; lamprey, salmon, frog, turtle, quail and hamster eyes. A specific reaction was also seen in the pineal organ of all the vertebrates examined, albeit weak in turtle and quail. In addition, several structures associated with photoreceptor organs, including the reduced frontal eyes of crayfish, the organ of Bellonci in crayfish eyestalk, and bipolar cells resembling those giving rise to Landolt's clubs in quail and golden hamster retinae, were immunopositive. Immunochemical studies revealed the presence of a single immunopositive band of protein which was similar but not identical in size in all vertebrate eyes and pineal organs (except that of chicken pineal) and invertebrate tissue examined. The wide distribution of positive reaction in photoreceptive tissue indicates that the retinal S-antigen determinant has been highly conserved during evolution.

Animals↗
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

Photoneural regulation of the mammalian pineal gland.

Mammalian pineal function appears to be controlled primarily through the release of noradrenaline from the terminals of nerves whose cell bodies lie in the superior cervical ganglia. This is the final segment of the following neural pathway: retina----retinohypothalamic projection----suprachiasmatic nuclei----paraventricular nuclei----intermediolateral cell column----superior cervical ganglia----nervi conarii----pineal gland. Noradrenaline acts on pinealocytes through alpha- and beta-adrenoceptors in an atypical manner. Beta-Adrenergic activation is an absolute requirement for the stimulation of both cyclic AMP and cyclic GMP production, and by itself produces a sixfold increase in the former and a twofold increase in the latter. Alpha-Adrenergic activation potentiates the beta-adrenergic stimulation of cyclic AMP production 10-fold, and that of cyclic GMP production about 200-fold. The mechanism of alpha- and beta-adrenergic interaction is being examined, and progress is being made in understanding the adrenergic control of cyclic AMP. It appears that alpha-adrenergic agonists act through the alpha 1-subclass of adrenoceptors to stimulate phospholipid turnover and the production of a breakdown product of phosphatidylinositol, diacylglycerol. This compound promotes the association of protein kinase C with membranes, which leads to the marked phosphorylation of one protein. The precise identity of this protein remains a mystery. This interaction leads to a larger cyclic AMP response but does not appear to be involved in the mechanism of potentiation of the cyclic GMP response. Changes in chronic neural stimulation produce reciprocal changes in the magnitudes of cyclic AMP and cyclic GMP responses. Chronic denervation results in a supersensitive cyclic AMP response and nearly complete disappearance of the cyclic GMP response. This is termed 'see-saw' signal processing. All the available evidence indicates that melatonin production is regulated by cyclic AMP. This nucleotide not only increases the activity of serotonin N-acetyltransferase (more correctly called arylalkylamine N-acetyltransferase) but also stabilizes the enzyme and prevents its inactivation.

Animals↗
PubMed1985

Immunocytochemical demonstration of retinal S-antigen in the pineal organ of four mammalian species.

By means of immunocytochemistry retinal S-antigen is selectively demonstrated in retinal photoreceptor cells of the rat and in pinealocytes of the hedgehog, rat, gerbil and cat. Brain areas surrounding the pineal organ are immunonegative. The immunoreactive material is evenly distributed in the perikarya of the cells. Occasionally, inner segments of retinal photoreceptors and processes of pinealocytes are also stained. The outer segments of retinal photoreceptors display a strong immunoreaction. In both pinealocytes and retinal photoreceptors the intensity of the immunoreaction varied considerably among individual cells. The immunocytochemical demonstration of retinal S-antigen in mammalian pinealocytes indicates that these cells still bear characteristics of photoreceptors. This finding is in accord with the concept that mammalian pinealocytes are derived from pineal photoreceptor cells of poikilothermic vertebrates.

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↗