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Biomedical subjects

D C Klein

Publications and source records attributed to D C Klein.

At least 19 recordsLinked to original sources
PubMed1992

Recoverin in pineal organs and retinae of various vertebrate species including man.

Recoverin is a recently discovered 26 kDa calcium-binding protein, which activates guanylate cyclase in retinal photoreceptors when the intracellular concentration of free calcium drops upon photoexcitation. In this study we examined the distribution of recoverin in retinae and pineal organs of Xenopus laevis larvae, 1-day-old chicken, adult pigeon, albino rat, sheep and man by means of immunocytochemistry. Recoverin immunoreaction was found in all species investigated except for the chicken. In the retina, recoverin immunoreaction was restricted to photoreceptors; all other cell types were immunonegative. In the pineal organ, the recoverin immunoreaction labeled 'pinealocytes of the sensory line', i.e. classical pineal photoreceptors of Xenopus laevis larvae, modified pineal photoreceptors of pigeon, and pinealocytes of mammals. The number of recoverin immunoreactive pinealocytes varied considerably among species of mammals: very few cells were stained in the rat pineal organ, whereas in rabbit, sheep and man, numerous pinealocytes were found to be recoverin-immunoreactive. No immunocytochemical staining was observed after preabsorption of the recoverin antibody with the recombinant protein. Immunoblotting experiments showed that the immunoreaction is due to a protein of 26 kDa in both retina and pineal tissue. Thus, recoverin appears to belong to the family of proteins which are expressed in both retina and pineal organ and are highly conserved in the course of phylogeny. Recoverin may be involved in phototransduction in the directly light-sensitive pineal organs of poikilothermic vertebrates and birds. However, the functional role of recoverin in the mammalian pineal organ, which is not photosensitive, remains unknown.

Animals↗
PubMed1992

The pineal adrenergic----cyclic GMP response develops two weeks after the adrenergic----cyclic AMP response.

Pineal metabolism is regulated primarily by noradrenergic innervation. Stimulation of the adult gland with norepinephrine elevates both cyclic AMP and cyclic GMP production, through remarkably similar mechanisms requiring activation of both beta- and alpha 1-adrenergic receptors. As described here, however, the adrenergic stimulation of cyclic GMP is first detectable about 2 weeks after the cyclic AMP response can be detected. This indicates there is a profound difference in when cyclic AMP- and cyclic GMP-regulated processes can be adrenergically regulated.

Animals↗
PubMed1992

Development of MEKA (phosducin), G beta, G gamma and S-antigen in the rat pineal gland and retina.

Pinealocytes and retinal photoreceptor cells contain an unusual cytoplasmic complex composed of the G beta gamma dimer of GTP-binding regulatory proteins (G-proteins) tightly bound to an acidic 33 kDa phosphoprotein termed MEKA or phosducin; MEKA is a substrate of cyclic AMP-dependent protein kinase. This study characterized the developmental appearance of these and two related proteins, G gamma and S-antigen, in pineal and retinal tissue. MEKA was absent in the pineal gland prior to birth, at a time when it was possible to detect G beta in pineal cytoplasm, indicating that the appearance of G beta in the cytoplasm precedes that of MEKA and does not appear to require the presence of MEKA. The absence of MEKA at this time indicates that the cyclic AMP stimulation of pineal serotonin N-acetyltransferase activity is not mediated by MEKA, which has been considered as a possible role of MEKA. After postnatal day 7, pineal MEKA and cytoplasmic G beta increased in a parallel manner, with peak values occurring at about postnatal day 21. Thereafter, both proteins in the pineal gland decreased in a parallel fashion to 10 and 35% of their peak values, respectively; in contrast, the cytoplasmic protein S-antigen and membrane associated G beta remained at maximal levels after this time. Whereas both MEKA and G beta decreased late in development in the pineal gland, these proteins either increased or remained constant in the retina. These tissue-specific patterns were found to differ from those of another cytosolic protein found exclusively in the pineal gland and retina, S-antigen, which remained constant after day 21 in the pineal gland but decreased in the retina late in life.

Aging↗
PubMed1992

Bovine hydroxyindole-O-methyltransferase. Significant sequence revision.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step in melatonin synthesis. The nucleotide and deduced amino acid sequences of bovine HIOMT have been reported. Our laboratory recently isolated a cDNA clone encoding human HIOMT. Comparison of the human and bovine nucleotide sequences revealed several discrepancies which prevented perfect alignment and produced defined regions of virtually no homology in the deduced amino acid sequence. Consequently, we repeated sequence analysis of the original bovine HIOMT cDNA clone, the results of which are reported here. The revised nucleotide sequence includes 23 differences from the published sequence. This completely changes the deduced amino acid sequence in two regions, encompassing a total of 96 residues, or 28% of the protein. The revised deduced amino acid sequence predicts different post-translational modifications as compared to that of the original deduced sequence. This information will make it possible in future investigations of HIOMT to design improved polymerase chain reaction primers, peptides for the generation of antisera, and probes for various types of analysis and screening of libraries.

Acetylserotonin O-Methyltransferase↗
PubMed1992

Rat pineal Gsa, Gia and Goa: relative abundance and development.

The adult rat pineal gland contains relatively high concentrations of Gsa, low amounts of both Gia and Goa, and undetectable levels of GTa. During development the amounts of 45 kDa Gsa and of Gia remain constant. In contrast, 42 kDa Gsa and Goa are nearly absent at birth and increase in abundance markedly thereafter. GTa is undetectable at any age. It would appear that multiple mechanisms regulate the expression of G-proteins in the pineal gland.

Animals↗
PubMed1992

Immunocytochemical demonstration of rod-opsin, S-antigen, and neuron-specific proteins in the human pineal gland.

The aim of this study was to examine whether rod-opsin and S-antigen immunoreactions were present in the pineal organ of adult man and how these immunoreactions were correlated with neuronal markers, e.g., synaptophysin, and neurofilaments L, H and M. Three perfusion-fixed epithalamic regions including the pineal organ and five pineal glands obtained at routine autopsy were used. The specimens were taken from female or male patients, 25 to 85 years of age. All immunoreactions were performed using highly specific, well-characterized antibodies. Rod-opsin and S-antigen-immunoreactive pinealocytes occurred in all pineal organs investigated; however, the immunoreaction was restricted to small subpopulations of pinealocytes (rod-opsin immunoreaction: approximately 3%-5%; S-antigen immunoreaction: approximately 5%-10% of the total population). In contrast, immunoreactions for synaptophysin and neurofilaments M and H were present in numerous pinealocytes. Immunoreactivity for neurofilament L was not found. These data suggest that the cellular composition of the human pineal organ is heterogeneous. Moreover, the presence of rod-opsin and S-antigen immunoreactions in the human pineal organ indicates that it may be affected by autoimmune retinal diseases that are provoked by antibodies against these proteins, as is the case in rodents and non-human primates.

Adult↗
PubMed1992

Stimulus deprivation increases pineal Gs alpha and G beta.

Denervation and other forms of stimulus deprivation cause an increase in the magnitude of subsequent responses, a phenomenon commonly referred to as denervation supersensitivity. This has been well demonstrated with the cyclic AMP response to norepinephrine in the pineal gland. In the present report, we address the question of whether stimulus deprivation alters alpha and beta subunits of the GTP binding regulatory protein that stimulates adenylyl cyclase activity (Gs). Stimulus deprivation of the pineal gland was produced by denervation (superior cervical ganglionectomy), decentralization of the superior cervical ganglia, or by exposure of the animal to continuous lighting. All increased both the alpha and beta subunits of Gs (Gs alpha and G beta) by up to fourfold, as estimated using semiquantitative western blot technology. These effects were detectable after 1 day of stimulus deprivation and were sustained for 2 weeks. The stimulatory effects of constant light-induced stimulus deprivation were also apparent by measuring cholera toxin-dependent ADP-ribosylation of Gs alpha, which revealed a four-fold increase in the amount of labeled substrate. The results of in vivo studies were confirmed with in vitro studies, which demonstrated a spontaneous increase in both Gs alpha and G beta during 72 h of organ culture. The constant light-induced increases in both Gs alpha and G beta were prevented by continuous administration of isoproterenol (0.3 mg/kg/day), supporting the suggestion that adrenergic stimulation controls the levels of Gs alpha and G beta. These studies indicate that stimulus deprivation increases both Gs alpha and G beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1992

Melatonin inhibits gonadotropin-releasing hormone-induced elevation of intracellular Ca2+ in neonatal rat pituitary cells.

GnRH stimulates LH release by increasing intracellular Ca2+ ([Ca2+]i). Melatonin is known to inhibit GnRH-stimulated LH release from neonatal rat pituitary cells. In the present report, the issue of whether melatonin acts through [Ca2+]i was addressed. [Ca2+]i was studied in cells in suspension, using Fluo-3 as a fluorescent indicator. In neonatal rat pituitary cells, melatonin inhibited the GnRH-induced [Ca2+]i increase in a dose-dependent manner; the GnRH-induced increase in [Ca2+]i was inhibited 40% by 100 nM melatonin. The relative potencies of several indoles as inhibitors of the GnRH stimulation of [Ca2+]i in neonatal pituitary cells (2-iodo-melatonin greater than melatonin greater than 6-hydroxymelatonin) correlate with their known potencies to inhibit LH release and with their binding affinity to high affinity melatonin receptors, which indicates that these receptors probably mediate the effects of melatonin. Further support for this interpretation comes from the observation that melatonin does not inhibit the GnRH effect on [Ca2+]i in cells obtained from adolescent rat pituitary glands, which lack melatonin receptors and are insensitive to melatonin as an inhibitor of GnRH-stimulated LH release. The possible involvement of an inhibitory G-protein was also investigated by studying the effects of pertussis toxin. Pretreatment with pertussis toxin antagonized the effects of melatonin on [Ca2+]i and LH release. This indicates that melatonin may inhibit the GnRH-induced increase in [Ca2+]i through a mechanism involving a pertussis toxin-sensitive G-protein. To examine the role of extracellular Ca2+ in this effect, the effects of melatonin were examined in a low Ca2+ medium. Under these conditions, the effect of melatonin was markedly reduced, which indicates that melatonin may act by inhibiting Ca2+ influx. These observations indicate that melatonin inhibits GnRH stimulation of [Ca2+]i in neonatal rat gonadotrophs, and this probably explains the inhibitory action of melatonin on GnRH stimulation of LH release.

Aging↗
PubMed1992

Characterization of alpha 2-adrenergic receptors on rat pinealocytes.

alpha 2-Adrenergic receptors in rat pineal membranes were characterized using p-[125I]iodoclonidine, a highly selective, high specific activity ligand. Binding was rapid (association constant rate = 0.0462 nM/min-1) and reversible after the addition of phentolamine (apparent dissociation rate constant = 0.04 min-1). Saturation experiments indicate the presence of a single class of noncooperative binding sites, with an equilibrium binding constant (Kd) of 1.1 +/- 0.3 nM and a binding capacity (Bmax) of 69 +/- 9 fmol/mg protein. Analysis of the relative potency of selected adrenoreceptor agonists and antagonists in competition studies with p-[125I]iodoclonidine indicates that the ligand is binding to a member of the family of alpha 2-adrenergic receptors that has a high affinity for oxymetazoline, phentolamine, and (-)norepinephrine and a low affinity for prazosin, similar to the recently described alpha 2-adrenergic receptor present in the bovine pineal gland, classified as belonging to the newly described alpha 2D-adrenergic receptor subtype. Rat pineal alpha 2-adrenergic receptors were unaltered after nerve endings degenerated. This observation and the recent finding that alpha 2-adrenergic agonists potentiate N6,2'-O-dibutyryl-cAMP or isobutylmethylxanthine stimulation of arylalkylamine N-acetyltransferase in the rat pineal gland establish that alpha 2D-like adrenergic receptors are located on pinealocytes.

Animals↗
PubMed1992

Sodium-dependent effects of melatonin on membrane potential of neonatal rat pituitary cells.

Melatonin inhibits GnRH-stimulated release of LH from neonatal rat pituitary cells, probably by inhibiting GnRH-induced elevation of intracellular Ca2+. This effect of melatonin seems to involve inhibition of Ca2+ influx through voltage-sensitive channels. Accordingly, it is possible that melatonin could act by hyperpolarizing pituitary cells, which would close these channels. This issue was addressed here by determining if melatonin influences membrane potential. Membrane potential and intracellular Ca2+ were studied in neonatal rat pituitary cells in suspension, using bis-oxonol and Fluo-3 as fluorescent indicators, respectively. It was found that treatment with melatonin alone causes membrane hyperpolarization and that it has a repolarizing effect after GnRH-induced membrane depolarization. This effect on membrane potential appears to be mediated by high affinity melatonin receptors and a pertussis toxin-sensitive Na(+)-dependent mechanism; it is not dependent upon Ca2+, Cl-, or bicarbonate. This may be the molecular basis of action of melatonin in other tissues with high affinity melatonin receptors.

Aging↗
PubMed1991

Norepinephrine stimulates potassium efflux from pinealocytes: evidence for involvement of biochemical "AND" gate operated by calcium and adenosine 3',5'-monophosphate.

Biochemical studies of K+ efflux from rat pinealocytes revealed for the first time that norepinephrine (NE) increases 86Rb+ and 42K+ efflux. The effects of NE depend upon concurrent activation of both alpha 1- and beta-adrenoceptors. This effect is mediated by cAMP and Ca2+, which appear to act in conjunction to control K+ efflux; studies with charybdotoxin and tetraethylammonium indicate that a Ca2(+)-sensitive K+ channel (K(Ca] appears to be involved. Patch clamp studies identified a large conductance (approximately 100 psec) K+ channel. This study also revealed for the first time that NE treatment increases the fraction of time that this channel was open. Studies of inside-out pineal membrane patches indicated that increasing Ca2+ at the cytoplasmic surface of the membrane increased the frequency of channel opening, as is typical of K(Ca) channels in this type of preparation. Outward K+ currents were almost completely blocked by tetraethylammonium (10 mM) and scorpion venom (L. quinquestriatum; 100 ng/ml). Cell-attached studies confirm that the effects of NE are mediated by intracellular second messengers. These investigations suggest that NE elevates K+ flux, probably through a large conductance K(Ca) channel, that NE acts through alpha 1- and beta-adrenergic receptors, and that Ca2+ and cAMP act together through a biochemical "AND" gate to mediate the effects of receptor activation. Activation of this K(Ca) channel would have a hyperpolarizing influence and might contribute to the adrenergic hyperpolarization of pinealocytes.

Animals↗
PubMed1991

Photoneural control of the synthesis and phosphorylation of pineal MEKA (phosducin).

MEKA is an acidic 33-kilodalton phosphoprotein found in the retina and pineal gland. It is of interest because it forms a cytoplasmic heterotrimer with the beta gamma-complex of GTP-binding regulatory proteins (G proteins). Accordingly, MEKA may play a role in signal transduction. MEKA is phosphorylated on Ser73 by cAMP-dependent protein kinase. In the present report, MEKA was studied using an antiserum (Anti-32) against MEKA65-96, which can be used to estimate total MEKA and the phosphorylation state of MEKA. It was confirmed that MEKA is rapidly phosphorylated by adrenergic stimulation of pineal glands in organ culture. In addition, total (dephosphorylated) MEKA was observed to increase after a 6-h treatment with norepinephrine or (Bu)2 cAMP, an effect which was dependent upon new protein synthesis. In in vivo studies, it was found that the total amount of MEKA and MEKA phosphorylation were increased at night in the dark, a time when the pineal gland is adrenergically stimulated. The high level of phosphorylation was rapidly reduced when animals were exposed to light, which blocks neural stimulation of the gland. This report provides the first in vivo evidence that MEKA phosphorylation is under physiological control, and that MEKA synthesis is controlled by an adrenergic----cAMP mechanism which requires protein synthesis.

Animals↗
PubMed1990

Noradrenergic control of the synthesis of two rat pineal proteins.

Pineal physiology is controlled by norepinephrine released from sympathetic nerves terminating in the gland. In the present study, the effect of norepinephrine on the labelling of specific proteins was investigated by incubating glands with [35S]methionine and then resolving the proteins by two-dimensional polyacrylamide gel electrophoresis; the patterns were analyzed by computer-assisted image analysis. The most prominent effects of norepinephrine were distinct and consistent increases in the labelling of two proteins (37 kDa, pI = 6.0, 50 kDa, pI = 6.0), designated adrenergically induced protein (AIP 37/6 and AIP 50/6). In both cases, norepinephrine was effective at low concentrations (EC50 = 10 nM). Pharmacological studies indicated that the effects of norepinephrine on both proteins involved a beta-adrenergic receptor, and that cyclic AMP was the second messenger. Pulse-chase labelling experiments revealed that these effects of norepinephrine did not involve post-translational modification of previously labelled precursor proteins, but depended upon de novo synthesis of protein. An inhibitor of mRNA synthesis, actinomycin-D, was found to block the effect of norepinephrine on AIP 50/6 but not on AIP 37/6, suggesting that norepinephrine acted on AIP 50/6 via a transcriptional mechanism and on AIP 37/6 via a translational mechanism. These in vitro studies were extended into in vivo investigations by measuring silver-stained AIP 37/6 in the two-dimensional gels. Changes in the amount of AIP 37/6 in pineal glands were studied in response to treatments which block the adrenergic stimulation of the gland, including exposure to constant lighting or removal of the superior cervical ganglia. Both treatments reduced AIP 37/6 by 50-75% in 8 weeks. These observations, together with those from in vitro studies, suggest that the amount of AIP 37/6 in the pineal gland is regulated by norepinephrine; and further, that norepinephrine acts through a beta-adrenergic-cyclic AMP mechanism to control AIP 37/6 synthesis at a translational level.

Adrenergic Fibers↗
PubMed1990

Pineal transduction. Adrenergic----cyclic AMP-dependent phosphorylation of cytoplasmic 33-kDa protein (MEKA) which binds beta gamma-complex of transducin.

Adrenergic regulation of phosphorylation of pineal proteins was studied. Norepinephrine treatment of intact pinealocytes incubated with 32Pi enhanced phosphorylation of a 33-kDa phosphoprotein (33PP). The effect of NE was rapid, sustained, and appeared to be mediated by a beta-adrenergic----cyclic AMP mechanism. Studies using broken cell preparations revealed that 33PP was phosphorylated by cyclic AMP-dependent protein kinase (PKA). It was also possible to demonstrate PKA-dependent phosphorylation of the 33-kDa protein in cytosol from rat retina and in cow and sheep pineal glands. Two-dimensional polyacrylamide gel electrophoresis revealed that 33PP is acidic (pI congruent to 4.5), appears to exist as two isoforms with slightly different charge, and has the same mobility as the retinal 33-kDa PKA substrate. Immunological analysis indicated 33PP in both tissues is a previously reported 33-kDa protein (MEKA); this protein is a PKA substrate which has been reported to form a cytoplasmic complex with the beta gamma complex of transducin. Consistent with this, it was possible to identify the beta-subunit in pineal cytoplasm and in the same congruent to 70-kDa gel permeation fraction which contained the 33-kDa protein identified as MEKA. Thus, it appears possible that MEKA is present in pineal cytoplasm in a 70-kDa complex with G beta gamma, as is the case in retina. The finding of MEKA in the pineal makes it the latest addition to a family of retinal/pineal proteins which are thought to have evolved from a common ancestral photochemical transduction system.

Animals↗
PubMed1990

Differential expression and enzymatic properties of the Na+,K(+)-ATPase alpha 3 isoenzyme in rat pineal glands.

We have used immunoblotting and biochemical techniques to analyze expression of Na+,K(+)-ATPase alpha and beta subunits in rat pineal glands. Western blot analysis of pineal microsomal membrane fractions with antisera specific for each of the three rat alpha and two rat beta subunits revealed similar levels of expression of alpha 1 and alpha 3 subunits in pineal glands of 5-day-old rats. High levels of alpha 3 and beta 2 subunits and low levels of alpha 1 subunits were detected in adult glands. No alpha 2 or beta 1 subunits were detectable at either developmental stage. Examination of the enzymatic properties of the pineal gland alpha 3 isoform suggests that this enzyme is a ouabain-sensitive ATPase whose activity is dependent upon Na+ and K+. This ATPase exhibited a lower apparent Km for Na+ than the kidney alpha 1 isoenzyme and did not show positive cooperative Na+ activation. Our results suggest that the activity of the Na+,K(+)-ATPase alpha 3 isoenzyme may be adapted to function under conditions of hyperpolarizing transmembrane potentials.

Aging↗
PubMed1989

Evidence of alpha 1-adrenergic----protein kinase C----Na+/H+ antiporter-dependent increase in pinealocyte intracellular pH. Role in the adrenergic stimulation of cGMP accumulation.

The regulation of intracellular pH (pHi) in isolated rat pinealocytes was studied using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Resting pHi was 7.09 when the extracellular pH (pHe) was 7.2. Treatment of pinealocytes with the physiological regulator of pineal function, norepinephrine, resulted in a concentration-dependent increase in pHi. Further analysis indicated that norepinephrine is probably acting via an alpha 1-adrenergic----[Ca2+]i----Ca2+/phospholipid- dependent protein kinase (protein kinase C) mechanism to activate the Na+/H+ antiporter, thereby causing cytoplasmic alkalization. A potential influence of cytosolic alkalization on the responsiveness of cyclic nucleotides to adrenergic agonists was also studied. Five analogs of the antiporter inhibitor amiloride reduced norepinephrine stimulation of cGMP accumulation with the same relative potency as they act on the antiporter. In contrast, although inhibitory effects of these compounds on cAMP accumulation were detectable, they occurred at 10-100-fold higher concentrations, and the relative potency of these inhibitors did not indicate they were acting via the antiporter. These findings provide evidence that 1) alpha 1-adrenergic receptor activation increases pinealocyte pHi through Ca2+----protein kinase C-dependent activation of the Na+/H+ antiporter; and 2) norepinephrine stimulation of cGMP accumulation is pHi-dependent. It would appear that alpha 1-adrenergic regulation of pHi via the Na+/H+ antiporter may be of general importance in the control of cGMP accumulation.

Adrenergic alpha-Agonists↗
PubMed1989

Forskolin stimulates pinealocyte cGMP accumulation. Dramatic potentiation by an alpha 1-adrenergic----[Ca2+]i mechanism involving protein kinase C.

The effect of forskolin on cGMP regulation was investigated using dispersed rat pinealocytes. Forskolin stimulated cGMP accumulation in a concentration-dependent manner; this response was strongly potentiated by an alpha 1-adrenergic----[Ca2+]i mechanism involving protein kinase C. These findings provide further evidence that activation of two receptor-regulated signal transduction mechanisms may be commonly required for maximal stimulation of cGMP accumulation, and establish a new experimental approach to the study of cGMP regulation.

Adenylyl Cyclases↗