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D C Klein

Publications and source records attributed to D C Klein.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1989

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence.

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.

Amino Acid Sequence↗
PubMed1989

[3H]AHN 086 acylates peripheral benzodiazepine receptors in the rat pineal gland.

AHN 086, an isothiocyanato derivative of Ro 5-4864 (4'-chlorodiazepam), inhibits radioligand binding to peripheral benzodiazepine receptors with characteristics of an irreversible (acylating) ligand. We now report that [3H]AHN 086 labels a approximately 30 kDa protein in the rat pineal gland determined by both SDS-polyacrylamide gel electrophoresis and gel filtration high-performance liquid chromatography of digitonin-solubilized membranes. Specific incorporation of [3H]AHN 086 into this protein was inhibited by preincubating membranes with excess AHN 086. Moreover, significant specific binding of [3H]AHN 086 was not observed in either bovine pineal gland (which does not possess high-affinity binding sites for Ro 5-4864) or ovalbumin. These findings suggest that the approximately 30 kDa protein labeled by [3H]AHN 086 in rat pineal gland is associated with peripheral benzodiazepine receptors in this tissue.

Acylation↗
PubMed1989

Adenosine stimulates adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate accumulation in rat pinealocytes: evidence for a role for adenosine in pineal neurotransmission.

Adenosine produces a concentration-dependent increase in pinealocyte cAMP (EC50, approximately 0.3 nM) and cGMP accumulation (EC50, approximately 0.7 nM). Maximal increases in both nucleotides are evident 10 min after treatment; 1 h later values return to pretreatment levels. Concentration-dependent effects on cAMP are also observed with N6-(L-2-phenylisopropyl)adenosine (EC50, approximately 0.75 nM), 5'-N-ethylcarboxy aminoadenosine (EC50, approximately 0.75 nM), and 2-chloroadenosine (EC50, approximately 2.0 nM); the EC50 values for stimulation of cGMP with these agents are higher by a factor of 2-10. In the case of 5'-N-ethylcarboxy amidoadenosine, the concentration-response curve is biphasic, with a significant effect evident within the range of 1-100 pM. The stimulatory nature of this response and the relative potency of the agonists tested are consistent with the involvement of an A2-like adenosine receptor. Comparison of adenosine and the selective beta-adrenergic agonist isoproterenol indicated that their maximal EC50 values were generally similar. Studies with antagonists revealed that both 8-(p-sulfophenyl)theophylline (1 microM) and the xanthine amine congener (8-[4-[[[(2-aminoethyl)carbonyl]methyl]oxy]phenyl]1,3- dipropylxanthine (1 microM) inhibited the effects of adenosine (1 nM to 1 microM), but xanthine amine congener was more potent; the latter was markedly effective at 0.1 nM, whereas 8-(p-sulfophenyl)theophylline was nearly ineffective at this concentration. It was also determined that pineal cells generate extracellular adenosine from extracellular ATP. ATP is thought to be released along with catecholamines during neurotransmission. Hence, these studies support the view that adenosine could participate in the transsynaptic regulation of pineal function.

Adenosine↗
PubMed1989

Immunoreactive S-antigen in cerebrospinal fluid: a marker of pineal parenchymal tumors?

This investigation evaluated the possibility that the occurrence of S-antigen in cerebrospinal fluid (CSF) might be used as a preoperative marker of pineal parenchymal tumors (pineoblastoma and pineocytoma). Such a marker could provide a means of preoperatively differentiating these neoplasms from pineal region tumors of other origin. The S-antigen, also known as the 48-kD protein or arrestin, is a highly antigenic protein originally found in the retina and pineal gland. In the retinal photoreceptors and submammalian pineal photoreceptors the protein is thought to be involved in phototransduction; its function in the mammalian pinealocyte is unknown. S-Antigen immunoreactivity also occurs in certain neoplastic cells of retinoblastomas, pineocytomas, pineoblastomas, and cerebellar medulloblastomas. This study included a group of 13 patients with tumors of the pineal region. Samples of CSF were obtained preoperatively and analyzed for the S-antigen using western blot technology. Tumor biopsy material was classified according to conventional neurohistological criteria and was also examined by immunocytochemical techniques for the presence of the S-antigen. S-Antigen immunoreactivity was found in the preoperative CSF of the one patient found to have pineocytoma; tumor tissue removed from this patient was the only neoplastic tissue examined in this study which contained S-Antigen immunoreactive tumor cells. Furthermore, hydroxyindole-O-methyltransferase activity was detectable in the pineocytoma but not in three other pineal tumors, and melatonin levels in the CSF of the pineocytoma patient were the highest in the patient group examined. These preliminary results suggest that testing for S-antigen in CSF might be useful in characterizing and treating tumors of the pineal region and, when identified in conjunction with other markers, it might also help to better define pineal parenchymal tumors. This study needs confirmation with a larger number of patients. If this approach is eventually found to be a reliable predictor of pineal cell tumors, it may supplant the need for surgical biopsies before initiating appropriate adjunctive therapy.

Antigens↗
PubMed1988

Protein kinase C: subcellular redistribution by increased Ca2+ influx. Evidence that Ca2+-dependent subcellular redistribution of protein kinase C is involved in potentiation of beta-adrenergic stimulation of pineal cAMP and cGMP by K+ and A23187.

Phenylephrine is known to stimulate translocation of protein kinase C in rat pinealocytes (Sugden, D., Vanecek, J., Klein, D.C., Thomas, T.P., and Anderson, W. B. (1985) Nature 314, 359-361). In the present study, the receptor mediating this effect was found to belong to the alpha 1-adrenoceptor subclass. Activation of this receptor is also known to produce a sustained increase in [Ca2+]i by increasing net influx (Sugden, A. L., Sugden, D., and Klein, D. C. (1985) J. Biol. Chem. 261, 11608-11612), which points to the possible importance of Ca2+ influx in the subcellular redistribution (activation) of protein kinase C in intact cells. This possibility was investigated by reducing extracellular Ca2+ ((Ca2+]o) with EGTA or by inhibiting Ca2+ influx with inorganic Ca2+ blockers. These treatments reduced alpha 1-adrenoceptor-mediated translocation of protein kinase C. This suggested that elevation of Ca2+ influx alone triggers activation of protein kinase C. In support of this, it was found that treatments which elevate Ca2+ influx, including increased extracellular K+ and addition of the Ca2+ ionophore A23187, cause redistribution of protein kinase C. The effect of K+ was blocked by nifedipine and that of A23187 by EGTA, indicating that effects of these agents are Ca2+-dependent. The possible role of phospholipase C activation in these effects was examined by measuring the formation of [3H]diacylglycerol by cells labeled with [3H]arachidonic acid. Although [3H]diacylglycerol formation was easily detected in the presence or absence of an effective concentration of an inhibitor of diacylglycerol kinase, none of the agents which cause rapid translocation of protein kinase C were found to cause a rapid increase in the generation of [3H]diacylglycerol. These findings establish that an increase in Ca2+ influx is sufficient to trigger translocation of protein kinase C. In addition, we found that a very close correlation exists between translocation of protein kinase C by phenylephrine, K+, and A23187 and their ability to potentiate beta-adrenergic stimulation of cAMP and cGMP accumulation. This provides strong support to the proposal that translocation of protein kinase C is required for potentiation of beta-adrenergic stimulation of pinealocyte cAMP and cGMP accumulation.

Adrenergic beta-Antagonists↗
PubMed1988

Effects of protein kinase inhibitor (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on protein kinase C activity and adrenergic stimulation of cAMP and cGMP in rat pinealocytes.

Protein kinase C is thought to be involved in the adrenergic regulation of pineal function. In this tissue, norepinephrine increases cAMP and cGMP accumulation through a synergistic dual receptor mechanism involving alpha 1- and beta-adrenergic receptors; the available evidence indicates that the alpha 1-adrenergic stimulation activates protein kinase C, and that this potentiates beta-adrenergic stimulation of pineal cAMP. The role of protein kinase C in the regulation of cGMP is unclear. In the present report, we determined whether an inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibits pineal protein kinase C and the adrenergic stimulation of pineal cAMP and cGMP. H7 (10(-4) M) reduced soluble protein kinase C activity by 40%. Treatment of intact pinealocytes with H7 for 0-240 min reduced the effects of subsequent norepinephrine (NE) stimulation of cAMP and cGMP accumulation by at least 25%. H7 also inhibited 25-30% the maximum stimulation of both cAMP and cGMP produced by concurrent treatment with isoproterenol and two agents which elevate intracellular Ca2+, ouabain and A23187. However, H7 did not reduce the effects of selective beta-adrenergic activation, indicating that H7 was probably inhibiting the effects of NE by blocking alpha 1-adrenergic potentiation of beta-adrenergic stimulation, not beta-adrenergically activated mechanisms. H7 also reduced the stimulation of cAMP accumulation produced by the combined treatment of isoproterenol and an activator of protein kinase C, 4-beta-phorbol 12-myristate, 13-acetate, which is consistent with the view that H7 is acting by inhibiting protein kinase C activity. These observations are in agreement with the conclusion that potentiation of beta-adrenergic stimulation of cAMP by alpha 1-adrenergic agonists, protein kinase C activators, or [Ca2+]i elevating agents involves protein kinase C. In addition, these results are of special interest because they point to the possibility that protein kinase C is involved in the regulation of cGMP accumulation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Activators of protein kinase C act at a postreceptor site to amplify cyclic AMP production in rat pinealocytes.

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Permissive role of calcium in alpha 1-adrenergic stimulation of pineal phosphatidylinositol phosphodiesterase (phospholipase C) activity.

Activation of alpha 1-adrenergic receptors increases [Ca+2]i and phosphatidylinositol phosphodiesterase (phospholipase C) activity in the pinealocyte. In this report the receptor involved in the stimulation of phospholipase C activity was further characterized, and the role of Ca2+ in this effect was investigated in some detail. Phospholipase C activity was estimated by measuring the production of [3H]inositol phosphates by [3H]inositol-labelled dispersed pinealocytes in suspension culture. Norepinephrine stimulated [3H]inositol monophosphate production severalfold; this was blocked by alpha 1-adrenergic antagonists, including prazosin, WB 4101, and phenoxybenzamine, but by neither an alpha 2- nor a beta-adrenergic antagonist, confirming that an alpha 1-adrenoceptor is involved in the regulation of phosphatidylinositol hydrolysis. Treatment with the Ca2+ chelator, EGTA, or with inorganic Ca2+ blockers, including Co2+, Mn2+, and La3+, reduced the norepinephrine-stimulated response, suggesting that the alpha 1-adrenergic stimulation of phospholipase C activity is Ca2+ dependent. However, phospholipase C activity was not increased by elevating intracellular Ca2+ with either the Ca2+ ionophore A23187 or with depolarizing concentrations of K+. These results indicate that although Ca2+ is necessary for alpha 1-adrenergic stimulation of phospholipase C activity, an increase in [Ca2+]i alone is not sufficient to stimulate the activity of this enzyme, and that effects which A23187 and depolarizing concentrations of K+ have on pineal function probably do not involve stimulation of phospholipase C activity.

Adrenergic alpha-Agonists↗