Whole-cell recording of neuroblastoma x glioma cells during downregulation of a major substrate, 80K/MARCKS,of protein kinase C

Summary Differentiated neuroblastoma cells exhibit both the delayed rectifier potassium current (I _K) and the M-current (I _M). The present study was designed to determine the roles of protein kinase C (PKC) and of the calmodulin-binding protein 80K/MARCKS, a prominent substrate for PKC and possible regulator of these currents. Neuroblastoma x glioma (NG108-15) hybrid cells transfected with m1 muscarinic receptors were grown with 1% fetal bovine serum (FBS) without the prostaglandin E₁ (PGE₁) and isobutylmethylxanthine (IBMX) usually added in preparation for electrophysiological studies. Under these conditions, the usual pleomorphism was largely abolished, leaving two populations of small cells with stellate and spherically symmetrical geometries. Whole-cell patch clamping indicated that the two cell types had identical electrophysiological properties, displaying: I _k, a small current through a T-like Ca²⁺ channel, and no M-current.Stimulation with carbachol shifted the distribution of cells to a more stellate morphology within 24 hr and later (after 48 hr) reduced the PKC substrate 80K/MARCKS by 22±7%. In contrast to the stimulation of I _k observed with cardiac cells, PKC activation produced only a small inhibition of I _k, which was independent of carbachol pretreatment. Thus, PKC and 80K/MARCKS can be dissociated from the regulation of I _k in neuroblastoma cells.Supported in part by research grants from the National Institutes of Health (DK-40145 and EY-08343) and from the U.K. Medical Research Council.We thank Dr. Peter J. Parker for his generous gift of PKC, and Yvonne Vallis for her skillful assistance with the cultures and harvesting of the NG108-15 transfected cells.     

Striatal L-DOPA Decarboxylase Activity in Parkinson's Disease In Vivo: Implications for the Regulation of Dopamine Synthesis

Abstract: L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radidabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.     

Changes in Sympathetic Nerve Terminals in the Heart of Cold-Exposed Rats

Abstract: Changes in sympathetic nerve terminals of the heart after varying periods of exposure of rats to 4°C were investigated. Two indices were used for changes in the number of noradrenaline storage vesicles, i.e., vesicular dopamine β-hydroxylase (DBH) activity and noradrenaline storage capacity. The latter was obtained after uptake of [³H]noradrenaline; endogenous content, uptake of exogenous noradrenaline, and degree of saturation of the vesicles were calculated using the specific activity of the [³H]noradrenaline. As a measure of tyrosine hydroxylase activity, whole ventricular noradrenaline, dopamine, and dihydroxyphenylacetic acid content were used. After 4 h of cold exposure there was an increase in vesicular endogenous noradrenaline content, uptake, storage capacity, and DBH activity as well as a large increase in whole ventricular dopamine. After 6 h in the cold, vesicular endogenous noradrenaline content, storage capacity, and DBH activity were decreased. The results suggest that during cold exposure there is an initial increase followed by a decrease in the number of functional vesicles in the nerve terminal, which could explain the fluctuations in the rate of noradrenaline release.     

Effect of an n-3 Fatty Acid-Deficient Diet on the Adenosine-Dependent Melatonin Release in Cultured Rat Pineal

Abstract: We studied the effect of a diet deficient in n-3 fatty acids on the adenosine-dependent melatonin release from cultured rat pineal gland after stimulation by 5'- N -ethylcarboxamidoadenosine (NECA), an A₂ adenosine agonist. Experiments were conducted with 2-month-old rats raised on semipurified diets containing either peanut oil (n-3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n-3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n-3-deficient rats. This was compensated for partially by an increase in 22:4 n-6 and 22:5 n-6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N -acetylserotonin and melatonin release in the medium, was lower after stimulation by 10^-5 mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n-6/n-3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane-bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R_1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R_1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-t20 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R_1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R_1A and ANF-R_1C subtypes. A10 and NIH cells which express high density of ANF-R₂ receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R_1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R_1A and ANF-R_1C subtypes are modulated in the same manner. In the presence of Mn²⁺, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg²⁺ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R_1A, the ANF-R_1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R_1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R_1A subtype.Abbreviation ANF atrial natriuretic factor - BNP brain natriuretic peptide - CNP C-type natriuretic peptide - ATP adenosine-5-triphosphate - IBMX 3-isobutyl-1-methylxanthine - TPA 12-O-tetradecanoyl-phorbol-13-acetate - FKL forskolin - PKC calcium-phospholipid-dependent protein kinase - PKA cAMP-dependent protein kinase - PKG cGMP-dependent protein kinase - C-ANF [Cys¹¹⁶]-ANF-(102-116)-NH₂ - CC chromaffin cells     

Demonstration of melatonin-binding sites in cyclohexylamine-formaldehyde-fixed brain tissues

This study shows for the first time that perfusion of rat or hamster brain with a cyclohexylamine-paraformaldehyde mixture makes possible the observation by autoradiography of melatonin binding sites in structurally well-preserved fixed tissues. This result is a first step in the identification of melatonin-receptor-containing cell types by cytoautoradiography.     

Octopamine receptor subtypes and their modes of action

Octopamine receptor subclasses were first proposed to explain differences in the pharmacological profiles of a range of physiological responses to octopamine obtained in the extensor-tibiae neuromuscular preparation of the locust. Thus, OCTOPAMINE₁ receptors which inhibit an endogenous myogenic rhythm, increase intracellular calcium levels. Also OCTOPAMINE₂ receptors which modulate neuromuscular transmission in this preparation, increase the level of adenylate cyclase activity. The current status of this classification is reviewed by examining the pharmacology of responses to octopamine in a range of preparations. It is concluded that the distinction between OCTOPAMINE₁ and OCTOPAMINE₂ receptor types is still valid, but that OCTOPAMINE₂ receptors exhibit some tissue specific variations. Studies on a clonedDrosophila octopamine/tyramine (phenolamine) receptor are discussed and illustrate many of the difficulties presently encountered in making a definitive classification of octopamine receptors. These include the possibilities that single receptors may activate multiple second messenger systems and that different agonists may differentially couple the same receptor to different second messenger systems.