The use of [125I]iodocyanopindolol as a specific probe for beta-adrenergic receptors in differentiating cultured rat skeletal muscle

In order to examine more precisely the role of beta-adrenergic receptors in the process of differentiation we used the new radioligand iodocyanopindolol ([125I]ICYP), which we found to be a very useful probe to identify beta receptors. Binding characteristics conformed to those expected for a physiologically relevant beta receptor. L6E9 cells grown in horse serum, which allows differentiation, exhibit increased beta receptor density in intact cells as a function of age. In contrast, cells grown in fetal calf serum, which does not allow differentiation, exhibit constant beta receptor density. In broken cells, however, both differentiating and non-differentiating cells show an increase in beta receptors. These results suggest that the process of differentiation is associated with an unmasking of beta receptors which are increasing but cryptic in undifferentiated cells.     

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

Specific labelling by [125I]helodermin of high-affinity VIP receptors in rat liver membranes

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.     

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron.

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.     

The degradation of [125I]iodoinsulin by cytosol from rat liver and the formation of high-molecular-weight products. A study by gel chromatography

Degradation of [125I]iodoinsulin by cytosol from rat liver (as judged by the appearance of perchloric acid soluble counts) occurs rapidly and generates both high- and low-molecular-weight labelled products on chromatography on AcA54 or AcA202. One of the low-molecular-weight products can be converted to a high-molecular-weight product by further incubation with fresh cytosol. Degradation is not inhibited by chloroquine, but is affected by bacitracin. In the presence of bacitracin most of the radioactivity elutes in the void volume and possibly represents interaction of undegraded insulin with other cytoplasmic components.     

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropeptide Y Y1 receptors in the rat forebrain: Autoradiographic demonstration of [125I][Leu31,Pro34]-NPY binding sites and neurons expressing Y1 receptor mRNA

Abstract

Using the specific monoiodinated NPY analog [Leu³¹,Pro³⁴]-NPY we have localized NPY binding sites of the Y₁ type in forebrain areas of the rat. The resulting receptor autoradiograms were compared with the regional distribution and cellular localization of the mRNA encoding Y₁ receptor as demonstrated by in situ hybridization histochemistry. High densities of Y₁ binding sites were present in the cerebral cortex, the claustrum, the thalamus and the medial mammillary nucleus, while moderate densities of Y₁ binding sites were observed in the amygdalahippocampal complex. Lower binding densities were observed in septal nuclei, most hypothalamic nuclei and the circumventricular organs. High levels of Y₁ mRNA were observed in the granula cell layer of the hippocampal dentate gyrus, several thalamic nuclei and the hypothalamic arcuate nucleus, while moderate levels of Y₁ mRNA were seen in the frontoparietal cortex, several thalamic nuclei, the hippocampal pyramidal layers, the subiculum, the olfactory tubercle, the claustrum and a number of hypothalamic nuclei. Using the hypothalamic arcuate nucleus as an example, the distribution of immunoreactive NPY, Y₁ mRNA and Y₁ binding sites was compared, and possible implications of Y₁ mediated actions within this nucleus are discussed. The present study further enlightens the anatomical distribution of NPY binding sites of the Y₁ type within the central nervous system of the rat, and extends the understanding of central actions of NPY mediated via this type of receptor.     

Biochemical studies on histones of the central nervous system. I. Characterization and partial identification of the label of histones from rat brain following intraventricular administration of 1-[14C]-acetate

Rat brain histones were acetylated in vivo by intraventricular injection of [14C]-acetate. More than 90% of the label is the result of a true acetylation. Enzymatic proteolysis of the labelled histone fraction and subsequent chromatographic investigation of the digestion products showed about 60% of the recovered radioactive material to be epsilon-acetyl lysine, whereas 22% of the radioactivity was found in an unidentified spot.