Secretogranin II: Regulation of Synthesis and Post-Translational Proteolysis in Bovine Adrenal Chromaffin Cells

Secretogranin M (Sgll), also called chromogranin C, is an acidic tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurones. Although less abundant than chromogranin A (CGA) and chromogranin B (CGB), Sgll is found in adrenal medullary chromaffin granules. In the present study we investigated the regulation of Sgll biosynthesis in bovine chromaffin cells maintained in primary culture. Cellular proteins were labelled with [³⁵S]methionine and the heat stable chromogranin enriched fraction was isolated. Following electrophoretic separation, the 86 kDa Sgll band was identified by sequence analysis using the Edman degradation procedure. The radioactivity incorporated in the 86 kDa Sgll band was used as an index of the Sgll synthesis rate. We found that stimulation of chromaffin cells with nicotine and histamine and to a smaller extent with angiotensin II and bradykinin significantly enhanced the rate of Sgll synthesis. In contrast direct depolarization with K⁺ had no effect on Sgll synthesis suggesting that the raise of cytosolic calcium evoked by high K⁺ may not be sufficient to induce modifications in Sgll synthesis. The possible second messenger pathways involved in the control of Sgll biosynthesis were investigated by using protein kinase C and adenylate cyclase activators. We observed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and forskolin increased the basal rate of Sgll synthesis. Incubation with both TPA and forskolin was required to obtain an effect comparable to that produced by nicotine or histamine suggesting that these secretagogues recruit both protein kinase C- and cyclic AMP-dependent mechanisms to stimulate Sgll synthesis. Our results indicate that within chromaffin granules, the rates of CGA, CGB and Sgll synthesis are independently regulated and suggest a close relationship between the cell secretory activity and the biosynthesis of Sgll. The biological role of Sgll is unknown but it has been suggested that the protein may function as a precursor of potentially bioactive peptides. Here we identified a Sgll derived peptide corresponding to the C-terminal residues 582–586 in the soluble core of purified chromaffin granules. This peptide was released together with catecholamines upon stimulation of cultured chromaffin cells indicating that the peptide was present within the storage complex of chromaffin granules and was not the result of some artefactual proteolytic degradation of Sgll during the course of granule purification. We propose that this peptide is a specific product of the post-translational processing of Sgll. By analogy with peptides derived from CGA and CGB, it may possess some specific biological activity that remains to be identified.     

Localization of binding sites for insulin-like growth factor-I (IGF-I) in the rat brain by quantitative autoradiography

In vitro quantitative autoradiography was used to localize IGF-I binding sites in rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.01 nM ¹²⁵I[Thr⁵⁹]IGF-I, alone or mixed with 10 nM unlabeled [Thr⁵⁹]IGF-I or insulin, for 22 h at 4 °C and apposed to LKB Ultrofilm. Measurement of labeled [Thr⁵⁹]IGF-I binding by computer digital image analysis of the autoradiographic images indicated that high affinity IGF-I binding sites are widely distributed at discrete anatomical regions of the brain microarchitecture. The highest concentration of specific binding sites was in the choroid plexus of the lateral and third ventricles. Unlabeled porcine insulin was less potent than unlabeled IGF-I in competing for binding sites on brain slices. Regions of the olfactory, visual, and auditory, as well visceral and somatic sensory systems were labeled, in particular the glomerular layer of the olfactory bulb, the anterior olfactory nucleus, accessory olfactory bulb, primary olfactory cortex, lateral-dorsal geniculate, superior colliculus, medial geniculate, and the spinal trigeminal nucleus. High concentrations of IGF-I-specific binding sites were present throughout the thalamus and the hippocampus, (dentate gyrus, Ca1, Ca2, Ca3). The hypothalamus had moderate binding in the paraventricular, supraoptic, and suprachiasmatic nucleus. Highest binding in the hypothalamus was in the median eminence. The arcuate nucleus showed very low specific binding, approaching the levels found in optic chiasm and white matter regions. Layers II and VI of the cerebral cortex also had moderate IGF-I binding. The results suggest that the development and functions of brain sensory and neuroendocrine pathways may be regulated by IGF-I.     

Endogenous Histamine in Cultured Bovine Adrenal Chromaffin Cells

Histamine releases catecholamines and opioids in primary cultured bovine adrenal medullary (BAM) chromaffin cells. We have studied whether histamine is synthesized and localized in BAM cells, and whether it can be released upon activation with secretagogues. In BAM cells histamine is immunohistochemically co-localized with tyrosine hydroxylase in 45 ± 8% of all cells. Only histamine immunoreactivity was observed in 8 ± 2% of all BAM cells. No mast-cell-like cells were observed in our system. Histamine can be released from BAM cells by high potassium (56 mM K⁺) in a calcium-dependent manner. Compound 48/80 did not release histamine from BAM cells but nicotine caused a dose-dependent liberation of the amine. Cultured BAM cells have histidine decarboxylase activity which is inhibited by α-fluoromethylhistidine. These results indicate that endogenous histamine is synthesized, stored and released in BAM chromaffin cells in vitro.     

Chromogranin A: Secretion of Processed Products from the Stimulated Retrogradely Perfused Bovine Adrenal Gland

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N-terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N-terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS-PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA_1–40 and CGA_44–76 detected a 7 kD protein whose release was increased during stimulation. This component co-migrated with synthetic CGA_1–76, was not immunoreactive to antisera to CGA_79–113 or CGA_124–143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an ～ 18 kD component, which stained with antisera to CGA_1–40, CGA_44–76 and CGA_79–113, but not to chromostatin (CGA_124–143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA_1–40, CGA_44–78 and CGA_79–113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed Chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells. The major fragments secreted appear to be the N-terminal fragments of CGA, CGA_1–76 and CGA_1–113, which would arise as a result of processing of CGA at the first and second pairs of basic amino acids. A number of larger CGAFs, possibly containing the chromostatin sequence CGA_124–143 at their N-terminal, and components similar in size to intact CGA and to proteoglycan forms of CGA, are also secreted from the perfused bovine adrenal gland during stimulation.     

Cyclic AMP-induced depolarization measured by bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells

Summary Effects of cyclic AMP on membrane potentials were examined by measuring the changes of bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells. 8-Bromo cyclic AMP (8Br-cAMP) or forskolin caused a gradual and long lasting increase of the fluorescence intensity. The effects of 8br-cAMP was blocked by cyclic AMP-dependent protein kinase inhibitor, adenosine-3, 5-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS) and there was no further increase in the fluorescence by 8br-cAMP in the cells depolarized with 56 mM KCl or gramicidin D. Ouabain or the removal of extracellular K⁺ ([K⁺]₀free) which block Na⁺, K⁺-ATPase also increased the fluorescence. The effect of 8br-cAMP on the fluorescence was counteracted by ouabain or [K⁺]₀ free and was blocked in the absence of extracellular Na⁺ but not by tetrodotoxin or the removal of Ca²⁺ from the medium. These results may suggest that cyclic AMP causes the membrane depolarization by accumulating Na⁺ through the inhibition of Na⁺, K⁺-ATPase in adrenal chromaffin cells.     

Modulation of the nicotinic α-bungarotoxin site in chromaffin cells in culture by a factor(s) endogenous to neuronal tissue

An endogenous factor(s) which affects the in vitro binding of α-bungarotoxin (α-BGT) to rat brain membranes has previously been found in brain supernatant. This fraction, as well as a partially purified preparation of this material from bovine brain, is here shown to affect the binding of α-BGT to chromaffin cell membranes. To study possible long term effects, the supernatant extract was added to adrenal medullary chromaffin cells in culture. The cells were incubated for several days and at the end of this time, the medium bathing the cells, which contained the endogenous factor(s), was removed and α-BGT binding to the cells measured. Binding to control cultures had shown that α-BGT bound to the chromaffin cells in a saturable manner, with high affinity (K_d = 1.5 nM) and the specificity of a nicotinic receptor ligand. After incubation of the cells with supernatant factor, a marked decline in the number of a α-BGT binding sites was observed with no change in affinity. This does not appear to be due to a detrimental effect on the cells as cell number did not appear to be decreased in the cultures preincubated with the supernatant extract and the DNA and protein content were similar in the control and treated cultures. The possibility that there was some non-specific detrimental effect to the chromaffin cell membrane was considered; however, the stimulated release of noradrenaline from the cells was not affected by treatment of the cultures in the presence of the supernatant fractions. In addition, tyrosine hydroxylase activity was significantly increased in the treated cultures. d-Tubocurarine, an antagonist at the acetylcholine receptor, caused an increase in α-BGT binding after 7 days of treatment, while the agonist nicotine and choline had no effect. These results suggest that in brain supernatant there may exist an endogenous factor(s), which may function in the regulation of the nicotinic-like α-BGT receptors in neuronal cell.     

Basic Fibroblast Growth Factor in the Adrenal Gland

The importance of trophic agents for the development and maintenance of neurons and their presence in mesenchyme-derived neuronal target organs such as muscle is well exemplified by the protein nerve growth factor (NGF) and its synthesis in target areas of sympathetic and sensory nerves. Stringent conceptualization of target organ-regulated neuronal maintenance would imply that neurons were able to provide trophic support to their presynaptic counterparts. We present data suggesting that basic fibroblast growth factor (bFGF), a mitogen and trophic factor for several neuron populations in vitro, may be such a protein involved in retrograde trophic neuron - neuron interaction. Basic FGF or a closely related protein is present in the adrenal medulla and its sympathetic neuron-like chromaffin cells. A polyclonal antibody specific for bFGF recognizes an 18 kD band in Western blots of bFGF-enriched bovine adrenal medulla extracts and immunostains isolated bovine chromaffin cells. This antibody also blocks the bFGF-like activity present in adrenal medullary extracts and chromaffin granule extracts that both promote in vitro survival of embryonic chick ciliary ganglionic neurons. Furthermore, like bFGF, the soluble proteins of bovine chromaffin granules are mitogenic for cultured bovine aorta endothelial cells. Electrothermal unilateral destruction of the adrenal medulla causes the disappearance of 25% of Nissl-stained neurons in the ipsilateral intermediolateral column (IML) of the spinal cord between levels Th7 and L1, which contains the preganglionic neurons projecting to the adrenal medulla. Substitution of the adrenal medulla by gel foams soaked with bFGF prevents neuron losses in the IML. The effects are specific in that NGF and cytochrome C are ineffective. Our results suggest that bFGF is located in chromaffin cells and maintains target-deprived autonomic spinal cord neurons, thus possibly acting as an interneuronal trophic messenger in vivo.     

Up-regulation of functional voltage-dependent sodium channels by cyclic AMP-dependent protein kinase in adrenal medulla

Treatment of cultured bovine adrenal chromaffin cells with dbcAMP increased [³H]STX binding with an EC₅₀ of 126 μM and a half-effective time of 12 h; dbcAMP (1 mM × 18 h) raised theB_max approximately 1.5-fold without altering theK_d value. Forskolin (0.1 mM) or IBMX (0.3 mM) also increased [³H]STX binding, while dbcAMP had no effect. Effects of dbcAMP and forskolin were abolished by H-89, an inhibitor of cAMP-dependent protein kinase. Cycloheximide (10 μg/ml) and actinomycin D (10 μg/ml), inhibitors of protein synthesis, nullified the stimulatory effect of dbcAMP, whereas tunicamycin, an inhibitor of protein glycosylation, had no effect. Treatment with dbcAMP augmented veratridine-induced²²Na influx,⁴⁵Ca influx via voltage-dependent Ca channels and catecholamine secretion, while the same treatment did not alter⁴⁵Ca influx and catecholamine secretion caused by high K (a direct activation of voltage-dependent Ca channels) [25]. Na influx via single Na channel calculated from²²Na influx and [³H]STX binding was quantitatively similar between non-treated and dbcAMP-treated cells. Brevetoxin allosterically enhanced veratridine-induced²²Na influx approximately 3-fold in dbcAMP-treated cells as in non-treated cells. These results suggest that cAMP-dependent protein kinase is involved in the modulation of Na channel expression in adrenal medulla.     

Neuropeptide FF in the Rat Adrenal Gland: Presence, Distribution and Pharmacological Effects

Neuropeptide FF (NPFF, FLFQPQRFamide) is an FMRFamide-like octapeptide exhibiting antiopiate activity. The presence of both NPFF-immunoreactivity (NPFF-IR) and NPFF-specific receptors has been described in the mammalian central nervous system (CNS). The peripheral effects of NPFF indicate that NPFF-IR material is present outside the CNS. Biochemical and immunohistochemical methods enabled us to determine the presence and distribution of NPFF-IR in the rat adrenal gland. The amount of NPFF-IR material in whole gland was estimated by radioimmunoassay to be 19.00±4.00  fmol/gland. High performance liquid chromatography analysis of adrenal extracts revealed a single molecular form which coeluted with authentic NPFF. Demedullation decreased adrenal NPFF-IR content, indicating that NPFF-IR was present in both cortex and medulla. Light microscopy revealed NPFF-IR in beaded fibers confined in the outer part of the cortex and in medullary cells. Double-labeling with antityrosine-hydroxylase and anti-NPFF antibodies showed NPFF-IR in cortical catecholaminergic postganglionic fibers restricted to the subcapsular and glomerulosa zonae. NPFF-IR was also located in medullary chromaffin cells and in rays and islets of chromaffin cells dispersed throughout the cortex. Insulin-induced hypoglycemia did not alter NPFF-IR content. Denervation lowered adrenal NPFF-IR content. These data indicate that this peptide is present in nerve fibers of extrinsic origin. In vitro approaches using adrenal slices have shown that NPFF inhibited aldosterone release in a dose-dependent manner. Taken together, these data suggest that NPFF may participate in the control of aldosterone production and adrenal blood supply.     

Localization and Characterization of Insulin-Like Growth Factor-I Receptors in Rat Brain and Pituitary Gland Using in vitro Autoradiography and Computerized Densitometry* A Distinct Distribution from Insulin Receptors

In order to identify likely sites of action of insulin-like growth factor-I (IGF-I) in rat brain and pituitary gland, we have used the technique of in vitro autoradiography and computerized densitometry to map, characterize and quantify its receptors in coronal and sagittal sections. A discrete and characteristic distribution of IGF-I receptor binding was demonstrated, with specific binding representing 85% of total binding. Displacement and specificity competition curves in the olfactory bulb were typical for authentic IGF-I receptors and computer analysis indicated a single class of binding site with a dissociation constant (K_d) of 13 nM for the choroid plexus and 5.1 nM for the olfactory cortex. IGF-I receptor density was very high in the choroid plexus in ail ventricles, but the binding in other circumventricular organs was variable, with high levels in the median eminence and the sub-fornical organ, and low levels in the organum vasculosum of the lamina terminalis. Highest binding was seen in the glomerular layer of the olfactory bulb and its associated regions the taenia tecta and anteromedial olfactory nucleus. The preoptic and septal regions showed moderate binding, while the hypothalamus, with the exception of the median eminence, showed low IGF-I binding. The pituitary gland showed very high binding density in both anterior and posterior lobes, similar to the median eminence. The thalamus had high IGF-I binding density, while it was low in basal ganglia. In the limbic system the hippocampal CA2, CAS, CA4 layers showed high binding, with little in CA1, while binding was high also in the adjacent amygdala. Binding was low in the mid and hindbrain, with the exception of the geniculate bodies, and the sensory nucleus of the trigeminal nerve. Binding was high in the primary olfactory and endopyriform cortex and in specific superficial layers. Cerebellar binding was also high in the molecular layer. Fibre layers showed no binding. Comparison with insulin receptors revealed common distribution in the choroid plexus, paraventricular nucleus, cerebellum, entorhinal cortex and amygdala, with receptor density three- to five-fold higher for IGF-I than for insulin. In contrast, in the hippocampus, insulin binding was high in the CA1 field, and low in CA2, CA3, CA4 while for IGF-I binding the converse was seen. The arcuate nucleus showed prominent insulin labelling and minimal IGF-I binding, while the median eminence showed low insulin and high IGF-I binding. The hypothalamus was more widely labelled with insulin, while in the thalamus the converse was true. Olfactory bulb laminae were labelled with differing intensity by insulin and IGF-I. In common with insulin receptor distribution was the high density of IGF-I receptors over areas of extensive dendritic arborizations which receive rich synaptic inputs, in the cerebellum, hippocampus and olfactory bulb. We conclude that IGF-I receptors are widespread throughout rat brain and pituitary gland, with concentration in regions concerned with olfaction, autonomie and sensory processing, as well as in regulation of growth hormone release, via feedback at the median eminence and pituitary gland. Many of these regions have in common high rates of metabolic and synthetic activity, which may be mediated by IGF-I and its receptors.     

Insulin-like growth factor I: A mitogen for rat schwann cells in the presence of elevated levels of cyclic AMP

To develop effective procedures for improving the regeneration of peripheral nerves and for preventing the formation of neurofibromas, it is necessary to identify the different mitogens that stimulate the proliferation of Schwann cells. Insulinlike growth factor I (IGF-I), which is a potent autocrine growth factor in many tissues, is synthesized by proliferating Schwann cells. However, the role of IGF-I in stimulating their division is still uncertain. Here we show that nanomolar concentrations of IGF-I stimulate the growth of Schwann cells in primary culture. IGF-I alone was uneffective but in the presence of forskolin (5 μM) or dibutyryl cyclic AMP (dbcAMP, 10 μM), it became a potent mitogen. Neither IGF-II nor epidermal growth factor (EGF) were effective, even in the presence of forskolin. Insulin also stimulated Schwann cell proliferation in the presence of forskolin, but only at micromolar concentration. Receptors for IGF-I were visualized on the Schwann cell surface by indirect immunofluorescence staining using anti-human IGF-I receptor antibodies. Their presence was also assessed by binding assays using [¹²⁵I]-IGF-I as a ligand. Scatchard analysis showed a single class of high-affinity receptors (K_d = 1.5 nM). Competition studies with unlabeled IGF-I or insulin indicated a half-maximal displacement of [¹²⁵I]-IGF-I by IGF-I at about 5 nM, while insulin was about 500-fold less effective. The number of binding sites for IGF-I was increased by exposing cells for 3 days to forskolin (- forskolin: about 5,100; + forskolin: about 12,200 binding sites/cell). These results suggest that forskolin increases available receptors for IGF-I, which is consistent with the synergism between cAMP and IGF-I in stimulating Schwann cell growth. © 1993 Wiley-Liss, Inc.     

Specific binding of 2-[I]iodomelatonin by rat splenocytes: characterization and its role on regulation of cyclic AMP production

In the present paper we show that pineal hormone melatonin interacts with rat splenocytes through high-affinity binding sites. Binding of 2-[¹²⁵I]iodomelatonin ([¹²⁵I]MEL) by splenocytes fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, specificity, and increased under constant light exposure. Results suggest binding to a single class of binding sites without cooperative interactions. The dissociation constant (K_d) for the single site was 0.34 nM with a binding capacity of 2.25 fmol/107 cells. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived value of the dissociation constant was 0.20 nM. The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, pharmacological doses of melatonin also inhibited cyclic AMP production stimulated by forskolin, a potent activator of adenylate cyclase system. The demonstration of [¹²⁵I]MEL binding sites in the spleen, in addition to those described in blood mononuclear cells and thymus, provides evidence to support a direct mechanism of action of melatonin on immune system.     

Selective block of Ca2+-dependent K+ current in crayfish neuromuscular system and chromaffin cells by sea anemone Bunodosoma cangicum venom

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studied in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca²⁺ -dependent K⁺ current (I_K(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca²⁺ or K⁺ currents. Furthermore, the venom also reduced I_K(Ca) in chromaffin cells, without modifying voltage-gated Na⁺, Ca²⁺, or K⁺ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic I_K(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca²⁺ -dependent K⁺ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers. © 1995 Wiley-Liss, Inc.     

Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca-ATPase inhibitor in adrenal chromaffin cells

Long-term (≥12 h) treatment of cultured bovine adrenal chromaffin cells with thapsigargin (TG), an inhibitor of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), caused a time (t_1/2=16.3 h)- and concentration (IC₅₀=37.8 nM)-dependent decrease of cell surface ¹²⁵I-insulin binding by 35%, but did not change the K_d value. TG caused a sustained increase of cytoplasmic concentration of Ca²⁺ ([Ca²⁺]_c) in a biphasic manner, and the effect of TG on ¹²⁵I-insulin binding was abolished by BAPTA-AM. Western blot analysis showed that TG lowered insulin receptor (IR) β-subunit level in membrane, but did not alter total cellular levels of IR precursor and IR β-subunit. Internalization of cell surface IR, as measured by using brefeldin A, an inhibitor of vesicular exit from the trans-Golgi network (TGN), was not changed by TG. These results suggest that inhibition of SERCA by TG and the subsequent increase of [Ca²⁺]_c down-regulates cell surface IR by retarding externalization of IR from the TGN.     

Inhibition by neuroprotective drug NS-7 of nicotine-induced Na influx, Ca influx and catecholamine secretion in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀=15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na⁺,K⁺-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of ²²Na⁺ without altering the EC₅₀ value of nicotine. Also, NS-7 diminished nicotine-induced ⁴⁵Ca²⁺ influx via nicotinic receptors and voltage-dependent Ca²⁺ channels (IC₅₀=14.1 μM) and catecholamine secretion (IC₅₀=19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.     

α2-Adrenergic receptors inhibit catecholamine secretion from bovine adrenal medulla

Catecholamine secretion evoked by carbymlcholine from isolated bovine adrenalinal medullary cells was inhibited byα ₂-agonist, clonidine, in a dose-dependent manner with IC₅₀ value of 2.8 × 10⁻⁵ M. [³H]Clonidine bound to adrenal medullary membranes with high affinity and saturable characteristics. These results suggest thatα ₂-adrenergic receptors which exist on adrenal medullary cells have inhibitory effect on the secretion of catecholamine from the cells     

Specific [H]strychnine binding associated with glycine receptors in bovine retina

Saturable, specific [³H]strychnine binding can be demonstrated in homogenates of bovine retina. Scatchard plots revealed only one set of binding sites with a dissociation constant (K_d) of about 60 nM and a maximal number of binding sites of about 1.5 pmol/mg protein. The structural specificity of [³H]strychnine binding sites in bovine retina parallels the properties found for [³H]strychnine binding sites in the spinal cord of several vertebrates. Thus, the data do not give any evidence that specific [³H]strychnine binding in bovine retina labels taurine rather than glycine receptors and favors glycine rather than taurine as inhibitory neurotransmitter in bovine retina. The subcellular distribution of specific [³H]strychnine binding in bovine retina parallels that of sodium-dependent, high-affinity uptake of glycine and taurine. All 3 parameters are mainly found in the P₂ fractions of bovine retina homogenates, containing conventional synaptosomes, most abundant in the inner plexiform layer, but can also be found in the P₁ fractions, containing large synaptosomes from the photoreceptor cell layer.     

Triiodothyronine Regulates Insulin-Like Growth Factor-I Binding to Cultured Rat Pituitary Cells*

Triiodothyronine (T₃) stimulates the synthesis of growth hormone and enhances the growth of neoplastic rat pituitary somatomam-motrophs (GH cells) in culture. Moreover, T₃ has been shown to stimulate the production and secretion of an autocrine growth factor by these cells. We have previously demonstrated the presence of specific receptors for insulin-like growth factors (IGF) on GH cells. Since GH₃ cells contain mRNA encoding IGF-I, it has been suggested that IGF-I might act in an autocrine fashion in these cells. Therefore, it was of interest to learn how T₃ affects IGF-I binding to GH₃ cells. T₃ increased [¹²⁵I]IGF-I binding in a time - and dose-dependent manner. After 48 h of exposure to T₃, an increase in IGF-I binding was seen with 10^?11M T₃, maximizing with 10^?8M T₃. When cells were exposed to 10^?8 T₃, [¹²⁵I]IGF-I binding reached a maximum of 218 ± 20.8% of control (±SEM, P < 0.002) after 72 h of incubation. Scatchard analysis indicated that T₃ did not alter the K_d of IGF-I for its receptor, but that the total receptor number was increased. Dexamethasone (10^?7M) inhibited the T₃-induced increase in IGF-I binding, but glucocorticoid alone did not substantially alter receptor number. No significant change in insulin or IGF-II binding was seen after hormone treatment. 10^?8 M T₃ or IGF-I increased the growth of the GH₃ cells by ≥30%. Our data indicate that T₃ upregulates IGF-I binding in GH₃ cells without altering insulin binding and thereby provides a means for enhancing potential autocrine regulation in this cell line.     

Contrasting effect of a brain supernatant extract on neuronal vs neuromuscular α-bungarotoxin receptors

The effects of a rat brain supernatant extract and a partially purified supernatant preparation from bovine brain were determined on the binding of [¹²⁵I]α-bungarotoxin (α-BGT) to muscle membranes, as well as to membranes prepared from brain. In agreement with previous work, the supernatant preparations inhibited α-BGT binding to brain membranes in a dose-dependent fashion, (Brain Research, 245 (1982) 57–67); however, no significant effect of either of the preparations was observed on the binding of the toxin to muscle membranes. As well, the supernatant preparations did not affect binding of radiolabelled α-BGT to muscle cells in culture in competition binding experiments. The effect of long-term incubation of cells in culture with the supernatant preparations was subsequently determined. These studies showed that the binding of [¹²⁵I]α-BGT increased markedly (300%) in the presence of a crude rat brain supernatant preparation, while incubation of the muscle cells in the presence of the partially purified bovine supernatant extract had no significant effect on radiolabelled toxin binding. In contrast, both the rat and bovine supernatant preparations significantly decreased (up to 65%) radiolabelled toxin binding to a cultured neuronal cell population, adrenal medullary chromaffin cells. These results suggest that an endogenous factor(s), present in brain extracts, differentially regulates the neuronal as compared to the neuromuscular nicotinic α-bungarotoxin binding sites.     

Bovine adrenal chromaffin cells: high-yield purification and viability in suspension culture

A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded ～ 1–2 × 10⁸ chromaffin cells per adrenal medulla at a purity of 95% or higher.On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.