Role of late maternal thyroid hormones in cerebral cortex development: an experimental model for human prematurity.

Hypothyroxinemia affects 35-50% of neonates born prematurely (12% of births) and increases their risk of suffering neurodevelopmental alterations. We have developed an animal model to study the role of maternal thyroid hormones (THs) at the end of gestation on offspring's cerebral maturation. Pregnant rats were surgically thyroidectomized at embryonic day (E) 16 and infused with calcitonin and parathormone (late maternal hypothyroidism [LMH] rats). After birth, pups were nursed by normal rats. Pups born to LMH dams, thyroxine treated from E17 to postnatal day (P) 0, were also studied. In developing LMH pups, the cortical lamination was abnormal. At P40, heterotopic neurons were found in the subcortical white matter and in the hippocampal stratum oriens and alveus. The Zn-positive area of the stratum oriens of hippocampal CA3 was decreased by 41.5% showing altered mossy fibers' organization. LMH pups showed delayed learning in parallel to decreased phosphorylated cAMP response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) expression in the hippocampus. Thyroxine treatment of LMH dams reverted abnormalities. In conclusion, maternal THs are still essential for normal offspring's neurodevelopment even after onset of fetal thyroid function. Our data suggest that thyroxine treatment of premature neonates should be attempted to compensate for the interruption of the maternal supply.

Acetylcholine receptor subunit homomer formation requires compatibility between amino acid residues of the M1 and M2 transmembrane segments.

The neuronal nicotinic acetylcholine receptor (nAChR) subunits alpha3 and alpha7 have different assembly behavior when expressed in heterologous expression systems: alpha3 subunits require other subunits to assemble functional nAChRs, whereas alpha7 subunits can produce homomeric nAChRs. A previous analysis of alpha7/alpha3 chimeric constructs identified a domain comprising the first putative membrane-spanning segment, M1, as essential to homomeric assembly. The present study dissected further this domain, identifying three amino acid residues, which are located at the most intracellular third of the M1 transmembrane segment, as important in the assembly of homomers. Moreover, formation of homooligomeric complexes seems to require a compatible accommodation between this region and certain residues of the second transmembrane segment, M2. Thus, compatibility between defined domains of the M1 and M2 transmembrane segments appears as a determinant factor governing homomer association of nAChR subunits.

A residue in the middle of the M2-M3 loop of the beta4 subunit specifically affects gating of neuronal nicotinic receptors.

An aspartate residue in the M2-M3 loop of neuronal nicotinic receptor alpha7 subunits is a major determinant of the channel functional response. This residue is conserved in most beta4 subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of alpha3beta4 receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.

Separate [3H]-nitrendipine binding sites in mitochondria and plasma membranes of bovine adrenal medulla.

1. Two binding sites for the 1,4-dihydropyridine (DHP) derivative [3H]-nitrendipine have been found in the bovine adrenal medulla. The high-affinity site (Kd = 0.48 nM and Bmax = 128 fmol mg-1 protein) was specifically located in purified plasma membranes. The low-affinity site (Kd = 252 nM and Bmax = 169 pmol mg-1 protein) was located only in mitochondria. Chromaffin granule membranes lacked specific binding sites for [3H]-nitrendipine. 2. Kinetic analysis of the rates of association and dissociation of [3H]-nitrendipine, saturation isotherms and displacement experiments with unlabelled nitrendipine and PN200-110 revealed single, homogeneous populations of high- and low-affinity sites in plasma and mitochondrial membranes, respectively. 3. The high affinity site was sensitive to Ca2+ deprivation and heating; it was practically unaffected by changes in ionic strength of the medium and its optimal pH was slightly alkaline. This site exhibited a strong DHP stereoselectivity; diltiazem increased and verapamil decreased the affinity of [3H]-nitrendipine. 4. In contrast, binding of [3H]-nitrendipine to the low affinity site was more heat resistant and less affected by Ca2+ removal. Its optimal pH was slightly acid and the increase in ionic strength enhanced the number of available sites. The site had no DHP stereoselectivity. Verapamil decreased the dissociation constant of [3H]-nitrendipine acting in a non-competitive manner; diltiazem did not affect equilibrium binding parameters of [3H]-nitrendipine. 5. These results suggest that both biding sites reflect different receptor entities. The high-affinity binding site corresponds to the dihydropyridine receptor associated with the L-type calcium channel. The function of the mitochondrial, low-affinity binding site is, at present, unknown.

The low-affinity dihydropyridine receptor and Na+/Ca2+ exchanger are associated in adrenal medullary mitochondria.

The effect of Ca2+ channel-acting drugs on bovine adrenal mitochondria Ca2+ movements was investigated. Mitochondrial Ca2+ uptake is performed by an energy-driven Ca2+ uniporter with a Km of 20.9 +/- 3.2 microM and Vmax of 148.1 +/- 7.2 nmol 45Ca2+ min-1 mg-1. Ca2+ release is performed through an Na+/Ca2+ antiporter with a Km for Na+ of 4.2 +/- 0.5 mM, a Vmax of 7.5 +/- 0.4 nmol 45Ca2+ min-1 mg-1, and a Hill coefficient of 1.4 +/- 0.2 Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger was inhibited by several dihydropyridines (nitrendipine, felodipine, nimodipine, (+)isradipine) and by the benzothiazepine diltiazem with similar potencies. In contrast, neither CGP 28392, Bay-K-8644, amlodipine, nor verapamil had any effect on Ca2+ efflux. Nitrendipine at 20 microM modified neither the Km nor the Hill coefficient for Na+, whereas the Vmax was reduced to 2.9 nmol 45Ca2+ min-1 mg-1, thus demonstrating noncompetitive modulation of the Na+/Ca2+ exchanger. None of the Ca2+ channel-acting drugs assayed at 100 microM affected Ca2+ influx through the uniporter. Ca2+ channel blockers inhibited the Na+/Ca2+ antiporter and displaced the specific binding of [3H]nitrendipine to intact mitochondria with Ki values similar to the IC50s obtained for the inhibition of the Ca2+ efflux. Ca2+ channel-acting drugs that did not inhibit the Na+/Ca2+ exchanger (amlodipine, CGP 28392, Bay-K-9644, and verapamil, at concentrations of 100 microM or higher) had no effect on [3H]nitrendipine binding. These results suggest that the adrenomedullary mitochondrial dihydropyridine receptor is associated with the Na+/Ca2+ exchanger.

Density of apamin-sensitive Ca(2+)-dependent K+ channels in bovine chromaffin cells: relevance to secretion.

Three objectives were defined when planning this study: (i) to identify binding sites for [125I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 microM for 10 sec), histamine (10 microM for 10 sec) or high K+ (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca2+ concentrations [Ca2+]i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [125I]-apamin gave a saturation curve whose Scatchard transformation produced a Kd of 132 pM and a Bmax of 0.72 fmol/10(6) cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K+ channels) did not inhibit [125I]apamin binding. Binding was blocked by apamin and by d-tubocurarine, two blockers of small-conductance Ca(2+)-activated K+ channels (SK channels). The number of binding sites for [125I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per micron 2 surface area. Apamin (1 microM) enhanced the secretory response to histamine (10 microM), DMPP (1 or 5 microM) and high K+ (20 or 35 mM) by 2-3-fold. The response to 70 mM K+, however, was unaffected. Apamin also enhanced the peak [Ca2+]i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca2+ channels, the access of Ca2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.

Stimulus frequency affects c-fos expression in the rat visual system.

We have characterised the c-fos expression patterns in various centers of the visual pathway of adult rats monocularly stimulated either by continuous or flickering light at different frequencies. Results show different immunocytochemical patterns in all centers studied, the geniculate lateral complex (LGC), superior colliculus (SC) and primary visual cortex (Oc1), depending on the physical characteristics of the stimulus (blinking frequency and light wavelength). After stimulation of the left eye, the ipsilateral pathway presents a substantial density of immunoresponsive cells, which is greater than expected with respect to the number of fibers that project ipsilaterally from the retina to the LGC and the superficial layers of the SC. A surprisingly high positive immunoresponsiveness is obtained in all cases with coherent light stimulation in the red spectrum (634 nm).

Activity of the nicotinic acetylcholine receptor alpha5 and alpha7 subunit promoters in muscle cells.

The acetylcholine receptor alpha5 and alpha7 subunits are components of different nicotinic receptor subtypes expressed in the nervous system. However, they are also present in non-neuronal tissues. We have detected alpha5 and alpha7 transcripts in mouse C2C12 muscle cells. Moreover, on differentiation of myoblasts into myotubes, the amount of alpha7 transcripts increased significantly, whereas alpha5 remained unchanged. In order to explore how the expression of these neuronal genes is regulated in muscle, we have characterized their promoter activities. Deletion and mutagenesis analysis with transfected reporter genes showed that transcriptional activity was controlled by regulatory elements also operative in neuronal-like cells. Thus, the activity of the alpha5 subunit core promoter decreased to approximately 50% on alteration of one, two, or three of the five Sp1 binding sites present in this region and was almost abolished when four or five sites were mutated simultaneously. In the case of the alpha7 subunit promoter, the upstream stimulatory factor and the early growth response gene transcription factor were involved in regulating its transcriptional activity. In addition, the alpha7 promoter was activated during the differentiation process, in a mechanism partially dependent on the mentioned factors.

Interaction of opioid drugs with human platelet alpha 2-adrenoceptors.

Morphine, naltrexone and naloxone inhibited the binding of [3H]clonidine to the alpha 2-adrenoceptors in human platelet membranes, provided that Mg2+ was present in the medium. In the presence of 5'-guanylyl imidophosphate (Gpp(NH)p) or in the absence of Mg2+ morphine did not modify the binding of [3H]clonidine. Neither [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAGO), nor [D-Pen2,D-Pen5]enkephalin (DPDPE), nor dynorphin-(1-17) affected the [3H]clonidine binding. The presence of 1 mM naloxone did not alter the affinity of either [3H]clonidine or [3H]yohimbine, but reduced the number of binding sites of [3H]clonidine, having no effect on [3H]yohimbine. Naloxone inhibited the binding of adrenaline to high- but not low-affinity sites. It is concluded that morphine and semisynthetic antagonist derivatives interact with alpha 2-adrenoceptors only in the high-affinity state.

Sensitivity to µ-opioid receptor-mediated anti-nociception is determined by cross-regulation between µ- and δ-opioid receptors at supraspinal level.

BACKGROUND AND PURPOSE: The perception of pain and its inhibition varies considerably between individuals, and this variability is still unexplained. The aim of the present study is to determine whether functional interactions between opioid receptors are involved in the inter-individual variability in the sensitivity to µ-opioid receptor agonists. EXPERIMENTAL APPROACH: Anti-nociceptive tests, radioligand binding, stimulation of [(35) S]GTP-γ-S binding, inhibition of cAMP production and co-immunoprecipitation experiments were performed in two strains of rat (Sprague-Dawley bred at our university - SDU - and Wistar) that differ in their sensitivity to opioids. KEY RESULTS: The increased anti-nociceptive potency of µ-opioid receptor agonists in SDU rats was reversed by the δ-opioid receptor antagonist, naltrindole. Inhibition of the binding of [(3) H] naltrindole by µ-opioid receptor agonists was different in brain membranes from SDU and Wistar rats. Differences were also evident in the effect of δ-opioid receptor ligands on the binding of [(35) S]GTP-γ-S stimulated by µ-opioid receptors agonists. No strain-related differences were detected in spinal cord membranes. The potency of morphine to inhibit cAMP production in brain membranes varied between the strains, in the presence of deltorphin II and naltrindole. Co-immunoprecipitation experiments demonstrated that δ-opioid receptors were associated with µ-opioid receptors to a higher extent in brain synaptosomal fractions from SDU than in those from Wistar rats. CONCLUSIONS AND IMPLICATIONS: There was increased supraspinal cross-talk between µ and δ-opioid receptors in SDU, as compared with Wistar rats. This was related to an enhanced sensitivity to anti-nociception induced by µ-opioid receptor agonists.

Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera.

To test the hypothesis that cell-dependent expression of alpha7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat alpha7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of alpha7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. (125)I-alpha-bungarotoxin (alphaBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of alpha7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant alpha-BGT binding compared to transfection with GFP. In contrast, alpha7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without alphaBGT binding. Flow cytometry of cells transfected with alpha7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with alpha7-GFP did not correlate with amounts of cell surface or immunoprecipitable alphaBGT binding. Therefore, GFP folding at the C-terminal of the alpha7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of alpha7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled alpha7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for alpha7 receptor assembly.

Pharmacological characterization of alpha-bungarotoxin-sensitive acetylcholine receptors immunoisolated from chick retina: contrasting properties of alpha 7 and alpha 8 subunit-containing subtypes.

At least three subtypes of alpha-bungarotoxin-sensitive acetylcholine receptors (alpha Bgt-sensitive AChRs) exist in chick brain and retina. All may contain previously unknown structural subunits. One subtype contains alpha 7 subunits. Another contains alpha 8 subunits. A third contains both alpha 7 and alpha 8 subunits. In this article, we describe, for the first time, the pharmacological characterization of alpha 7 AChRs and alpha 8 AChRs immunoisolated from chick retina. Pharmacologically, the alpha 8 AChRs exhibit two classes of binding sites, the high affinity of which have higher affinity for most cholinergic ligands than do alpha 7 AChRs. These differences are most accentuated for ACh (approximately 5400-fold), decamethonium (approximately 1400-fold), 1,1,-dimethyl-4 phenylpiperazinium (approximately 200-fold), atropine (approximately 200-fold), nicotine (approximately 100-fold), and tetramethylammonium (approximately 100-fold). The alpha 8 AChR low affinity sites exhibit affinities that are similar but not identical to that of alpha 7 AChRs. Many of the pharmacological differences between the alpha 7 AChRs and alpha 8 AChRs can be attributed to the limited differences between the amino acid sequences of the N-terminal region of the alpha 7 and alpha 8 subunits because expressed alpha 7 homomers and alpha 8 homomers also exhibit these characteristic differences.

M2 muscarinoceptor-associated ionophore at the cat adrenal medulla.

Atropine and pirenzepine displaced 3H-quinuclydinyl-benzylate binding and inhibited methacholine-evoked catecholamine release with a similar order of potencies, atropine being 200 fold more potent than pirenzepine. In contrast to high-K, methacholine-evoked 45Ca uptake or catecholamine release were not blocked by (+)PN200-110. Bay-K-8644 did not modify the secretory response to methacholine either in the presence of Ca or Sr but potentiated K-evoked secretion. In depolarized glands, methacholine still evoked its usual secretory response. The results suggest that muscarinic stimulation of cat adrenal chromaffin cells stimulates Ca entry though an ionophore other than voltage-dependent Ca channels; such ionophore seems to be chemically operated through a M2 muscarinoceptor.

Secretory and radioligand binding studies on muscarinic receptors in bovine and feline chromaffin cells.

1. Muscarinic agonists enhanced catecholamine release from perfused cat adrenal glands with the following relative order of potencies: methacholine greater than oxotremorine greater than McN-A-343 greater than pilocarpine greater than bethanechol greater than muscarine. Because a continuous online electrochemical detection system was used to monitor catecholamine release, this sequence could be obtained at concentrations much lower (1-10 microM) and during much shorter stimulation times (3-30 s) than in previous reports. 2. All muscarinic agonists used secreted adrenaline preferentially over noradrenaline. Methacholine evoked a sustained, non-desensitizing response in the cat adrenal, which declined to basal levels of secretion immediately after Ca2+ removal: upon Ca2+ restoration secretion was restored to the previous plateau. 3. In addition to evoking a direct secretory response, low concentrations of methacholine, pilocarpine, bethanechol or muscarine clearly potentiated cat adrenal secretory responses evoked by pulses of nicotine (2 microM for 30 s) or high K+ (17.7 mM for 30 s). 4. [3H]Quinuclydinyl benzylate (QNB) specifically bound to cat adrenomedullary membranes with a saturating monophasic curve, suggesting a single binding site with a KD of 23 pM and a Bmax of 67 fmol (mg protein)-1. Preferential displacement by atropine over pirenzepine suggests that the binding site is associated to a M2-type muscarinoceptor. 5. Methacholine (3-300 microM) did not enhance the spontaneous catecholamine release from perfused bovine intact adrenal glands or superfused chromaffin cells. Neither did the drug affect secretion evoked by dimethylphenylpiperazinium (10 microM for 3 s) or K+ (35 mM for 3 s) from isolated superfused bovine adrenal chromaffin cells. 6. [3H]QNB bound to purified bovine adrenomedullary plasma membranes with a KD of 29 pM and a Bmax of 89 fmol (mg protein)-1. Displacement by pirenzepine suggests the presence of two binding sites (Hill coefficient = 0.64) with Ki1 of 39 nM and Ki2 of 2734 nM. 7. Because the ionophore A23187 enhanced K(+)-evoked secretion in both, bovine and cat adrenals, it seems that a similar cytosolic Ca2+ rise induced by muscarinic stimulation might constitute the underlying mechanism both to cause a secretory response per se as well as the potentiation of catecholamine release evoked by nicotinic or high K+ stimulation. However, it is unclear why the bovine behaves differently from the feline chromaffin cell as far as the muscarine-evoked effects are concerned.(ABSTRACT TRUNCATED AT 400 WORDS)

Phorbol ester activation of the neuronal nicotinic acetylcholine receptor alpha7 subunit gene: involvement of transcription factor Egr-1.

alpha-Bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells are up-regulated by long-term exposure to phorbol esters. The rise in receptor density is paralleled by an increase in transcripts corresponding to the alpha7 subunit, which is a component of this receptor subtype. Transcriptional activation of the alpha7 subunit gene is evidenced in reporter gene transfection experiments, in which phorbol esters increase alpha7 promoter activity by up to 14-fold. About 80% of this activation is abolished when at least two of the three sites for the immediate-early transcription factor Egr-1, present in the proximal promoter region of the alpha7 subunit gene, are mutated simultaneously. In addition, phorbol esters elevate both Egr-1 mRNA and Egr-1 protein levels in chromaffin cells, whereas electrophoretic mobility shift assays show that the Egr-1 component of the complexes that originate at the alpha7 promoter increases in cells treated with phorbol esters. These results suggest that the transcription factor Egr-1 is involved in triggering expression of alpha-bungarotoxin-sensitive nicotinic receptors in response to external stimuli, such as the ones resulting from phorbol ester treatment, and support our previous hypothesis that the alpha7 subunit gene is one of the specific targets for Egr-1.

GC- and E-box motifs as regulatory elements in the proximal promoter region of the neuronal nicotinic receptor alpha7 subunit gene.

The alpha7 subunit is a component of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (-77 to +43) including all of these elements gave rise to approximately 70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the alpha7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, alpha7 promoter activity increased by up to 5-fold when alpha7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the alpha7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells.

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and 45Ca2+ uptake, however H8 had no effect. The partial [3H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.

A two-dimensional electrophoresis study of phosphorylation and dephosphorylation of chromaffin cell proteins in response to a secretory stimulus.

Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two-dimensional electrophoretograms were studied with the aid of computer-assisted image analysis (CAIA). A database map of 32P-labeled proteins was constructed; approximately 500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and isoelectric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+-dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC12 cells.

Solubilization, characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla.

1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/protein ratio of 2.5, during 45 min at 4 degrees C. 2. From the rate constants of association (8.10 +/- 0.25 x 10(4) M-1 min-1) and dissociation (0.022 +/- 0.001 min-1) a Kd of 275 nM was calculated, while from saturation experiments a Kd of 270 +/- 30 nM and a density of receptors of 106 +/- 9 pmol/mg protein was obtained. 4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds. 5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [3H]Azidopine.