Identification and characterization of glycoproteins after extraction of bovine chromaffin-granule membranes with lithium di-iodosalicylate. Purification of glycoprotein II from the soluble fraction.

Chromaffin-granule membranes were separated into insoluble and soluble fractions after extraction with lithium di-iodosalicylate (LDIS). These fractions were characterized by one- and two-dimensional gel electrophoresis, and glycoproteins were detected after electroblotting with peroxidase-labelled concanavalin A and wheat-germ agglutinin (WGA). The LDIS-insoluble fraction contained components identified as glycoproteins III, H, J and K (carboxypeptidase H). Microsequence analysis indicated that component J is an N-terminally extended form of glycoprotein K. A major glycoprotein, GpII (Mr 80,000-100,000), present in the LDIS-soluble fraction was purified by affinity chromatography on WGA-Sepharose. This was characterized by one- and two-dimensional gel electrophoresis with Coomassie Blue staining, by amino acid analysis and automated N-terminal sequence analysis. Extraction of chromaffin-granule membranes with LDIS is a simple and rapid procedure that facilitates studies concerned with the structure and function of membrane glycoproteins from these and other secretory granules.

Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids.

A 74-kDa protein (adseverin) derived from adrenal medulla severs actin filaments and nucleates actin polymerization in a Ca2(+)-dependent manner but does not form an EGTA-resistant complex with actin monomers, which is different from the gelsolin-actin interaction. The dissociation of gelsolin-actin complexes by phosphatidylinositol 4,5-bisphosphate (PIP2) and the inhibitory effect on actin filament severing by gelsolin was recently reported. This study shows that the activity of adseverin is inhibited not only by PIP2 but also by some common phospholipids including phosphatidylinositol (PI) and phosphatidylserine (PS). Other phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) showed no effect. The addition of PC or PE to PI diminished the inhibitory effect of PI. Triton X-100 and neomycin were also found effective in suppressing the effect of PI, suggesting that the arrangement of polar head groups is important in exerting the inhibitory effect. Ca2(+)-dependent binding of adseverin to PS liposomes but not to PC or PE liposomes was observed by a centrifugation assay.

Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA.

Secretogranin II is a very acidic, tyrosine-sulfated protein found in secretory granules of cells belonging to the diffuse neuroendocrine system. It gained more general importance recently as a universal immunohistochemical marker for endocrine neoplasms. Sequence information was obtained from secretogranin II isolated from bovine anterior pituitaries, allowing the isolation of cDNA clones and deduction of its primary structure. Bovine secretogranin II is a 586-amino acid protein of 67,455 Da which is preceded by a signal peptide of 27 residues and contains 9 pairs of basic amino acids in its sequence which are used as potential cleavage sites for generation of physiologically active peptides. Moderately abundant mRNA levels were found in adrenal medulla, pituitary, hippocampus, and caudate. Secretogranin II message was absent from parathyroid gland, adrenal cortex, kidney, liver, and spleen. Depolarization of isolated chromaffin cells by various secretagogues significantly up-regulated secretogranin II mRNA levels by mechanisms distinct from those established for chromogranins and neuropeptides, components maintained along with secretogranin II in neuroendocrine storage vesicles.

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2.

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.

Secondary structure characteristics of proenkephalin peptides E, B, and F.

The conformations of three adrenal medullary enkephalin containing polypeptides (ECPs) were investigated to gain an understanding of their potential structure-activity relationships. Secondary structure characteristics of peptides E, B, and F were examined by circular dichrosim (CD) under conditions designed to mimic both the soluble state and the anisotropic environment which exists at the biological effector site. Conformational differences between the three peptides were further examined by Fourier Transform Infrared Spectroscopy (FTIR) and by empirical predictions for conformation and hydrophobic periodicity. Although all three peptides have a similar structure, existing in random configurations in aqueous solutions, they do exhibit unique individual potentials to assume secondary structure in less polar environments. These conformational differences may be important factors in determining their unique individual biological activities.

Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of neurofilament (NF) proteins; and for microtubule-associated proteins were applied. Morphologically, two major cell types could be distinguished, i.e., "large" cells with pale nuclei and ill-defined cytoplasm, which were present from 9 weeks GA on, and clusters of "small," primitive appearing cells, present from 14 weeks GA on. The large cells were immunoreactive for chromogranin A, synaptophysin, tyrosine hydroxylase, and NF proteins, similar to adult chromaffin cells. In contrast, small cells expressed NF proteins and tyrosine hydroxylase, but not chromogranin A or synaptophysin, more resembling ganglion cells in the adult adrenal medulla. At the latest developmental stages large cells were observed in the center of the clusters of "small" cells, which morphologically resembled immature ganglion cells and expressed NF proteins in their perikarya. These observations indicate that chromaffin and ganglion cells establish their immunophenotype early in embryogenesis. They suggest that "large" and "small" cells are progenitors of the chromaffin and the ganglion cells, respectively, of the mature adrenal medulla.

Presence of pancreatic trypsin inhibitor in adrenal medullary chromaffin cells.

A bovine pancreatic trypsin inhibitor was isolated from bovine adrenal medullary chromaffin granules. Its N-terminal sequence is: arg-pro-asp-phe-cys-leu-glu-pro-pro-tyr-thr-gly-pro-cys-lys-ala-arg-ile- arg-tyr- phe-tyr-asn-ala-lys-ala-gly-leu-cys-gln-thr-phe-val-tyr-gly-gly-cys-arg- ala-lys-arg-asn-asn-phe-lys- which corresponds precisely with the N-terminus of Bovine Pancreatic Trypsin inhibitor. The presence of this inhibitor in these granules suggests another method of regulating the prohormone proteases present there.

The distribution of galanin message-associated peptide-like immunoreactivity in the pig.

Using a radioimmunoassay (RIA) developed to the N-terminal part of the predicted sequence of porcine galanin message-associated peptide (GMAP), we have confirmed the existence of GMAP-like immunoreactivity (-LI) in normal porcine tissues. GMAP-LI was found to parallel the distribution of galanin-immunoreactivity (-IR), although consistently the concentrations detected were, on a molar ratio, significantly less than those measured for galanin throughout the gastrointestinal tract, brain, spinal cord, adrenal and pituitary gland. As cleavage of the prohormone would be expected to produce galanin and GMAP on an equimolar basis, it is possible that the endogenous, intact GMAP peptide does not fully cross-react with the antibody raised to the N-terminal GMAP sequence. Gel chromatography of tissue extracts revealed a single molecular form of galanin-IR in the gut and four distinct molecular forms in the adrenal gland. GMAP-LI eluted as a single immunoreactive component in the gut, and in the adrenal gland there were two major molecular forms, one of which was apparently also detected by the galanin assay, and a small amount of N-terminal fragment. This molecular heterogeneity seems likely to be a result of the various possible prohormone cleavage products and/or posttranslational processing modifications. Further analysis of the galanin gene products needs to be undertaken in order to confirm this.

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.

Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum.

Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.

Purification and primary structure of pro-aldosterone secretion inhibitory factor from bovine adrenal chromaffin cells.

This report documents the purification and the complete primary structure of bovine aldosterone secretion inhibitory factor precursor (pro-ASIF). ASIF-(1-103) contains at position 69-103 of its carboxy-terminal end the formely identified 35-amino acid biologically active form, hence confirming the endogenous character of ASIF in the adrenal medulla. Compared to atrial natriuretic factor (ANF)-related peptide precursors, bovine ASIF displays 65% homology at the carboxy-terminal while the remaining amino-terminal part shows much more variability. Bovine pro-ASIF exhibits 73% homology with porcine pro-brain natriuretic peptide (BNP), a situation reminiscent of the relationship of pro-ANF in various species. When ANF- and BNP-related COOH-termini of bovine, porcine, human, rat, and chicken are compared, it appears that bovine ASIF and porcine BNP are closely related and belong to the same family which however appears to be much more heterogenous than the ANF-related family. These results strongly suggest that bovine ASIF is encoded by a precursor gene similar to the gene of BNP but different from the one encoding ANF.

Localisation of chromogranin A and B, met-enkephalin-arg6-gly7-leu8 and PGP9.5-like immunoreactivity in the developing and adult rat adrenal medulla and extra-adrenal chromaffin tissue.

The localisation of chromogranins A and B, met-enkephalin-arg6-gly7-leu8 (met-enk 8) and protein gene product 9.5 (PGP 9.5) in the adrenal medulla and extra-adrenal chromaffin tissue has been studied in the developing rat by immunogold-silver staining. In the adult rat adrenal the cytoplasm of all medullary chromaffin cells showed a positive response with chromogranin A and B; in each case occasional groups of cells with a low reactivity that may have been NA cells were seen. Chromogranin A was first detected in adrenal medullary and extra-adrenal chromaffin cells at 18 days of gestation whilst chromogranin B was not detected in animals younger than 7 days. In 15 days old animals the adrenal medullary response to A and B was of the same intensity as that seen in the adult. Less than 1% of adult medullary chromaffin cells were responsive to met-enk 8 staining and medullary cells were unreactive in the fetus, with only extra-adrenal chromaffin tissue responding prenatally. During the first postnatal week immunoreactive cells appeared in the adrenal medulla in considerably greater proportions than in the adult gland. In contrast, positively stained nerve terminals associated with chromaffin cells and abundant in the adult adrenal were not detected during the first week of life. Immunoreactive nerve terminals were first seen early in the second week of life at a time when positive chromaffin cells were becoming less common. PGP 9.5 was located in all chromaffin cells of the adult adrenal and was readily detected in chromaffin cells in the adrenal and in extra-adrenal locations of the earliest stage examined (E16). Our findings suggest that the ontogenesis of the chromogranin-like immunostaining reflects the maturation of chromaffin granules and the PGP 9.5 immunostaining detected a protein common to cells of neuronal origin and expressed at an early stage of differentiation. The reciprocal relationship between the presence of enkephalins in chromaffin cells and in their presynaptic terminals merits further investigation.

Subcellular distribution of 65,000 calmodulin-binding protein (p65) and synaptophysin (p38) in adrenal medulla.

Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.

Immunocytochemical localization of neuropeptides in the adrenal medulla.

The distribution of neuropeptides exhibits pronounced interspecies heterogeneity. Neuropeptides may function as hormones secreted from chromaffin cells or as neurotransmitters/neuromodulators released from nerve terminals. However, other possible functions such as trophic or intracellular effects should also be considered. Thus, to understand the role of neuropeptides, it is important to explore their localization in different species. The distribution of enkephalins, neurotensin, neuropeptide Y, calcitonin gene-related peptide, and galanin in the adrenal medulla of rat, cat, hamster, and mouse is presented in detail.

In vivo demonstration of a paracrine, inhibitory action of Met-enkephalin on adrenomedullary catecholamine release in the rat.

The present study was an attempt to assess the inhibitory effect of methionine-enkephalin (Met-Enk) on adrenal catecholamine release under in vivo conditions employing a microdialysis system. One adrenal gland of intact male rats was implanted with a microdialysis system. One day after surgery, the adrenal dialysis system was connected to a perfusion pump and Ringer solution or a Ringer solution containing Met-Enk, naloxone (Nal), or a combination of Met-Enk and Nal was used for dialysis; dialysate fractions were collected at 5-min intervals. Catecholamine secretion was stimulated by an iv injection of 1.4 mumol (200 micrograms) acetylcholine (Ach). Met-Enk-immunoreactive material in adrenal medulla extracts and dialysate fractions was analyzed by reverse phase HPLC combined with Met-Enk RIA. Under resting conditions, adrenal release rates of norepinephrine and epinephrine into the Ringer solution were constant. After Ach application secretion of both catecholamines increased about 2.7-fold. Within 10 min after the injection, catecholamine levels returned to baseline levels. Intra-adrenal application of Met-Enk reduced Ach-stimulated epinephrine, but not norepinephrine, secretion significantly; application of Nal did not affect Ach-stimulated catecholamine secretion in the initial fraction after Ach injection, but significantly prolonged amine secretion after the cholinergic stimulus. Application of Nal followed by a combined application of Met-Enk and Nal was without an effect on the amount of catecholamines released in the initial fraction after Ach injection compared to that in the control group. Thus, naloxone prevented the inhibitory effect of Met-Enk on Ach-stimulated CA release. HPLC analysis of adrenal medulla extracts followed by Met-Enk RIA revealed that several forms of Met-Enk are present in chromaffin cells, whereas in adrenal dialysates only one, albeit broad, signal of Met-Enk immunoreactivity was detectable. We demonstrate in vivo a paracrine or autocrine action of Met-Enk on Ach-stimulated catecholamine release by applying the peptide directly into the adrenal gland via a microdialysis system. We conclude that neuropeptides endogenous to the adrenal gland not only reduce the amplitude of catecholamine secretion in response to an Ach stimulus but, in addition, modulate the duration of catecholamine secretion. This is demonstrated by a prolonged catecholamine secretion if opiate receptors are blocked by Nal.

Immunohistochemistry of chromogranin A and B, and secretogranin II in the canine endocrine pancreas.

The chromogranins are large acidic proteins contained in secretion granules of adrenal medullary cells. These proteins are also presumed to be regular constituents of other endocrine cells, e.g., pancreatic endocrine cells. In our previous immunohistochemical studies performed in the endocrine pancreas of 10 mammalian species, only canine endocrine cells lacked any immunoreactivity for chromogranin A. Therefore, this problem was reinvestigated in the present study. Antisera against bovine and rat chromogranin A, B, and C (= secretogranin II) were applied under various conditions of the immunohistochemical protocol. Upon meeting certain requirements, chromogranin-immunoreactivities were found also in canine pancreatic B- (insulin), PP- (pancreatic polypeptide), and EC- (enterochromaffin) cells. The immunohistochemical data suggest that canine chromogranins partially differ structurally or immunologically from bovine chromogranins and completely from rat chromogranins. In addition, the present findings confirm our previous findings about both interspecies differences in the cellular localization of chromogranins in the endocrine pancreas and the peculiar localization of secretogranin II to pancreatic PP-cells. Finally, the present methodological studies have shown that even the buffer used in the immunohistochemical protocol may be decisive for a false-positive or false-negative immunostaining.

Acute peripheral catecholaminergic changes in rat after MPTP and MPP+ treatment.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the peripheral catecholaminergic system of the rat were investigated. MPTP and MPP+ injections (20 mg/kg i.p.) caused a marked acute depletion of heart noradrenaline, up to 75% twelve hours after the administration, and a decrease of adrenal gland adrenaline. The time-course of the effect of MPTP and MPP+ is reported, together with a decrease in the tyrosine hydroxylase activity after MPTP treatment, more evident in the adrenal glands. Pargyline (50 mg/kg i.p.) is not able to prevent such a neurotoxic peripheral effect.

Effect of atrial natriuretic factor (ANF) on nicotinic acetylcholine receptor channels in bovine chromaffin cells.

Bovine chromaffin cells have binding sites for rat atrial natriuretic factor (ANF), as demonstrated autoradiographically by using the 125I-labelled peptide. Patch-clamp recording revealed that ANF reduces acetylcholine (ACh)-induced membrane currents in chromaffin cells at physiological membrane potentials. The effect was dose-dependent with the IC50 value being 5.2 microM ANF and the Hill coefficient close to 1. The channel block was absent at positive membrane potentials, indicating a non-competitive interaction of ANF with the open ACh receptor channel. Fragments of ANF had a much less pronounced action, which is possible due to their structure and molecular charge being different to ANF. The block of nicotinic ACh receptor channels may enable ANF to control the secretion of catecholamines from adrenal chromaffin cells.

An increased pool of secretory hormones and peptides in adrenal medulla of stroke-prone spontaneously hypertensive rats.

Secretory components of the adrenal medulla were compared in normotensive Wistar-Kyoto (WKY) rats and in stroke-prone spontaneously hypertensive rats (SHRSP) at both 4 and 12 months of age. Noradrenaline, adrenaline, dopamine, neuropeptide Y, and chromogranins A and B were significantly higher in adrenal glands of SHRSP than those of WKY rats at 4 months. At 12 months, the levels of these components in SHRSP had increased even more (about 200% in WKY rats). There was no change in the relative composition of the adrenal "secretory cocktail." Neither the chromogranin A/chromogranin B ratio nor their apparent proteolytic processing in chromaffin granules differed between SHRSP or WKY rats. The lack of a significant change in membrane-bound cytochrome b561 and the small increase in dopamine beta-hydroxylase suggest that the higher levels of secretory components in SHRSP are not simply caused by an increase in the number of chromaffin granules, but possibly by a selective increase in the secretory content of these organelles providing a larger package for quantal release by exocytosis. This may be relevant for the elevation of blood pressure in this strain. The immunological methods described in this paper allow for the first time a determination of the secretory quantal levels in catecholamine storage. This should be useful for further studies in hypertensive models.