Biomedical subjects

D Aunis

Publications and source records attributed to D Aunis.

At least 109 records · Page 6Linked to original sources

PubMed1994

Insulin-like growth factor-I stimulates c-fos and c-jun transcription in PC12 cells.

We analyzed the effects of insulin-like growth factor-I (IGF-I), a polypeptide growth factor which exerts mitogenic effects via specific membrane receptors. The control of IGF-I on c-fos and c-jun transcription was studied in PC12 cells. Gel mobility shift assays with a labeled AP1 consensus binding sequence (TRE: TGACTCA) showed an increase in specific binding upon trIGF-treatment. Gene transfer studies revealed that the increase in AP1 binding is functional since IGF-I stimulates transcription from a reporter gene containing the minimal TRE linked to the chloramphenicol acetyl transferase (CAT) reporter gene. To further characterize the molecular mechanism by which IGF-I increases AP1 activity, we analysed the transcription regulation of c-fos and c-jun using reporter genes containing the respective promoters or specific regulatory elements. Deletion studies with the c-jun promoter, showed that IGF-I stimulates c-jun transcription via a cis acting element(s) localized within the 132 base pairs prior to the transcription start site; possibly the AP1 like element TGACATCA. Similar studies revealed that c-fos stimulation by IGF-I requires the presence of a regulatory sequence spanning the dyad symmetry element (DSE) and the fos AP1-like sequence (FAP). Further experiments using specific elements linked to the minimal unresponsive c-fos promoter, showed that the DSE is the main target for c-fos induction by IGF-I.

Animals↗

PubMed1994

Stimulation of the cyclic GMP pathway by NO induces expression of the immediate early genes c-fos and junB in PC12 cells.

Stimulation of several second messenger pathways induces the expression of immediate early genes such as c-fos, c-jun, junB, and junD, but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c-fos and junB but not of c-jun or junD is increased upon activation of cyclic GMP pathway. c-fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8-p-chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.

Animals↗

PubMed1994

Distinct heterotrimeric GTP-binding-proteins act in series to control the exocytotic machinery in chromaffin cells.

Regulated exocytosis requires both calcium and MgATP. Although the biochemical events responsible for ATP-dependent calcium-activated secretion have not been elucidated yet, some progress has been made in determining the relative order of the ATP- and calcium-dependent steps. Studies on permeabilized secretory cells have shown that MgATP acts before calcium and maintains the secretory apparatus in a "primed" state. In this paper, we examine the possible role of heterotrimeric G-proteins in these two steps of exocytosis in permeabilized chromaffin cells. We show that mastoparan and other activators of heterotrimeric G-proteins inhibit the MgATP-dependent reaction, but stimulate the late calcium-dependent step of exocytosis. Non-hydrolyzable GTP analogues (GTP-gamma-S and GMP-PNP) mimic the dual effects of mastoparan on secretion, but with different potencies, suggesting the involvement of two distinct heterotrimeric G-proteins in regulated exocytosis. GPAnt-2, a substance P related peptide known to inhibit the stimulation of Gi and Go by mastoparan, reverses, in a dose-dependent manner, both the inhibitory and stimulatory effects of mastoparan on secretion. These results indicate that two distinct heterotrimeric G-proteins from the Gi/o family may act in series in the exocytotic pathway in chromaffin cells: one controls the ATP-dependent priming step, whereas the second is involved in the late calcium-dependent fusion step which does not require ATP.

Adenosine Triphosphate↗

PubMed1993

Intracellular and extracellular processing of chromogranin A. Determination of cleavage sites.

Chromogranins are a family of acidic soluble proteins which exhibit widespread distribution in endocrine cells and neurons. Chromogranin A (CGA), the major soluble component of the secretory granules in chromaffin cells of the adrenal medulla, is a single polypeptide chain of 431 residues with an apparent molecular mass of 70-75 kDa and a pI of 4.5-5. In mature bovine chromaffin granules about 50% of the CGA has been processed. In the present paper, the structural features of the proteolytic degradation mechanism have been characterized with regard to the possible function of CGA as a prohormone, as suggested by recent studies. CGA-derived components present in chromaffin granules were subjected to either two-dimensional gel electrophoresis or HPLC and the N-terminal of each fragment was sequenced. Immunoblotting with antisera to specific sequences within the CGA molecule were used to characterize these fragments further at their C-terminal. In addition, a similar approach was performed to characterize CGA-derived fragments released into the extracellular space from directly depolarized bovine cultured chromaffin cells. Our results identified several proteolytic cleavage sites involved in CGA degradation. Intragranular processing occurs at 12 cleavage sites along the peptide chain located in both N- and C-terminal moieties of the protein; a preferential proteolytic attack in the C-terminal part was noted. We found that CGA processing also occurs in the extracellular space after release, generating new shorter fragments. The proteolytic cleavage sites identified in this study were compared with the cleavage points which are thought to be involved in generating CGA fragments with specific biological activity: pancreastatin, chromostatin and N-terminal vasostatin fragments. In addition, a new 12-amino-acid CGA-derived peptide corresponding to the sequence 65-76 was identified in the soluble core of purified chromaffin granules. This short peptide was released, together with catecholamines, after stimulation of cultured chromaffin cells suggesting its presence within the storage complex of chromaffin granules. The specific biological activity of this CGA-derived fragment remains to be determined.

Adrenal Medulla↗

PubMed1993

Human chromostatin inhibits endothelin-1-induced contractures in human blood vessels.

Isolated endothelium-denuded segments of the human internal thoracic artery (ITA) and saphenous vein (SV) have been used for characterization of vasoinhibitory effects of the chromostatin (hChs) sequence of human chromogranin A (CGA124-143). In SV preincubation with hChs inhibited the response to depolarizing high K+ in Ca(2+)-free medium in a concentration dependent manner (EC50 approximately 2 nM). At 200 nM hChs the tension response to high K+ (80 mM) was inhibited by 44% (n = 8) and the tension response to noradrenaline (2.6 microM) was inhibited by 20% (n = 6), but the tension response to endothelin-1 (65 nM) (ET-1) was not affected. In ITA no effect of hChs was observed on tension response to K+ or ET-1 in Ca(2+)-free medium. On the other hand, in Ca(2+)-containing medium the tension evoked by 65 nM ET-1 was no longer sustained in segments preincubated with 200 nM hChs and declined spontaneously to 76 +/- 12% (n = 6) of maximal tension after 6 min. A vascular function for the Chs sequence of the human CGA is thus indicated, inhibiting different components of vasoconstrictor responses in the human SV and ITA segments.

Calcium↗

PubMed1993

Exocytosis in chromaffin cells. Possible involvement of the heterotrimeric GTP-binding protein G(o).

The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, we have examined the effect of mastoparan, an activator of heterotrimeric G proteins, on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibited catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibited calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that was similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocked both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reversed in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibited calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. We conclude that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells.

Adrenal Glands↗

PubMed1993

Chromostatin, a chromogranin A-derived bioactive peptide, is present in human pancreatic insulin (beta) cells.

Chromogranin A (CGA) is a secretory protein present in the adrenal medulla and in a variety of endocrine organs. This protein may serve as precursor for pancreastatin (PST) and for other biologically active peptides. Recently, chromostatin (CST), a CGA derivative, has been identified that possesses high biological activity. The cellular distribution of CST in various endocrine organs is completely unknown. Using immunohistochemistry on plastic sections, we investigated the occurrence and cellular distribution of CST, PST, and CGA in human endocrine pancreas of healthy and diseased states and in the adrenal medulla. In the normal and diabetic pancreas, CST immunoreactivity was localized exclusively in beta cells, which were mostly unreactive for PST and CGA. Both latter peptides were confined mainly to glucagon (alpha) cells. Insulinoma cells displayed strong insulin, PST, and CGA immunoreactivities, but they were faintly immunoreactive for CST or unreactive. Adrenal chromaffin cells exhibited strong immunoreactivity for CGA but lacked CST and PST immunoreactivities. Based on the peculiar distributive pattern of CST, PST, and CGA, we suggest that CGA is differentially processed in chromaffin and islet tissues and in insulinoma cells. The unique cellular localization of CST in the endocrine pancreas of normal and pathological conditions may indicate that CST is involved in beta-cell function.

Adrenal Medulla↗

PubMed1993

Posttranslational processing of proenkephalins and chromogranins/secretogranins.

Posttranslational processing of peptide-precursors is nowadays believed to play an important role in the functioning of neurons and endocrine cells. Both proenkephalins and chromogranins/secretogranins are considered as precursor molecules in these tissues, resulting in posttranslationally formed degradation products with potential biological activities. Among the proteins and peptides of neuronal and endocrine secretory granules, the enkephalins and enkephalin-containing peptides have been most extensively studied. The characterization of the post-translationally formed degradation products of the proenkephalins have enabled the understanding of their processing pathway. Chromogranins/secretogranins represent a group of acidic glycoproteins, contained within hormone storage granules. The biochemistry, biogenesis and molecular properties of these proteins have already been studied for 25 years. The chromogranins/secretogranins have a widespread distribution throughout the neuroendocrine system, the adrenal medullary chromaffin granules being the major source of these storage components. Recent data provide evidence for a precursor role for all members of the chromogranins/secretogranins family although also several other functions have been proposed. In this review, some of the methods applied to study proteolytic processing are described. In addition, the posttranslational processing of chromogranins/secretogranins and proenkephalins, especially the biochemical aspects, will be discussed and compared. Recent exciting developments on the generation and identification of potential physiologically active fragments will be covered.

Amino Acid Sequence↗

PubMed1993

Differences in vimentin distribution in glial cells in culture revealed with an antibody against a phosphorylated epitope.

We have previously described that spatial and temporal changes in the organization of vimentin that are correlated with protein kinase C (PKC)-induced phosphorylation of vimentin can be detected with the mouse monoclonal antibody B3 in cultures of amoeboid microglia [Ciesielski-Treska et al. (1991) J. Neurosci. Res. 29, 362-378]. The antibodies were generated to native form of vimentin-containing filaments and antibody B3 reveals a restricted immunostaining of vimentin in glial cells from human, rat and mouse origin. In the present study we show the distribution of epitope B3 analyzed by immunofluorescence within defined populations of rat glial cells. Relatively high immunoreactivity was found in Type 1 astrocytes, Type 2 astrocytes and oligodendrocytes had low immunoreactivity. Although the results suggested that in Type 1 astrocytes the phosphorylated epitope is prominent, its phosphorylation was not found to be cell cycle-dependent, and appeared unrelated to the organizational changes of intermediate filaments associated with the morphological conversion of polygonal to stellate astrocytes. As expected, the immunofluorescence was increased by exposition of astrocyte cultures to an activator of PKC, confirming our previous conclusion that the immunoreactivity of the epitope B3 depends on PKC-mediated phosphorylation. In addition, the finding that the immunofluorescence of vimentin was more homogeneous in quiescent, serum-deprived astrocytes and also in astrocytes exposed to an inhibitor of protein synthesis, cycloheximide, may suggest that phosphorylation of the epitope B3 depends on a protein factor present in fetal calf serum. The immunofluorescence studies on cultures enriched in Type 2 astrocytes and in oligodendrocytes indicate that the epitope B3 is hypophosphorylated in glial cells of this lineage and becomes dephosphorylated after terminal differentiation of oligodendrocytes. These observations suggest that in Type 2 astrocytes and in oligodendrocytes the low level of phosphorylation of vimentin could be related to the down regulation in vimentin expression.

Animals↗

PubMed1993

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 CGA1-40 and CGA44-76 detected a 7 kD protein whose release was increased during stimulation. This component co-migrated with synthetic CGA1-76, was not immunoreactive to antisera to CGA79-113 or CGA124-143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an approximately 18 kD component, which stained with antisera to CGA1-40, CGA44-76 and CGA79-113, but not to chromostatin (CGA124-143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1-40, CGA44-76 and CGA79-113. 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1993

Secretogranin II: regulation of synthesis and post-translational proteolysis in bovine adrenal chromaffin cells.

Secretogranin II (SgII), 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), SgII is found in adrenal medullary chromaffin granules. In the present study we investigated the regulation of SgII 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 SgII band was identified by sequence analysis using the Edman degradation procedure. The radioactivity incorporated in the 86 kDa SgII band was used as an index of the SgII 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 SgII synthesis. In contrast direct depolarization with K+ may not be sufficient to induce modifications in SgII synthesis suggesting that the raise of cytosolic calcium evoked by high K+ may not be sufficient to induce modifications in SgII synthesis . The possible second messenger pathways involved in the control of SgII 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 SgII 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 SgII synthesis.

Adenylyl Cyclase Inhibitors↗

PubMed1992

Chromogranin A processing in sympathetic neurons and release of chromogranin A fragments from sheep spleen.

Chromogranin A (CGA) has been localized to the large dense cored vesicles (LDV) of sympathetic neurons. SDS-PAGE and immunoblotting of soluble LDV proteins from ox and dog adrenergic neuronal cell bodies, axons and nerve terminals, revealed an increasing number of CGA-immunoreactive forms, consistent with proteolytic processing during axonal transport. Splenic nerve electrical stimulation (10 Hz, 2 min) revealed that, apart from CGA, these CGA-processing products are released from the sheep spleen. The secretion of CGA-derived fragments from sympathetic neurons might suggest a role in the regulation of synaptic transmission.

Adrenergic Fibers↗

PubMed1992

Cellular localization of the neural cell adhesion molecule L1 in adult rat neuroendocrine and endocrine tissues: comparisons with NCAM.

The tissue distribution and cellular localization of the neural cell adhesion molecule L1 was determined by immunocytochemistry at the optical and ultrastructural levels in adult rat neuroendocrine tissues and pancreatic endocrine cells. L1 was found to be abundant in the neurohypophysis but undetectable in the rest of the pituitary gland. It was barely detectable in the normal rat endocrine pancreas, but a rat pancreatic insulinoma cell line was found by immunofluorescence to express low levels of L1. In the adrenal medulla, it was present on a sub-population of chromaffin cells and its density appeared to be lower on surfaces exposed to the extracellular matrix. Double immunolabelling showed this sub-population to consist of noradrenergic chromaffin cells. Adrenergic chromaffin cells were found not to express L1. In addition, the tissue distribution and cellular localization of NCAM mRNAs was determined by in situ hybridization, extending our previous studies on the cellular expression of NCAM proteins in endocrine and neuroendocrine tissues. This confirmed that the NCAM message has a wider cellular distribution than L1 within the hypophysis and the adrenal gland. In addition to secretory cells, L1 immunoreactivity was detected in glial cells, in particular in the pituicytes of the neurohypophysis, which further distinguishes them from astrocytes, their counterparts in the central nervous system. These data are discussed in terms of the different embryological origins of the various endocrine tissues examined and also in terms of the specific design constraints imposed on these tissues during their development.

Adrenal Glands↗

PubMed1992

Expression of cell adhesion molecules and catecholamine synthesizing enzymes in the developing rat adrenal gland.

Cell adhesion molecules play a major role in determining tissue architecture during histogenesis. This immunocytochemical study of the adrenal gland examines the embryonic and early postnatal cellular expression of two neural cell adhesion molecules, NCAM and L1, which are widely expressed in brain and have been found also to be expressed in the adult rat adrenal gland. In parallel, antibodies directed against two neuroendocrine cell markers, tyrosine hydroxylase and phenylethanolamine N-methyltransferase, were employed to verify the phenotypic nature of developing chromaffin cells in order to correlate cell adhesion molecule expression with the state of chromaffin cell differentiation. NCAM was found to be expressed by chromoblasts within extra-adrenal blastema (i.e. before their migration into the cortical primordium) at the 16th day of embryonic life. It continued to be expressed by all developing chromaffin cells after their infiltration into the developing adrenal gland at all ages. L1 was also expressed by chromoblasts in extra-adrenal sites, but was found only in a subpopulation of chromaffin cells within the cortical primordium from the 16th embryonic day onwards. Those chromoblasts which expressed L1 constituted relatively large compact cell clusters within the gland at this stage, while intra-adrenal chromaffin cells not expressing L1 were dispersed in small cell groups. L1 was also strongly expressed by nerve fibres (and their surrounding Schwann cells) which appeared to innervate cell groups as early as the 16th embryonic day. Both extra- and intra-adrenal chromoblasts expressed tyrosine hydroxylase, but the large L1-positive cell aggregates were less intensely immunoreactive for tyrosine hydroxylase than were cells in small groups. PNMT expression was restricted to L1-negative intra-adrenal chromoblasts present in small groups. Ultrastructural observations demonstrated that cells expressing L1 contained few secretory granules at the 18th embryonic day. It is concluded from these data that these chromoblasts are the precursors of the noradrenergic cells found in the mature gland. In addition, the arrangement of noradrenergic chromaffin cells in the form of homotypic cell groups throughout the course of histogenesis of the adrenal medulla is likely to be a direct consequence of the exclusive co-expression of both NCAM and L1 by this subpopulation of maturing chromaffin cells.

Adrenal Glands↗

PubMed1992

Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.

Adenosine Diphosphate↗

PubMed1992

Chromostatin inhibits catecholamine secretion in adrenal chromaffin cells by activating a protein phosphatase.

Chromostatin is a 20-residue peptide derived from chromogranin A (CGA), the major soluble component of secretory granules in adrenal medullary chromaffin cells. One known biological function of chromostatin is to inhibit the secretagogue-evoked catecholamine secretion from chromaffin cells. Putative receptors are present on the chromaffin-cell plasma membrane, and the activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels. We report here that exposure of chromaffin cells to chromostatin modifies neither cAMP and cGMP levels nor protein kinase C activity but does provoke the activation of soluble protein phosphatase (PPase) type 2A in a dose-dependent manner compatible with the peptide concentration inhibiting catecholamine secretion. The activation of the PPase as well as the inhibition of both secretagogue-induced Ca2+ entry and catecholamine secretion by chromostatin were all blocked by okadaic acid, a specific PPase inhibitor. We suggest that chromostatin directly or indirectly stimulates PPase-2A, dephosphorylating a target protein and lowering its activity in the secretory process.

Adrenal Medulla↗

PubMed1992

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.

One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.

Adrenal Glands↗

PubMed1992

Voltage-gated Ca entry in isolated bovine capillary endothelial cells: evidence of a new type of BAY K 8644-sensitive channel.

Isolated bovine capillary endothelial cells have been examined for voltage-dependent Ca entry. All cells displayed a low threshold activity, with the main characteristics of a T-type transient current, when examined using whole-cell recording for activation and inactivation and cell-attached conditions or inside-out patches for the elementary conductance (8 pS). 25% of the cells displayed an additional sustained current in 5 mM CaCl2 above -40 mV, which was enhanced by application of BAY K 8644, but almost insensitive to superfusion with nicardipine. Two types of channels (2.8 and 21 pS, in 110 mM BaCl2) were shown to have a BAY K 8644 sensitivity. The large conductance channels were L-type channels. The smaller events were elicited at more hyperpolarized potentials (by some 30 mV). Their mean open time was 16 ms in control conditions. In presence of BAY K 8644, additional long open times were observed (up to 100 ms as compared to 7.8 ms for the time constants of the slow mode of the L-type channel). We refer to these channels as SB channels: of small conductance and sensitive to BAY K 8644. In the presence of nicardipine, SB channels are not noticeably modified, in contrast to the L-type openings which are abolished. Also, SB open times are close to control values when nicardipine is added after a BAY K 8644 application. We suggest that, at physiological concentrations of divalent ions, an SB-type activity is elicited above -40 mV which generates the low threshold sustained current.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗