The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. An electron microscope study.

The authors of this study examine the question of whether the so-called enterochromaffin or argentaffin cells of the gastrointestinal tract should be considered as a single cell type. The systematic application of purely morphologic methods has led to the conclusion that the epithelium of the gastrointestinal mucosa comprises endocrine cells of several types. This conclusion is primarily based on the uneven and characteristic distribution of the various cell types along the intestinal tract, an observation precluding the interpretation that the different types correspond to diverse functional stages of the same cell. A specific endocrine function may be attributed to each of the given cell types recognized so far on account of their appearance and their localization in characteristic areas of the gastrointestinal tract. It is acknowledged, however, that a purely morphological study leaves room for doubt. The first cell type is probably responsible for the formation of 5-hydroxytryptamine. Cells of type II are morphologically comparable to the pancreatic A cells and may, therefore, be called intestinal A cells. Cell type III comprises intestinal D cells since their appearance corresponds to that of pancreatic D cells. Cell type IV might well be responsible for catecholamine production, whereas gastrin is in all probability produced in endocrine cell type V. As yet, the thorough morphological study of the gastrointestinal epithelium does not provide information as to additional distinct cellular sites of production of the several other hormones isolated from different parts of the gut.

Characterization of an unusual catecholamine-containing cell type in the toad hypothalamus. A correlated ultrastructural and fluorescence histochemical study.

A nucleus of catecholamine-containing cells bordering the preoptic recess of the toad hypothalamus has been studied by both fluorescence histochemical and electron microscopic methods. The perikarya of these cells form one to three rows immediately subjacent to the ependyma. They send brightly fluorescent apical processes between the ependymal cells to the ventricular surface, and also give rise to long basal processes, the proximal portions of which are also fluorescent. These cells contain two distinctive constitutents: juxtanuclear bundles of tightly packed filaments, the members of which are separated from one another by only approximately 100 A, and large numbers of dense-cored vesicles (400-2200 A in diameter), which appear to arise from an agranular tubular reticulum distinct from the Golgi apparatus. Axons containing either clear vesicles alone or clear and dense-cored vesicles form synapses on the subependymal cells, but no evidence has been found that the subependymal cells themselves form presynaptic contacts, or that axons originate from them. The cytological characteristics of these catecholamine-containing cells, plus the fact that they border directly on the cerebrospinal fluid, suggest that they may be more closely related to peripheral chromaffin cells than to the other cell types intrinsic to the central nervous system, and the name "encephalo-chromaffin cells" is therefore proposed for them. The possible functions of such cells in the central nervous system are discussed.

Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.

We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.

Nerve growth factor-induced changes in the intracellular localization of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells.

High levels of the neuron-specific protein kinase C substrate, B-50 (= GAP43), are present in neurites and growth cones during neuronal development and regeneration. This suggests a hitherto nonelucidated role of this protein in neurite outgrowth. Comparable high levels of B-50 arise in the pheochromocytoma PC12 cell line during neurite formation. To get insight in the putative growth-associated function of B-50, we compared its ultrastructural localization in naive PC12 cells with its distribution in nerve growth factor (NGF)- or dibutyryl cyclic AMP (dbcAMP)-treated PC12 cells. B-50 immunogold labeling of cryosections of untreated PC12 cells is mainly associated with lysosomal structures, including multivesicular bodies, secondary lysosomes, and Golgi apparatus. The plasma membrane is virtually devoid of label. However, after 48-h NGF treatment of the cells, B-50 immunoreactivity is most pronounced on the plasma membrane. Highest B-50 immunoreactivity is observed on plasma membranes surrounding sprouting microvilli, lamellipodia, and filopodia. Outgrowing neurites are scattered with B-50 labeling, which is partially associated with chromaffin granules. In NGF-differentiated PC12 cells, B-50 immunoreactivity is, as in untreated cells, also associated with organelles of the lysosomal family and Golgi stacks. B-50 distribution in dbcAMP-differentiated cells closely resembles that in NGF-treated cells. The altered distribution of B-50 immunoreactivity induced by differentiating agents indicates a shift of the B-50 protein towards the plasma membrane. This translocation accompanies the acquisition of neuronal features of PC12 cells and points to a neurite growth-associated role for B-50, performed at the plasma membrane at the site of protrusion.

CNTF, FGF, and NGF collaborate to drive the terminal differentiation of MAH cells into postmitotic neurons.

The differentiation of neuronal cell progenitors depends on complex interactions between intrinsic cellular programs and environmental cues. Such interactions have recently been explored using an immortalized sympathoadrenal progenitor cell line, MAH. These studies have revealed that depolarizing conditions, in combination with exposure to FGF, can induce responsiveness to NGF. Here we report that CNTF, which utilizes an intracellular signaling pathway distinct from that of both FGF and NGF, can collaborate with FGF to promote efficiently the differentiation of MAH progenitor cells to a stage remarkably reminiscent of NGF-dependent, postmitotic sympathetic neurons. We also find that similar collaborative interactions can occur during transdifferentiation of normal cultured chromaffin cells into sympathetic neurons.

Mechanisms determining the time course of secretion in neuroendocrine cells.

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.

Opioid peptide modulation of Ca(2+)-dependent K+ and voltage-activated Ca2+ currents in bovine adrenal chromaffin cells.

Opioid peptides are abundantly expressed in the adrenal medulla, and there is evidence that they may be released presynaptically or as medullary paracrine agents. To assess the physiological relevance of these observations, we investigated opioid effects on ionic currents from cultured bovine adrenal chromaffin cells. Under whole-cell path-clamp conditions, opioid peptides, acting via a mu-type opioid receptor, strongly potentiated the large conductance Ca(2+)-dependent K+ (BK) channel current. Opioids also inhibited voltage-activated Ca2+ currents. Application of opioid peptides to the extracellular face of outside-out patches also increased opening activity of single BK channels, suggestive of tight receptor-channel coupling. This potentiating effect on BK current, combined with the inhibition of Ca2+ current, indicates that opioids may have an inhibitory influence on secretory activity of the adrenal medulla. The widespread distribution of the BK channel class suggests that the significance of its modulation by opioids could also extend beyond the adrenal gland.

Chromatin structure as a molecular marker of cell lineage and developmental potential in neural crest-derived chromaffin cells.

Adrenal medullary chromaffin cells have the capacity to transdifferentiate into sympathetic neurons. We show here that SCG10, a neural-specific gene that is induced during this transdifferentiation, is maintained in mature chromaffin cells in a potentially active chromatin conformation marked by two DNAase I hypersensitive sites (HSS). A low level of transcription is associated with this conformation. The HSS are also present in neurons expressing high levels of SCG10, but not in nonneuronal cells. Experiments using transgenic mice suggest that these HSS can in principle form in any cell type expressing the gene, but that a cis-repression mechanism normally prevents their assembly in nonneuronal cells. We suggest that the SCG10 HSS may represent a molecular marker of the lineage and phenotypic plasticity of chromaffin cells.

Recombinant scinderin enhances exocytosis, an effect blocked by two scinderin-derived actin-binding peptides and PIP2.

The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca(2+)-dependent F-actin-severing protein) potentiated Ca(2+)-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP1 and Sc-ABP2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous gamma-actin, or phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 effect was blocked by peptide Sc-PIP2BP (with sequence corresponding to a PIP2-binding site of scinderin). Truncated scinderin254-715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP1, Sc-ABP2, and gamma-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.

The Wnt-1 proto-oncogene induces changes in morphology, gene expression, and growth factor responsiveness in PC12 cells.

The product of the Wnt-1 proto-oncogene is a secreted glycoprotein that is normally produced in regions of the embryonic neural tube. We show here that expression of mouse Wnt-1 cDNA in the rat PC12 pheochromocytoma cell line causes a dramatic conversion from a round to a flat cell morphology. In addition, PC12 cells expressing Wnt-1 (PC12/Wnt-1) fail to extend neurites after treatment with NGF, despite the presence and activation of high affinity NGF receptors encoded by the trk gene and the induction of early response genes. Furthermore, PC12/Wnt-1 cells fail to express several neuron- and chromaffin-specific genes, indicating that PC12/Wnt-1 cells have assumed a new phenotype. Although NGF and FGF utilize similar signal transduction pathways in PC12 cells, only FGF is capable of inducing a morphological response and synthesis of transin mRNA in PC12/Wnt-1 cells.

Meta-analysis of microarray-derived data from PACAP-deficient adrenal gland in vivo and PACAP-treated chromaffin cells identifies distinct classes of PACAP-regulated genes.

Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes also regulated by either TNFalpha or TGFbeta, two first messengers acting on transcription mainly through NFkappaB and Smads, respectively.

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.

Down-regulation and recycling of the nitrobenzylthioinosine-sensitive nucleoside transporter in cultured chromaffin cells.

The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h.

Stimulation of cAMP formation by prostaglandin D2 in primary cultures of bovine adrenal medullary cells: identification of the responsive population as fibroblasts.

In primary cultures of bovine adrenal medulla, chromaffin cells responded to prostaglandin (PG) E2 by stimulating phosphoinositide metabolism (Yokohama et al. (1988) J. Biol. Chem. 263, 1119-1122). In contrast, nonchromaffin cells were found to respond to PGD2 by elevating their intracellular cAMP level. The formation of cAMP was detected at as low as 0.1 nM PGD2 and increased more than 100-fold over the basal level at 0.1 microM, and the response was specific for PGD2 (greater than PGE1 greater than PGE2 greater than PGF2 alpha = PGI2). The magnitude of cAMP formation and its specificity to PGD2 were retained throughout a 40-day culture period. Based on the inhibitory effect of cis-4-hydroxy-L-proline, an inhibitor of collagen synthesis, on cAMP formation, morphology, and immunoreactivity of cells to anti-collagen type I antiserum, the responsive cells were identified as fibroblasts. These results taken together demonstrate that the adrenal medulla is composed of chromaffin and nonchromaffin cells, which respond to PGE2 and PGD2, respectively, by two different signal transduction pathways. The cAMP formation by PGD2 was also observed in fibroblasts from bovine embryonic trachea among cell lines tested, suggesting that some populations of fibroblasts responsive to PGD2 exist in various tissues and may discriminate the signal from that of PGE1 or PGE2.

Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

Dose-dependency in spatial dynamics of [Ca2+]c in adrenal chromaffin cells.

The spatial dynamics of cytosolic Ca2+ concentration, [Ca2+]c, in guinea pig adrenal chromaffin cells was monitored by a digital image analysing technique using Fura-2. When a freshly isolated cluster of cells was stimulated with lower concentrations of carbachol (CCh; 0.3-1 microM), the [Ca2+]c began to increase in the region beneath the plasma membrane facing the extracellular environment. The [Ca2+]c increase depended on the presence of extracellular Ca2+ ([Ca2+]o). CCh at a higher concentration (100 microM), however, caused [Ca2+]c increase even in the absence of [Ca2+]o. These results are compatible with the view that the receptor activation with a physiological concentration of secretagogue accelerates Ca2+ entry, and that stimulation with a higher concentration of the secretagogue induces small transient Ca2+ release from intracellular stores and predominant continuous Ca2+ entry.

Ca2+ signalling in rat chromaffin cells: interplay between Ca2+ release from intracellular stores and membrane potential.

Cytosolic Ca2+ oscillations are physiologically important in a range of excitable and non-excitable cells. The combined techniques of whole-cell patch clamp and photometric measurement of cytosolic Ca2+ has enabled us to identify the components of Ca2+ spiking in rat chromaffin cells. We show that Ca2+ oscillations continue at a fixed membrane potential and that infusion of the InsP3 receptor antagonist, heparin, substantially blocked the cytosolic Ca2+ spikes. However, even in the presence of heparin we observed spikes of membrane potential depolarization due to the repetitive activation of a transient inward cation current. We conclude that Ca2+ oscillations are dependent on Ca2+ release from heparin sensitive Ca2+ stores and possibly on Ca2+ entry associated with the repetitive activation of a transient cation current. The depolarizing action of the cation current would, in turn, recruit voltage-sensitive Ca2+ channels and further Ca2+ entry would augment the cytosolic Ca2+ spikes. Our results demonstrate that Ca2+ oscillations in rat chromaffin cells are due to a complex interplay of Ca2+ entry and Ca2+ release.

[Ca2+]i oscillations in rat chromaffin cells: frequency and amplitude modulation by Ca2+ and InsP3.

Rat chromaffin cells in primary culture exhibit oscillations of cytosolic Ca2+ concentration, sustained by the rhythmic discharge of Ca2+ from specialized intracellular stores. Each Ca2+ spike starts from a discrete region of the cell (pacemaker), and then propagates across the entire cytosol. Spike initiation and propagation, governing the oscillation frequency and amplitude respectively, appeared to be controlled by different mechanisms. The pacemaker was found to be directly activated by increases of cytosolic Ca2+ concentration obtained by either K+ depolarization or nicotinic stimulation. On the other hand, muscarinic or B2 stimulation was required for an efficient spreading to occur, thus suggesting a key role of InsP3 in the signal propagation. The pacemaker displayed an autonomous activity, as documented by the presence of local Ca2+ discharges, which were not necessarily accompanied by spreading to the rest of the cell. This uncoupling could be stimulated by the selective increase of the pacemaker firing rate, due to the rise of the intracellular Ca2+ concentration. Modulation of Ca2+ spike amplitude by treatments affecting either the pacemaker or the spreading phase might be related to quantal Ca2+ release from functionally discrete stores.

Intracellular distribution of kinesin in chromaffin cells.

In this paper we examined the association of the microtubule motor protein kinesin with organelles in chromaffin cells. Approximately 15% of kinesin was associated with membranes as determined by differential and equilibrium centrifugation on sucrose gradients. Kinesin was not enriched in a particular organelle fraction but cofractionated with a variety of organelle markers including markers for early and late endosomes, smooth and rough endoplasmic reticulum (ER) and the Golgi apparatus. Surprisingly, low amounts of kinesin were present in fractions of purified chromaffin granules. The absence of kinesin from the bulk of chromaffin granules was also indicated by immunostaining of tissue sections. A polyclonal antibody that specifically recognized the 120 kDa kinesin heavy chain labeled predominantly a perinuclear region that is typical for most of the kinesin-binding organelles identified by cell fractionation (endosomes, Golgi, ER). Since these organelles are compartments with high membrane turnover, we speculate that kinesin might be involved in certain aspects of trafficking of these membrane systems.

Membrane events in adrenal chromaffin cells during exocytosis: a freeze-etching analysis after rapid cryofixation.

Catecholamine-storing chromaffin cells, isolated from bovine adrenal medullae by collagenase digestion, were stimulated with carbachol at 37 degrees C; aliquots for controls were kept at 37 degrees C: Starting from this temperature, cells were ultrarapidly frozen with the use of a sandwich-propane-jet procedure and freeze-fractured. The replicas were analysed quantitatively for exocytotic activity: After stimulation the cell membrane displayed a significant increase of exo-endocytotic openings varying in size from 20 to 300 nm. The number of openings increased in parallel to the catecholamine output. At no stage could a clearing of membrane-intercalated particles (MIPs) be observed. Openings of all size classes were etchable. Results from the PF-face were comparable with those of the EF-face. We conclude that (i) exocytosis in isolated chromaffin cells starts as a focal event; the smallest possible stages are about 10 nm in size, (ii) fusion proceeds without previous rearrangement of MIPs, and (iii) the opening starts without formation of a diaphragm.