Localization of G protein alpha-subunits in the human fetal adrenal gland.

The aim of the present study was to investigate the presence and localization of the main G protein alpha-subunits in the human fetal adrenal gland during the second trimester of gestation. Immunofluorescence studies conducted on sections from frozen glands obtained immediately after therapeutic abortion indicated that the alpha s subunit of the heterotrimeric Gs protein was detected in all adrenal cell types, except for endothelial cells. The other alpha-subunits had a more specific pattern of distribution. Indeed, the alpha il-2 protein was restricted to the definitive zone, whereas alpha i3 labeling was mainly expressed in the fetal zone. The alpha q protein subunit was localized in vascular endothelial cells at the periphery of the adrenal gland and in fetal cells at the center. Finally, chromaffin cells expressed alpha s, alpha q, and alpha o1, but not alpha o2 nor alpha i. Altogether, these results indicate that the human fetal adrenal gland is not only unique in its particular morphology and expression of steroidogenic enzymes, but also by the differential expression of G protein alpha-subunits. Such cell specific distribution in glands from midgestational fetuses may account for the absence or the different responses to stimuli, when compared with the adult adrenal gland.

Identification of a novel secretogranin II-derived peptide (SgII(187-252)) in adult and fetal human adrenal glands using antibodies raised against the human recombinant peptide.

Molecular cloning of secretogranin II (SgII) in phylogenetically distant species has recently revealed the existence of a highly conserved 66-amino acid peptide flanked by preserved pairs of basic residues. This observation suggested that this peptide, named EM66, which had not been described to date, could be an important processing product of SgII. The aim of the present study was to investigate the possible occurrence of EM66 in the human adrenal gland. The EM66 peptide was generated in Escherichia coli, which was programmed to make a fusion protein containing the human EM66 sequence. The affinity-purified fusion protein was used to raise polyclonal antibodies in rabbits. The free EM66 peptide was obtained by cleavage of the fusion protein followed by high performance liquid chromatography purification. Immunohistochemical analysis using the EM66 antibodies revealed intense labeling of adrenochromaffin cells in the adult adrenal medulla and the fetal adrenal gland. A sensitive and specific RIA was developed and applied to the detection of EM66-like immunoreactivity in extracts of adult adrenal medulla and whole fetal adrenal gland after high performance liquid chromatographic analysis. A major immunoreactive species exhibiting the same retention time as recombinant EM66 was detected in both adult and fetal adrenal extracts. Taken together, these data demonstrate that posttranslational processing of SgII actually generates EM66 in the adrenal gland. The strong conservation of the amino acid sequence of EM66 in the vertebrate phylum and the occurrence of the mature peptide in both fetal and adult chromaffin cells suggest that EM66 could play an important physiological role in the human adrenal gland.

Apoptosis in the adrenal gland of non-obese diabetic (NOD) mice.

Apoptosis appears to play an important role in the development of diabetes in the non-obese diabetic (NOD) mouse. Since the autoimmune process leading to the manifestation of insulin dependent diabetes mellitus (IDDM) can also affect the sympathochromaffin system, we analyzed the role of apoptosis and infiltration of the adrenal medulla as features of this autoimmune process in parallel with the development of diabetes. Prediabetic and diabetic NOD mice aged 3 to 30 weeks were studied and compared with control mice. Apoptosis was assessed by in situ end-labeling method and ultrastructural analysis. Adrenals were screened for lymphocytic infiltration by conventional hematoxylin-eosin staining. Chromaffin cells were characterized by immunohistochemical staining against synaptophysin and tyrosine hydroxylase. Apoptotic nuclei were detected in all mice studied at a very low level, mainly occuring within the connective tissue between medulla and cortex. The maximum score was achieved at 3 weeks (1.91+/-0.48 apoptotic cells/1000 counted cells; n = 4). There was no significant difference between NOD mice and control mice. No correlation could be found between blood glucose levels and apoptosis. On the ultrastructural level, apoptotic cells presented typical features of apoptosis, i.e. condensed nuclei and cytoplasm. Neither in NOD mice nor in controls lymphocytic infiltration or fibrosis of the adrenal was detected. Even NOD mice with overt diabetes did not exhibit morphological signs of medullitis. In summary, no signs of immune destruction of the adrenal medulla in NOD mice aged 3 to 30 weeks could be detected.

Microelectrodes for the measurement of catecholamines in biological systems.

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-micron diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that high-pass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.

Differential regulation of phenylethanolamine N-methyltransferase expression in two distinct subpopulations of bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.

Similarities and differences in supporting and chromaffin cells in the mammalian adrenal medullae: an immunohistochemical study.

BACKGROUND: The adrenal medulla is a typical paraganglion, having the same origin as the sympathetic ganglia, and contains at least two types of parenchymal cells: chromaffin cells and supporting cells. We previously reported that the extent of cellular association of chromaffin cells with supporting cells was remarkably higher in noradrenaline (NA)-than in adrenaline (A)-cell regions in the adrenal medullae of the rat and pig. METHODS: Cryostat sections of adrenal medullae of nine mammalian species fixed with Zamboni fluid for 24 h were immunostained by ABC methods using antisera to S-100 protein and PNMT. RESULTS: The distribution patterns of A and NA cells in the adrenal medullae were classified into four types. In the chipmunk and rabbit, adrenomedullary chromaffin cells consisted of A cells. S-100-immunoreactive cells were present more frequently in NA- than in A-cell regions in seven species (rat, golden hamster, cat, dog, pig, ox, and horse). These cells sent out cytoplasmic processes and formed a network by immunoreactive elements among NA cells. The cell-association patterns of S-100-positive cells with NA cell were classified into three types. In A-cell regions, only a few S-100-positive cells were seen in most of the species, although the frequency of S-100-labeled cells were exceptionally high in the horse. CONCLUSIONS: The close association of supporting cells with NA cells was commonly found in the adrenal medulla in many mammalian species, irrespective of the proportions and distribution patterns of A cells and NA cells. On the other hand, species differences existed in details of the cellular association between supporting cells and NA cells.

Human adrenal cells express tumor necrosis factor-alpha messenger ribonucleic acid: evidence for paracrine control of adrenal function.

Tumor necrosis factor (TNF) is gaining increasing importance in clinical medicine. It plays a role in the interaction of the immune system with the hypothalamic-pituitary-adrenal axis. In the present study various morphological methods, including immunohistochemistry, electron microscopy, and in situ hybridization were applied to characterize the localization and distribution of TNF in the human adrenal gland. Double immunostaining revealed an astonishing degree of intermingling of steroid-producing cells and chromaffin cells. Macrophages could be found in all regions of the adrenal gland, but particularly in the transition zone of cortex and medulla. The steroid-producing cells of the inner zone of the cortex express major histocompatibility complex class II molecules. On the ultrastructural level, immune cells, steroid cells, and catecholamine-producing cells were found in direct contact. The combination of immunohistochemistry and in situ hybridization was optimally suited to define the exact cellular source of TNF in the human adrenal. TNF is produced in macrophages, but above all in 17 alpha-hydroxylase-positive cells (steroid-producing cells) in the zona reticularis and medulla. No signal was found in chromaffin cells. TNF may induce major histocompatibility complex class II in human adrenal gland in a paracrine or autocrine manner. It is concluded that TNF may have an important role in normal human adrenal physiology.

Modulation of calcium current by ATP in guinea-pig adrenal chromaffin cells.

The effects of extracellular adenosine 5'-triphosphate (ATP) on voltage-dependent Ca2+ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 microM) reversibly suppressed Ca2+ currents in the presence of 5mM Ca2+ in the extracellular solution. The inhibitory effect of ATP on Ca2+ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10mM EGTA, on the other hand, the inhibitory effect did not change regardless of the amplitude of current responses to ATP. In the presence of 10 mM Ba2+, ATP (100 microM) reduced Ba2+ currents in a manner similar to Ca2+ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]; 1 mM) or guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]; 100 microM). A depolarizing prepulse to + 100 mV partly relieved ATP-induced reduction of Ba2+ currents. ADP, AMP and adenosine also reduced Ba2+ currents and the effect of adenosine was the most potent. Adenosine (0.5 and 1 mM) significantly inhibited adrenaline secretion induced by nicotine (50 microM). These results suggest that Ca2+ entry through ATP-activated non-selective cation channels results in the inactivation of voltage-dependent Ca2+ channels. In addition, ATP seems to modulate Ca2+ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.

Separation of nanoliter samples of biological amines by a comprehensive two-dimensional microcolumn liquid chromatography system.

A two-dimensional liquid chromatography system based on the combination of a charge separation mechanism, provided by anion exchange chromatography, with a hydrophobic separation mechanism, provided by reversed phase chromatography, is presented. A 90 cm long anion exchange microcolumn is coupled to a 3 cm long reversed phase microcolumn. Both microcolumns are interfaced by two electronically controlled valves. Effluent from the anion exchange microcolumn is collected in a sample loop and then concentrated onto the head of the reversed phase microcolumn. Analyte peaks consisting of tagged amines elute from the second column and are detected by laser-induced fluorescence. The entire operation is controlled by a personal computer. The resolving power of this system is demonstrated with a two-dimensional chromatogram of the peptides obtained from a tryptic digest of porcine thyroglobulin. The sensitivity of the system is demonstrated with a two-dimensional chromatogram of the contents of a single bovine chromaffin cell.

A new sensitive and simple method for detection of catecholamines from adrenal chromaffin cells.

A biosensor was used for the analysis of catecholamines in media and lysates of cultured bovine adrenal chromaffin cells. The sensor is composed of coimmobilised laccase and glucose dehydrogenase coupled with an oxygen electrode, using the catalytic effect of cate cholamines for glucose oxidation in this system. The analysis time is almost 5 min. The correlation between the biosensor and HPLC determination is 0.99.

Use of fluorogenic substrates for detection and investigation of ectoenzymatic hydrolysis of diadenosine polyphosphates: a fluorometric study on chromaffin cells.

A set of procedures to assay and investigate ectoenzymatic hydrolysis of diadenosine polyphosphates (ApnA) in both intact cell or plasma membrane preparations is described. Procedures are based on the use of the fluorogenic ApnA analogs, epsilon-(ApnA), as artificial substrates. It is shown that these fluorogenic analogs behave as excellent substrates of the ectoenzyme present in cultured chromaffin cells. The ectoenzyme hydrolyzed all epsilon-(ApnA) tested (n = 2-6), always producing epsilon-AMP and epsilon-Ado 5'(n - 1) phosphate moieties. These released nucleotide moieties were then further catabolized up to epsilon-Ado by other ectonucleotidases. Epsilon-(Ap4A) hydrolysis by cultured cells displayed Km and Vmax values of 4.1 +/- 1.5 microM and 13.2 +/- 1.3 pmol/min x 10(6) cells, respectively, as measured by continuous fluorometric assays and 3.5 +/- 1.6 microM and 10.0 +/- 1.9 pmol/min x 10(6) cells by chromatographic-fluorometric assays. Using plasma membranes, values of 2.5 +/- 0.8 microM and 669 +/- 59 pmol/min x mg protein for Km and Vmax, respectively, were obtained through continuous fluorometric assays. ApnA and GpnG behaved as competitors and Ki values for these dinucleotides ranged between 0.7 and 3.5 microM. The ectoenzyme was activated by Mg2+ and Ca2+ and achieved maximal activity in the pH range 8.5-9.0.

pH- and Ca(2+)-induced conformational change and aggregation of chromogranin B. Comparison with chromogranin A and implication in secretory vesicle biogenesis.

Chromogranins A and B have been known to undergo pH- and Ca(2+)-dependent aggregation, and this property is considered essential for the proper sorting of the vesicular matrix proteins. In the present study, purified native chromogranin B (CGB) from bovine adrenal medulla was used to study the pH- and Ca(2+)-dependent conformational changes and aggregation property. Similar to chromogranin A (CGA), which had been shown to undergo pH- and Ca(2+)-dependent conformational changes and to be composed of 60-65% random coil with 25-40% alpha-helicity, chromogranin B was also shown to consist of 65-70% random coil, 15-25% alpha-helix, and 10-15% beta-sheet structures. The high percentage of random coil suggests that CGB behaves hydrodynamically as an asymmetric molecule, thus explaining its anomalous migration on SDS-polyacrylamide gels. Further, CGB eluted from a gel filtration column in the volume indicative of a globular protein with molecular weight of approximately 200,000 at both the intravesicular pH of 5.5 and a near physiological pH of 7.5. Considering that dimeric CGA eluted from a gel filtration column in the position suggestive of a 300-kDa protein, this result indicated that CGB exists in a monomeric state at both pH levels. Like CGA, which exhibited greater aggregation at pH 5.5 than at pH 7.5 upon Ca2+ binding, CGB also aggregated much more readily at pH 5.5 than at pH 7.5. However, there was a marked difference in the aggregation properties of CGA and CGB with regard to their sensitivity to Ca2+: CGB was at least 2 orders of magnitude more sensitive to Ca2+ than CGA. This suggested that, in spite of the low concentration of CGB (approximately one-tenth that of CGA) in bovine adrenal chromaffin cells, CGB would start to aggregate well ahead of CGA in the trans-Golgi network. In view of the proposed importance of the pH- and Ca(2+)-induced chromogranin aggregation in vesicle biogenesis, the extreme sensitivity of CGB aggregation to Ca2+ appears to underline the potential importance of CGB aggregation in the early stages of vesicle biogenesis.

GABAB receptors increase intracellular calcium concentrations in chromaffin cells through two different pathways: their role in catecholamine secretion.

The activation of GABAB receptors of adrenal chomaffin cells produces an increase of [Ca2+]i measured by fura-2 AM techniques. GABAB agonists 3-aminopropylphosphinic acid or (-)baclofen, at concentrations of 0.5 mM, increased basal Ca2+ values 332 +/- 60.9 and 306 +/- 40.5 nM, respectively, in cells suspended in a 2.5 mM Ca2+ buffer. The GABAB-induced increase of [Ca2+]i seemed to have two different components. The first was due to an entry from the extracellular medium mainly through L-type voltage-dependent Ca2+ channels as the dihydropiridine nifedipine 50 microM was able to decrease it more than 60%, while omega-conotoxin, which blocks N-type channels, did not produce any change in the GABAB-evoked Ca2+ increment. The second component was due to a release of Ca2+ from intracellular pools and was about one-third of the total GABAB-induced increase of [Ca2+]i. GABAB receptors stimulated inositol 1,4,5-trisphosphate-sensitive and not the caffeine-sensitive Ca2+ store. In a low-Ca2+ buffer after treatment with 2 microM angiotensin II, neither 0.5 mM 3-APPA nor baclofen were able to produce an additional increase of [Ca2+]i, whereas 4 mM caffeine had no effect on GABAB response. This intracellular Ca2+ mobilization could be due to inositol 1,4,5-trisphosphate accumulation produced by the activation of GABAB receptors. In fact, the specific agonists after 10 minutes incubation produced a dose-dependent increase of inositol 1,4,5-trisphosphate. The maximal effect was obtained at 100 microM baclofen and 3-APPA, and it was 3.63 +/- 0.75 and 3.2 +/- 1.5 times the basal levels (7.3 +/- 0.3 pmol/10(6) cells), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucocorticoids enhance histamine-evoked catecholamine secretion from bovine chromaffin cells.

The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC50 for dexamethasone was approximately 1 nM. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H1 histamine receptor antagonist mepyramine but was not affected by the H2 antagonist cimetidine; thus, dexamethasone appeared to enhance an H1 receptor-mediated process. In the absence of glucocorticoids, H1 receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H1 receptor mRNA levels in both cell types. The increased expression of H1 receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.

Nitric oxide implication in the control of neurosecretion by chromaffin cells.

In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme L-arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium- and concentration-dependent (EC50 = 64 +/- 8 microM) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose-dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (< 3 microM; EC50 = 16 +/- 3 microM), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (> or = 30 microM; IC50 = 52 +/- 6 microM). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [14C]citrulline from [14C]arginine and formation of an NO-hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium- and mainly calmodulin-dependent and could be specifically blocked by the NOS inhibitor N-methyl-L-arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.

Histochemical study of chromaffin cells and nerve fibers in the adrenal gland of the flat snake (Waglerophis merremii).

The distributions of tyrosine hydroxylase (TH), protein gene product (PGP) 9.5, calcitonin gene-related peptide (CGRP), and peptide histidine isoleucine (PHI) have been examined immunohistochemically in the adrenal gland of the snake Waglerophis merremii. The morphology of chromaffin cells and the presence of ganglionic neurons in the gland revealed by means of the glutaraldehyde-silver technique and electron microscopy are also described. Two distinct types of TH-immunoreactive (-IR) cells are present in the dorsal noradrenergic ribbon: small chromaffin cells and a larger type identified as ganglionic neurons. Small, mostly round or fusiform cells often displayed long processes. Ganglionic cells, arranged in patches, had long processes entering the cortex of the gland. Chromaffin adrenergic cells, forming small groups of 4-7 cells, were scattered within the interrenal tissue and had a wide variety of shapes with processes that appeared to contact other chromaffin cells. Bundles of PGP 9.5-IR fibers occurred in the subcapsular zone of the adrenal gland with fibers entering the cortex and dorsal noradrenergic ribbon of the gland. Thick and thin TH-IR fibers were seen. Thick TH-IR fibers were nonvaricose and appeared to originate mainly in ganglionic neurons. Thin TH-IR fibers with small varicosities were numerous in the interrenal tissue and were frequently seen between clusters of adrenergic cells in close apposition to cortical cells and vessels. CGRP-IR fibers were present throughout the entire adrenal gland, whereas PHI-IR fibers had a preferential distribution in the interrenal tissue. Both CGRP- and PHI-IR fibers were closely associated with vessels and cortical cells.

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.

Pituitary adenylate cyclase-activating peptide in the adrenal gland of mammals: distribution, characterization and responses to drugs.

The occurrence, cellular distribution and nature of the 27-amino acid form of pituitary adenylate cyclase-activating peptide (PACAP-27) in the adrenal glands of mammals was investigated by means of immunohistochemistry, radioimmunoassay and chromatography. The concentrations (pmol/g wet weight) of PACAP-27-like immunoreactivity (LI) varied considerably between the species examined: mouse (n = 8), 12.1 +/- 2.0; hamster (n = 4), 9.1 +/- 1.5; rat (n = 13), 2.0 +/- 0.3; cow (n = 3), 0.8 +/- 0.1, and pig (n = 5), 0.7 +/- 0.1. Upon HPLC, the immunoreactivity in extracts of rat, mouse and hamster adrenals coeluted with synthetic PACAP-27 while the immunoreactivity in extracts of cow and pig adrenals eluted as less hydrophobic material. Immunohistochemistry revealed the presence of PACAP-27-LI in the noradrenaline-storing chromaffin cells of the adrenal medulla. No nerve fibers exhibiting PACAP-27-LI could be detected. The effects of drug administration in vivo on the stores of PACAP-27-LI in the rat adrenal were studied and compared with the effects on the adrenal stores of neuropeptide Y (NPY)-LI. Splanchnic activation following insulin-induced hypoglycemia elicited a 41% depletion of NPY-LI 2 h after insulin injection, while the concentrations of PACAP-27-LI remained unchanged. Two days after reserpine administration the stores of PACAP-27-LI and NPY-LI were depleted by 62 and 41%, respectively. Two days later, the concentrations of NPY-LI were increased to 216% of controls, while the concentrations of PACAP-27-LI were similar to that of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of vesicular monoamine transporter isoforms (VMAT1 and VMAT2) to endocrine cells and neurons in rat.

Polyclonal antipeptide antibodies have been raised against each of the two isoforms of the rat vesicular monoamine transporter, VMAT1 and VMAT2. Antibody specificity was determined by isoform-specific staining of monkey fibroblasts programmed to express either VMAT1 or VMAT2. The expression of VMAT1 and VMAT2 in the diffuse neuroendocrine system of the rat has been examined using these polyclonal antibodies specific for either VMAT1 or VMAT2. VMAT1 is expressed exclusively in endocrine/paracrine cells associated with the intestine, stomach, and sympathetic nervous system. VMAT2 is expressed in neurons of the sympathetic nervous system, and aminergic neurons in the enteric and central nervous systems. VMAT2 is expressed in at least two endocrine cell populations in addition to its expression in neurons. A subpopulation of chromogranin A (CGA)-expressing chromaffin cells of the adrenal medulla also express VMAT2, and the oxyntic mucosa of the stomach contains a prominent population of CGA- and VMAT2-positive endocrine cells. The expression of VMAT2 in neurons, and the mutually exclusive expression of VMAT1 and VMAT2 in endocrine/paracrine cell populations of stomach, intestine, and sympathetic nervous system may provide a marker for, and insight into, the ontogeny and monoamine-secreting capabilities of multiple neuroendocrine sublineages in the diffuse neuroendocrine system.

Cytological and biochemical studies on chromaffin cells in the head kidney of Gasterosteus aculeatus (Teleostei, Gasterosteidae).

Adrenal chromaffin cells in the head kidney of the stickleback Gasterosteus aculeatus were examined by ultrastructural analysis and determination, by HPLC, of the catecholamine content (adrenaline, noradrenaline, and dopamine). Chromaffin cells are characterized by the presence of membrane-bound vesicles and two cell types were identified: (i) those with vesicles containing a strongly electron-dense core; and (ii) those with vesicles that are completely electron lucent or containing smaller and less-dense granules. HPLC analysis revealed the presence of adrenaline and noradrenaline in both right and left head kidneys. Dopamine was not detected. Cytological and biochemical data suggest that the cells with electron-dense granules contain noradrenaline and those with electron-lucent vesicles contain adrenaline.