Diverse proportion in composite pheochromocytoma-ganglioneuroma may induce varied clinical symptom: comparison of two cases.

Composite pheochromocytoma-ganglioneuroma is extremely rare. We described two cases of composite pheochromocytomas in the adrenal medullar. Case 1 was a 70-year-old male presenting with lower abdominal pain and normal blood electrolytes. Case 2 was a 48-year-old female with palpitation and back tenderness. Biochemical investigations showed hypocalcium, hypokalemia and high level of vma. The histological images and the immunohistochemical staining demonstrated the two cases composed of pheochromocytoma and ganglioneuromoma components. Ganglioneuroma component in case 2 accounted for more proportion than that in case 1. We speculated that the varied clinical symptoms were related with the diverse proportions in composite pheochromocytome-ganglioneuroma.

An uncommon type of adrenal incidentaloma: a case report of a schwannoma of the adrenal medulla with cytological, histological, and ultrastructural correlation.

Benign nerve sheath tumors of the adrenal gland are an extremely uncommon cause of an incidentaloma. We report a case of a schwannoma of the adrenal medulla in an asymptomatic 51-year-old woman, which was discovered incidentally on a computed tomography scan after routine workup for her degenerative joint diseases of the lumbar spine. Because of the large size and unknown biologic nature of the tumor by clinical and radiographic studies alone, an adrenalectomy was performed. The gross specimen featured a well-circumscribed medullary based tumor with cystic degeneration. The diagnosis of a nerve sheath tumor was based on classic histological findings, supported by S-100 positivity, and ultrastructurally by the finding of typical Schwann cells. The cytological diagnosis from the fine-needle aspiration biopsy material obtained at the time of gross examination was much more challenging on retrospective review. The aspirated material showed a round- to oval-cell predominant smear with occasional striking anisonucleosis, intranuclear inclusions (so-called ancient change), and pigment deposition. A review of the histogenesis and differential diagnosis of this common nerve sheath tumor in this unusual location is discussed.

Histopathology and immunohistochemistry of adrenal medullary tumors and paragangliomas.

Paragangliomas arise from sympathetic or parasympathetic paraganglia and should now be defined by their site and type. The term pheochromocytoma is reserved for intra-adrenal tumors. This short review discusses the gross and microscopic features, the immunohistochemical profile, the problem of recognizing malignant potential, and the rare instances where a differential diagnosis has to be considered.

Increased susceptibility to transcriptional changes with novel stressor in adrenal medulla of rats exposed to prolonged cold stress.

The response to stress is influenced by prior experience with the same or different stressor. For example, exposure of cold pre-stressed rats to heterotypic (novel) stressors, such as immobilization (IMO), triggers an exaggerated release of catecholamines and increase in gene expression for adrenomedullary tyrosine hydroxylase (TH), the rate limiting catecholamine biosynthetic enzyme. To study the mechanism, we examined induction or phosphorylation of several transcription factors, which are implicated in IMO-triggered regulation of TH transcription, in rats exposed to cold (4 degrees C) for up to 28 days and then subjected to IMO. Levels of c-fos increased transiently after 2-6 h and returned to basal levels after 1-28 days cold stress. Fra-2, was unaffected by short term cold, but was induced about 2-fold by 28 days continual cold. In contrast, there were no significant changes in CREB phosphorylation or Egr1 induction. Rats, with and without pre-exposure to 28 days cold, were subjected to single IMO for up to 2 h. Phosphorylation of CREB after 30 min IMO was greater in cold pre-exposed rats. Induction of Egr1 was three times higher in cold pre-exposed rats and remained significantly elevated even 3 h after cessation of IMO. Exposure to IMO triggered a 10-20-fold elevation in Fra-2 in both groups, which was even higher 3 h after the IMO. However, Fra-2 was more heavily phosphorylated following IMO stress in cold pre-exposed animals. The results reveal that sensitization to novel stress in cold pre-exposed animals is manifested by exaggerated response of several transcription factors.

Rats with steroid-induced polycystic ovaries develop hypertension and increased sympathetic nervous system activity.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, abdominal obesity, hyperandrogenism, hypertension, and insulin resistance. METHODS: Our objectives in this study were (1) to estimate sympathetic-adrenal medullary (SAM) activity by measuring mean systolic blood pressure (MSAP) in rats with estradiol valerate (EV)-induced PCO; (2) to estimate alpha1a and alpha2a adrenoceptor expression in a brain area thought to mediate central effects on MSAP regulation and in the adrenal medulla; (3) to assess hypothalamic-pituitary-adrenal (HPA) axis regulation by measuring adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels in response to novel-environment stress; and (4) to measure abdominal obesity, sex steroids, and insulin sensitivity. RESULTS: The PCO rats had significantly higher MSAP than controls, higher levels of alpha1a adrenoceptor mRNA in the hypothalamic paraventricular nucleus (PVN), and lower levels of alpha2a adrenoceptor mRNA in the PVN and adrenal medulla. After exposure to stress, PCO rats had higher ACTH and CORT levels. Plasma testosterone concentrations were lower in PCO rats, and no differences in insulin sensitivity or in the weight of intraabdominal fat depots were found. CONCLUSION: Thus, rats with EV-induced PCO develop hypertension and increased sympathetic and HPA-axis activity without reduced insulin sensitivity, obesity, or hyperandrogenism. These findings may have implications for mechanisms underlying hypertension in PCOS.

Primary sequence characterization of catestatin intermediates and peptides defines proteolytic cleavage sites utilized for converting chromogranin a into active catestatin secreted from neuroendocrine chromaffin cells.

Catestatin is an active 21-residue peptide derived from the chromogranin A (CgA) precursor, and catestatin is secreted from neuroendocrine chromaffin cells as an autocrine regulator of nicotine-stimulated catecholamine release. The goal of this study was to characterize the primary sequences of high molecular mass catestatin intermediates and peptides to define the proteolytic cleavage sites within CgA that are utilized in the biosynthesis of catestatin. Catestatin-containing polypeptides, demonstrated by anti-catestatin western blots, of 54-56, 50, 32, and 17 kDa contained NH(2)-terminal peptide sequences that indicated proteolytic cleavages of the CgA precursor at KK downward arrow, KR downward arrow, R downward arrow, and KR downward arrow basic residue sites, respectively. The COOH termini of these catestatin intermediates were defined by the presence of the COOH-terminal tryptic peptide of the CgA precursor, corresponding to residues 421-430, which was identified by MALDI-TOF mass spectrometry. Results also demonstrated the presence of 54-56 and 50 kDa catestatin intermediates that contain the NH(2) terminus of CgA. Secretion of catestatin intermediates from chromaffin cells was accompanied by the cosecretion of catestatin (CgA(344)(-)(364)) and variant peptide forms (CgA(343)(-)(368) and CgA(332)(-)(361)). These determined cleavage sites predicted that production of high molecular mass catestatin intermediates requires cleavage at the COOH-terminal sides of paired basic residues, which is compatible with the cleavage specificities of PC1 and PC2 prohormone convertases. However, it is notable that production of catestatin itself (CgA(344)(-)(364)) utilizes more unusual cleavage sites at the NH(2)-terminal sides of downward arrow R and downward arrow RR basic residue sites, consistent with the cleavage specificities of the chromaffin granule cysteine protease "PTP" that participates in proenkephalin processing. These findings demonstrate that production of catestatin involves cleavage of CgA at paired basic and monobasic residues, necessary steps for catestatin peptide regulation of nicotinic cholinergic-induced catecholamine release.

Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury.

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40-50%, prevented gastric ulcer formation by 70-80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-alpha and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-alpha and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.

Ret protein expression in adrenal medullary hyperplasia and pheochromocytoma.

Ret is a developmentally regulated tyrosine kinase involved in formation and maintenance of the nervous system. Ret mutations predisposing to pheochromocytomas and medullary thyroid carcinomas occur in multiple endocrine neoplasia (MEN) syndromes 2A and 2B. Biochemical studies have demonstrated overexpression of Ret mRNA and protein in pheochromocytomas compared to normal adrenal medulla. However, the cellular distribution of Ret in the normal human adrenal and in hyperplastic lesions that antecede pheochromocytomas are unclear. The present investigation was undertaken to resolve the histological distribution of Ret in the normal human adrenal, in pheochromocytomas evolving from adrenal medullary hyperplasia in MEN2A and in sporadic pheochromocytomas. Ret expression was studied by immunohistochemistry using both a polyclonal and a monoclonal antibody, with confirmation by immunoblotting of representative cases. Only occasional cells stained for Ret in the normal adrenal, consistent with the distribution in adult adrenals of other species. Heterogeneous, progressively increased Ret expression was observed during the evolution of pheochromocytomas. In both normal and neoplastic adrenal, the most intense immunoreactivity was observed in cells with neuron-like features. Our finding that Ret is not expressed at high levels in the early stages of disease suggests that elucidation of mechanisms that regulate Ret expression is required for understanding the pathobiology of MEN2A. The association of high-level Ret expression with neuronal morphology suggests that the variable overexpression of Ret in pheochromocytomas might in part be an epiphenomenon, reflecting the known phenotypic plasticity of these tumors.

Modulation of secretion by the endoplasmic reticulum in mouse chromaffin cells.

The endoplasmic reticulum (ER) has been suggested to modulate secretion either behaving as a Ca2+ sink or as a Ca2+ source in neuronal cells. Working as a Ca2+ sink, through ER-Ca2+ pumping, it may reduce secretion induced by different stimuli. Instead, working as a Ca2+ source through the Ca2+ induced Ca2+ release (CICR) phenomenon, it may potentiate secretion triggered by activation of plasma membrane Ca2+ channels. We have previously demonstrated the presence of CICR in bovine chromaffin cells, but we now find that mouse chromaffin cells almost lack functional caffeine-sensitive ryanodine receptors in the ER and, consistently, no CICR from the ER could be observed. In addition, inhibition of ER Ca2+ pumping with ciclopiazonic acid or thapsigargin strongly stimulated high-K+-evoked catecholamine secretion and cytosolic [Ca2+] ([Ca2+]c) transients. Surprisingly, 5 mm caffeine reduced high-K+-induced [Ca2+]c peaks but considerably potentiated secretion induced by high-K+ stimulation. However, this potentiation was insensitive to ryanodine and additive to that induced by emptying the ER of Ca2+ with thapsigargin, suggesting that it is unrelated to the activation of ryanodine receptors. We conclude that, in mouse chromaffin cells, CICR is not functional and the ER strongly inhibits secretion by acting as a damper of the [Ca2+]c signal.

Beta-amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for beta-secretase activity in chromaffin vesicles.

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

Cellular localization of the diazepam binding inhibitor in glial cells with special reference to its coexistence with brain-type fatty acid binding protein.

The diazepam binding inhibitor (DBI) was originally isolated from the brain as an intrinsic ligand of the benzodiazepine binding site on the type-A gamma-aminobutyric acid receptor (GABA(A) receptor). Its wide-spread distribution in non-neural tissues outside the brain suggests that DBI has various functions other than GABA-mediated neurotransmission. Since DBI is identical with the acyl-CoA binding protein, which has the ability to bind long chain acyl-CoA esters, the major function of DBI may possibly be related to lipid metabolism. This idea was supported by our previous study showing the consistent coexpression of DBI and fatty acid binding proteins (FABPs) in epithelia throughout the gastrointestinal tract. The present histochemical study focused on the distribution of DBI in neural tissues, and revealed a definite existence of DBI in non-neuronal supporting cells in both the central and peripheral nervous systems. In the brain, intense immunoreactivity for DBI was detected in the cerebellar Bergmann glia, olfactory ensheathing glia, subgranular layer of the dentate gyrus, and retinal Muller cells. In the peripheral nervous system, satellite cells in sensory/autonomic ganglia, Schwann cells, and sustentacular cells in the adrenal medulla were immunoreactive to a DBI antibody. Moreover, the colocalization of DPI and brain-type FABP (B-FABP) was observed in most of the non-neuronal supporting cells mentioned above, indicating that DBI and B-FABP are cooperatively involved in the energy metabolism of astrocytes and related cells, which are thought to support neuronal development and functions.

Synaptotagmin I-DeltaC2B. A novel synaptotagmin isoform with a single C2 domain in the bovine adrenal medulla.

Synaptotagmin I is a 65 kDa type 1 membrane glycoprotein found in secretory organelles that plays a key role in regulated exocytosis. We have characterised two forms (long and short) of synaptotagmin I that are present in the bovine adrenal medulla. The long form is a type I integral membrane protein which has two cytoplasmic C2 domains and corresponds to the previously characterised full-length synaptotagmin I isoform. The short-form synaptotagmin I-DeltaC2B has the same structure in the lumenal and transmembrane sequences, but synaptotagmin I-DeltaC2B is truncated such that it only has a single cytoplasmic C2 domain. Analysis of synaptotagmin I-DeltaC2B expression indicates that synaptotagmin I-DeltaC2B is preferentially expressed in the bovine adrenal medulla. However, it is absent from the dense core chromaffin granules. Furthermore, when expressed in the rat pheochromocytoma cell line PC12 bovine synaptotagmin I-DeltaC2B is largely absent from dense core granules and synaptic-like microvesicles. Instead, indirect immunofluorescence microscopy reveals the intracellular location of synaptotagmin I-DeltaC2B to be the plasma membrane.

Undifferentiated ganglioneuroblastoma in a sheep.

Ganglioneuroblastoma, presumably originating from the adrenal medulla, was found in an 18-month-old sheep. Histologically, the tumour was mainly composed of undifferentiated neuroblasts with neuropil and occasional ganglion cells. The morphological and immunohistochemical features with respect to intermediate filament proteins and the neuroendocrine nature of the tumour are described. The neoplastic cells were positive for neuron-specific enolase, synaptophysin and vimentin.

Increased expression of tenascin in pheochromocytomas correlates with malignancy.

Tenascin is a significant extracellular matrix glycoprotein, which is upregulated in various neoplasias and pathologic processes. Pheochromocytomas are rare tumors of the sympathoadrenal system, whose malignancy is almost impossible to predict. There are no histologic or chemical markers available that would define the malignant behavior of these tumors, except the discovery of metastases. In our search for new markers, we investigated the immunohistochemical expression of tenascin in a large number of pheochromocytomas and paragangliomas. Seven tumors were metastasized and were thus considered malignant. Normal adrenal medulla was tenascin negative. A striking difference was seen between malignant and benign pheochromocytomas. All malignant pheochromocytomas expressed stromal tenascin strongly or moderately, whereas most benign pheochromocytomas (28 of 37, 70%) showed no or only weak immunopositivity. The staining was strong or moderate also in 13 of 28 (46%) of the tumors that showed histologically suspicious features, here called borderline tumors. Paragangliomas showed a more heterogeneous staining pattern, and no significant difference was found between benign and malignant paragangliomas. To our knowledge, this is the first study to demonstrate the expression of tenascin in pheochromocytomas and particularly the enhanced expression in malignant pheochromocytomas. We therefore suggest that tenascin may be associated with the malignant transformation and metastasis of pheochromocytomas. It is also a potential marker predicting more aggressive behavior in pheochromocytomas.

On the occurrence of multiple isoprenylated cysteine methyl ester hydrolase activities in bovine adrenal medulla.

Rab proteins intervene in the controlled exocytosis of catecholamines by chromaffin cells from the adrenal medulla. These proteins are posttranslationally modified by digeranylgeranylation and carboxymethylation. Reversible carboxymethylation terminating the isoprenylation pathway may play an important role in both the functioning and the subcellular housing of small G-proteins. Controlled methylation infers a rational interplay between the two enzymes involved i.e., the protein-S-prenylcysteine methyltransferase and the opposing esterase. Previously we have identified a methyltransferase type III in chromaffin cells. In this paper we focus on the corresponding demethylase. The methyl ester hydrolase activity was monitored using AFCM and AGGCM as artificial substrates while p-nitrophenylacetate was adopted as a pseudosubstrate for nonspecific esterase action. Based on subcellular fractionation experiments, kinetic studies and screening a battery of potential effectors, including a series of metallic ions and metal chelators, multiple sulphydryl reagents and host of specific protease/esterase inhibitors, it is suggested that at least two prenylcysteine carboxymethyl esterase isoenzymes are operational in bovine adrenal medulla. These isoenzymes are distinctly different from the nonspecific esterase.

[(35)S]GTPgammaS autoradiography reveals a wide distribution of G(i/o)-linked ADP receptors in the nervous system: close similarities with the platelet P2Y(ADP) receptor.

No G(i)-linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [(35)S]guanosine 5'-[gamma-thio]-triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G-protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto-/heteroreceptor function. In the white matter, activated G proteins appeared as 'hot spots' (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2-methylthio-ADP activated G proteins approximately 500-fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP-dependent G-protein activity was reversed by 2-hexylthio-AdoOC(O)Asp(2), a non-phosphate ATP analogue, whereas selective P2Y(1) receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of G(i/o)-linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2Y(ADP) receptor.

Spatial and temporal expression patterns of the cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 during mouse development.

The cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p57Kip2 are thought to regulate progression of the cell cycle. We have previously shown that the phenotypes of p27-/- mice are substantially different from those of p57-/- mice, suggesting that spatial and temporal expression patterns of p27Kip1 and p57Kip2 might be distinct. In this study, the roles of p27Kip1 and p57Kip2 in development were examined by characterizing their expression patterns during mouse embryogenesis by immunohistochemical analysis. Whereas certain organs and tissues (brain, lens, ganglion, lung, heart, liver, skin and kidney) expressed both proteins, others expressed only p27Kip1 (thymus, spleen, retina, testis and ovary) or only p57Kip2 (gut, palate, pancreas, cartilage and skeletal muscle). In addition, some organs expressed both p27Kip1 and p57Kip2 but showed mutually exclusive patterns of distribution among tissues. Thus, in the adrenal gland, p57Kip2 was expressed in the cortex but not in the medulla, whereas p27Kip1 was expressed in the medulla but not in the cortex. Whereas the expression of p57Kip2 in most tissues was restricted to embryogenesis, expression of p27Kip1 in many tissues was maintained in adult animals. Double-label immunofluorescence staining with either anti-p27Kip1 or anti-p57Kip2 and anti-BrdU revealed that the expression of p27Kip1 and p57Kip2 was inversely correlated with cell proliferation, suggesting that p27Kip1 and p57Kip2 are expressed exclusively in postmitotic cells. These complex spatial and temporal patterns of expression are consistent with the phenotypes of mice deficient in p27Kip1 or p57Kip2, and they suggest that these proteins might play important roles in tissue development.

Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

Repeated 2-deoxy-D-glucose-induced glucoprivation attenuates Fos expression and glucoregulatory responses during subsequent glucoprivation.

A condition of reduced responsiveness to hypoglycemia, known as hypoglycemia-associated autonomic failure (HAAF), occurs in diabetic patients in the wake of a prior hypoglycemic episode. This condition suggests that hypoglycemia alters central glucose-sensing mechanisms. This experiment examined the effects of repeated 2-deoxy-D-glucose (2DG)-induced glucoprivation on subsequent 2DG-induced feeding and hyperglycemic responses in rats. Fos immunoreactivity (ir) in adrenal medulla and brain sites involved in these responses was also examined. Rats were injected daily for 10 days with 2DG (200 mg/kg) or saline (0.9%) or were handled. On day 11, rats were injected with 2DG (200 mg/kg). After injection, food intake was measured in one group. In another group, food was withheld, and multiple blood samples were collected for glucose determination. In a third group, food was withheld, and rats were killed after 2 h for evaluation of Fos-ir. Prior repeated glucoprivation reduced subsequent feeding and hyperglycemia responses to 2DG to baseline levels. Double-label immunohistochemistry showed that Fos-ir was reduced or abolished in catecholamine cell groups A1, A1/C1, C1, C3, and A6 and in the paraventricular nucleus of the hypothalamus and adrenal medulla. In other brain sites, 2DG-induced Fos-ir was diminished or unaffected by prior glucoprivation. Sites in which Fos-ir was abolished have been implicated previously in glucoprivic control of feeding and adrenal medullary secretion. Therefore, the present findings may identify crucial neuroanatomical sites that are altered by prior glucoprivation and that mediate some of the physiological deficits observed in HAAF.

Identification of morphine in the adrenal medullary chromaffin PC-12 cell line.

Morphine was identified in the adrenal medulla chromaffin PC-12 cell line by reversed-phase HPLC, following liquid and solid extraction. The morphine corresponding HPLC fractions (1.746+/-0.615 ng of morphine/million cells) were further analyzed by gas chromatography-mass spectrometry and found to be identical to synthetic morphine. Furthermore, using primers derived from the human neuronal mu 1 opiate receptor, we used RT-PCR to detect expression of mu transcripts from this cell line. The transcript was absent. The study conclusively proves morphine, but not a mu opiate receptor, is constitutively expressed in the adrenal medulla chromaffin PC-12 cell line.