Quantal release at a neuronal nicotinic synapse from rat adrenal gland.

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.

Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia.

To study the involvement of cyclin D1 in epithelial growth and differentiation and its putative role as an oncogene in skin, transgenic mice were developed carrying the human cyclin D1 gene driven by a bovine keratin 5 promoter. As expected, all squamous epithelia including skin, oral mucosa, trachea, vaginal epithelium, and the epithelial compartment of the thymus expressed aberrant levels of cyclin D1. The rate of epidermal proliferation increased dramatically in transgenic mice, which also showed basal cell hyperplasia. However, epidermal differentiation was unaffected, as shown by normal growth arrest of newborn primary keratinocytes in response to high extracellular calcium. Moreover, an unexpected phenotype was observed in the thymus. Transgenic mice developed a severe thymic hyperplasia that caused premature death due to cardio-respiratory failure within 4 months of age. By 14 weeks, the thymi of transgenic mice increased in weight up to 40-fold, representing 10% of total body weight. The hyperplastic thymi had normal histology revealing a well-differentiated cortex and medulla, which supported an apparently normal T-cell developmental program based on the distribution of thymocyte subsets. These results suggest that proliferation and differentiation of epithelial cells are under independent genetic controls in these organs and that cyclin D1 can modulate epithelial proliferation without altering the initiation of differentiation programs. No spontaneous development of epithelial tumors or thymic lymphomas was perceived in transgenic mice during their first 8 months of life, although they continue under observation. This model provides in vivo evidence of the action of cyclin D1 as a pure mediator of proliferation in epithelial cells.

Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.

The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors.

The nude mutation (nu) causes athymia and hairlessness, but the molecular mechanisms by which it acts have not been determined. To address the role of nu in thymogenesis, we investigated whether all or part of the nude thymic epithelium could be rescued by the presence of wild-type cells in nude <--> wild-type chimeric mice. Detailed immunohistochemical analyses revealed that nude-derived cells could persist in the chimeric thymus but could not contribute to cortical or medullary epithelial networks. Nude-derived cells, present in few clusters in the medulla, expressed markers of a rare subpopulation of adult medullary epithelium. The thymic epithelial rudiment of nude mice strongly expressed these same markers, which may therefore define committed immature thymic epithelial precursor cells. To our knowledge, these data provide the first evidence that the nu gene product acts cell-autonomously and is necessary for the development of all major subpopulations of mature thymic epithelium. We propose that nu acts to regulate growth and/or differentiation, but not determination, of thymic epithelial progenitors.

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms.

Intermittent electrical footshock induces c-fos expression in parvocellular neurosecretory neurons expressing corticotropin-releasing factor and in other visceromotor cell types of the paraventricular hypothalamic nucleus (PVH). Since catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla make up the dominant loci of footshock-responsive cells that project to the PVH, these were evaluated as candidate afferent mediators of hypothalamic neuroendocrine responses. Rats bearing discrete unilateral transections of this projection system were exposed to a single 30-min footshock session and sacrificed 2 hr later. Despite depletion of the aminergic innervation on the ipsilateral side, shock-induced up-regulation of Fos protein and corticotropin-releasing factor mRNA were comparable in strength and distribution in the PVH on both sides of the brain. This lesion did, however, result in a substantial reduction of Fos expression in medullary aminergic neurons on the ipsilateral side. These results contrast diametrically with those obtained in a systemic cytokine (interleukin 1) challenge paradigm, where similar cuts ablated the Fos response in the ipsilateral PVH but left intact the induction seen in the ipsilateral medulla. We conclude that (i) footshock-induced activation of medullary aminergic neurons is a secondary consequence of stress, mediated via a descending projection transected by our ablation, (ii) stress-induced activation of medullary aminergic neurons is not necessarily predictive of an involvement of these cell groups in driving hypothalamic visceromotor responses to a given stressor, and (iii) despite striking similarities in the complement of hypothalamic effector neurons and their afferents that may be activated by stresses of different types, distinct mechanisms may underlie adaptive hypothalamic responses in each.

Adrenocortical suppression blocks the memory-enhancing effects of amphetamine and epinephrine.

This study examined glucocorticoid-adrenergic interactions in modulating acquisition and memory storage for inhibitory avoidance training. Systemically (s.c.) administered amphetamine (1 mg/kg), but not epinephrine (0.1 mg/kg) or the peripherally acting amphetamine derivative 4-OH amphetamine (2 mg/kg), given to rats shortly before training facilitated acquisition performance in a continuous multiple-trial inhibitory avoidance (CMIA) task. Adrenocortical suppression with the 11beta-hydroxylase inhibitor metyrapone (50 mg/kg; s.c.), given to rats 90 min before training, did not block the effect of amphetamine and did not affect acquisition performance of otherwise untreated animals. Retention of CMIA and one-trial inhibitory avoidance was enhanced by either pre- or posttraining injections of amphetamine as well as 4-OH amphetamine and epinephrine. The finding that injections of amphetamine and epinephrine have comparable effects on memory is consistent with the view that amphetamine may modulate memory storage, at least in part, by inducing the release of epinephrine from the adrenal medulla. Metyrapone pretreatment blocked the memory-enhancing effects of amphetamine, 4-OH amphetamine, and epinephrine but did not affect retention performance of otherwise untreated animals. Posttraining injections of different doses of epinephrine (ranging from 0.0001 to 1.0 mg/kg) produced a dose-dependent memory enhancement for inhibitory avoidance training and metyrapone blocked the memory-enhancing effects of all these doses. These findings provide further evidence that the sympathoadrenal and adrenocortical systems are intimately coupled during processes of memory storage.

Angiotensin II type 2 receptor mediates programmed cell death.

The function of the recently discovered angiotensin II type 2 (AT2) receptor remains elusive. This receptor is expressed abundantly in fetus, but scantily in adult tissues except brain, adrenal medulla, and atretic ovary. In this study, we demonstrated that this receptor mediates programmed cell death (apoptosis). We observed this effect in PC12W cells (rat pheochromocytoma cell line) and R3T3 cells (mouse fibroblast cell line), which express abundant AT2 receptor but not AT1 receptor. The cellular mechanism appears to involve the dephosphorylation of mitogen-activated protein kinase (MAP kinase). Vanadate, a protein-tyrosine-phosphatase inhibitor, attenuated the dephosphorylation of MAP kinases by the AT2 receptor and restored the apoptotic changes. Antisense oligonucleotide to MAP kinase phosphatase 1 inhibited the AT2 receptor-mediated MAP kinase dephosphorylation and blocked the AT2 receptor-mediated apoptosis. These results suggest that protein-tyrosine-phosphatase, including MAP kinase phosphatase 1 activated by the AT2 receptor, is involved in apoptosis. We hypothesize that this apoptotic function of the AT2 receptor may play an important role in developmental biology and pathophysiology.

Alterações em adrenais de cães relacionadas à morte súbita ou agônica: análise morfométrica, histopatológica, imunoistoquímica e correlação com os níveis de adrenalina e noradrenalina na medula adrenal

A necropsia é essencial para a identificação da causa de morte e dos processos que culminaram no óbito do animal. O presente estudo visou à pesquisa da real ocorrência de morte súbita e inesperada em cães submetidos à necropsia, e à determinação da discrepância entre as suspeitas dos proprietários em relação ao estado de saúde prévio e à causa de morte de seus cães, quando comparadas às conclusões necroscópicas. Este trabalho também focou nas alterações morfológicas e funcionais nas glândulas adrenais de cães necropsiados, a fim de se estudar a aplicação de possíveis marcadores da resposta adrenal ao estresse sofrido pelo animal na iminência do óbito. Foram utilizados os dados de 82 cães necropsiados na FMVZ-USP para análise da ocorrência real de morte súbita nestes animais. As alterações morfológicas nas adrenais de 46 cães necropsiados foram avaliadas através de análises morfométricas e histopatológicas. Também foram avaliados os índices de proliferação e apoptose nas células do córtex adrenal em relação à causa de morte do animal, através da marcação imunoistoquímica para o antígeno nuclear de proliferação celular (PCNA) e para BAX e Bcl-2, proteínas envolvidas na regulação da apoptose. A análise das alterações funcionais sofridas pelas adrenais focou na quantificação das concentrações das catecolaminas adrenalina e noradrenalina na medula adrenal dos cães por cromatografia líquida de alta eficiência com detecção eletroquímica (CLAE-DE). Dentre os resultados encontrados, a suspeita do proprietário de que seu cão sofreu uma morte súbita e inesperada é muito maior do que a real ocorrência deste tipo de morte em cães, sendo o óbito por decorrência de complicações de doenças muito mais frequente. As características morfométricas das adrenais dos cães apresentaram maior influência pelo peso corpóreo do animal e pela presença ou ausência de hiperplasia cortical do que pela associação com doenças crônicas ou condições agudas. Cães que vieram a óbito em decorrência de complicações de doenças crônicas exibiram fibrose em região corticomedular e focos de infiltrado inflamatório, ausentes nos animais com morte súbita ou doenças agudas, além de maior ocorrência de hiperplasia adrenocortical. Cães que sofriam de alterações cardíacas crônicas apresentaram alterações histopatológicas significativas mais marcantes em suas adrenais, como necrose, fibrose e depleção vacuolar cortical. Por outro lado, a congestão severa nas adrenais foi um achado mais frequente nos animais previamente saudáveis que sofreram morte súbita ou que vieram a óbito por doenças agudas. A avaliação dos índices de proliferação celular e apoptose no córtex das adrenais através da marcação imunoistoquímica para PCNA e BAX e Bcl-2, respectivamente, não apresentou potencial relevante para o estudo dos efeitos do estresse por doenças crônicas sobre as adrenais de cães. As concentrações de adrenalina e noradrenalina na medula adrenal se mostraram muito maiores em cães machos quando comparados às fêmeas. Os achados deste estudo podem auxiliar nas conclusões da necropsia, sendo especialmente relevantes em casos médico-legais, nos quais todos os achados possíveis devem ser relatados e analisados a fim de se prover um diagnóstico preciso, seguro e incontroverso

Sobrecarga e restrição de cloreto de sódio durante a gestação: repercussão sobre a estrutura cardíaca e renal no neonato

Introdução: Diversos estudos indicaram consequências de alterações na nutrição materna durante a gestação sobre a saúde da prole adulta, tais como: hipertensão, doenças cardiovasculares, resistência à insulina, diabete melito e doença renal. No entanto, a literatura é pobre em avaliações decorrentes de modificações nutricionais maternas sobre a prole logo após o nascimento. Métodos: Ratas Wistar durante o período gestacional foram alimentadas com dieta hipossódica (HO - 0,15% de NaCl), normossódica (NR - 1,3% de NaCl) ou hipersódica (HR - 8% de Na Cl). Após o nascimento, nas primeiras vinte e quatro horas foram coletados rins e coração dos neonatos machos e fêmeas (n=6- 8/grupo) para verificar as possíveis alterações na estrutura cardíaca e renal pelo método de estereologia. Também foi avaliada a expressão proteica e gênica dos componentes do sistema renina angiotensina (SRA) no coração e rins através do método ELISA indireto e RT-qPCR. Resultados: O peso ao nascimento foi menor em machos e fêmeas da prole de mães alimentadas com dieta hipossódica durante a gestação quando comparado NR e HR. Não houve diferença no volume renal, volume de seus compartimentos (córtex, medula e pelve) e número de glomérulos entre os grupos experimentais (HO, NR e HR). No entanto, o número de glomérulos foi maior em fêmeas comparado aos machos nos três grupos experimentais. O diâmetro transverso do núcleo dos cardiomiócitos no ventrículo esquerdo e no ventrículo direito de machos da prole HR foi maior do que na prole NR. A expressão proteica do receptor AT1 no rim de machos da prole foi menor no grupo HO do que no grupo NR e HR. A expressão proteica do receptor AT2 também foi menor em machos do grupo HO do que no grupo NR. Não houve diferença entre os grupos na expressão proteica dos receptores AT1 e AT2 no rim das fêmeas. Conclusão: O presente estudo detectou alterações na estrutura cardíaca de neonatos machos, mas não em neonatos fêmeas decorrentes de sobrecarga de sal durante a gravidez. As alterações observadas na expressão dos receptores AT1 e AT2 no rim de neonatos machos podem ser responsáveis por alterações na função renal

Plant species, biomass and environmental characteristics of relevés along the North America Arctic bioclimate gradient

Question: How do interactions between the physical environment and biotic properties of vegetation influence the formation of small patterned-ground features along the Arctic bioclimate gradient? Location: At 68° to 78°N: six locations along the Dalton Highway in arctic Alaska and three in Canada (Banks Island, Prince Patrick Island and Ellef Ringnes Island). Methods: We analysed floristic and structural vegetation, biomass and abiotic data (soil chemical and physical parameters, the n-factor [a soil thermal index] and spectral information [NDVI, LAI]) on 147 microhabitat releves of zonalpatterned-ground features. Using mapping, table analysis (JUICE) and ordination techniques (NMDS). Results: Table analysis using JUICE and the phi-coefficient to identify diagnostic species revealed clear groups of diagnostic plant taxa in four of the five zonal vegetation complexes. Plant communities and zonal complexes were generally well separated in the NMDS ordination. The Alaska and Canada communities were spatially separated in the ordination because of different glacial histories and location in separate floristic provinces, but there was no single controlling environmental gradient. Vegetation structure, particularly that of bryophytes and total biomass, strongly affected thermal properties of the soils. Patterned-ground complexes with the largest thermal differential between the patterned-ground features and the surrounding vegetation exhibited the clearest patterned-ground morphologies.

Calcareous nannofossils of ODP Sites 149-897 and 149-901

Mesozoic calcareous nannofossil assemblages recovered during Ocean Drilling Program Leg 149 from the Iberia Abyssal Plain off the coast of Portugal were examined to determine the age of the rifting processes that affected the western Iberia Margin. Dark carbonaceous claystones (black shales) recovered from Site 901 contain highly diverse and abundant Tithonian calcareous nannofossil assemblages. Careful examination and documentation of this material has extended the ranges of numerous Jurassic and Cretaceous species and detailed a significant Late Jurassic assemblage turnover observed in the calcareous nannofossil record. The Lower Cretaceous sequence consists of intervals of serpentinized peridotite intercalated between various breccias and dark claystones. With the exception of a few samples, calcareous nannofossils are few and moderately preserved. The age of nannofossils within these varied sedimentary lithologies ranges from the late Barremian to the late Aptian. Eight new species are described: Ansulasphaera covingtonii, Clepsilithus meniscus, Conusphaera sinespina, Crepidolithus parvulus, Diazomatholithus galicianus, Percivalia arata, Rotelapillus pleoseptatus, and Tranolithus incus. Also proposed are five new combinations.

(Table 1) Distribution of Lower Cretaceous calcareous nannofossils at DSDP Hole 93-603B

Upper Berriasian to lower Aptian calcareous nannofossil assemblages have been studied from a siliciclastic deep-sea fan complex and a subjacent limestone sequence drilled beneath the lower continental rise in the western North American Basin, 270 miles (435 km) off Cape Hatteras, North Carolina (USA). Sharp lithologic facies changes and reworking by turbidites complicate the biostratigraphic interpretation, but provide an excellent opportunity to better distinguish "nearshore" from open-ocean nannofossil species, and to investigate the introduction of neritic taxa into the deep-see environment, a phenomenon that appears to have been widespread within the circum-North Atlantic during Neocomian times. Well-preserved assemblages in dark, carbonaceous claystones were probably displaced from the oxygen minimum zone along the upper slope or outer shelf. Neritic, continental margin species prevalent in this facies include the holococcolith Zebrashapka vanhintei n. gen., n. sp., Lithraphidites alatus magnus n. spp., Pickelhaube furtiva n. gen., and a host of nannoconids and micrantholiths. A qualitative evaluation of widely used guide fossils suggests that the triad of proposed markers for the base of Roth's Zone NC3 make their first appearances in the following (ascending) order: Diadorhombus rectus, TUbodiscus verenae, Calcicalathina oblongata. Of these, we chose the nominative species for the zone, T. verenae, to mark its base and to approximate the Berriasian/Valangian boundary. Cyclagelosphaera deflandrei is strongly affected by diagenesis and is therefore not a reliable index species for the base of Zone NC4 near the Valanginian/Hauterivian boundary (the last occurrence of T. verenae is also not suitable there). In addition, Lithraphidites bollii, a form apparently confined to the low latitudes of the Tethyan region, was absent at the more temperate Site 603 and not available as a subzonal marker for the upper Hautervian-lower Barremian (mid-NC4 and mid-NC5, respectively). Cruciellipsis cuvillieri, however, provides a reliable datum just below the Hauterivian/Barremian boundary (base of NC5), despite the potential for reworking in this section. Nannoconids tend to be reworked in this section, and do not provide trustworthy forms to mark the Barremian/Aptian boundary (base of NC6). Hayesites irregularis n. comb, probably does provide a useful first appearance datum within the lower Aptian, if it is not confused with a more birefringent and globular form, Rucinolithus terebrodentarius n. sp. Rhagodiscus angustus is mimicked by a similar form (Zeughrabdotusl pseudoangustus n. sp.), which apparently ranges down to the Hauterivian, thus Lithastrinus floralis provides a more useful first appearance datum for the base of the middle-upper Aptian Rhagodiscus angustus Zone (NC7). Aside from the new taxa mentioned above, the following are also described: Cretarhabdusl delicatus n. sp. and Cyclagelosphaera jiangii n. sp.