RESUMO
We have addressed the molecular mechanism(s) of hyperalgesia, which depends on increased excitability of dorsal horn neurons and on sensitization of primary afferent nociceptors, during peripheral inflammation. Following unilateral adjuvant-induced inflammation in the rat hind paw, time-course changes in behavioral hyperalgesia and functional activities of Ca2+/phospholipid-dependent protein kinase C isozymes were examined. Inflammation was characterized by increase in paw diameter, and behavioral hyperalgesia was quantified as paw withdrawal latency from a radiant heat source. Behavioral hyperalgesia on the injected paw was significantly increased. This was accompanied by a significant increase in total functional membrane-associated protein kinase C activity, whereas total cytosolic protein kinase C activity was unchanged on the sides of the lumbar spinal cord both contralateral and ipsilateral to the inflammation. Importantly, on the side of lumbar cord ipsilateral to the inflamed paw, the activity of membrane-associated protein kinase CbetaII was increased following the same time-course as the paw withdrawal latency decrease, suggesting an increased translocation of protein kinase Cbetall to the membrane related to behavioral hyperalgesia. A defined mixture of purified gangliosides, which inhibits intracellular protein kinase C translocation and activation, decreased inflammation-induced paw withdrawal latency, and specifically decreased the activity of membrane-associated protein kinase Cbetall on the side of the spinal cord ipsilateral to the inflammation. Quantitative immunohistochemical analyses demonstrated intensified protein kinase CbetaII-like immunoreactivity on the side of the spinal cord ipsilateral to the inflammation. Time-course for increases in the activity of membrane-associated protein kinase CbetaII, and in intensity of protein kinase CbetaII-immunoreactivity, paralleled inflammation-mediated changes in paw withdrawal latency and paw diameter. Our findings indicate an apparent involvement of protein kinase CbetaII isozyme specifically in the molecular mechanism(s) of thermal hyperalgesia.
Assuntos
Hiperalgesia/enzimologia , Inflamação/enzimologia , Isoenzimas/metabolismo , Neurônios/enzimologia , Proteína Quinase C/metabolismo , Medula Espinal/enzimologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Citosol/efeitos dos fármacos , Citosol/enzimologia , Edema/induzido quimicamente , Edema/fisiopatologia , Pé/inervação , Pé/patologia , Pé/fisiopatologia , Adjuvante de Freund/farmacologia , Gangliosídeos/farmacologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Nociceptores/citologia , Nociceptores/enzimologia , Medição da Dor/efeitos dos fármacos , Proteína Quinase C beta , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacosRESUMO
Gabapentin (Neurontin, Pfizer Global R & D) is a novel anticonvulsant, antihyperalgesic, and antinociceptive agent with a poorly understood mechanism of action. In this study, we show that gabapentin (EC50 2 microM) inhibited up to 70 to 80% of the total K+-evoked Ca2+ influx via voltage-dependent calcium channels (VD-CCs) in a mouse pituitary intermediate melanotrope clonal mIL-tsA58 (mIL) cell line. mIL cells endogenously express only gamma-aminobutyric acid type B (GABA(B)) gb1a-gb2 receptors. Moreover, activity of the agonist gabapentin was dose dependently and completely blocked with the GABA(B) antagonist CGP55845 and was nearly identical to the prototypic GABA(B) agonist baclofen in both extent and potency. Antisense knockdown of gb1a also completely blocked gabapentin activity, while gb1b antisense and control oligonucleotides had no effect, indicating that gabapentin inhibition of membrane Ca2+ mobilization in mIL cells was dependent on a functional GABA(B) (gb1a-gb2) heterodimer receptor. In addition, during combined whole cell recording and multiphoton Ca2+ imaging in hippocampal neurons in situ, gabapentin significantly inhibited in a dose-dependent manner subthreshold soma depolarizations and Ca2+ responses evoked by somatic current injection. Furthermore, gabapentin almost completely blocked Ca2+ action potentials and Ca2+ responses elicited by suprathreshold current injection. However, larger current injection overcame this inhibition of Ca2+ action potentials suggesting that gabapentin did not predominantly affect L-type Ca2+ channels. The depressant effect of gabapentin on Ca2+ responses was coupled to the activation of neuronal GABA(B) receptors since they were blocked by CGP55845, and baclofen produced similar effects. Thus gabapentin activation of neuronal GABA(B) gb1a-gb2 receptors negatively coupled to VD-CCs can be a potentially important therapeutic mechanism of action of gabapentin that may be linked to inhibition of neurotransmitter release in some systems.
Assuntos
Acetatos/farmacologia , Aminas , Analgésicos/farmacologia , Anticonvulsivantes/farmacologia , Canais de Cálcio/efeitos dos fármacos , Ácidos Cicloexanocarboxílicos , Agonistas dos Receptores de GABA-B , Células Piramidais/efeitos dos fármacos , Ácido gama-Aminobutírico , Animais , Baclofeno/farmacologia , Canais de Cálcio/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Agonistas GABAérgicos/farmacologia , Gabapentina , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/fisiologiaRESUMO
Studies of gamma-aminobutyric acid (GABA)(B) receptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G-protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABA(B) receptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABA(B), D(2) and corticotrophin releasing factor receptors. Here, we report on characterization of the GABA(B) receptors. Solution hybridization-nuclease protection assays reveal the presence of GABA(B(1)) and GABA(B(2)) transcripts. Western blots show GABA(B(1a)) and one of two GABA(B(2)) proteins. Addition of the GABA(B) agonist baclofen to cultured mIL-tsA58 (mIL) cells inhibits high voltage activated Ca(2+) channels, as measured by agonist-induced inhibition of the K(+)-depolarization-stimulated increase in Ca(2+) influx. CGP55845, a GABA(B) antagonist, blocks the response to baclofen. Knockdown of either GABA(B(1)) or GABA(B(2)) subunits with selective antisense oligodeoxynucleotides reduced GABA(B) protein levels and completely abolished the GABA(B) receptor response in the mIL cells. Taken together, these results indicate that functionally active GABA(B) receptors in mIL cells require the constitutive expression of both GABA(B) genes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABA(B) receptor expression, regulation and function.
Assuntos
Regulação da Expressão Gênica/genética , Hipófise/metabolismo , Receptores de GABA-B/biossíntese , Receptores de GABA-B/genética , Animais , Western Blotting , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular , Agonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Ensaios de Proteção de Nucleases , Oligonucleotídeos Antissenso/farmacologia , Hipófise/citologiaRESUMO
Somatostatin-14 was first detected on gestational day 17 in radially-oriented, bipolar cells spanning the width of the intermediate lobe of the rat pituitary. Cells were prominent, and constituted approximately 50% of the lobe area. The presence of vimentin, the cellular shape, and the localization identified these cells as glia. At postnatal day 6, somatostatin-14 and vimentin staining appeared in stellate-shaped cells. This is in agreement with the change from bipolar to stellate shape these glia undergo after the onset of innervation ([13] Gary et al. Int. J. Devl. Neurosci. 13, 555-565, 1995). Glia were more abundant, relative to melanotropes, throughout embryonic and early postnatal development compared to adulthood. Reverse transcription-polymerase chain reaction data showed a high level of prosomatostatin mRNA in the intermediate lobe, compared to the anterior and neural lobes from postnatal day 2 animals, and a significant drop in intermediate lobe content in the adult. The correlation between the number of glia and high expression of somatostatin in neonatal relative to adult tissue, together with the close apposition of incoming axons to the abundant, radially oriented glia during innervation of the lobe, support a neurotrophic function of glia-derived somatostatin.
Assuntos
Neuroglia/metabolismo , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Somatostatina/biossíntese , Somatostatina/fisiologia , Animais , Astrócitos/metabolismo , Axônios/fisiologia , Axônios/ultraestrutura , Feminino , Imuno-Histoquímica , Neuroglia/ultraestrutura , Hipófise/citologia , Gravidez , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Dopamine D2 receptors both acutely and chronically inhibit high-voltage-activated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D2L (long) and D2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D2L and D2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D2-receptor regulation of Ca2+ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca2+ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D2-agonist quinpirole (250 microM) to the K+/CXR solution significantly increased the lag times for D2-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D2L- and D2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.
Assuntos
Canais de Cálcio Tipo N , Canais de Cálcio/genética , Melanócitos/química , Proteínas de Membrana/genética , Receptores de Dopamina D2/genética , Animais , Ligação Competitiva/fisiologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Condutividade Elétrica , Expressão Gênica/fisiologia , Ativação do Canal Iônico/fisiologia , Isomerismo , Masculino , Melanócitos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodos , Nifedipino/farmacologia , Hipófise/citologia , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Tempo de Reação/fisiologia , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Transdução de Sinais/fisiologia , Espiperona/farmacologia , Transfecção , TrítioAssuntos
Axônios/fisiologia , Bromocriptina/farmacologia , Haloperidol/farmacologia , Plasticidade Neuronal/fisiologia , Hipófise/fisiologia , Receptores de Dopamina D2/fisiologia , Animais , Axônios/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Hipófise/citologia , Hipófise/efeitos dos fármacos , Ratos , Receptores de Dopamina D2/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Adrenergic markers and neuropeptide Y (NPY) were examined in Dahl NaCl-sensitive and -resistant outbred male rats, fed either 0.35% or 8% NaCl diets for 8 weeks. The high salt diet caused left ventricular hypertrophy in sensitive rats but not in the resistant strain. Norepinephrine stores were not affected by high salt intake, but tyrosine hydroxylase, and dopamine beta-hydroxylase were elevated in the salt-induced hypertrophied left ventricle in conjunction with increased levels of nerve growth factor and p75 neurotrophin receptor. In contrast, high salt intake reduced ventricular neuropeptide Y in both Dahl salt-resistant and -sensitive rats.
Assuntos
Adrenérgicos/metabolismo , Miocárdio/metabolismo , Neuropeptídeo Y/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Animais , Hipertrofia Ventricular Esquerda/patologia , Masculino , Miocárdio/patologia , Fatores de Crescimento Neural/metabolismo , Norepinefrina/metabolismo , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta/efeitos adversos , Sistema Nervoso Simpático/metabolismoRESUMO
GABA(B) and dopamine D2 receptors, both of which acutely inhibit adenylyl cyclase and high voltage-activated Ca2+ channels (HVA-CCs), are found in high levels in the melanotrope cells of the pituitary intermediate lobe. Chronic D2 receptor agonist application in vitro has been reported to result in inhibition of HVA-CC activity by down-regulation. Here we report that chronic GABA(B), but not GABA(A), agonist treatment also resulted in HVA-CC inhibition. Two GABA(B) receptor variants have been cloned and shown to inhibit adenylyl cyclase in HEK-293 cells. We have constructed an antisense deoxynucleotide knockdown-type probe that is complementary to 18 bp from the point at which the two sequences first become homologous. Chronic coincubation with baclofen and GABA(B) antisense nucleotide completely eliminated the inhibition of the channels by baclofen alone but had no reversing effect on HVA-CC inhibition by the D2 agonist quinpirole. A scrambled, missense nucleotide also had no reversing effect. Incubation with a D2 antisense knockdown probe eliminated the ability of a D2 agonist to inhibit the channels but had no effect on baclofen blockade. These results show the existence an R1a/R1b type of GABA(B) receptor, which, like the D2 receptor, is coupled to chronic HVA-CC inhibition in melanotropes.
Assuntos
Canais de Cálcio/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Hipófise/metabolismo , Receptores de GABA-B/genética , Animais , Baclofeno/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Eletrofisiologia , Masculino , Hipófise/citologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/efeitos dos fármacos , Receptores de GABA-B/fisiologia , Fatores de TempoRESUMO
The biosynthetic activity of rat intermediate lobe melanotropes in vivo is inhibited by stimulation of dopamine D2 receptors. Individual melanotropes are innervated differentially by dopaminergic axons and vary in their levels of pro-opiomelanocortin (POMC) mRNA. We tested the hypothesis that placement of the lobe in primary culture, which removes the inhibitory innervation, would increase POMC mRNA levels and abolish the heterogeneity in POMC expression. POMC mRNA levels increased successively in untreated melanotropes when tested on culture Days 10, 16, and 20; however, some heterogeneity in POMC expression persisted. If treated with a D2 receptor agonist (1 microM bromocriptine) from culture Day 1, POMC mRNA levels were decreased significantly throughout the testing period when compared to untreated cells with the same time in culture. Although some melanotropes still expressed high POMC levels, preparations appeared more homogeneous by Day 20. Melanotrope responses were reversible, since POMC mRNA levels were down-regulated by application and up-regulated by withdrawal of a D2 receptor agonist. A short agonist treatment resulted in subpopulations that responded differently to the agonist, possibly representing a mechanism for fine-tuning peptide hormone release.
Assuntos
Bromocriptina/farmacologia , Agonistas de Dopamina/farmacologia , Hipófise/metabolismo , Pró-Opiomelanocortina/biossíntese , Receptores de Dopamina D2/agonistas , Animais , Técnicas de Cultura , Denervação , Hipófise/citologia , Hipófise/inervação , Pró-Opiomelanocortina/genética , RNA Mensageiro/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Stimulation of melanotrope dopamine D2 receptors decreases mitotic rate, calcium channel activity, and the biosynthesis of several proteins. This study demonstrates that D2 receptor activation also affects GABA(A) receptor beta2/beta3 subunit immunoreactivity. Following chronic treatment with haloperidol, a D2 receptor antagonist, GABA(A) receptor immunoreactivity increased, whereas it decreased after chronic treatment with bromocriptine, a dopamine D2 receptor agonist. Thus, these data indicate that D2 function regulates GABA(A) receptor expression in melanotropes, a mechanism by which peptide release may be modified.
Assuntos
Hormônios Estimuladores de Melanócitos/metabolismo , Receptores de Dopamina D2/fisiologia , Receptores de GABA-A/fisiologia , Animais , Bromocriptina/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Haloperidol/farmacologia , Imuno-Histoquímica , Masculino , Hipófise/efeitos dos fármacos , Hipófise/fisiologia , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/agonistasRESUMO
Dopamine and GABA were detected in intermediate lobe axons around birth, and early axons were closely apposed to glial cells and processes, possibly using them for guidance. In the adult, axons containing colocalized dopamine and GABA were distributed in a distinct pattern within the lobe, with plexuses located dorsally and ventrally. Axons preferentially followed glial processes in interlobular septa, yet were also interspersed between melanotropes. Individual melanotropes were contacted by varying numbers of axon terminals, with some devoid of contacts. Boutons contained both small clear vesicles and large dense-cored vesicles; membrane specializations were not well-developed. From these findings we concluded that in addition to direct synaptic inhibition, dopamine and GABA could stimulate their receptors by mechanisms similar to "parasynaptic" [Schmitt (1984) Neuroscience, 13:991-1001] or "volume" [Agnati et al. (1995) Neuroscience, 69:711-726] transmission as proposed for the CNS. Humoral agents passing into the intermediate lobe from portal vessels, thus acting as classical hormones, further regulate the melanotropes. Moreover, approximately 50% of the axonal elements were closely apposed to glia, suggesting that glia could have regulatory roles. Previous studies from our laboratory [Chronwall et al. (1987) Endocrinology, 120:1201-1211; Chronwall et al. (1988) Endocrinology, 123:1992:1202] demonstrated heterogeneity in proopiomelanocortin (POMC) biosynthesis among individual melanotropes, prompting the hypothesis that the degree of innervation could govern the expression of certain molecules. We combined immunohistochemistry and in situ hybridization histochemistry to evaluate whether melanotrope molecular heterogenity is spatially correlated with axons and terminals. Tentatively, melanotropes expressing low levels of POMC and alpha1A subunit P/Q type Ca2+ channel mRNAs often were apposed to axons, whereas those with low levels of D2L receptor mRNA rarely were contacted by axons, suggesting that innervation could be one of the factors inducing and maintaining heterogeneity.
Assuntos
Melanóforos/fisiologia , Hipófise/embriologia , Hipófise/inervação , Fatores Etários , Animais , Axônios/química , Axônios/enzimologia , Axônios/ultraestrutura , Canais de Cálcio/genética , Dopamina/fisiologia , Feminino , Proteína Glial Fibrilar Ácida/análise , Glutamato Descarboxilase/análise , Masculino , Microscopia Eletrônica , Sistema Nervoso Parassimpático/embriologia , Sistema Nervoso Parassimpático/enzimologia , Sistema Nervoso Parassimpático/ultraestrutura , Hipófise/ultraestrutura , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Sinapses/química , Sinapses/fisiologia , Sinapses/ultraestrutura , Tirosina 3-Mono-Oxigenase/análise , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/fisiologiaRESUMO
The two isoforms of the dopamine D2 receptor, the D2short and the D2long differ in a 29 amino acid insert in the third cytoplasmic loop with which G proteins interact. We have previously reported that in rat melanotropes, expression of D2short increases markedly at the end of the first postnatal week which is concurrent with innervation of the intermediate lobe. Using immunohistochemistry, this study examined expression of G alpha i1/2, G alpha i3, G alpha o and G alpha s proteins before and after dopaminergic innervation. G alpha i3 increased through gestational day 20, and then remained level to postnatal day 6. At this time, coinciding with the induction of D2short expression, G alpha i3 immunoreactive intensity increased markedly, possibly indicating co-regulation of these proteins. On postnatal day 6, G alpha s immunoreactive intensity increased in some, but not all, melanotropes. The resulting heterogeneity in Gs expression persisted in the adult. G alpha i1/2 immunoreactivity did not change and G alpha o was detected only subsequent to the event of innervation. Thus, dopamine released from axons and acting through D2 receptor stimulation could increase G alpha i3 immunoreactivity and decrease G alpha s immunoreactive intensity in some melanotropes.
Assuntos
Proteínas de Ligação ao GTP/genética , Hipófise/química , Hipófise/citologia , Animais , Feminino , Imunofluorescência , Proteínas de Ligação ao GTP/análise , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/fisiologia , Hibridização In Situ , Neuroglia/química , Neuroglia/citologia , Hipófise/embriologia , Gravidez , Pró-Opiomelanocortina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Stimulation of dopamine D2 receptors inhibits melanotrope pro-opiomelanocortin (POMC) biosynthesis and alpha-melanocyte-stimulating hormone (MSH) secretion. These effects are mediated by G-protein alpha i- and alpha o-subunits and are reversed by stimulating receptors linked to activation of G alpha s protein. Melanotrope activity is increased by haloperidol, a D2 receptor antagonist, and decreased by bromocriptine, a D2 receptor agonist. Both the short and long isoforms of the D2 receptor mRNA and protein increase following chronic haloperidol treatment. After chronic bromocriptine treatment the short isoform is downregulated, whereas the long isoform is upregulated. Our hypothesis is that specific G protein alpha- subunits alter in pattern of expression similarly to the receptor isoforms. Using immunohistochemistry and in situ hybridization, this study examined changes in G alpha i, G alpha o, and G alpha s protein and mRNA expression following chronic treatments with bromocriptine or haloperidol. G alpha i3 and G alpha o immunoreactivities increased following bromocriptine treatment, whereas G alpha s and G alpha i1/2 did not change. Gs immunoreactivity increased after haloperidol treatment, whereas G alpha i1/2, G alpha i3, and G alpha o did not change. G alpha i and G alpha o mRNA increased following bromocriptine and decreased following haloperidol treatments, whereas the inverse results were observed with G alpha s mRNA. These results suggest D2 receptor activation can specifically increase G alpha i3 and G alpha o expression, and D2 receptor blockade increases G alpha s expression.
Assuntos
Proteínas de Ligação ao GTP/biossíntese , Hipófise/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Autorradiografia , Sequência de Bases , Bromocriptina/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Expressão Gênica/efeitos dos fármacos , Haloperidol/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Hipófise/citologia , Hipófise/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/efeitos dos fármacosAssuntos
Hipófise/metabolismo , Pró-Opiomelanocortina/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Cloreto de Sódio na Dieta/administração & dosagem , alfa-MSH/metabolismo , Animais , Imuno-Histoquímica , Hibridização In Situ , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Neuropeptide Y (NPY) and somatostatin immunoreactivities are present in neural lobe axons of the rat pituitary. Both peptides are upregulated during lactation, because NPY gene expression increases in the hypothalamus and plasma concentrations of somatostatin are elevated. However, the effects of lactation on NPY and somatostatin in the neural lobe are unknown. Although NPY immunoreactivity increases in the neural lobe following salt loading of male rats, the somatostatin response is unknown. To answer these questions, NPY and somatostatin immunoreactivities in the neural lobe were examined during lactation and salt loading using immunohistochemistry and image analysis. On day 2 of lactation, the area covered by immunoreactivity, a combined measurement of axon density and size of axonal swellings, of both NPY and somatostatin increased compared to ovariectomized rats. The increase in NPY was four- to fivefold greater than that of somatostatin. By day 10 of lactation, values returned to those of ovariectomized rats. Following 10 days of salt loading, the area covered by NPY immunoreactivity increased approximately 10-fold over control male rats, whereas somatostatin remained unchanged. NPY and somatostatin were not colocalized in neural lobe axons in either paradigm, demonstrating that two different neuronal populations were involved in both cases. These data indicate that NPY and somatostatin were regulated similarly during lactation, but differentially following salt loading.
Assuntos
Lactação/metabolismo , Neuropeptídeo Y/metabolismo , Neuro-Hipófise/metabolismo , Cloreto de Sódio/administração & dosagem , Somatostatina/metabolismo , Animais , Axônios/metabolismo , Feminino , Imuno-Histoquímica , Masculino , Neuro-Hipófise/efeitos dos fármacos , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Cultures of pituitary neurointermediate lobe cells were established from rats aged 1, 12, and 42 days to identify the types and assess the activities of Ca2+ channels present in melanotropes, glial-like cells, and fibroblasts during development. Day 12 represents the time at which dopaminergic axons have become distributed throughout the lobe, glial cells begin to lose their radial orientation, and melanotropes robustly express the short isoform of the dopamine D2 receptor. Thus, we studied Ca2+ channels in relation to the event of innervation of melanotropes. Real-time fluorescence video microscopy, in the presence of pharmacological agents, which block L-, N-, P-, and T-type channels, was used as an indirect measurement of channel activity. Assessment of cell type was verified by triple-label fluorescence immunohistochemistry. In melanotropes, extracellular Ca2+ addition caused Ca2+ influx through omega-conotoxin GVIA-sensitive, N-type channels on days 1 and 12 but not on day 42. The K+ depolarization induced an increase in intracellular Ca2+ concentration in all age-groups. This effect was decreased by nifedipine, an L-type channel blocker, at all ages, and by omega-agatoxin IVa, a P-type blocker, only on day 42. These results demonstrate that the predominance of N- or P-type channels on melanotropes is age-dependent and can be correlated with other developmental changes. The T-type blocker, NiSO4, had no effect. In glial-like cells of all ages, extracellular Ca2+ addition resulted in an increase in intracellular Ca2+ concentration, which was inhibited only by NiSO4. The percentage of responsive glial-like cells was equally high in days 1 and 12 cultures, then declined by day 42. The K+ depolarization had no effect on glial-like cells. Fibroblasts did not respond significantly to extracellular Ca2+ or K+ depolarization, indicating little detectable activity by this methodology from functional voltage-operated Ca2+ channels.
Assuntos
Canais de Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Microscopia de Fluorescência , Microscopia de Vídeo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Hipófise/citologia , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/metabolismoRESUMO
This study measured melanotrope mRNA and protein expression for the dopamine D2 receptor, and its long isoform, in relation to the appearance of dopamine in axons of the postnatal rat pituitary intermediate lobe. At postnatal day 2, prior to the onset of dopaminergic innervation, D2 receptor (D2T) mRNA was expressed heterogeneously in a subpopulation of melanotropes which also expressed the long isoform (DL). The D2L mRNA appeared to be predominant during early postnatal development, since the D2T probe, which did not discriminate between the isoforms, and the D2L probe hybridized generally to the same cells, as demonstrated in serial sections. Immunohistochemical methods, using two different antisera for the D2T receptor, however, indicated a low level of protein in most melanotropes. Localization of D2L protein corresponded well to D2T receptor mRNA distribution. At day 10, representing a time when dopamine is present in axons throughout the lobe, both D2T receptor mRNA and protein were detected in a significantly larger population of melanotropes than those expressing D2L mRNA and protein. This suggests the appearance of detectable short isoform (D2S) mRNA in virtually all melanotropes and implicates dopamine as a possible signal for increasing D2S isoform mRNA expression.
Assuntos
Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Receptores de Dopamina D2/biossíntese , Animais , Sequência de Bases , Feminino , Imuno-Histoquímica , Hibridização In Situ , Isomerismo , Microscopia de Fluorescência , Dados de Sequência Molecular , Hipófise/citologia , Gravidez , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Vimentina/metabolismo , beta-Endorfina/metabolismoRESUMO
Calcitonin (CT) is known to inhibit basal and TRH-stimulated prolactin release in cultured anterior pituitary cells in vitro and pituitary CT-like peptide (pit-CT) is synthesized and released by isolated anterior pituitary cells. However, the specific cell type containing pit-CT has not been identified. To determine this, double label immunohistochemistry was performed on pituitary sections from male rats using antisera for specific marker peptides of gonadotrophs, thyrotrophs, lactotrophs, somatotrophs, corticotrophs, and folliculo-stellate cells. CT was only colocalized with gonadotroph-specific markers and the distribution of pit-CT immunoreactive (IR) cells followed the patterns of gonadotroph distribution in male and female rats. Double and triple label immunohistochemistry using antiserum for CT, FSH, and PRL showed an apposition of calcitonin-like peptide containing gonadotrophs to cup-shaped lactotrophs. To examine whether pit-CT IR was altered, similarly to gonadotrophs, with known changes in PRL serum levels, studies were extended to ovariectomized, pregnant, and lactating rats. The area covered by pit-CT immunoreactivity and the tissue content of pit-CT significantly differed between physiological states and the pit-CT level was inversely related to the known PRL status. Pit-CT containing gonadotrophs were in all cases apposed to cup-shaped lactotrophs. These results provide histological support for previous studies proposing that pit-CT serves as a paracrine inhibitor of PRL release.
RESUMO
This study was conducted to determine if intermediate lobe glial-like cells are affected by compounds that regulate melanotrope biosynthetic activity via the dopamine D(2) receptor. Glial-like cells were stellate, and expressed glial fibrillary acidic protein (GFAP) and vimentin in cell bodies and processes as revealed by immunohistochemistry. Following bromocriptine and quinpirole treatments, the number of cell bodies and processes expressing vimentin did not change, whereas those expressing GFAP increased, although never to exceed the number of vimentin containing structures. The percent of cells and processes coexpressing GFAP and vimentin also increased. The GFAP response was reversible by haloperidol treatment following the agonist treatment. Haloperidol treatment alone did not change GFAP expression. Thus, following D(2) receptor agonist treatment, GFAP was induced in pre-existing vimentin-positive glial cells. Dopamine D(2) receptor mRNA and protein were detected in melanotropes, but not in cells expressing GFAP or vimentin. Although glial-like cells may express dopamine D(2) receptor mRNA and protein below the detection levels of our methods, the possibility of an indirect effect via dopamine D(2) receptor agonists acting on melanotropes needs to be considered.
RESUMO
This study demonstrated morphological changes in glial-like cells of the rat pituitary intermediate lobe during early postnatal development, and a subsequent shift in protein expression from vimentin to GFAP. Vimentin immunoreactivity was detected in the lobe at embryo day 14 and was localized in radially-oriented, bipolar cells whose processes spanned the thickness of the intermediate lobe. At electron microscopical resolution, processes contained intermediate filaments, cell nuclei were indented while secretory vesicles characteristic of the endocrine cells were not found. Vimentin immunoreactive intensity began to decrease at postnatal day 5. By postnatal day 7, vimentin-positive, stellate cells were observed, with few radial processes found by day 10. The intensity of vimentin immunoreactivity decreased through day 25. Within the lobe parenchyma, vimentin was localized in glial-like cells since double-label immunohistochemistry revealed no colocalization of beta-endorphin and vimentin, or fibronectin and vimentin. Dopamine-containing axons were in close apposition to vimentin-positive processes. GFAP immunoreactivity first appeared on postnatal day 20 and, by day 25, stellate cell bodies with three to six extended processes were evident. Cells were primarily distributed in the caudal third of the lobe. The characteristic adult pattern of cell clusters in latero-dorsal and ventral portions of the lobe was fully established by postnatal day 55. The transition from vimentin to GFAP expression and concurrent morphological changes resemble those described for radial glial during cerebral cortical development.