Turnover of mouse intestinal brush border membrane proteins and enzymes in organ culture. A direct evaluation from studies on the evolution of enzyme activities during the culture.

The turnover of mouse intestinal brush border membrane enzymes has been studied by kinetic analysis of the evolution of enzyme activities during organ culture. By comparing the results obtained in these studies with the predictions from a mathematical model of enzyme synthesis and degradation in organ cultures, it has been possible to reach the following conclusions: (1) There is no degradation of brush border membrane enzymes during culture and the rate of synthesis of each enzyme is directly measurable from the kinetics of total enzyme accumulation (tissue + media). (2) Brush border membrane enzymes are released in culture media by two complementary processes. The first one involves a differential solubilization of enzymes but its exact nature cannot be exactly stated. The second one involves a microvesiculation of brush border membranes, the importance of which in vivo is seen in the possible conciliation between urinary membrane synthesis and heterogeneous turnover of membrane components.

Is atrial natriuretic peptide synthesized and internalized by gonadotrophs?

The present study was designed to determine whether atrial natriuretic peptide (ANP) could be both synthesized and internalized by rat anterior pituitary gonadotrophs. ANP synthesis was assessed by in situ hybridization of ultrathin frozen sections of anterior pituitary to a biotinylated 30-base oligonucleotide to rat ANP mRNA. As revealed by the immunogold technique, only gonadotrophs were labeled by the probe. At the subcellular level, ANP mRNA was observed at both the nuclear and cytoplasmic levels in gonadotrophs, and labeling of the latter compartment was quantitatively more intense. Internalization of ANP was investigated by an in vivo ultrastructural autoradiographic approach. Intravenous injection of [125I]ANP resulted in rapid labeling within 1 min of the plasma membrane, cytoplasmic matrix, secretory vesicle, and mitochondrial compartments and the Golgi apparatus; these compartments were labeled throughout the remainder of the time course studied (1-30 min). Peak labeling of the plasma membrane compartment was at 1 min and diminished from that point; labeling in the Golgi apparatus peaked 5 min postinjection, while in the other compartments labeling was fairly uniform over the time course. The lysosomal compartment was also radiolabeled; however, only 2 and 5 min after injection of [125I]ANP. The findings demonstrate that gonadotrophs can both synthesize and internalize extracellular ANP. These observations can be extended to suggest that ANP has both autocrine and paracrine actions in the anterior pituitary gland. Since the peptide neither stimulates nor antagonizes the release of any anterior pituitary hormone, these actions are probably unrelated to the adenohypophyseal secretory function.

Synthesis, internalization, and localization of atrial natriuretic peptide in rat adrenal medulla.

Some, though not all studies, have indicated that atrial natriuretic peptide (ANP) can bind to adrenal medullary cells. ANP-like immunoreactivity (ANP-LI) has also been identified in catecholamine-secreting cells. Together, these findings suggest that ANP may be taken up and/or synthesized in the adrenal medulla. The present study was designed to ascertain, by in situ hybridization, whether adrenal chromaffin cells could synthesize ANP, to define by an in vivo ultrastructural autoradiographic approach, whether ANP could, in fact, bind to rat adrenal medulla cells, to determine whether there was a cellular [noradrenaline (NA) vs. adrenaline (A)] selectivity in the binding process, and to establish whether extracellular [125I]ANP could be internalized by these cells. The cellular and subcellular distribution of endogenous ANP-LI was also investigated in both cell types by cryoultramicrotomy and immunocytochemical approaches. The in situ hybridization studies indicate the presence of mRNA to ANP in about 15% of adrenal medullary cells. Intravenous injection of [125I]ANP resulted in a 3-fold, preferential and specific radiolabeling of A-as compared to NA-containing cells. In A-containing cells, plasma membranes were significantly labeled 2 and 5 min post injection; cytoplasmic matrix, mitochondria, and secretory granules throughout the time course studied (1-30 min post injection). Lysosomes, rough endoplasmic reticulum, Golgi apparatus, and nuclei were not labeled. ANP-LI was identified in both NA- and A-containing cells; in the former, it was almost exclusively localized in secretory vesicles, in the latter it was detected in plasma membranes, cytoplasmic matrix, nuclear euchromatin, some mitochondria and relatively fewer granules than in NA-containing cells. The findings suggest that ANP may be synthesized primarily in NA-containing cells and that A-containing cells primarily bind and internalize the extracellular (endogenous or exogenous) atrial peptide. The data suggest that ANP secreted by adrenal medullary chromaffin cells may have distal paracrine actions or interactions with coreleased catecholamines and neuropeptides. Binding and internalization may reflect an action of ANP on the secretory function of A-containing cells.

Quantitative autoradiographic localization of [125I]insulin-like growth factor I, [125I]insulin-like growth factor II, and [125I]insulin receptor binding sites in developing and adult rat brain.

Insulin-like growth factors I and II (IGF I and IGF II) and insulin itself, which are structurally related polypeptides, play an important role in regulating brain growth and development as well as in the maintenance of its normal functions during adulthood. In order to provide a substrate for the better understanding of the roles of these growth factors, we have investigated the anatomical distribution as well as the variation in the density of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites in developing and adult rat brain by in vitro quantitative autoradiography. The distributional profile of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites showed a widespread but selective regional localization throughout the brain at all stages of development. The neuroanatomic regions which exhibited relatively high density of binding sites with each of these radioligands include the olfactory bulb, cortex, hippocampus, choroid plexus, and cerebellum. However, in any given region, receptor binding sites for IGF I, IGF II, or insulin are concentrated in anatomically distinct areas. In the cerebellum, for example, [125I]IGF II receptor binding sites are concentrated in the granular cell layer, [125I]insulin binding sites are localized primarily in the molecular layer, whereas [125I]IGF I receptor binding sites are noted in relatively high amounts in granular as well as molecular cell layers. The apparent density of sites recognized by each radioligand also undergoes remarkable variation in most brain nuclei, being relatively high either during late embryonic (i.e., IGF I and IGF II) or early postnatal (i.e., insulin) stages and then declining gradually to adult levels around the third week of postnatal development. These results, taken together, suggest that each receptor-ligand system is regulated differently during development and thus may have different roles in the process of cellular growth, differentiation, and maintenance of the nervous system. Furthermore, the localization of [125I]IGF I, [125I]IGF II, and [125I]insulin receptor binding sites over a wide variety of physiologically distinct brain regions suggests possible involvement of these growth factors in a variety of functions associated with specific neuronal pathways.

Localization of sulfated glycoprotein-2/clusterin mRNA in the rat brain by in situ hybridization.

Sulfated glycoprotein-2 (SGP-2) gene expression seems to be constitutively expressed in a variety of tissues and organs, although levels of expression vary widely from one tissue to the other. SGP-2, also known as clusterin, has been reported to be expressed in the central nervous system (CNS). Some possible roles for brain SGP-2 have been postulated. In order to provide a substrate for a better understanding of the functions of this glycoprotein in the CNS, we investigated the detailed anatomical and cellular distribution of SGP-2 mRNA in the adult rat brain as well as the variation in its cellular expression after excitotoxin lesion. Transcripts for SGP-2 were found to be distributed throughout the rat CNS, although regional differences in their prevalence were readily observed. The ependymal lining of the ventricles showed the highest level of expression followed by various gray matter areas, some of which contained very intensively labeled cells. These cells were mostly found among several hypothalamic and brainstem nuclei, the habenular complex, as well as in the ventral horn of the spinal cord, which displayed striking hybridization signals over motoneurons. Occasional cells expressing high levels of SGP-2 transcripts were found in fiber tracts. Highly SGP-2 mRNA-positive resting glial cells were mainly located near the glial limitans and blood vessels. Two areas of relatively low constitutive SGP-2 mRNA expression are shown to produce strong hybridization signals 10 days after the local administration of the excitotoxin kainic acid. This overexpression of SGP-2 transcripts appears to involve GFAP-positive cells. Taken together, these results indicate that in the intact adult rat CNS, various cell populations, including neurons, constitutively express SGP-2 transcripts, whereas in the injured brain, reactive astrocytes become the major producers.

[Epidermal growth factor: structure, location, phosphorylation and regulation of its receptor]. / Le facteur de croissance épidermique: structure, localisation, phosphorylation et régulation de son récepteur.

Epidermal growth factor (EGF) is a Mr 6045 polypeptide first characterized for its ability to stimulate mitogenesis in epidermal and epithelial cells. The first step in the action of the growth factor is its binding to specific, high affinity membrane receptors. These receptors have been studied in a number of tissues and cell culture lines. The level of EGF receptors is modulated by many agents. EGF down-regulates its receptor. In addition, the number of EGF receptors is decreased by other growth factors (platelet-derived growth factor; transforming growth factor), by many tumor promoters and by viral transformation. Several hormones also can regulate EGF binding in its target tissues.

Acetylcholine induces neuritic outgrowth in rat primary olfactory bulb cultures.

The rat olfactory bulb is innervated by basal forebrain cholinergic neurons and is endowed with both nicotinic and muscarinic receptors. The development of this centrifugal cholinergic innervation occurs mainly in early postnatal stages. This developmental time-course and the demonstration that acetylcholine can modulate some aspects of neuronal proliferation, differentiation or death, suggests the possible involvement of cholinergic afferents in the morphogenesis and/or plasticity of the olfactory bulb. The purpose of the present work was to assess whether acetylcholine could modulate neuronal morphogenesis in the olfactory bulb. Toward this aim, we developed a primary culture model of rat olfactory bulbs. Three major cell types were identified on the basis of their morphological and immunocytochemical phenotype: neuronal-shaped cells expressing the neuronal markers neuron specific enolase, microtubule associated protein 2, neural cell adhesion molecule and beta-tubulin III; glial-like cells immunoreactive for glial fibrillary acidic protein and flattened cells immunolabelled with antibodies against beta-tubulin III and nestin, most likely neuronal precursors. After three to six days of treatment with 100-microM carbachol, a cholinergic agonist, significant increase in neuritic length was observed in cultured olfactory bulb neurons. The neurite outgrowth effect of carbachol was abolished by co-treatment with 1 microM alpha-bungarotoxin, an alpha 7 subunit nicotinic receptor antagonist, but was not affected by the addition of 10 microM atropine, a general muscarinic antagonist. The effect of carbachol was also mimicked by the nicotinic agonists, nicotine (100 microM) and epibatidine (10 microM). This pharmacological profile suggested the involvement of nicotinic receptors of the alpha 7-like subtype as confirmed using 125I-alpha-bungarotoxin receptor autoradiography.Taken together, these data argue for a role for nicotinic receptors in neuritic outgrowth in the rat olfactory bulb and provide a cellular support to the previously described effects of acetylcholine on olfactory bulb plasticity in vivo.

Expression of calcitonin gene-related peptide, substance P and protein kinase C in cultured dorsal root ganglion neurons following chronic exposure to mu, delta and kappa opiates.

The mechanisms involved in morphine tolerance are poorly understood. It was reported by our group that calcitonin gene-related peptide (CGRP)-like immunoreactivity (IR) was increased in the spinal dorsal horn during morphine tolerance [Ménard et al. (1996) J. Neurosci. 16, 2342-2351]. More recently, we observed that it was possible to mimic these results in cultured dorsal root ganglion (DRG) neurons allowing for more detailed mechanistic studies [Ma et al. (2000) Neuroscience 99, 529-539]. The aim of the present series of experiments was to further validate the DRG cell culture model by establishing which subtypes of opioid receptors are involved in the induction of CGRP in cultured rat DRG neurons, and to examine the signaling pathway possibly involved in the induction of CGRP-like IR following repeated opiate treatments. Other neuropeptides known to be expressed in DRG neurons, such as substance P (SP), neuropeptide Y (NPY) and galanin, were investigated to assess specificity. Following treatment with any of the three opioid agonists (mu, DAMGO; delta, DPDPE; kappa, U50488H), the number of CGRP- and SP-IR cultured DRG neurons increased significantly, and in a concentration-dependent manner, with the effects of kappa agonist being less pronounced. NPY and galanin were not affected.Double-immunofluorescence staining showed that the three opioid receptors were co-localized with both CGRP- and SP-like IR.Protein kinase C (PKC)-like IR was found to be significantly increased following a repetitive treatment with DAMGO. Double-immunofluorescence staining showed the co-localization of PKCalpha with CGRP- and SP-IR in cultured DRG neurons. Moreover, a combined treatment with DAMGO and a PKC inhibitor (chelerythrine chloride or Gö 6976) was able to block the effects of the opioid on increased CGRP-like IR. These data suggest that the three opioid receptors may be involved in the induction of CGRP and SP observed following chronic exposure to opiates, and that PKC probably plays a role in the signaling pathway leading to the up-regulation of these neuropeptides. These findings further validate the DRG cell culture as a suitable model to study intracellular pathways that govern changes seen following repeated opioid treatments possibly leading to opioid tolerance.

Systemic administration of kainic acid induces selective time dependent decrease in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in adult rat hippocampal formation.

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1-CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [125I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [125I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [125I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [125I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [125I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1-CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment. These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.

Impact of neonatal kainate treatment on hippocampal insulin-like growth factor receptors.

The insulin-like growth factors-I and -II have neurotrophic properties and act through specific membrane receptors. High levels of binding sites for these growth factors are distributed discretely throughout the brain, being concentrated in the hippocampal formation. Functionally, the insulin-like growth factors, in addition to their growth-promoting actions, are considered to play important roles in normal cell functions, as well as in response to pharmacological or surgical manipulations. In adult rats, we have previously shown that systemic injection of kainate produces an overall decrease, in a time-dependent manner, in insulin-like growth factor-I and -II receptor binding sites in the hippocampus [Kar S. et al. (1997) Neuroscience 80, 1041-1055]. Given the evidence that insulin-like growth factors play a critical role during the early stages of brain development, the present study is a logical extension of this earlier report and established the effect of neonatal kainate injection on the developmental profile of insulin-like growth factor receptors. We have evaluated the time-course alteration of these receptors following systemic injection of kainate to newborn rats. After injection of a sublethal dose of kainate (5 mg/kg, i.p.) to postnatal one-day-old pups, [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin binding sites were studied at different postnatal days (7, 14, 21, 28 and 35) using receptor autoradiography. In the developing hippocampus, insulin-like growth factor-I and insulin binding sites are concentrated primarily in the dentate gyrus and the CA2/CA3 subfields, whereas insulin-like growth factor-II binding is discretely localized to the pyramidal layer and the granular layer of the dentate gyrus. Following kainate injection, we observed a slight increase in insulin-like growth factor-I binding sites in given hippocampal subfields starting at postnatal day 14, being significant at day 21. At later days, a progressive decrease was noted. This transient increase may represent an attempt for neuronal plasticity by up-regulating receptor levels. In contrast, insulin-like growth factor-II and insulin receptor binding sites are found to be decreased in various regions of the hippocampus in kainate-treated pups. Taken together, these results provide further evidence for the existence and differential alterations of insulin-like growth factor-I, insulin-like growth factor-II and insulin receptors in the developing rat hippocampus following kainate-induced lesion, suggesting possible involvement of these growth factors in brain plasticity.

Localization and modulation of calcitonin gene-related peptide-receptor component protein-immunoreactive cells in the rat central and peripheral nervous systems.

Calcitonin gene-related peptide (CGRP) is widely distributed in the central and peripheral nervous system. Its highly diverse biological activities are mediated via the G protein-coupled receptor that uniquely requires two accessory proteins for optimal function. CGRP receptor component protein (RCP) is a coupling protein necessary for CGRP-receptor signaling. In this study, we established the anatomical distribution of RCP in the rat central and peripheral nervous system and its relationship to CGRP immunoreactivity. RCP-immunoreactive (IR) perikarya are widely and selectively distributed in the cerebral cortex, septal nuclei, hippocampus, various hypothalamic nuclei, amygdala, nucleus colliculus, periaqueductal gray, parabrachial nuclei, locus coeruleus, cochlear nuclei, dorsal raphe nuclei, the solitary tractus nucleus and gracile nucleus, cerebellar cortex, various brainstem motor nuclei, the spinal dorsal and ventral horns. A sub-population of neurons in the dorsal root ganglia (DRG) and trigeminal ganglia were strongly RCP-IR. Overall, the localization of RCP-IR closely matched with that of CGRP-IR. We also determined whether RCP in DRG and dorsal horn neurons can be modulated by CGRP receptor blockade and pain-related pathological stimuli. The intrathecal injection of the antagonist CGRP(8-37) markedly increased RCP expression in the lumbar DRG and spinal dorsal horn. Carrageenan-induced plantar inflammation produced a dramatic bilateral increase in RCP expression in the dorsal horn while a partial sciatic nerve ligation reduced RCP expression in the ipsilateral superficial dorsal horn. Our data suggest that the distribution of RCP immunoreactivity is closely matched with CGRP immunoreactivity in most of central and peripheral nervous systems. The co-localization of RCP and CGRP in motoneurons and primary sensory neurons suggests that CGRP has an autocrine or paracrine effect on these neurons. Moreover, our data also suggest that RCP expression in DRG and spinal cord can be modulated during CGRP receptor blockade, inflammation or neuropathic pain and this CGRP receptor-associated protein is dynamically regulated.

Knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) alleviates pain and restores opioid efficacy after nerve injury in rats.

1. Nerve injury often produces long-lasting spontaneous pain, hyperalgesia and allodynia that are refractory to treatment, being only partially relieved by clinical analgesics, and often insensitive to morphine. With the aim of assessing its therapeutic potential, we examined the effect of antisense oligonucleotide knockdown of spinal metabotropic glutamate receptor 1 (mGluR(1)) in neuropathic rats. 2. We chronically infused rats intrathecally with either vehicle, or 50 microg day(-1) antisense or missense oligonucleotides beginning either 3 days prior to or 5 days after nerve injury. Cold, heat and mechanical sensitivity was assessed prior to any treatment and again every few days after nerve injury. 3. Here we show that knockdown of mGluR(1) significantly reduces cold hyperalgesia, heat hyperalgesia and mechanical allodynia in the ipsilateral (injured) hindpaw of neuropathic rats. 4. Moreover, we show that morphine analgesia is reduced in neuropathic rats, but not in sham-operated rats, and that knockdown of mGluR(1) restores the analgesic efficacy of morphine. 5. We also show that neuropathic rats are more sensitive to the excitatory effects of intrathecally injected N-methyl-D-aspartate (NMDA), and have elevated protein kinase C (PKC) activity in the spinal cord dorsal horn, two effects that are reversed by knockdown of mGluR(1). 6. These results suggest that activity at mGluR(1) contributes to neuropathic pain through interactions with spinal NMDA receptors and PKC, and that knockdown of mGluR(1) may be a useful therapy for neuropathic pain in humans, both to alleviate pain directly, and as an adjunct to opioid analgesic treatment.

Synthesis and internalization of atrial natriuretic factor in anterior pituitary cells.

Atrial natriuretic factors (ANF) are a family of peptides originally identified in atrial cardiocytes and having natriuretic, diuretic and vasorelaxatory properties. ANF was recently reported to be synthesized in anterior pituitary cells. In the current study, the cell-specific sites of binding and internalization of ANF in the adenohypophysis, a site of ANF action, were investigated. By in vitro autoradiographic techniques, [125I]ANF was found to bind specifically to both anterior and posterior lobes of the pituitary, but not to the intermediate lobe. 5 min following intravenous injection of [125I]ANF, radiolabel was detected by ultrastructural autoradiography on gonadotrophs, corticotrophs and lactotrophs, but not thyrotrophs or somatotrophs. Label was found at both the plasma membrane level and cytoplasmic matrix, but not in the nuclei, of all three cell types. The cellular and subcellular localization of endogenous ANF-like immunoreactivity was also investigated. Specific immunoreactivity was also detected in gonadotrophs, corticotrophs and some lactotrophs. At the subcellular level in all three cell types, immunoreactivity was localized in the cytoplasmic matrix, on secretory granule membranes, and in mitochondria. Less dense staining was observed in nuclear euchromatin; endoplasmic reticulum and Golgi apparatus were not labelled in any of the cells. The data suggest that notwithstanding their reported ability to synthesize ANF, exogenous ANF can also bind and be internalized by adenohypophyseal gonadotrophs; the binding and presence of endogenous ANF-like immunoreactivity in corticotrophs and lactotrophs suggest that these cells may be sites of action for the atrial peptide.

Distribution of epidermal growth factor receptors in the rat ovary.

The distribution of epidermal growth factor (EGF) was studied in the ovary using light microscope radioautography which was performed at different time intervals (2-60 min) after intravenous (i.v.) injection of [125I]EGF into adult rat at random stages of the estrous cycle and also after topical localization of iodinated EGF on slide-mounted frozen ovarian sections. After i.v. injection of label, the labeling was mostly observed in the theca interna cells of secondary, preovulatory and atretic follicles and luteal cells of corpus luteum. Primordial and primary follicles did not show any significant labeling. The time-course study performed on luteal cells showed that, 2 min after injection, most silver grains were found at the periphery of the cells. At the 10 min time interval, silver grains were found both at the periphery and over the cytoplasm of these cells. The number of grains was very reduced over the cytoplasm at the 60 min time interval. In the in vitro study, a positive radioautographic reaction was seen in the same cellular elements as found in vivo, with the additional labeling of the granulosa cells of growing and preovulatory follicles. Control experiments indicated that the radioautographic labeling was due to specific interaction of [125I]EGF with its receptor. These results clearly indicate that EGF binding sites are present in luteal, thecal and granulosa cells, and provide support for the inhibitory and stimulatory actions of EGF on different parameters of ovarian cells.

Immunocytochemical localization, binding, and effects of atrial natriuretic peptide in rat adipocytes.

The metabolic effects of atrial natriuretic peptide (ANP) have not been widely investigated. Since adipocyte cells represent a model system extensively used to examine the metabolic actions of many peptide hormones, we sought to establish whether ANP could bind to adipocyte membranes, alter cyclic nucleotide metabolism, and affect spontaneous or hormone-stimulated lipolysis. Using in vitro autoradiographic techniques, radiolabelled ANP was found to bind specifically to mammary gland fat cells. Additionally, endogenous ANP-like immunoreactivity could be localized in the plasma membrane compartment and cytoplasmic matrix of fat cells, but not in fat vacuoles. [125I]ANP bound to single high affinity sites (Kd = 0.72 nM) in fat cell membranes. The binding was rapid (equilibrium within 1 min at 25 degrees C) and specific. The atrial peptide was capable of stimulating a time- and concentration-dependent increase in cGMP accumulation in isolated adipocytes, but had no effect on spontaneous or stimulated [-)-isoproterenol, ACTH, forskolin) cAMP formation. ANP did not alter the increase in glycerol production stimulated by l-epinephrine in isolated fat cells. While i.v. infusion of ANP stimulated a marked increase in circulating levels of cGMP, the atrial peptide did not alter plasma triglyceride levels. These data demonstrate the presence of specific ANP binding sites on adipocyte membranes and internalization of ANP-associated immunoreactivity. These receptors are biochemically functional given the ability of ANP to augment cGMP formation. The peptide, however, does not exert an action on adipocyte lipolysis. Adipocytes, therefore, represent an ANP target tissue in which the physiological action of the peptide is yet to be defined.

Expression of the purported sigma(1) (sigma(1)) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy.

Sigma (sigma) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of sigma receptor binding sites (sigma(1) and sigma(2)) is now well established. The recent cloning of the mouse, guinea pig and human sigma(1) receptors has allowed the study of the discrete distribution of the sigma(1) receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific sigma(1) receptor mRNA signals were in accordance to the anatomical distribution of sigma(1) receptor protein first established by quantitative receptor autoradiography. Specific sigma(1) receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I-II) and deeper (IV-VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of sigma(1) receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the sigma(1) receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of sigma(1) receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV-VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the sigma(1) receptor mRNA. Antisense oligodeoxynucleotides against the purported sigma(1) receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific sigma(1) receptor antisense into the third ventricle (0.4 nmol/h for 5 days), sigma(1)/[3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the sigma(1) receptor antisense treatments (5 nmol/injection, every 12 hx3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the sigma(1) receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported sigma(1) receptor gene reduced specific sigma(1)/[3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Ontogenic profile of the expression of the mu opioid receptor gene in the rat telencephalon and diencephalon: an in situ hybridization study.

The developmental profile of mu (mu) opioid receptor gene expression has been characterized in the embryonic, postnatal and adult rat brain by in situ hybridization histochemistry. By ED12, mu opioid receptor mRNA was detectable in the deep neuroepithelium of the cortical plate. In the developing rat central nervous system (ED13-PD40), transcripts were seen over numerous telencephalic and diencephalic structures, such as the olfactory bulb, caudate-putamen, nucleus accumbens, amygdaloid complex, hippocampal formation, hypothalamus and thalamus. In the vast majority of brain regions examined, the developmental profile of the mu opioid receptor gene expression is similar to that of its translated protein as established using receptor autoradiography. Once a hybridization signal is detected in the prenatal period, it gradually increased to reach maximal levels during the second and third postnatal weeks. By the end of the third postnatal week, mu opioid receptor mRNA levels decreased to reach amounts seen in adulthood. Our study demonstrates that mu opioid receptor gene expression is seen very early on in the embryonic rat brain with transient increases observed during the critical period of neurogenesis, neuronal migration and synaptogenesis, suggesting a role of this opioid receptor subtype in brain developmental processes.

Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland.

The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.

Insulin-like growth factor-1 (somatomedin-C) receptors in the rat brain: distribution and interaction with the hippocampal cholinergic system.

The present work characterizes the autoradiographic distribution of insulin-like growth factor-1 (IGF-1)/somatomedin-C binding sites in neonatal and adult rat brain, and attempts to correlate the distribution of IGF-1 sites, in certain regions of the rat brain, with functional IGF-1 receptors. In neonatal brain, [125I]IGF-1 binding sites are especially concentrated in superficial cortical layers, nucleus accumbens and hippocampus. In the adult rat brain, the distribution of IGF-1 sites is broader, with a high density of sites observed in superficial and deep cortical layers, olfactory bulb, endopiriform nucleus, basomedial nucleus of the amygdala, thalamic nuclei and hippocampus. Specific binding of [125I]IGF-1 to its sites in these brain regions was almost completely inhibited by 100 nM nonradioactive IGF-1. In contrast, similar concentrations of either IGF-2 or insulin did not significantly alter [125I]IGF-1 binding to its sites. Therefore, under our incubation conditions, [125I]IGF-1 appears to label specifically the type-I IGF receptor. In the hippocampus, which is highly enriched with specific [125I]IGF-1 binding sites in both neonatal and adult rat brain, IGF-1 significantly altered the potassium-evoked (25 mM) release of acetylcholine (ACh) from slices of adult, but not immature (6- and 18-day-old), rat brain. This IGF-1-induced decrease in ACh release from adult rat brain slices was concentration-dependent and appeared to be specific to hippocampus; ACh release from frontal cortical slices was not affected by this GF. The spontaneous release of ACh in the presence of IGF-1 in either tissue was not significantly different from control.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of epidermal growth factor binding sites in the adult rat pancreas by light microscopic autoradiography.

The distribution of epidermal growth factor (EGF) receptors was studied in the pancreas using light microscopic autoradiography, which was performed at different time intervals (2-60 min) after injecting 125I-labeled EGF intravenously into the adult rat. In the exocrine pancreas, a labeling was found to occur over the pyramidal cells of the acini and cells lining the intercalated ducts. Moreover, substantial binding of EGF to cells of the islets of Langerhans was also revealed. At the 2-min time interval, most silver grains were found at the periphery of the target cells. The localization, as well as the diminution of silver grains over the cytoplasm of these cells, between 7 and 60 min, suggested the internalization and degradation of 125I-labeled EGF. Control experiments indicated that the autoradiography reaction was due to specific interaction of 125I-labeled EGF with its receptor. These results clearly indicate that EGF receptors are present in the acinar cells and the cells of intercalated ducts of the exocrine pancreas, as well as the cells of the endocrine pancreas. Finding that there are EGF binding sites in pancreatic acinar cells supports the physiological role of EGF in the regulation of pancreatic exocrine function. The presence of EGF receptors in cells of the islets of Langerhans suggests that EGF may play a role in the regulation of the endocrine pancreas.