[Hospital nurses' perception of public health]. / La perception de la santé publique des infirmières à l'hôpital.

The objective of this work was to study hospital nurses' perceptions of public health based on a pedagogical exercise. A qualitative study, lead by students of a training institute for nursing care (IFSI), was conducted using semi-directed interviews of hospital nurses from different departments of a provincial hospital. The main results show that for one-third of the nurses interviewed, the hospital is not a setting for public health practice. In terms of their definition of public health, it is closely linked to the concept of prevention, with primarily individual approaches. This definition is very close to the public health activities that they conduct at the hospital and which are centred on information and disease prevention, on education related to pathologies and the relationship between the provision of care and listening to the patients and their families. Few nurses place their public health activities within the scope of the areas of hospital cleanliness, the welcoming of the patients, the organisation of services, and the improvement of the quality of care. The potential tracks which have emerged from this work lead to the need for the strengthening of training in patient education, in the hospital's work networking with external partners, and in better development of public health activities undertaken in the hospital setting.

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

Characterization and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites in the brain of the frog Rana ridibunda.

The biochemical characteristics and the distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites have been investigated in the brain of the frog Rana ridibunda by using [(125)I]PACAP27 as a radioligand. Membrane-binding studies revealed the existence of high-affinity receptors for frog PACAP38 and PACAP27. In contrast, the [Des-His(1)]PACAP38 analogue had a much lower affinity and vasoactive intestinal polypeptide did not produce any displacement of the binding. Autoradiographic labeling of frozen brain sections revealed that the highest concentrations of PACAP receptors were located in the olfactory bulb, pallium, striatum, habenular nuclei, ventromedial thalamic nucleus, corpus geniculatum, posterior tubercle, dorsal part of the magnocellular preoptic nucleus, tectum, and the molecular cell layer of the cerebellum. Moderate binding was observed in the septum, in most parts of the thalamus, the dorsal hypothalamic nucleus, the median eminence, the ventral nuclei of the tegmentum, the torus semicircularis, and the interpeduncular and isthmi nuclei. The present data provide the first biochemical characterization and anatomic distribution of PACAP binding sites in the brain of a nonmammalian vertebrate species. The widespread distribution of specific PACAP receptors in the frog brain suggests that the peptide does not act solely as a hypophysiotropic factor, but likely fulfills neurotransmitter functions, neuromodulator functions, or both.

[Strategy for identification of new neuropeptides]. / Stratégie d'identification de nouveaux neuropeptides.

Neuropeptides play a crucial role in cell communication as neurotransmitters, neuromodulators or neurohormones, and are involved in a number of biological activities including neuroendocrine regulations, control of neurovegetative functions, trophic effects and modulation of the immune response. The number of neuropeptides that have been fully characterized so far is rather limited, as compared to the number of precursor proteins that are actually expressed in nerve cells. Owing to the development of powerful analytical and structural identification methods, and the rapid advance in molecular biology techniques, a number of novel neuropeptides have been characterized during the last decade, in both vertebrates and invertebrates. The aim of the present review is to provide a comprehensive coverage of the different approaches which are currently used to identify novel neuropeptides.

Effects of the two somatostatin variants somatostatin-14 and [Pro2, Met13]somatostatin-14 on receptor binding, adenylyl cyclase activity and growth hormone release from the frog pituitary.

Two isoforms of somatostatin from frog brain have been recently characterized, namely somatostatin-14 (SS1) and [Pro2, Met13]somatostatin-14 (SS2). The genes encoding for the precursors of these two somatostatin variants are expressed in hypothalamic nuclei involved in the control of the frog pituitary. The aim of the present study was to investigate the effect of SS1 and SS2 on adenohypophysial cells. Autoradiographic studies using [125I-Tyr, D-Trp8] SS1 as a radioligand revealed that somatostatin binding sites are evenly distributed in the frog pars distalis. The SS2 variant was significantly (P < 0.01) more potent than SS1 in competing with the radioligand (IC50= 1.2 +/- 0.2 and 5.6 +/- 0.6 nM, respectively). Both SS1 and SS2 induced a modest but significant reduction in cAMP formation in dispersed distal lobe cells but did not affect spontaneous growth hormone (GH) release. Synthetic human GRF (hGRF) induced a significant increase in cAMP accumulation and GH release in this system. Both SS1 and SS2 inhibited the stimulatory effects of hGRF on cAMP formation and GH secretion. These data show that the SS1 and SS2 variants can regulate adenohypophysial functions. The fact that GH cells are exclusively located in the dorsal area of the frog adenohypophysis, while somatostatin receptors are present throughout the pars distalis, indicates that the two somatostatin isoforms may control the secretion of pituitary hormones additional to GH in amphibians.

Localization and characterization of PACAP receptors in the rat cerebellum during development: evidence for a stimulatory effect of PACAP on immature cerebellar granule cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are abundant in the brain and particularly in the cerebellum of adult rats. In contrast, the occurrence of PACAP binding sites has not been investigated during ontogenesis. The aim of the present study was to determine the distribution and biochemical characteristics of PACAP binding sites in the rat cerebellum during postnatal development, and to examine the effect of PACAP on immature cerebellar granule cells. Autoradiographic studies revealed that PACAP binding sites are transiently expressed in a germinative matrix of the cerebellar cortex, the external granule cell layer, and in the medulla, from postnatal days 8 to 25. A population of PACAP binding sites persisted in the internal granule cell layer in the mature cerebellum. Emulsion-coated cytoautoradiography, performed on cultured immature granule cells from eight-day-old rat cerebellum, demonstrated that transient PACAP binding sites are expressed by cerebellar immature granule cells. Biochemical characterization of binding revealed the occurrence of two classes of PACAP recognition sites exhibiting, respectively, high (Kd = 0.39 +/- 0.08 nM) and low (Kd = 21.2 +/- 9.4 nM) affinity for PACAP27. The two naturally occurring forms PACAP38 and PACAP27 were equipotent in competing for [125I]PACAP27 binding. In contrast, the [Des-His1]PACAP38 analog was eight times less efficient and vasoactive intestinal polypeptide only induced weak displacement of the binding. Exposure of cultured immature granule cells to PACAP27 resulted in a dose-dependent stimulation of the production of cAMP, indicating that PACAP binding sites represent authentic receptors positively coupled to adenylate cyclase. These results show that PACAP receptors are actively expressed in the cerebellum of rats during postnatal development. The presence of functional PACAP receptors in the external granule cell layer suggests that PACAP may play a role in the control of proliferation and/or differentiation of granule cells.

Neuroanatomical and physiological evidence for the involvement of pituitary adenylate cyclase-activating polypeptide in the regulation of the distal lobe of the frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ribibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluorescence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50 = 2 x 10(-8) M). At the maximum dose tested (5 x 10(-6) M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine-vasopressin in anterior pituitary cells: in situ hybridization of mRNA and ultrastructural localization of immunoreactivity.

The hypothalamic nonapeptide arginine-vasopressin (AVP) exerts several distinct receptor-mediated actions on pituitary cells. Although hypothalamic AVP reaches the anterior pituitary via well-defined pathways, there is now accumulating evidence that AVP may also be produced endogenously in anterior pituitary cells. Using in situ hybridization, we demonstrate here the presence of AVP mRNA in the anterior pituitary of the rat. The observed grain density over pituitary cells was, however, greater than 10-fold lower than the one observed over AVP producing neurons present in the supraoptic and paraventricular nuclei of the hypothalamus. Immunoelectron microscopic analysis using two different AVP-specific antibodies revealed that the distribution of AVP-like immunoreactivity (AVP-LI) in the anterior pituitary is cell-specific. AVP-LI is most abundant in corticotrophs, followed by lactotrophs, gonadotrophs and thyrotrophs. On the other hand, there is complete absence of AVP-LI from somatotrophs. Interestingly, all pituitary cells in which AVP-LI is detected also represent potential target sites for AVP action. A minor fraction of AVP-LI was found to be membrane-associated and may originate, at least in part, from extrapituitary sources. This fraction likely represents receptor-bound peptide. The bulk of AVP-LI, however, was present in the cellular cytoplasm, not associated with any specific ultracellular structure. Specifically in corticotrophs, AVP-LI was excluded from secretory granules. However, our finding of AVP mRNA in anterior pituitary cells indicates that intracellular AVP-LI includes endogenously produced peptide, suggesting a paracrine and/or autocrine action.

Atrial natriuretic peptide stimulates submandibular gland synthesis and secretion of cGMP.

Binding of atrial natriuretic peptide (ANP) to rat submandibular gland and its effect on guanosine 3',5'-cyclic monophosphate (cGMP) formation and salivary secretion were investigated. Membranes rapidly and specifically bound 125I-ANP. Binding was inhibited by unlabeled ANP (IC50 approximately 1.6 nM), but not by atriopeptin I, other COOH- and NH2-terminal deleted ANP fragments, or agents such as pilocarpine or substance P. Scatchard analysis revealed a single class of high-affinity sites (dissociation constant 0.74 +/- 0.25 nM; maximal binding capacity 20.5 +/- 6.3 pmol/mg protein). Intravenous infusion of ANP with pilocarpine caused a significant dose-dependent increase in the levels of cGMP detected in plasma and saliva. Because salivary cGMP may have originated in plasma, the effect of ANP on cGMP formation was evaluated in dispersed cells. ANP evoked a concentration-dependent increase in both cGMP synthesis and secretion (EC50 approximately 1.7 x 10(-8) M). The atrial peptide did affect basal or l-isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate synthesis in dispersed cells. When infused by itself and/or with pilocarpine, ANP did not alter the rate of spontaneous or pilocarpine-induced salivary flow, secretion of chloride, or protein release. The data demonstrate the presence of guanylate cyclase-coupled ANP receptors in submandibular gland; the atrial peptide, however, does not exert an effect of the secretory function of the gland.

4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid inhibits adrenocorticotropin secretion from anterior pituitary cells.

Mouse clonal ACTH-secreting corticotrophs (AtT-20 cells) possess a membrane Ca2+-activated Cl- conductance which is partially blocked by the disulfonic stilbene derivative 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). In the current study the effect of SITS on the ACTH secretory process was evaluated. SITS markedly blocked basal and forskolin-stimulated ACTH secretion from AtT-20 cells (IC50 = 2.7 x 10(-4) M). Both CRF-induced ACTH secretion and forskolin-stimulated GH secretion from acutely dispersed rat anterior pituitary cells were inhibited by SITS (IC50 = 2.4 and 1.3 x 10(-4) M, respectively). SITS did not alter unstimulated or forskolin-elicited cAMP synthesis in AtT-20 cells, and in fact, could inhibit ACTH secretion in response to cAMP-independent agonists such as the calcium channel activator BAY-K-8644 or the protein kinase-C activator 12-tetradecanoyl-phorbol-13-acetate (IC50 = 2.6 and 2.4 x 10(-4) M, respectively). SITS did not alter the secretion of amylase from isolated exocrine pancreatic acinar cells. Its action was also fully reversible; after its removal from the incubation medium, cells secreted ACTH without a change in response to forskolin activation. Increasing extracellular Ca2+ or the addition of up to 10(-3) M tetraethylammonium or 4-aminopyridine did not reverse the inhibitory pattern of SITS action, suggesting that its inhibitory effect is most likely not due to hyperpolarization of AtT-20 cell membranes. The inability of amiloride to inhibit ACTH secretion further suggests that inhibition of ACTH secretion provoked by SITS is not due to a blockade of Cl-/HCO3- exchange. On the other hand, SITS was able to block 44% of basal 36Cl uptake by AtT-20 cells. Exchange of incubation medium chloride for gluconate or a reduction in the osmotic strength of the medium reduced both basal and secretagogue-stimulated ACTH secretion. The data suggest that SITS may modulate chloride-dependent, osmotically driven secretion from AtT-20 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.

Internalization of atrial natriuretic peptide by adrenal glomerulosa cells.

Internalization of 125I-labelled atrial natriuretic peptide ([ 125I]ANP) by rat adrenal glomerulosa cells in vivo was investigated by means of an ultrastructural autoradiographic approach. One to 30 min after IV injection of [125I]ANP, silver grains were found, at the light microscope level, over all glomerulosa cells; coinjection of 20 micrograms of unlabelled ANP inhibited this binding by 64%. At the electron microscope level, the time-course study indicated maximal silver grain densities in plasma membranes 1 min after IV injection; grains were detected in mitochondria (external membranes and matrix) 2 min after injection, with maximal labelling at 15 min. The cytoplasmic matrix was labelled only 30 min after injection. During the time-course, labelling of nuclei, Golgi apparatus, and lysosomes was minimal. The data suggest that after binding to plasma membranes ANP is rapidly internalized and distributed within glomerulosa cells. The association of radioactivity with mitochondria suggests that ANP may have intracellular sites of action complementary to those on plasma membranes.

ANF and exocrine pancreas: ultrastructural autoradiographic localization in acinar cells.

Atrial natriuretic factor (ANF) binding sites have been recently demonstrated to be present in exocrine pancreas by an in vitro autoradiographic approach. An autoradiographic study was carried out to identify the exocrine cells containing ANF binding sites and to monitor the fate of 125I-labeled ANF in acinar cells after removal of pancreas at specific time intervals (1-30 min) after intravenous administration. At the light microscopic level, silver grains were found over acinar and centroacinar cells. Concomitant injection of an excess of unlabeled ANF inhibited the binding of labeled peptide by approximately 60%. At the electron microscopic level, the time-course study in acinar cells has revealed that of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria, and zymogen granules, the nucleus had distinct labeling patterns. Plasma membrane was maximally labeled 1 and 2 min after injection with 125I-ANF. Golgi apparatus was significantly labeled from 2 to 30 min after injection, mitochondria from 1 to 30 min after injection, zymogen granules at 1 and 15 min, and the nucleus only at 30 min. The lysosomal compartment was not labeled during the 30-min observation period. These results suggest that after binding to the plasma membrane, ANF is rapidly internalized and distributed to the intracellular organelles as a function of time. Labeling of the zymogen granules suggests that they may bind ANF and that the atrial peptide may be secreted by acinar cells. The significance of association of radioactivity with mitochondria and nuclei remains to be elucidated but may represent intracellular sites of action of ANF complementary to those on plasma membranes.

Evidence that AVP receptors in AtT-20 corticotrophs are not coupled to secretion of POMC-derived peptides.

This study reports the presence in AtT-20 corticotrophs of high affinity-low capacity receptors for arginine-vasopressin (AVP), whose binding capacity was considerably enhanced by the divalent metal ion nickel. These binding sites, when analyzed in the presence of nickel, showed high affinity for AVP, vasotocin and oxytocin, but recognized to a lesser extent the V2-agonist 1-deamino-AVP, as well as V1-antagonists. Surprisingly, AVP failed to alter secretion of proopiomelanocortin (POMC)-derived peptides from the cells or corticotropin-releasing factor (CRF)-induced cAMP synthesis, as reported in normal corticotrophs. Exposure of cells to CRF elicited an increase in mRNAPOMC levels, while, in contrast, AVP was without significant effect. It thus appears that in AtT-20 tumor cells, the AVP receptors are not coupled to either the biochemical or biological cellular response.

Characterization and modulation of corticotropin-releasing factor in the neurointermediate pituitary gland.

The present study attempts to determine whether part of the corticotropin-releasing factor (CRF)-like materials present in the 'posterior pituitary' is composed of authentic CRF and examines whether the concentration of that peptide may be modulated by circulating glucocorticoids. Analysis of crude extracts of neurointermediate lobes (NIL) of rat pituitaries by reverse-phase HPLC, coupled with a specific radioimmunoassay (RIA), revealed the presence of a major component eluting with the same retention time as rat CRF (rCRF) and, importantly, which was indistinguishable by RIA from the synthetic peptide. Also, two minor forms eluted earlier than rCRF upon HPLC; one of these forms matched the elution position of r[Met(O)21,38]CRF. All three species did show biological activity and stimulated ACTH release from pituitary cells. Essentially the same elution profile was generated by median eminence (ME) extracts. Immunoreactive CRF (CRFi) content of the NIL was about 3% of that of the ME and was found to undergo a significant increase as a result of long-term adrenalectomy while, in contrast, CRFi content of the ME was decreased. This effect of adrenalectomy was completely antagonized by dexamethasone treatment. This study thus provides strong evidence for the presence of authentic CRF within the NIL of the rat pituitary and also shows that tissue concentration of that peptide was modulated by glucocorticoids.