G. G. Nussdorfer

Effects of oxytocin on the function and morphology of the rat adrenal cortex: in vitro and in vivo investigations

The effects of oxytocin (OX) on the function and morphology of the rat adrenal cortex were studied in vivo and in vitro. OX exerted a potent stimulatory action on basal, but not 10−8 M ACTH-stimulated corticosterone (B) secretion of dispersed rat inner (zona fasciculata and zona reticularis) adrenocortical cells (maximal effective concentration: 10−9 M); in contrast, at higher concentrations (10−7/10−6 M) OX inhibited maximally ACTH-stimulated B output. A single subcutaneous (s.c.) injection of 1.2 nmol/100 g body weight OX resulted in a longlasting (up to 12 h) rise in plasma B concentration (PBC). The prolonged administration of OX (daily s.c. injections of 0.6 or 1.2 nmol/100 g for 10 days) caused a marked lowering in the adrenal weight and volume of all adrenocortical zones, that in turn was due to a decrease in the number of their parenchymal cells; however, the average volume of inner adrenocortical cells was significantly increased. Basal PBC was lowered, but its response to ether stress was unchanged in comparison with control rats. Prolonged OX treatment did not change B secretion by adrenal slices, but it markedly raised that of dispersed inner adrenocortical cells. Our present findings clearly show that the effects of OX on the adrenal cortex depend on the experimental model employed (in vitro versus in vivo) and the duration of treatment (acute versus chronic). Taken together they allow us to conclude that OX exerts an acute direct stimulatory effect on the rat adrenal cortex, and a chronic inhibitory one, that at least in part could be due to the interference of OX with the mechanism(s) of intracellular transduction of the ACTH secretagogue signal.

Cerebellin stimulates the secretory activity of the rat adrenal gland: in vitro and in vivo studies.

Cerebellin is a 16-aminoacid peptide widely distributed in the central nervous system, where it exerts neuromodulatory functions. Cerebellin is contained in human adrenal medulla, and it has been recently demonstrated that cerebellin elicits catecholamine release by human adrenal in vitro. Aim of the present study was to ascertain whether cerebellin affects adrenal function in the rat. Cerebellin concentration-dependently (from 10(-9)to 10(-7)M) increased norepinephrine (but not epinephrine) and cyclic-AMP production by adrenomedullary tissue in vitro. The norepinephrine response to 10(-7)M cerebellin was blocked by the protein kinase (PK) A inhibitor H-89, but not by the phospholipase C inhibitor U-73122 or the PKC inhibitor calphostin-C. Cerebellin did not affect aldosterone and corticosterone secretion of dispersed zona glomerulosa and zona fasciculata-reticularis adrenocortical cells. Cerebellin concentration-dependently (from 10(-8)to 10(-7)M) enhanced norepinephrine release by in situ perfused rat adrenals. Cerebellin (10(-7)M) also elicited a significant rise in aldosterone and corticosterone output, and this effect was annulled by either the beta1-adrenoceptor antagonist l -alprenolol or H-89. Collectively, the present findings allow us to conclude that cerebellin 1) directly stimulates norepinephrine release via the adenylate cyclase/PKA-dependent signaling pathway; and 2) indirectly enhances adrenocortical secretion in vivo, through a paracrine mechanism involving medullary catecholamine release.

Effects of neuropeptide-Y and substance-P on the secretory activity of dispersed zona-glomerulosa cells of rat adrenal gland

Neuropeptide-Y (NPY) and substance-P (SP), two peptides contained in the chromaffin granules of adrenal medullary cells, were found to partially inhibit both basal ACTH-stimulated release of aldosterone and 18-hydroxy-corticosterone by isolated rat zone-glomerulosa cells, without affecting the overall post-pregnenolone yield or basal progesterone output. Conversely, the exposure to both peptides increased 11-deoxy-corticosterone and corticosterone secretion. These data indicate that NPY and SP are able to exert a direct suppression of 18-hydroxylase activity in rat zona-glomerulosa cells, without conceivably altering the earlier steps of aldosterone synthesis. The possible physiological implications of these findings are discussed in light of previous studies suggesting a net adrenoglomerulotrophic effect of NPY and SP in vivo.

Effects of substance P on the rat adrenal zona glomerulosa in vivo

Substance P (SP) acutely enhanced the plasma concentration of aldosterone in rats whose hypothalamo-hypophyseal-adrenal axis and renin-angiotensin system were pharmacologically interrupted. The maximal response was obtained with a dose of 100 micrograms/kg. A prolonged (7 days) subcutaneous infusion with SP (50 micrograms/kg/hr) caused a notable hypertrophy of zona glomerulosa cells associated with significant rises in both basal and angiotensin-stimulated plasma levels of aldosterone. Zona fasciculata and the blood concentration of corticosterone were not affected. These findings suggest that SP is specifically involved in the stimulation of the growth and secretory activity of the rat zona glomerulosa.

Investigations on the turnover of adrenocortical mitochondria

SummaryThe effects of chronic administration of dexamethasone (for up to 15 consecutive days) on both the morphology and DNA-synthesis of the mitochondria of the rat adrenal zona fasciculata were investigated by stereologic and autoradiographic techniques. Up to the 3rd day of continuous dexamethasone treatment, the average volume of mitochondria did not change, whereas the number of mitochondria per cell was significantly decreased. From the 3rd to the 15th day of hormonal administration both the volume and number of mitochondria were found to decrease in proportion to the duration of treatment. Autoradiography showed that after the 3rd day of dexamethasone administration there is virtually no incorporation of 3H-thymidine into the mitochondrial compartment. These findings are discussed in the light of evidence indicating that dexamethasone blocks ACTH-release by inhibiting the hypothalamo-hypophyseal axis. The results confirm the view that ACTH controls the maintenance of growth and proliferation of rat adrenocortical mitochondria.

Order and disorder in the vascular network

Embryonic blood vessel patterns are formed by the combined action of both deterministic and random processes. During development, an abundant capillary meshwork is formed initially and an increasing number of branches and bifurcations is generated that connect the various capillary beds to their stem vessels. Vascular network formation proceeds along three main stages: (i) migration and early network formation; (ii) network remodelling; and (iii) differentiation in tubular structures and the development of capillary networks, characterized by typical intercapillary distances ranging from 50 to 300mm, which is instrumental for optimal metabolic exchanges. Several studies have been performed to understand the logic of vascular network growth. For example, a theoretical model for in vitro angiogenesis has been previously proposed based on experiments performed with bovine aortic endothelial cells focusing on cellular network reorganization driven by cellular traction. More recently, Serini et al provided a model including chemoattraction as a fundamental mechanism for cell to cell communication in order to identify key parameters in the complexity of the formation of vascular patterns. They showed that nonlinear mechanics and chemotactic cellular dynamics fit into a model able to reproduce with great accuracy the formation of capillary networks in vitro. The chick chorioallantoic membrane (CAM) is characterized by two distinct growth periods: before incubation day 11, it expands to cover the egg from inside, but after day 11, it can expand no further. The CAM capillary network undergoes three phases of development characterized by very different growth processes. In the early phase (days 5–7), sprouting of new capillaries represents the dominant angiogenic mechanism. Between days 8 and 12, the capillary network expands mainly by intussusceptive microvascular growth. In the later stages (days 13 and 14), the CAM capillary network undergoes a simple expansion with not much increase in network complexity. The analysis of mitotic rates in the CAM fits well with the concept that early sprouting is followed by intussusception: mitotic rates were very high early in the CAM and decreased to very low levels after day 11. The vascular tree of the CAM develops as a two-dimensional (2D) structure because of its confinement within the allantois, the inner layer of the bilaminar CAM. The capillary plexus develops via arteriolar/venular connections within the chorion, the outer membrane layer of the CAM, which lies adjacent to the shell. Angiogenic factors have been delivered to the CAM via encapsulation within a delivery device, such as a gelatin sponge, or methyl cellulose disk, direct placement of factorcontaining tissue or tissue-like substances (eg collagen gel) on the CAM or adsorption of the factor to a polymer window. These methods create interesting models of gradient or regional stimulation of angiogenesis. Blood vessel patterns in the chick CAM during normal development and after growth factor application have previously been characterized with measurements of vessel length density, endothelial proliferation intensity, and complexity and by fractal generation branching morphometry. A structural feature of particular interest in order to get a better understanding of tissue perfusion, and therefore of CAM efficiency, is the way the pattern of growing vessels fills the available space. To address this point, we have developed automatic image analysis methods allowing a quantitative evaluation of several parameters (fractal dimension, lacunarity and nonfractal order–disorder parameters, such as positional, topological and orientational order) characterizing the level of spatial order/disorder exhibited by the vascular network of the CAM in basal conditions and after treatment for 96 h with an angiogenic cytokine, such as fibroblast growth factor-2 (FGF-2), or with an angiostatic molecule, such as vinblastine. At day 12, CAM’s areas adjacent to the gelatin sponges soaked with 500 ng of FGF-2 or 1 pmol vinblastine were photographed in ovo with a stereomicroscope connected to an image analysis system at a primary magnification of 16. Greylevel images were then saved as TIFF files, processed and analysed by using a computer-assisted image analysis software and macro routines specifically developed by the authors. After brightness inversion, an adaptive discrimination procedure was applied to select vessel profiles virtually exclusive of all background. This method operated with a local threshold: the mean grey value of a neighbouring region was calculated for every pixel (by a 20 20 pixel low-pass filter); this value plus an offset threshold constant (40 grey levels) defined the local threshold for each pixel. A 3 3 median filter was then applied to clean the resulting binary image. By using binary thinning procedures, the skeleton of this image was finally derived and pruned to remove eventually present small artefactual branches. Vascular density was estimated from the thresholded image by evaluating the area fraction (AA) covered by the vessels. The ‘box counting’ method at multiple origins was applied to the image of the binary skeleton to estimate fractal dimension (D) and lacunarity (L) of the vascular network, well-known global indices of morphological complexity and structural nonuniformity, respectively. In addition, three types of order exhibited by the CAM vessels were considered: (i) positional order, associated with the arrangement of the network in the membrane; (ii) topological Received 1 July 2004; accepted 20 August 2004 Correspondence: Professor D Ribatti, Department of Human Anatomy and Histology, University of Bari Medical School, Policlinico, Piazza Giulio Cesare, 11, Bari I-70124, Italy; Fax: þ 39 080 54 78 310; E-mail: ribatti@anatomia.uniba.it Leukemia (2004) 18, 1745–1750 & 2004 Nature Publishing Group All rights reserved 0887-6924/04

Further investigations on the effects of neuropeptide Y on the secretion and growth of rat adrenal zona glomerulosa

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Effects of neuromedin U-8 on the secretory activity of the rat adrenal cortex: evidence for an indirect action requiring the presence of the zona medullaris

NPY is a regulatory peptide, high levels of which are contained in adrenal glands of several mammals and which is co-released with catecholamines during various stressful conditions. The acute and chronic effects of NPY on adrenocortical secretion and growth were studied in the rat. NPY concentration-dependently increased aldosterone (ALDO), but not corticosterone (B) secretion of adrenal slices (maximal effective concentration was 10(-7) M). Two competitive inhibitors of NPY receptors, named PYX-1 and PYX-2, were found to dose-dependently inhibit ALDO response of adrenal preparations to 10(-7) M NPY; PYX-2 was more efficient than PYX-1, and at a concentration of 10(-5) M completely annulled the effect of 10(-7) M NPY. The acute bolus intraperitoneal (i.p.) injection of NPY (3 nmol/kg) raised plasma ALDO concentration (PAC), but not that of B (PBC); this effect of NPY was blocked by the simultaneous injection of PYX-2 (300 nmol/kg). The prolonged i.p. infusion with NPY (3 nmol/kg/h for 7 days) increased PAC (but not PBC) and induced a marked hypertrophy of the zona glomerulosa (ZG) and its parenchymal cells; dispersed ZG cells obtained from NPY-infused rats displayed a significantly enhanced basal and maximally agonist-stimulated ALDO production. The simultaneous infusion with PYX-2 (300 nmol/kg/h) completely annulled all these effects of NPY. The acute or chronic administration of PYX-2 alone did not evoke any apparent effect on the ZG secretion and growth. In light of these findings the following conclusions can be drawn: (i) NPY is able to stimulate not only the secretion, but also the growth of adrenal ZG in rats, via a receptor-mediated mechanism (since this effect is blocked by PYX-2); (ii) endogenous NPY does not play a prominent role in the physiological maintenance of secretion and growth of rat ZG (since PYX-2 alone is ineffective); (iii) NPY may play a crucial role in the fine tuning of the ZG functions in conditions requiring an increased release of mineralocorticoid hormones.

Effects of neuromedin-N on the pituitary-adrenocortical axis of dexamethasone-suppressed rats

The acute effect of increasing concentrations (from 10−8 to 10−6M) of neuromedin U-8 (NMU-8) on steroid secretion of rat adrenal gland was investigated in vitro by high-pressure liquid chromatography. The production of the following steroids was measured: pregnenolone (PREG), progesterone (PROG), 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18OH-DOC), 18-hydroxycorticosterone (18OH-B) and aldosterone (ALDO). NMU-8 had no effects on either dispersed adrenocortical cells or fragments of adrenocortical autotransplants lacking medullary chromaffin cells. Conversely, NMU-8 exerted concentration-dependent secretagogue effects on adrenal slices, including both cortex and medulla. At all concentrations tested, NMU-8 increased the production of both PREG and total post-PREG steroids. The increase in total post-PREG steroid output induced by low concentrations of NMU-8 (10−8M) was due to similar rises in the production of non-18-hydroxylated steroids (PROG, DOC and B) and 18-hydroxylated hormones (18OH-DOC, 18OH-B and ALDO); conversely, that provoked by higher concentrations of the neuropeptide (10−7 to 10−6M) was almost exclusively caused by the rise in the yield of 18-hydroxylated steroids. The stimulating effect of NMU-8 on PREG output was blocked by both α-helical-CRH and corticotropin-inhibiting peptide, which are competitive inhibitors of CRH and ACTH, respectively. The following conclusions have been drawn: (1) NMU-8 affects adrenal steroid secretion indirectly by acting on the medullary chromaffin cells, which in turn may paracrinally stimulate the cortical ones; (2) at all concentrations tested, NMU-8, by stimulating the intramedullary CRH/ACTH system, causes a net rise in the activity of the early ratelimiting step of steroidogenesis, with the consequent increase in the output of the entire spectrum of post-PREG steroids; and (3) at higher concentrations (over 10−8M), NMU-8 also elicits the release from chromaffin cells of a factor (not yet known) that specifically enhances 18-hydroxylase activity.

Effects of galanin on the secretory activity of the rat adrenal cortex: in vivo and in vitro studies.

Neuromedin-N (NMN) (6 micrograms/100 g body weight for 2 d) partially reversed the dexamethasone (Dx)-induced inhibition of ACTH release and the consequent adrenal atrophy and decrease in glucocorticoid (corticosterone) plasma concentration in rats. Dx administration did not alter the level of circulating mineralocorticoid (aldosterone), but NMN (2 or 6 micrograms/100 g body weight for 2 d) significantly increased it. These findings suggest that the mechanism underlying the glucocorticoid (but not the mineralocorticoid) secretagogue action of NMN involves the stimulation of hypophyseal ACTH release. The hypothesis is advanced that the potent mineralocorticoid secretagogue effect of NMN may be mediated either by a direct action on zona glomerulosa cells or by the enhanced release of other regulatory peptides exerting aldosterone stimulating effect.