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Biomedical subjects

G Alonso

Publications and source records attributed to G Alonso.

At least 19 recordsLinked to original sources
PubMed1992

Dipyridamole stimulates types II cAMP-dependent protein kinase in vitro.

Dipyridamole activates in vitro type II cAMP-dependent protein kinase. This agent stimulates the autophosphorylation of the regulatory subunit in the presence of cAMP but not so in the absence of the cyclic nucleotide. The activation was also observed with exogenous substrates such as casein, histone 2A and MAP2. This stimulation did not seem to be related to the cAMP binding to the R II subunit of the enzyme. Competition binding experiments showed that dipyridamole does not compete with adenosine for the A1 receptor. The results suggest that the reported regulatory properties of dipyridamole on lipid metabolism (González-Nicolás et al. Int J Biochem 21: 883-888, 1989) might be mediated through a direct action--an activation--on the catalytic subunit of a cAMP-dependent protein kinase.

Adipose Tissue↗
PubMed1992

Effects of litter removal on the lipolytic response and the regulatory components of the adenylate cyclase in adipocytes isolated from lactating rats.

The effects of litter removal on the status of different components of the hormone-sensitive adenylate cyclase system were analysed in plasma membranes of rat adipocytes. These effects were correlated with the decreased lipolytic response of adipose tissue. No changes in total number of A1 adenosine receptors or their affinity were detected in response to litter removal. In contrast, beta-adrenergic receptors showed a decrease (35%) in total number of receptors, without any significant change in their affinity. The status of alpha-GS and alpha-Gi, the alpha-subunits of G proteins which mediate stimulation and inhibition respectively of adenylate cyclase, were probed by cholera- and pertussis-toxin-catalysed ADP-ribosylation respectively and by immunoblot. Associated with litter removal, decreases of 63% and 62% in the incorporation of [alpha 32P]ADP-ribose catalysed by cholera toxin and pertussis toxin into alpha-Gs and alpha-Gi respectively were detected. Immunoblotting using RM/1 (anti-alpha-Gs) and AS/7 (anti-alpha-Gi) antisera also showed decreases in the levels of alpha-Gs (52%) and alpha-Gi (55%) in adipocyte membranes from litter-removed rats compared with lactating rats. Alterations in the status of hormone-sensitive adenylate cyclase components, such as those described herein, may be biochemical mechanism(s) by which adipose tissue shows a decreased lipolytic response during recovery from lactation.

Adenylyl Cyclases↗
PubMed1992

Identification of new world Leishmania using ribosomal gene spacer probes.

DNA probes from the nontranscribed ribosomal spacer (NTS), of Leishmania garnhami and Leishmania braziliensis were constructed and tested for sensitivity and specificity against different Leishmania isolates. The L. garnhami probes were species-specific under hybridization conditions of high stringency, but displayed specificity for the mexicana complex under conditions of intermediate stringency. The L. braziliensis probes showed 'complex' specificity. RFLP for the nontranscribed spacer within the braziliensis complex revealed very homogeneous patterns even for organisms currently accepted as different species. A PCR assay for the detection of Leishmania from the braziliensis complex is presented.

Animals↗
PubMed1992

[Decrease of pineal AMPc and TOH activity in the superior cervical ganglia after ablation of submaxillary glands in rats].

In order to investigate a possible functional relationship between the submandibular salivary gland (SSG) and the central nervous system (CNS), we have extirpated the salivary organs from thirty male rats. Twenty days after ablation both the pineal glands and the cervical superior ganglions (CSG) were dissected, homogenized and frozen until AMPc and TOH were assayed respectively. We observed a significant decrease in pineal AMPc (53.9 +/- 6.2 vs 76.1 +/- 7.6% of maximum value; p less than 0.02) which seems to be linked with a significant drop in TOH activity measured at CSG level (1.5 +/- 0.6 vs 3.7 +/- 0.9 nmoles of DOPA/h/pair GCS; p less than 0.03). Our results suggest that both findings might be due to the lack of NGF normally reaching the CSG from SSG. This data reinforces the idea of a functional link between SSG and CNS via the pineal gland.

Animals↗
PubMed1991

Microinjection of oxytocin into the dorsal vagal complex decreases pancreatic insulin secretion.

Microinjections of oxytocin and of an oxytocin antagonist into the dorsal vagal complex of the medulla oblongata were performed in order to study the possible role of the oxytocin containing axons that innervate this region in the regulation of pancreatic insulin secretion. No significant effect was produced by the intramedullary injection of the oxytocin vehicle alone or of 0.04 pM oxytocin. Injections of 4 and 20 pM oxytocin produced a reversible decrease of plasmatic insulin levels which fall to 59% of basal levels 15 min after the injection. Such an effect was abolished when 4 pM oxytocin was injected to animals which have been previously bilaterally vagotomized. In contrast to oxytocin, intramedullary injection of a specific antagonist of oxytocin to intact animals induced a marked increase of plasmatic insulin levels which raised 131% of basal levels 15 min after the injection. In animals receiving such an injection of oxytocin antagonist, a secondary injection of 4 pM oxytocin produced a slight but not significant decrease of plasmatic insulin levels. These data strongly suggest that the hypothalamic neurons producing oxytocin that densely project to the dorsal vagal complex may be involved in an inhibitory control of the vagal preganglionic neurons that innervate the pancreas.

Animals↗
PubMed1991

Differences in the immunoreactivity to phenylethanolamine-N-methyltransferase in the central adrenergic neurons of four strains of rats.

Immunocytochemistry was used to compare the immunoreactivity of adrenergic neurons to a well characterized specific immunoserum to phenylethanolamine-N-methyltransferase (PNMT) in different strains of rats commonly used in research studies. In adult animals, marked differences were found in the PNMT-immunoreactivity of neurons between Wistar rats and other strains, resulting in a lower PNMT-immunostaining intensity (i) within neuronal perikarya of the medulla oblongata, and (ii) more strikingly, within nerve fibers and terminals located in various brain regions. This low PNMT-immunoreactivity of nerve fibers was detected both in 14- and 35-day-old Wistar rats. On the other hand, the HPLC measurement of catecholamines, in particular of adrenaline in the hypothalamus and the medulla oblongata, did not show any difference between adult Wistar and Sprague-Dawley rats. These data suggest that the low PNMT-immunoreactivity observed in central adrenergic neurons of the Wistar rats is related to the poor recognition of the antigen by the PNMT-antibody used. Possibly, these nerve cells mainly display an isoform of the enzyme that is immunologically different from the PNMT contained within the adrenergic neurons of other rat strains.

Adrenergic Fibers↗
PubMed1990

Adrenergic innervation of the dorsal vagal motor nucleus: possible involvement in inhibitory control of gastric acid and pancreatic insulin secretion.

Morphological and physiological approaches were used to investigate the possible role of an adrenergic innervation of the dorsal vagal complex in the control of basal gastric acid and pancreatic insulin secretion in the rat. The use of retrograde-tracing methods with injections of True Blue or of wheat-germ agglutinin into the stomach or pancreas first confirmed that most vagal preganglionic neurons innervating these two viscera are localized in the dorsal motor nucleus of the vagus, a number of them connected to both viscera. Light- and electron-microscopic investigation of the organization of adrenergic neuronal structures immunoreactive to phenylethanolamine-N-methyltransferase within this medullary nucleus further revealed: (i) that adrenergic axons establish profuse synaptic connections of the symmetrical type with perikarya and dendrites of this nucleus, and (ii) that several of these adrenergic fibers are connected with retrogradely labeled neurons innervating the stomach and/or pancreas. Lastly, measurements of basal gastric acid output and plasma insulin clearly indicated that both visceral secretions are rapidly and conspicuously decreased by local infusion of 2 nM adrenaline within the dorsal vagal complex. Taken together, these data strongly suggest that the adrenergic innervation of the dorsal medulla oblongata is involved in direct synaptic inhibition of the parasympathetic preganglionic neurons of the vagus that control secretion of gastric acid and pancreatic insulin.

Adrenergic Fibers↗
PubMed1990

Some aspects of metabolic adaptations in lipid metabolism during starvation are mimicked by epinephrine in rat adipocytes.

1. The effects of fasting on the neutral lipid synthesis to insulin and/or epinephrine in isolated fat cells have been examined using [1-14C]glucose. 2. The ability of adipocytes from starved rats to synthesize fatty acids from both labeled substrates was markedly diminished compared to adipocytes from control rats. 3. The response of lipogenic stimulation to insulin at all concentrations tested was greatly diminished in adipocytes from 24 hr starved rats. 4. [1-14C]glucose utilization rates in the absence or in the presence of insulin were not significantly different in adipocytes from 24 hr starved rats as compared with control adipocytes, although basal and insulin stimulated glyceride-glycerol synthesis were significantly higher in starved adipocytes. 5. Epinephrine acutely inhibited [1-14C]acetate incorporation into fatty acids for insulin-stimulated lipogenesis in control adipocytes, in contrast, this lipolytic agent strongly increased [1-14C]glucose conversion to triacylglycerols. 6. In both cases, the differences in lipid synthesis capacities found in both nutritional states were abolished by epinephrine.

Acetates↗
PubMed1989

Comparative immunocytochemical study of the catecholaminergic and peptidergic afferent innervation to the dorsal vagal complex in rat and guinea pig.

Light and electron microscopic immunocytochemistry was used to study the fine structural organization of the catecholaminergic and hypothalamic peptidergic innervation of the dorsal vagal complex of the medulla oblongata in the rat and guinea pig, the latter of which is known to lack central adrenergic neurons. In the rat, adrenergic fibers immunoreactive to phenylethanolamine-N-methyltransferase were concentrated in the dorsal motor nucleus of the vagus, where they established frequent symmetric synapses with dendrites and perikarya. On the other hand, the density of both oxytocin- and corticotropin-immunoreactive fibers appeared far lower in this nucleus than in the dorsal regions of the nucleus of the tractus solitarius, where they formed asymmetric synapses with small dendrites. In tissue treated for the dual labeling of two neuronal antigens, oxytocin- or corticotropin-reactive fibers were in close contact with adrenergic neurons in this dorsal medullary region. In the guinea pig, unlike the rat, the dorsal motor nucleus of the vagus contained large amounts of oxytocin- and corticotropin-reactive fibers, which formed many symmetric synapses with perikarya and dendrites. Taken together, these data suggest that the control of vagal preganglionic neurons by hypothalamic peptidergic neurons involves a bisynaptic neuronal pathway including adrenergic medullary neurons in the rat, whereas it is direct in the guinea pig, which lacks this adrenergic relay.

Adrenergic Fibers↗
PubMed1989

The organization of prolactin-like-immunoreactive neurons in the rat central nervous system. Light- and electron-microscopic immunocytochemical studies.

The localization and distribution of prolactin-like-immunoreactive perikarya and nerve fibers in the rat central nervous system have been studied by a preembedding immunoperoxidase method using well-characterized specific immunsera to rat prolactin. Although the localization of labeled neuronal structures in a number of brain areas correlates with the data of previous immunocytochemical studies, we found prolactin-immunoreactive neurons in various regions not previously reported. In untreated animals, the highest concentrations of prolactin-fibers were observed: (i) in the external layers of the median eminence where they exhibited close contact with blood vessels, and (ii) in the bed nucleus of the stria terminalis and in the central nucleus of the amygdala where they closely surrounded unlabeled perikarya. Dense networks of finely varicose prolactin fibers were also observed in the organum vasculosum of the lamina terminalis, in the subfornical organ, and in the dorsolateral regions of the medulla oblongata and the spinal cord. Lastly, a number of large, varicose, intensely immunoreactive fibers were found in the olfactory bulb, the cingulum, and the periventricular regions of the hypothalamus and central gray, whereas isolated fibers could be detected in the caudate nucleus and in the cerebral cortex. In animals treated with colchicine, prolactin-immunoreactive perikarya were essentially located within the periventricular and perifornical regions of the hypothalamus, and within the bed nucleus of the stria terminalis. Although corticotropin (ACTH 17-39)-immunoreactive fibers could be detected in several regions found to contain prolactin fibers, the distribution and organization of both fiber types clearly differed in numerous brain regions, and the regions containing the corresponding perikarya did not overlap.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1989

Combined use of immunoperoxidase and radioimmunocytochemistry for double immunocytochemical labeling of neurons at light and electron microscopic level.

Complexes formed by binding 125I- or 3H-labeled neuropeptides to one of the two binding sites of their specific antibodies allowed specific and sensitive labeling of various peptidergic neurons, which could be detected by classical autoradiographic methods. To visualize two neuronal antigens on the same material at both light and electron microscopic level, we used a new technique of double immunocytochemical labeling, combining immunoperoxidase and radioimmunocytochemistry. The main steps of the process included: (a) indirect labeling of the first antigen by its specific antibody and by a peroxidase-labeled Fab immunoglobulin fragment directed against the primary antibody; (b) direct labeling of the second antigen by a radiolabeled peptide-antibody complex; (c) revealing of the first label in the presence of peroxidase substrate; and (d) revealing of the second label by autoradiographic treatment of tissue sections. Compared with other known techniques of double immunostaining, this technique offers major advantages for combined visualization of two neuronal antigens at the electron microscopic level: (a) two neuron types can be labeled by a pre-embedding approach, allowing highly sensitive detection of neuronal antigens throughout the 50-microns thickness of vibratome sections; (b) two primary antibodies obtained in the same species can be used to label the two antigens without any risk of crossreactions between the two successive labelings; and (c) the two labels can easily be differentiated, even when they are co-localized within the same neuron structures. Application of this double immunostaining technique is illustrated by data obtained in rat hypothalamus concerning the relationships among a variety of identified neurons and the co-localization of different neuropeptides within the same neuron system.

Animals↗
PubMed1988

Effects of colchicine on the intraneuronal transport of secretory material prior to the axon: a morphofunctional study in hypothalamic neurosecretory neurons of the rat.

The effects of colchicine on neurosecretory neurons of the rat hypothalamus were studied by immunocytochemistry, high-resolution radioautography, and conventional electron microscopy. In control rats, intraneuronal immunocytochemical labeling of vasopressin, oxytocin and somatostatin occurred essentially in the Golgi apparatus, the neurosecretory granules and to a lesser extent, the endoplasmic reticulum. These immunostaining patterns were dramatically modified 24 h after the administration of colchicine: immunoreactive peptides were located in granular or tubular structures accumulated at the periphery of the perikarya, but the Golgi stacks were not immunostained. Two h after the administration of tritiated leucine, quantitative analysis of radioautographic labeling of supraoptic perikarya revealed large amounts of radioactive protein in the Golgi saccules of neurosecretory neurons in control rats, but in the neurons of colchicine-treated rats, radioautographic labeling was mainly located in granular structures accumulated at the periphery of the perikarya, with no significant labeling on the Golgi stacks. Lastly, 3 noteworthy effects of colchicine on the ultrastructural morphological features of these neurosecretory neurons consisted in: (1) a dramatic disorganization of the Golgi complexes, (2) an accumulation of electron-dense proteic material within the lumen of cisternae of both the rough and smooth endoplasmic reticulum and, (3) a marked depolymerization of perikaryal microtubules, specifically those associated with the Golgi stacks. Taken together, these data do not fit the prevailing concept that the colchicine-induced accumulation of secretory material within the perikarya of neurosecretory neurons essentially results from the blockade of axoplasmic transport mechanisms. Instead, they support the idea that the effects of colchicine are related to the inhibition of the intraneuronal transport of newly synthesized secretory material from the endoplasmic reticulum to the Golgi apparatus, suggesting that the microtubules associated with the Golgi stacks are possible sites of colchicine action.

Animals↗
PubMed1988

Peptidergic neurohormonal systems in the basal hypothalamus of the ferret and the mink: immunocytochemical study of variations during the annual reproductive cycle.

The hypothalamic systems secreting corticotropin-releasing hormone (CRF), somatostatin, oxytocin, vasopressin and luteinizing hormone-releasing hormone (LHRH) were characterized using immunochemistry, and variations were studied in relation to the recrudescence of testicular activity in the ferret and the mink, two species with opposite photoregulation of their annual reproductive cycles. Under the present conditions of study, the immunoreactivity of the CRF, somatostatin, and oxytocin systems showed no significant variation in either species. In contrast, in these two species, the immunoreactivity of the LHRH system varied considerably depending on the date of observation. The increase in the number and immunoreactivity of the LHRH-secreting neurons that occurred in November in the mink and in January in the ferret, is in agreement with previous results showing that the photoperiod plays an essential role in regulating the annual activity of the testis and that the photoperiodic environmental conditions required for the activation of the LHRH system differ between the species. Similarly, correlations could be found between an increase in immunoreactivity of the vasopressinergic axons projecting to the external median eminence and the recrudescence of testicular activity.

Animals↗
PubMed1988

Immunocytochemical ultrastructural study of hypothalamic neurons containing corticotropin-releasing factor in normal and adrenalectomized rats.

The neurons of the rat hypothalamus which secrete corticotropin-releasing factor were studied by using a pre-embedding immunocytochemical staining technique that improves both the penetration of immunoreagents within the tissue and the preservation of the ultrastructural morphology of labeled structures. Comparison was made between the subcellular location of corticotropin-releasing factor-41 in perikarya of the paraventricular nucleus and axons of the median eminence, both in intact and adrenalectomized animals either untreated or 24 h after the intracerebral injection of colchicine. Morphometric analysis of the numerical density and of the diameter of corticotropin-releasing factor immunoreactive neurosecretory granules in axons of the median eminence of rats not treated with colchicine, indicated that the main modifications induced by adrenalectomy concerned (1) the differential repartition of labeled granules within the preterminal and terminal axonal portions of the median eminence, and (2) the enlargement of the diameter of labeled granules contained in these axons (from 98 nm to 165 nm). In the hypothalamus of intact and adrenalectomized rats, colchicine treatment increased the number of corticotropin-releasing factor-immunoreactive granules in the neuronal perikarya and reduced their number in the axons, but both these variations were much more marked in adrenalectomized rats. Although the corticotropin-releasing factor immunoreactive granules that accumulated in the perikarya after colchicine treatment were slightly smaller than those in the corresponding axons, the diameter of perikaryal-labeled granules was larger in adrenalectomized than in intact animals (129 nm vs 93 nm). These findings fit the idea that adrenalectomy markedly stimulates both the synthesis and axonal excretion of secretory granules in the hypothalamic neurons secreting corticotropin-releasing factor. They also indicate that suppression of circulating corticosteroids induces qualitative modifications in these neurons leading to the visualization of larger neurosecretory granules, which may reflect differential synthesis and granular packing of synergistic peptides other than corticotropin-releasing factor and/or changes in the process of intragranular maturation of hormonal material.

Adrenalectomy↗
PubMed1988

Central catecholaminergic system stimulates secretion of CRH at different sites.

To explore a possible differential role of distinct catecholamine (CA) innervation sites in corticotropin-releasing hormone (CRH) secretion, especially under stress conditions, we compared the effects in adult female rats of selective CA denervation of either the whole hypothalamus, by a discrete pharmacological lesion of the ventral noradrenergic ascending bundle [VNAB; 3 micrograms of 6-hydroxydopamine (6-OHDA) in 0.2 microliter of vehicle, bilaterally] or of the paraventricular nuclei (PVN) alone (1 microgram of 6-OHDA in 0.2 microliter of vehicle, bilaterally). Although both procedures induced a similar dramatic fall in norepinephrine and epinephrine concentrations (-55 to -65%) measured by high-performance liquid chromatography in PVN punches, the VNAB lesion, unlike PVN denervation, depleted the median eminence (ME) of both amines (-80%). Concomitantly, the VNAB lesion led to a 97% reduction of the immunoreactive (ir) CRH-41 concentration in the hypophysial portal vessels, associated with a 64% fall in plasma adrenocorticotropic hormone (ACTH), and, in another group, with an 80% inhibition of ether stress-induced ACTH surge. The deletion of CA innervation of the PVN alone reduced irCRH-41 levels in the portal vessels by only 57% and plasma ACTH by 35%. This lesion did not significantly impair stress-induced ACTH release. These results suggest that the CA innervation of the hypothalamus exerts a stimulatory control on CRH-41-secreting neurons not only directly at the perikaryal level but also at other hypothalamic sites of VNAB innervation including peripheral contacts between the terminals of CA and CRH nerves in the external ME.

Adrenocorticotropic Hormone↗
PubMed1988

Axons containing a prolactin-like peptide project into the perivascular layer of the median eminence: an immunocytochemical light and electron microscope study in adult and infant rats.

A light and electron microscopic immunocytochemical study was undertaken to explore the fine structural organization of prolactin-immunoreactive axons in the rat median eminence. In adult intact males and females and in hypophysectomized females, light microscopic immunocytochemical labeling of the mediobasal hypothalamus revealed a marked concentration of prolactin-like immunoreactive fibers in the perivascular layer throughout the median eminence and the hypophysial stalk. At the electron microscopic level, immunostaining was associated with typical neurosecretory axons located either in the palisade layer where they displayed numerous contacts with tanycyte processes, or in the perivascular layer where they frequently contacted the perivascular space. Within the labeled axonal profiles, immunostaining was essentially located on secretory granules, 90-120 nm in diameter, whereas the microvesicles accumulated in some perivascular profiles constantly remained unlabeled. These data strongly suggest that most prolactin-immunoreactive axons of the median eminence release their content into the hypophysial portal vessels. In 1-day-old infant rats, intensely prolactin-like immunoreactive fibers were similarly localized in the most external layer of the median eminence, in which, contrary to adult animals, very slight if any tyrosine-hydroxylase-immunoreactive fibers were detected. Since earlier studies have provided evidence for a nondopaminergic prolactin-release-inhibiting factor in the hypothalamus of infant rats, and for an inhibitory effect of prolactin on pituitary mammotrophs, we propose that hypothalamic prolactin may contribute, as an additional prolactin-release-inhibiting factor, to the multifactorial control of pituitary mammotrophs.

Aging↗