Fermín C. Iturriza

Electron-microscopic study of the pars intermedia of the pituitary of the toad Bufo arenarum

Three consistent cell types were found in the pars intermedia of the pituitary of the toad Bufo arenarum: type I cells with concentrically arranged ergastoplasm and without secretory granules; type II cells with well-developed Golgi complex, small dense granules, and swollen ergastoplasmic sacs; and type III cells with large dense colloid masses located in confluent ergastoplasmic cisternae. A noncontinuous dense basement membrane partially separates the pars intermedia and pars nervosa; because of this arrangement close contact among parenchymatous cells of the pars intermedia and neurohypophysial axons exists. Neurosecretory axons were not found in the pars intermedia of the toad; instead nerve fibers whose endings contain clear synaptic vesicles, dense ones, and “ribosome-like granules” were found. Frequently these nerve endings were observed in close contact with the colloid masses. Based on previous studies, it is assumed that the masses are representative of stored melanocyte-stimulating hormone. It is suggested that the nerve endings in contact with the colloid masses offer an adequate anatomical basis for the opinion that inhibitory non-neurosecretory nerves control the storage of melanocyte-stimulating hormone.

The regulation of the corticomelanotropic cell activity in aves—II. Effect of various peptides on the release of ACTH from dispersed, perfused duck pituitary cells

We have estimated the corticotropin-releasing activity (CRA) of different neurohypophyseal peptides and synthetic corticotropin-releasing factor (CRF) in the duck, using perfused dispersed pituitary cells and an ACTH radioimmunoassay adapted to duck material. Log dose-response curves were obtained for different doses of arginine-vasopressin (AVP), arginine-vasotocin (AVT), mesotocin (MT), oxitocin (OT) and ovine CRF (oCRF) and compared to the response obtained with dilutions of duck median eminence extracts (DME). All peptides tested behaved as partial agonists compared to DME. AVT and MT were the most potent of all peptides tested, with a capacity of 60% relative to DME. CRF was a weak agonist together with AVP and OT. AVT and CRF perfused together at equal doses significantly potentiated the effect of each other, yielding a dose-response line whose slope approximated that of DME. A similar design was used to test the CRA of the same substances in the rat. The main difference in the pattern of response between the two species was the low potency displayed by all the neurohypophyseal peptides in the rat, compared with CRF which, in contrast with what occurred with the duck system, was the most potent secretagogue of all peptides tested. It is concluded that in birds, as in mammals, the control of ACTH secretion may be exerted by neurohypophyseal peptides and a CRF-like peptide acting synergistically upon the corticomelanotropic cell.

Monoamines and control of the pars intermedia of the toad pituitary

Monoamine-depleting drugs (reserpine; α-methyl-m-tyrosine) were administered to normal “bleached” Bufo arenarum toads and appreciable increases in the melanophore index (MI) were observed. These drugs failed to increase the MI of hypophysectomized animals. Monoamine-increasing drugs (nialamide and dihydroxyphenyl-alanine) produced marked decrease in the MI of normal “darkened” toads as well as in animals darkened by adrenalectomy, hypothalamic transection, and both adrenalectomy and hypothalamic transection. No bleaching action of these drugs was observed when administered in hypophysectomized toads darkened with ACTH. Guanethidine produced an initial decrease in the MI followed by an increase to pre-injection values. The importance of monoamines (specially adrenal and nervous catecholamines) in the regulation of the activity of the pars intermedia of the pituitary is emphasized. Evidence is discussed indicating that the control exerted by the hypothalamus on the intermediate lobe is achieved through monoamines released into the pars intermedia instead of by other neurosceretory products.

Further evidences for the blocking effect of catecholamines on the secretion of melanocyte-stimulating hormone in toads

Abstract Partes intermedias were autotransplanted into the anterior chamber of left eyes in toads. After postoperative day 15 either dopamine or noradrenalin was administered into these eyes and a fall in the melanophore index (MI) could be observed. The same catecholamines injected into the intact contralateral eyes did not produce any change in the MI. Solvent of catecholamines and heated hypothalamic extracts injected either into both right and left eyes or intravenously did not show effect on the melanocytes of the animals with grafts. Other partes intermedias were grafted into the kidney, some of them in contact with the adrenal gland, others distant from it. The toads bearing transplants in contact with the adrenal showed, on postoperative day 15, MI 2–3, the value of which decreased to 1 after stressing of the toads. This decrease could be abolished by the administration of reserpine or by destruction of the medulla oblongata. The animals with pars intermedia growing deep in the kidney tissue displayed MI 5 and the “bleaching effect” of stress could not be observed. Extirpation of the organs containing grafts caused maximal contraction of melanocytes, indicating that remnants of pars intermedia were not left in situ. These results support the previous assumption that catecholamines are the substances that in amphibians effect the hypothalamic inhibitory control of the pars intermedia.

Coexistence of α-melanocyte-stimulating hormone and adrenocorticotrophin in all cells containing either of the two hormones in the duck pituitary

Since birds lack the pars intermedia of the pituitary gland, the presence of ACTH-, α-MSH-, and β-MSH-containing cells in the pars distalis of the duck was investigated. By using the peroxidase-anti-peroxidase unlabeled antibody enzyme technique there were found cells reacting positively with antibodies against ACTH1–24, ACTH17–39, and α-MSH. Positive reactions could not be obtained with anti-bovine β-MSH serum. The coexistence of α-MSH and ACTH in all cells containing either of the two hormones could be established.

Hypothalamic-hypophysial relationships in the South American lungfish Lepidosiren paradoxa.

Abstract An analysis of the anatomical relations between the hypothalamus and the hypophysis of the South American lungfish Lepidosiren paradoxa , has shown the coexistence of peptidergic fibers which end on the portal vessels of the median eminence and a direct adrenergic innervation of the glandular cells of the adenohypophysis. According to the internal structure and size of the elementary neurosecretory granules revealed by electron microscopy, three different types of peptidergic fibers contacting the capillary portal vessels of the median eminence are distinguished: (a) endings which contain electron dense granules about 140 nm in diameter and irregular shape, (b) endings containing smaller neurosecretory granules 120–130 nm in diameter, and (c) endings having elementary granules 140 nm in diameter and a much paler core. Although all these fibers seem to originate in the preoptic nucleus, the cell bodies of this nucleus invariably contain only one type of elementary neurosecretory granule, characterized by a diameter of 140–190 nm. The adrenergic fibers react positively to the Falck-Hillarp technique for the demonstration of catecholamines and are identified at the electron microscope level as fibers containing dense-cored granulated vesicles (DGV), 90–100 nm in diameter. These fibers, which seem to originate in numerous small neurons present in the median eminence, end in contact with glandular cells of the partes distalis and intermedia and are also present in large numbers in the zones of the pars nervosa contacting the pars intermedia. These findings suggest that the structural organization of the hypothalamic-hypophysial relationships in Lepidosiren is reminiscent of the situation found in most vertebrates having an indirect, neurovascular control, and that of teleosts, which have a more or less direct innervation of pituitary cells.

Immunohistochemical investigation of tyrosine-hydroxylase in the islets of Langerhans of adult mice, rats and guinea pigs.

As an indirect way to establish whether catecholamines are actually elaborated in the islets of Langerhans of adult albino and pigmented mice and guinea pigs, or albino rats, the presence of tyrosine-hydroxylase was immunohistochemically investigated using a highly specific polyclonal serum against the enzyme. The antiserum was applied to paraffin sections of pancreases fixed either in acrolein or Bouins fluid. Such sections were then treated with the avidin-biotin method. Tyrosine-hydroxylase was found exclusively in the beta cells of rats and in those of the two strains of mice. Results in the guinea pig varied according to whether the animals were pigmented or albino. In pigmented specimens, the enzyme was detected in beta and non-beta cells, whereas in albino animals it was undetectable in any of the islet cells. All these observations were performed in material fixed in acrolein. Bouins fluid resulted a rather poor fixative for the detection of the enzyme in the islets. The results are compared with those of other authors who investigated the presence of monoamines in the islets of Langerhans. Since tyrosine-hydroxylase is a specific marker for catecholamine-synthesizing cells, it is concluded that insular cells have the ability to elaborate these substances.

HISTOCHEMICAL LOCALIZATION OF SOME α-MELANOCYTE STIMULATING HORMONE AMINO ACID CONSTITUENTS IN THE PARS INTERMEDIA OF THE TOAD PITUITARY

Wash ‘well in water (‘olloum 3() muiui to I hr with alcian yellow GX 0.5% bufiemed at pH 2.5 Wash in water Coumiterstain 5 mimi imi Kemnechtmot (opt iomial) Wash imi water, dehydrate, clear in xylene, mutoumit in (‘anada balsam. Resulls. Sulfated groups are coloured 1)lue, (.; boxvt groups yellow; sites contaimiimug acid! mnucopolysacchamides such as mucoitimi sulfuric or chondroitimi sulfuric acid, which possess both carb((xyl amid sulfated groups, are coloured imi blue-green. 1(1 obtain imidicat ions on the specificity of this niethod we have performed it also on these substances adsorbed on filter jmjem : Na chomidroitimi sulfate, lI\alur (Inc a(’id (obtained from Ntitmitional Biochem. Co.) amid sialic acid (kimidly supplied by Professor E. Klemik of the University of Cologne). Chondroitin sulfate gives a blue-green colour, hyaturomiic and sialic acids are coloured imi yellow. We obtained also positive results applying t his mnethod on various tissues after the mnethvlationsaponification sequence that hydrolyzes sulfated groups leaving unaltered only carboxyl groups. After this techmiic all sites contaimiing acid muco-

Catecholamine Metabolizing Enzymes and Synthesis of Dopamine in Normal and Grafted Pituitary Partes distales

The presence of enzymatic activity (tyrosine hydroxylase, dopa-decarboxylase, dopamine-beta-hydroxylase, monoamine oxydase and catechol-O-methyl transferase), as well as dopamine (DA) content and DA synthesis from tyrosine and dopa, were investigated in intact rats partes distales and in grafts (both estrogenized and nonestrogenized). Counts of prolactin cells showed the following regression in the number of these cells: estrogenized grafts greater than nonestrogenized grafts greater than intrasellar intact glands. Tyrosine hydroxylase activity was not found in intact glands, but this enzyme was detected in the two types of grafts. An approximate correlation could be established between the number of prolactin cells and the diverse enzyme activities. Dopamine was not synthesized from tyrosine in intact glands, but it occurred in the transplants. However, when dopa was used, both intact and grafted glands produced dopamine. Estrogen administration decreased dopamine content in all the glands investigated. The significance of these results in relation to the physiology of the pars distalis is discussed.

AN ELECTRON MICROSCOPIC STUDY OF THE TOAD PARS DISTALIS.

Abstract Based on the size and shape of the secretory granules and on the cell morphology, the following five cell types can be recognized in the pars distalis of Bufo arenarum with the electron microscope. Type I: Triangular cells which show granules of a mean diameter about 0.25μ. Type II: Spherical or cylindrical cells which have granules alveolar in aspect, the larger ones with a mean diameter of about 0.6 μ. Type III: Cells with two kinds of granules, one of them similar to those of Type II cells; the other is a very opaque type of granule with a mean diameter of about 0.3 μ. Type IV: Cells with granules fusiform or polygonal in shape. Type V: Cells with pale cytoplasm and very rare granules. Some aspects of the ultrastructure of the capillaries and the formation and extrusion of the secretory granules are described.