The role of dehydration and antidiuretic hormone on water exchange in Rana pipiens

• 1.1. Experiments were performed to evaluate the effects of 8-arginine vasotocin on the effective water potential and on the hydraulic conductance of leopard frogs (Rana pipiens) during water uptake through their ventral integument.
• 2.2. Vasotocin apparently decreases the effective water potential of intact frogs, thus bringing the effective water potential into close correspondence with the osmotic potential of extracellular fluids.
• 3.3. Thus, well-hydrated frogs, which release little AVT, have effective water potentials considerably higher (more positive) than the osmotic potentials of their plasma, and therefore, demonstrate a diminished capacity to absorb water.
• 4.4. Dehydrated frogs release AVT which causes their effective water potential to become essentially identical to the osmotic potential of their plasma.
• 5.5. We hypothesize that the action of AVT is to mobilize water from the site of absorption by the frog, thereby resulting in maintenance of a high water potential gradient between the environment and the frog. and in increased rates of water uptake.

     

Gonadotropin-releasing hormone release by superfused hypothalami in response to norepinephrine

We have been studying the release of gonadotropin-releasing hormone (GnRH) from adult male rat medial basal hypothalamus (MBH) utilizing a continuous flow superfusion system. This model system allows for direct application of modifying substances into the superfusion chambers and for continuous collection of effluent for radioimmunoassay of GnRH levels. Gonadotropin-releasing hormone is rapidly released in response to specific chemical stimuli. As demonstrated by others, pulses of KCl or prostaglandin E2 (PGE2) result in sharp peaks of GnRH release followed by rapid return to baseline. Forty millimolar KCl increases [GnRH] 3- to 4-fold, consistent with a membrane-associated secretory process for GnRH release. A 50-micrograms bolus of PGE2 results in a 2-fold rise in GnRH. Norepinephrine stimulates the release of GnRH in a log-linear dose-dependent manner in the range of 10(-9) to 10(-5) M norepinephrine (NE). At 10(-11) M, NE does not increase GnRH release above baseline, whereas at 10(-9) M NE GnRH release is increased 2-fold. The alpha-receptor blocker phentolamine significantly inhibits the NE-induced rise in GnRH. Propranolol, a beta-adrenergic receptor blocker, does not inhibit the GnRH response to NE. This study demonstrates a direct, dose-dependent, alpha-mediated stimulatory effect of NE on GnRH release from superfused male rat MBH, and establishes the potential of this system for the investigation of the GnRH response to other aminergic agents and their extraneural modifiers, including steroid hormones.     

Similar effects of inhibin and cycloheximide on gonadotropin release in superfused pituitary cell cultures

The actions of two inhibin preparations and cycloheximide on gonadotropin release were investigated in superfused pituitary cell cultures. Pituitary cells isolated from 18-day-old male rats were grown in Matrigel-coated superfusion chambers in chemically defined medium. After stationary culture for 4 days, the cell monolayers were superfused at a constant speed (0.25 ml/min) and were intermittently stimulated (6 min/h) with 10 nM gonadotropin-releasing hormone (GnRH). Groups of cultures were exposed to the test substances for varying time periods during stationary culture and/or during superfusion. Inhibitory effects of both inhibin preparations on the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in response to GnRH pulses were observed after 2 h of exposure and became maximal after about 6 h. Basal secretion of FSH between GnRH pulses was also suppressed, whereas the basal interpulse secretion of LH was not changed. When exposure to inhibin was discontinued, the secretion of both FSH and LH progressively increased and returned to control values by approximately 6 h. Cycloheximide (500 ng/ml) affected gonadotropin release with dynamics similar to those observed for the inhibin preparation. These data support the hypothesis that inhibition of gonadotropin synthesis may be an important step in the molecular mechanism of action by which inhibin regulates gonadotropin release.     

The cellular basis of the calcium dependence of GnRH-stimulated gonadotropin release from frog, Rana pipiens, pituitaries

An in vitro superfusion system was used in an attempt to identify the cellular systems involved in the Ca2+ dependence of gonadotropin-releasing hormone (GnRH) actions in the frog, Rana pipiens. Superfusion with 5 microM A23187 (a calcium ionophore) or phorbol myristate acetate (an analog of diacylglycerides) caused marked increases in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. Exclusion of Ca2+ from the medium prevented the stimulatory effects of PMA. The potent stimulator of adenylate cyclase, forskolin, caused only slight stimulation of LH and FSH secretion, which was also prevented by removal of Ca2+. The cytoskeletal disruptive agents colchicine, nocodazole, and cytochalasin B, and the calmodulin inhibitors, trifluoperazine and pimozide, had no significant effects on the action of GnRH. Overall, these results indicate that the major mechanisms of Ca2+ involvement in the response to GnRH by the frog pituitary are similar to that of mammals, with the possible exceptions of lesser roles for calmodulin and the cytoskeleton in the frog. The study suggests that polyphosphatidyl-inositol-diacylglyceride metabolism may be critical in understanding the mechanism of GnRH action in frogs.     

The ultimobranchial body in Rana pipiens

Summary Autoplastic transplants of ultimobranchial glands of male Rana pipiens were bilaterally or unilaterally placed in a homeotopic or heterotopic site. Serum calcium levels were maintained at normal values in bilateral autotransplants, while total ultimobranchialectomy resulted in hypercalcemia. Electron microscopy verified the viability and functional state of transplanted, denervated glands. During the periods of denervation, ergastoplasm and Golgi membranes exhibited hypertrophy which was reversed when unmyelinated nerves reappeared in the pericapillary space. Autotransplants under hypercalcemic conditions indicated that the process of secretion is primarily an intrinsic cellular activity and independant of innervation. The present evidence suggests that the sympathetic axons which innervate the parenchyma probably are inhibitory in nature and may allow depression of glandular functions during periods of hypercalcemia.The technical assistance of Mrs. Lilly Weeks is gratefully acknowledged. This project was supported by N. I. H. Grant No. AM-11795; The National Institutes of Arthritic and Metabolic Diseases.     

The ultimobranchial body in Rana pipiens

Summary Hypercalcemia was induced in male frogs by injection of Vitamin D₂ and maintaining animals in calcium chloride water. The fine structure of the Ultimobranchial gland was examined 3, 7 and 14 days after the initial injection. The initial response observed after the third day was a depletion of secretory granules in addition to an alteration of nuclear shape and cytoplasmic hypertrophy. After seven days secretory granule depletion continued and early cell types occurred which indicated an increase in mitotic activity. There was also a demonstrable increase in the amount of ergastoplasm and hypertrophy of the Golgi apparatus. On the fourteenth day, the height of the epithelium was markedly increased while the underlying vascular network was enlarged and more intimately associated with the secretory parenchyma. The homeostatic mechanisms of the Ultimobranchial gland appear to include both a rapid secretory response upon stimulation and a cellular renewal system to replace exhausted cells. This suggests that such a glandular system provides a mechanism to supply a rapidly expanding cell population to meet the demands of an excessive depletion of secretory materials. The response of this gland to hypercalcemia supports previous studies which suggest that the Ultimobranchial gland is the probable source of the hypocalcemic hormone, calcitonin.This project was supported in part by funds provided by the Department of Anatomical Sciences and National Institutes of Health, Grant No. AM-11795.     

Morphological changes in the skin of Rana pipiens in response to metabolic acidosis

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.     

In vitro effects of cycloheximide and luteinizing hormone on the estradiol-to-progesterone shift in follicular steroidogenesis

The objectives of this study were to establish a completely in vitro system that would simulate the in vivo effects of cycloheximide (cyclo) on preovulatory serum levels of estradiol (E2) (prolonged) and progesterone (P4) (reduced). Graafian follicles were removed from proestrous hamsters at 0900 h and incubated for a basal hour (Hour 1) with various doses of cyclo before the medium was replaced; in Hour 2, 100 ng luteinizing hormone (LH) was added with cyclo added every hour for 5 or 6 h. The endpoints were steroid levels/follicle/h per ml medium of P4, 17 alpha-hydroxyprogesterone (170HP), androstenedione (A), and E2. The goal was best accomplished with hourly addition of 400 ng cyclo, which reduced follicular protein synthesis by 76%. Cyclo suppressed P4 and 170HP and prolonged the accumulation of A and E2, in Hour 5 and Hour 6, correlated with sustained thecal C-17,20-lyase/17 alpha-hydroxylase as determined by enzyme assays. Cyclo therefore prevented the early demise of the enzyme complex after LH stimulation and hence prolonged the ability of the theca to provide androgens for conversion to E2 by the granulosa cells. Our earlier work established that one of the major effects of LH is to recruit the granulosa compartment as a source of C-21 steroids, and cyclo interferes with the availability of cholesterol to mitochondrial side-chain cleavage (Greenwald and Limback, 1984). Thus, cyclo affects follicular steroidogenesis through different mechanisms in theca and granulosa.     

The effects of in vivo and in vitro hyperosmolality on skeletal muscle performance in the amphibians Rana pipiens and Scaphiopus couchii

1. The effect of in vivo and in vitro hyperosmolality on skeletal muscle function was investigated in two species of anuarans Scaphiopus couchii and Rana pipiens. 2. Muscle contractile performance, measured as peak tetanic tension declined to greater degree when tissue dehydration occurred in vitro rather than in vivo, even though tissue water contents were greater in vivo. 3. The muscles from S. couchii, a more dehydration tolerant species than R. pipiens, maintained tension at lower tissue water contents than R. pipiens. 4. Data for the effects of in vivo dehydration on plasma sodium, urea and osmotic concentration, as well as tissue water contents, are also presented for both species.     

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens female X Rana esculenta male

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 +/- 0.85 microns/min for PIP but only 0.46 +/- 0.56 microns/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 +/- 0.38 microns/min.     

Immunohistochemical localization of thyrotropin-releasing hormone (TRH) in skin of Rana pipiens

Summary Using immunofluorescent techniques thyrotropin releasing hormone (TRH) is demonstrated in skin of Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer.We wish to thank Dr. Ronald DeLellis and Ms. Mary Blount for their expert advice and guidance in the immunohistochemical techniques.This investigation was supported by NIH National Research Service Award # 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.     

Interaction of beta-endorphin, naloxone and dopamine: effects on melanocyte-stimulating hormone secretion of amphibian pituitaries in vitro

Neurointermediate lobes from amphibians (Rana pipiens) were incubated in Medium 199 containing dopamine, beta-endorphin or dopamine plus beta-endorphin. Dopamine inhibited melanocyte-stimulating hormone (MSH) secretion as measured by bioassay in hypophysectomized frogs, an effect which was transiently reversed by beta-endorphin. The effects of endorphin were in turn partially suppressed by the opiate antagonist, naloxone hydrochloride. Cells treated with all three agents exhibited expanded rough endoplasmic reticulum and decreased secretory granule content, indicative of peptide release and new synthesis. Beta-Endorphin alone did not stimulate MSH secretion above control levels, and at one time period was seen to reduce MSH secretion. The findings indicate a complex interaction between beta-endorphin and dopamine directly upon MSH secretion at the level of the neurointermediate lobe.     

Isolation and characterization of luteinizing hormone from amphibian (Rana catesbeiana) pituitaries.

We report here the first isolation of an anterior pituitary hormone from an amphibian species, the bullfrog ($\text{Rana catesbeiana}$). Highly purified luteinizing hormone was isolated from alkaline extracts of bullfrog pituitaries by salt fractionation, chromatography on ion-exchangers and gel filtration. Characterization studies show the hormone to contain 9% carbohydrate and to possess an amino acid composition similar to ovine luteinizing hormone. Sedimentation-velocity experiments in the ultracentrifuge indicate that the bullfrog gonadotropin dissociates in acidic solution and is composed of subunits. Bullfrog luteinizing hormone is highly active in an $\text{in vitro}$ toad ovulation assay and also ellicits testosterone production $\text{in vitro}$ from isolated rat testis Leydig cells.     

Role for corticoids in mediating the response of Rana pipiens tadpoles to intraspecific competition.

Competition is known to decrease growth and development rate in tadpoles, but the physiological basis for this phenomenon is poorly understood. We hypothesized that competition results in increased production of stress hormones and that these hormones are responsible for the suppression of growth and development. To test this hypothesis, we measured whole-body corticosterone content in premetamorphic Leopard frog (Rana pipiens) tadpoles raised at two different population densities and three different food levels. Whole body corticosterone content was elevated in tadpoles subjected to either limited food (at low density) or high density. Within the low and intermediate food treatments, high density reduced tadpole growth and slowed development. Limited food slowed growth and development at all densities. Blocking corticoid synthesis by treating tadpoles with metyrapone (MTP) reversed the growth suppression caused by high density (tested in the intermediate food level treatment) but did not alter the effect of density on development rate. MTP treatment did not alter the depressive effect of limited resources on growth or development. Our results suggest that elevated corticoid biosynthesis mediates the negative effect of increased population density (i.e., increased intraspecific competition) on tadpole growth.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in vitro

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media which are known to affect differentially prolactin and growth hormone release. Ultrastructural examination of the prolactin cells after 24 h culture showed the Golgi bodies were markedly more abundant and widely distributed in hemi-pituitaries from the low sodium medium. Secretory granule release profiles and dense bodies were also more frequent, but the percentage of the cytoplasmic volume occupied by secretory granules was lower than on the high sodium medium. RER was only slightly modified. Significant differences were noted in the shape and processes of the non-granulated (stellate) cells of the RPD, but there were only slight differences in the ultrastructure of the somatotropes.