Static lung compliance during the development of the bullfrog, Rana catesbeiana

The static compliance of excised lungs was measured during the metamorphic development of the bullfrog, Rana catesbeiana. Absolute static compliance of excised lungs increased with developmental stage from 0.02(+/- 0.01 SD) ml/cm H2O at Taylor-Kollros (TK) stage III to 34.67(+/- 15.00 SD) ml/cm H2O for adults. When static lung compliance was standardized by dividing by the lung volume at 4 cm H2O transmural pressure (approximately maximal capacity), lung compliance was relatively constant during early development and increased at the onset of metamorphic climax (TK stage XX). Although the absolute lung compliances of developing tadpoles are small, the volume-specific compliances are three to four times greater than those of some mammals. The compliance-independent index of hysteresis also increased during development from 0.0008(+/- 0.0006 SD) cm at TK stage V to 1.36(+/- 1.21 SD) cm for adults suggesting increases in pulmonary structural complexity and/or an increase in the production and secretion of a pulmonary surfactant-like substance. These developmental changes in pulmonary compliance and hysteresis mirror the increasing utility of the lung for gas exchange during the metamorphosis of the bullfrog.     

Plasma 5 alpha-dihydrotestosterone and testosterone in the bullfrog, Rana catesbeiana: stimulation by bullfrog LH.

Effects of purified bullfrog (Rana catesbeiana) LH and FSH on plasma levels of the androgens, testosterone (T), and 5α-dihydrotestosterone (DHT), were studied using adult male bullfrogs. Rana LH was considerably more potent than Rana FSH in stimulating increased plasma androgen levels in hypophysectomized and intact animals. Simultaneous injection of Rana FSH or ovine PRL with Rana LH, over a 10-day period, did not alter the LH-induced increase in plasma androgens. More DHT than T was present in plasma after LH injection. Castration abolished plasma DHT and greatly reduced plasma T. Results indicate that DHT is a major testicular steroid, and that testicular androgen secretion is stimulated primarily by LH in the bullfrog.     

Ontogeny of regulation of gill and lung ventilation in the bullfrog, Rana catesbeiana

Gill and lung ventilatory frequencies at 20-23 degrees C were recorded in five different larval stages and in the adults of the bullfrog, Rana catesbeiana (n = 101, body mass 40 mg-90 g). Ventilatory frequencies in unanesthetized, unrestrained animals were determined (1) after normoxic acclimation, (2) during acute hypoxic and hyperoxic exposure, and (3) after brief but intense activity. Gill ventilation frequency (fG) under normoxic, resting conditions was 110-120 cycles X min-1 immediately after hatching, but fell to and remained at 40-50 cycles X min-1 for the remainder of larval development. Activity caused a sharp decrease in fG in newly hatched larvae, a sharp increase in larvae between stages IV-XIV, and no change in fG in all older larval states. Hypoxia increased fG in younger larvae up to developmental stage XIV, but had no effect upon fG in older larvae. Lung ventilation was rare in normoxic, resting larvae up to stage X. Thereafter until metamorphosis lung ventilation frequency (fL) was 2-6 breaths X h-1, with fL in adults being much higher at 1-3 breaths X min-1. Activity did not affect fL in any larval stage, but markedly increased fL in adults. Hypoxia had no significant effect on mean fL in larvae below stage XX. Mean values of fL increased during acute hypoxic exposure in most adults, but these changes were not significant. Collectively, these data indicate that progressive larval development is accompanied by a decline in reflex regulation of branchial ventilation frequency well before reabsorption of gills occurred. At the same time, respiratory responses are 'transferred' to the lung prior to metamorphosis and the attendant increasing dependence on air breathing.     

Biological activity of bullfrog growth hormone in the rat and the bullfrog (Rana catesbeiana).

Highly purified bullfrog growth hormone (GH) was tested for growth promoting activity in rats and bullfrogs (Rana catesbeiana). In repeated assays, the potency of bullfrog GH in the rat tibia assay was 1 X bovine GH, which is significantly greater than all other non-mammalian GHs tested. It also stimulated significant whole body growth in immature hypophysectomized rats injected daily for 16 days. In bullfrogs, the bullfrog GH (about 2 microgram/injection) stimulated growth and appetite in intact frogs, and improved survival in hypophysectomized frogs. In contrast, even relatively high doses of bovine GH (up to 200 microgram/injection) were inactive in the bullfrog.     

Four pancreatic endocrine cells in the bullfrog (Rana catesbeiana)

The pancreas of the bullfrog was studied by the immunoperoxidase method and electron microscopy for hormone-storing cells and radioimmunoassay for content of four pancreatic hormones. Immunoperoxidase staining disclosed four cell types, namely insulin, glucagon, somatostatin (SRIF), and pancreatic polypeptide (PP) cells with regional differences of distribution: the hepatic process contained relatively small islets with abundant insulin, glucagon, and PP cells, and a few SRIF cells, whereas the duodenal process contained relatively large islets with abundant insulin cells and scattered glucagon and SRIF cells. There were few PP cells in the duodenal process. The morphology of four distinct secretory granules was confirmed by electron microscopy. The amount of the four hormones was estimated by nonhomologous radioimmunoassay and compared with the levels of immunoreactive hormone measured in human pancreas.     

Calcitonin gene-related peptide in the bullfrog, Rana catesbeiana: localization and vascular actions

Calcitonin gene-related peptide (CGRP) was found using radioimmunoassay in the brain and spinal cord of the bullfrog, Rana catesbeiana. The brain extracts were found to contain 241.7 +/- 68.1 pg/mg tissue, and the spinal cord contained 1753.0 +/- 96.8 pg/mg tissue. An intense immunocytochemical reaction was observed in the dorsal spinal cord. Vascular studies using helical strips of the dorsal aorta, iliac artery, and femoral artery showed CGRP to exert a vasorelaxant effect which was most pronounced in the femoral artery and minimal in the aorta. As in the rat, CGRP was shown to exert its vasorelaxant effect by inhibiting the mobilization of intracellular calcium.     

The complete amino acid sequence of prolactin from the bullfrog, Rana catesbeiana

The complete amino acid sequence of prolactin (PRL) from an amphibian species, the bullfrog (Rana catesbeiana), has been determined and conserved residues and domains were analyzed by sequence comparison of PRLs from 15 species of five major vertebrate classes. The bullfrog PRL consists of 197 amino acid residues with three disulfide linkages formed between residues 4-11, 58-172, and 189-197. The bullfrog PRL shows the highest identity with sea turtle PRL (75%); lower identities with chicken PRL (72%), pig, horse, and fin whale PRLs (68%), human, cattle, sheep, and elephant PRLs (60-58%), and rat and mouse PRLs (50%); and significantly lower identity with teleost PRLs (about 30%). It is apparent that all tetrapod PRLs characterized so far contain three disulfide bonds in homologous positions and differ from teleost PRLs which lack the N-terminal disulfide loop. The tetrapod and teleost PRLs share 34 common residues and these conserved residues are clustered in six domains (PD1 to PD6), suggesting that these common residues, or at least part of them, are responsible for the activities common to all PRLs. On the other hand, PD5 is conserved significantly within tetrapod PRLs, but to a lesser extent in teleost PRLs, suggesting that the PD5 contributes to the activities specific to tetrapod PRLs.     

Chemoreceptors and control of episodic breathing in the bullfrog (Rana catesbeiana)

To test the hypothesis that the episodic breathing pattern of bullfrogs is necessarily caused by fluctuations of Pa_O2 and Pa_CO2/pH, the natural oscillations of blood gases associated with periods of ventilation and apnea were experimentally prevented by undirectional ventilation (UDV) of the lungs. UDV with air or a 50% O₂ in N₂ gas mixture eliminated breathing episodes; only sporadic single breaths were ever observed under these conditions. UDV with hypoxic or hypercarbic gas mixtures, however, produced episodic breathing despite the fact that UDV virtually eliminated fluctuations in pHa, Pa_CO2 and Pa_O2. Furthermore, the breathing patterns of animals with the same mean levels of blood gases and acid-base status, with (UDV) and without (non-UDV) phasic chemoreceptor input were identical. These data indicate that phasic chemoreceptor input plays little or no role in the control of the normal breathing pattern although some tonic level of chemoreceptor input is required for ventilation to occur. Animals on UDV were more sensitive to hypercarbic than hypoxic gases and hypoxemia and hypercapnia affected breathing pattern differently. This indicates that tonic chemoreceptor input also affects the length of the periods of apnea and ventilation but this must be through some mechanism other than an “on” or “off” threshold.     

Blockade of the pressor response to angiotensins I and II in the bullfrog, Rana catesbeiana

We examined the effects of three angiotensin II (AII) analogs, [Sar1,Ala8] AII, [Sar1,Ile8] AII, and [Sar1,Thr8] AII, phenoxybenzamine and captopril on the pressor response to angiotensins I (AI) and II (AII) and norepinephrine (NE) in the bullfrog, Rana catesbeiana. Injection of AI and AII at 0.25, 0.5, and 1.0 micrograms/kg, or NE at 3 micrograms/kg elicited dose-dependent rises in blood pressure. [Sar1,Ile8] AII (10 micrograms/kg/min) significantly blocked the pressor effects of all AI and AII doses. [Sar1,Ala8] AII blocked only the highest dose, and [Sar1,Thr8] AII produced no blockade. Captopril (0.1 mg/kg bolus + 0.5 mg/kg/hr) significantly reduced the response to AI, but not AII or NE. Phenoxybenzamine (5-10 mg bolus + 1 mg/kg/hr) blocked NE, and partially inhibited (36-49%) the pressor effects of AI and AII. These results demonstrate that (1) [Sar1,Ile8] AII is a potent angiotensin antagonist in the bullfrog, while [Sar1,Ala8] AII is partially effective and [Sar1,Thr8] AII is largely ineffectual; (2) captopril is an effective converting enzyme inhibitor; and (3) a portion of the angiotensin response can be inhibited by alpha-receptor blockade and is apparently due to catecholamine release.     

Immunohistochemical studies on peptide neurons in the hypothalamus of the bullfrog Rana catesbeiana

The hypothalamus of the bullfrod Rana catesbeiana was examined immunohistochemically by using the indirect immunoperoxidase and the peroxidase-antiperoxidase (PAP) methods. Seven kinds of bioactive peptides were demonstrated in different neuronal somata and fibers located in the preoptic and infundibular areas of the bullfrog. The preoptic nucleus contains β-endorphin-, substance P-, and calcitonin-immunoreactive cells and fibers in its anterior part. Neuronal somata immunoreactive for somatostatin are scattered in the magnocellular preoptic nucleus. Pancreatic polypeptide (PP)-, calcitonin-, somatostatin-, gastrin-, methionine (Met)-enkephalin-, and substance P-immunoreactive neurons and fibers are found in the nucleus infundibularis ventralis. Met-enkephalin-immunoreactive cells occurs also in the ventral part of the nucleus infundibularis dorsalis.Most of the peptide neurons in the bullfrog hypothalamus are multipolar in shape, whereas some are cerebrospinal fluid (CSF)-contacting in type. This type includes substance P- and somatostatin-immunoreactive neurons in the preoptic nucleus and calcitonin-immunoreactive ones in the nucleus infundibularis ventralis. Immunoreactive substances often are gathered to the knobbed end of the CSF-contacting neuronal process and this finding supports the view that neurosecretions may be released into the CSF. Some, if not all, of the above mentioned neurons containing Met-enkephalin-, β-endorphin-, calcitonin-, gastrin-, and somatostatin-like immunoreactivities send their axons to the median eminence, where they are beaded in shape surrounding the blood capillaries, suggesting the release these bioactive substances into the hypothalamo-hypophyseal portal circulation. Possible regulation of the adenohypophyseal secretion by these neuronal peptides are proposed. In the neurohypophysis, somatostatin-, and Met-enkephalin-like immunoreactivities are localized in the nerve terminals abutting on the blood capillaries. Through the general circulation these peptides presumably exert certain unknown actions in remote targets.     

Androgen metabolism in the brain and non-neural tissues of the bullfrog Rana catesbeiana.

By investigating steroid conversions in the bullfrog Rana catesbeiana, the present study tests the hypothesis that androgen metabolism by the central nervous system (CNS) is a primitive vertebrate characteristic. Parts of the brain containing the amygdala, preoptic area, and hypothalamus (AMY/HTH), remaining forebrain (RFB), midbrain and hindbrain (MB/HB), and also the ovary, testis, muscle, and fat were homogenized and incubated with [7α-³H]androstenedione and cofactors for 60 min at 25°. Products were isolated and characterized by thin-layer chromatography and recrystallized to constant specific activity; estrone (E₁) derived from brain incubates was also methylated. E₁, testosterone (T), and 5α-dihydrotestosterone (DHT) were the metabolic products in brain incubates. In addition to E₁, ovarian homogenates synthesized estradiol-17β (E₂-17β) and estradiol-17α (E₂-17α), but no aromatase activity was detectable in the testis, fat, or muscle. T was the primary metabolite in the testis. Radioimmunoassayable T, DHT, E₁, and E₂ were present in the plasma of male and female bullfrogs. Concentration of aromatase in the HTH/AMY of the bullfrog is consistent with a similar distribution of enzyme activity in the limbic system or its homologs in a reptile and mammals. Centers for the control of reproduction and cells which bind sex steroids have been identified in the same brain areas in other anuran species. The experiments reported here indicate that the conversion of androgen to estrogen and other neutral metabolites by the brain is a primitive tetrapod characteristic and suggest that metabolism is an integral component of brain-steroid interactions which has been conserved during the evolution of vertebrates.     

Changing respiratory importance of gills, lungs and skin during metamorphosis in the bullfrog Rana catesbeiana

Oxygen uptake (MO2) and carbon dioxide excretion (MCO2) by the skin, lungs and gills (if present) of Rana catesbeiana have been measured at 20 degrees C during 4 developmental stages - strictly water breathing tadpoles, air breathing tadpoles, post-metamorphic bullfrogs and 4-year-old adult bullfrogs. In aquatic tadpoles, branchial performance is comparable to that of teleost fishes, but a large skin area to body mass ratio, particularly for the tail, plus a thin and highly vascularized skin, presumably facilitates a large (60% of total MO2) cutaneous O2 uptake. As development proceeds, MO2 by the gills decreases and the lungs assume importance in O2 uptake, but the skin remains the major organ of O2 uptake until metamorphosis is nearly complete. Immediately after metamorphosis, O2 uptake by the lung is elevated to 80% of total MO2. Carbon dioxide excretion in both aquatic and air breathing tadpoles was also achieved mostly by the skin (60% of total MCO2, R = 0.9). The lungs of air breathing tadpoles excreted less than 2% of total MCO2, rising to a maximum of only 20% (R = 0.2) even in adult bullfrogs. The considerable importance of the skin to CO2 excretion thus rises even further with the degeneration of the gills at metamorphosis, with R for the skin rising from 0.8 before metamorphosis to 7.5 in adults. Thus, large adjustments in skin and lung gas exchange occur as the larval gills slowly degenerate, and lung ventilation is initiated and increased. Aquatic O2 uptake is rapidly superseded by the uptake of O2 from the air, while CO2 excretion largely remains a function of the aquatic respiratory surfaces throughout the life cycle of the bullfrog.     

Cloning, expression, and biological activity of growth hormone in bullfrog (Rana catesbeiana)

The cDNA (GenBank, AY251538) encoding bullfrog growth hormone (fGH) was cloned by RT-PCR from the total RNA of pituitary glands. Its sequence encoded a putative polypeptide of 215 amino acids, including a signal peptide of 25 amino acids with no change to those of other previous reported bullfrog GHs. The fGH precursor shares 98.1, 96.3, and 95.3% homologies to those of other bullfrog GHs (AAB24792, AAB19428, CAA31038) in amino-acid sequence and its nucleotide sequences of the coding region shares 99.1 and 98.5% homologies to those of previous bullfrog GH genes (S52027 and X12520). The fGH cDNA was also efficiently expressed in Escherichia coli carrying a plasmid pGfGH in which the cDNA was under the control of GST promoter of pGEX1-lambdaT. The expressed fusion protein GST-fGH is comprised about 29.3% of the total cellular protein in such bacteria. The purified GST-fGH cannot only showed a obvious dose-response curve when it reacted with the hepatic membrane receptor proteins from bullfrog, but also significantly increased the body weight and length of bullfrog after twice injection and such effects lasted two or three weeks after the last injection with purified GST-fGH.     

Respiratory gas transport by the incompletely separated double circulation in the bullfrog, Rana catesbeiana.

To investigate respiratory gas transport in the bullfrog, Rana catesbeiana (mean body weight 249 g. ambient temperature 25 degrees C), O2 uptake and CO2 output were determined, and blood gas parameters (PO2, PCO2, pH, O2 content, O2 capacity and hematocrit) were measured in blood samples taken from various heart cavities and blood vessels. Analysis of the data on the basis of a simplified circulatory gas transport model allowed to estimate the cardiac output and its distribution, and to describe the O2 and CO2 exchange in lungs, skin and tissues. The total cardiac output (average 20.5 ml/min) was estimated to be distributed about equally to the pulmocutaneous (56%) and systemic arterial vessels (44%), whereas the systemic venous return (62%) was larger than the pulmonary venous return (38%). The marked difference in oxygenation between aortic and pulmocutaneous arterial blood (average O2 saturation 85% and 47%, respectively) showed a highly effective separation of systemic venous and pulmonary venous blood in the ventricle and conus arteriosus. After enlargement of the ventricle produced by incision of the pericardium, the separation of arterialized and venous blood was markedly reduced, but not abolished.     

Antagonism of responses to arginine vasotocin by its structural analogs in the bullfrog, Rana catesbeiana

Antagonism of arginine vasotocin (AVT) actions in vivo by synthetic AVT analogs was studied in bullfrogs. In addition, one analog was examined for its in vitro effect on water flux in the urinary bladder and on vascular contraction in a dorsal aortic preparation. AVT and its analogs were injected into conscious bullfrogs while blood pressure and urine flow rate were recorded simultaneously. d(CH2)5Tyr(Me)AVT induced a slight, but not statistically significant, antidiuresis and a clear vasopressor response. d(CH2)5Tyr(Et)OVT and d(Et2)Tyr(Et)OVT did not show any intrinsic effects. These analogs partially blocked the antidiuretic effects of AVT and completely blocked the pressor effects of AVT. In studies in vitro, 1 microM d(Et2)Tyr(Et)OVT antagonized AVT-stimulated water flux, whereas 10 nM competitively inhibited (by about 50%) the vasocontraction induced by AVT. These results suggest that d(Et2)Tyr(Et)OVT has no intrinsic activity but retains a relatively high receptor affinity, thereby producing effective antagonism of AVT in target cells of vascular smooth muscle. On the other hand, this antagonist showed no detectable intrinsic activity and appeared to be a weaker antagonist in target cells of the urinary bladder. This suggests that there might be two types of AVT receptors in bullfrogs.     

The complete amino acid sequence of the follitropin beta-subunit of the bullfrog, Rana catesbeiana.

The amino acid sequence of the bullfrog, Rana catesbeiana, follitropin beta-subunit has been determined by sequencing the intact protein (residues 1-39) and peptides originated by lysyl endopeptidase and pepsin. Twelve cysteine residues and two sugar chain binding sites at Asn-5 and Asn-22 are positional identities with bullfrog and mammalian beta-subunits. The bullfrog FSH beta-subunit is composed of 107 amino acid residues with a molecular mass of 11,782 Da, including the six cystine bridges and excepting the sugar chain. The bullfrog FSH beta-subunit has approximately 60% sequence identity with that of mammals and 40% with the fish gonadotropin beta-subunit. Conserved sequences among mammals (residue numbers 33-55 and 66-71) extensively differed from those of the bullfrog.     

Seasonal and stress related changes in plasma gonadotropins, sex steroids, and corticosterone in the bullfrog, Rana catesbeiana

Studies of seasonal gonadal cycles combined with direct measurements of plasma levels of the two gonadotropins (FSH and LH), several gonadal steroids (estrogen, E; progesterone, P; testosterone, T; and 5 alpha-dihydrotestosterone, DHT), and the interrenal steroid corticosterone (B) were made in male and female bullfrogs in central California over a 5-year period (between 1976 and 1981). During the course of these studies, we discovered that levels of plasma gonadotropins and steroids are highly labile and particularly sensitive to the effects of captivity, especially in males. In animals captured and sampled repeatedly in the field over a 3-day period, hormone levels remained constant, but if held in collecting sacks, gonadotropin and gonadal steroids began to drop within 2--4 hr and usually reached "baseline" levels within 20 hr. This effect was apparent in all seasons, except occasionally in early spring when hormone levels dropped by only about half. Hormone levels were also generally depressed by the time commercially collected frogs reached local supply houses. Plasma B increased within 30 min of capture and remained high for days in captive animals. Blood samples taken from several hundred animals at the time of capture reveal pronounced seasonal cycles in all hormones measured. These changes are discussed in connection with other gross changes in gonadal condition and with regard to possible interactions among gonadotropins and steroids. Both sexes show a general elevation of hormone levels in spring and early summer, but the sexes differ somewhat both in timing and in magnitude of the changes, as well as in the nature of the dominant steroids. Pronounced "surges" in gonadotropins are evident around the time of gamate release in bot sexes, but the temporal pattern of these surges is not the same for ovulation and spermiation; an elevation in plasma P is associated with the periovulatory surge in gonadotropins. Results were not entirely consistent with expectation of pituitary--gonadal relationships. Levels of plasma gonadotropins and steroids did not show the reciprocal relationship expected from a simple negative feed-back between gonadal and pituitary secretion, nor did changes in gonadotropins and gonadal activities show the consistent positive correlation expected from a direct dependence of gonadal function on circulating gonadotropins. In females, plasma T, but not E, correlated with ovarian growth. Plasma T in females reached much higher levels than in males, but DHT was higher in males. Androgens were generally elevated during the period of sexual activity in males, but absolute levels did not correlate well with individual differences in sexual behavior. Thus, seasonal changes in testicular and ovarian activities cannot be accounted for solely by seasonal cycles in circulating gonadotropin levels.