Intact Toad Research Articles

Effects of exercise on plasma catecholamine levels in the toad, Bufo paracnemis: Role of the adrenals and neural control

Resting plasma epinephrine (E) and norepinephrine (N) concentrations for intact toads (Bufo paracnemis) were 5.57+/-1.0 and 0.88+/-0.38 ng/ml, respectively. Exercise induced a significant increase in heart rate, blood pressure and plasma epinephrine (about 4.3 times), whereas norepinephrine remained unchanged. The resting [E]/[N] ratio was 6.3 and increased to 32.9 during exercise. Adrenal denervation did not alter the basal plasma catecholamine or norepinephrine levels after exercise, but prevented the increase in epinephrine during exercise, suggesting that in the intact toad this increase is due to adrenal secretion whereas resting norepinephrine may be liberated by extra-adrenal chromaffin tissues. This also suggests that the adrenal glands can release selectively the two catecholamines. The increases in heart rate and blood pressure in denervated toads were not significantly different from those of intact animals, suggesting that during exercise the sympathetic nerves play the main role in inducing cardiovascular responses. Spinal transection induced a significant increase in basal norepinephrine levels, which remained elevated after exercise. Since spinal toads are unable to perform spontaneous movements it is possible that this increase may be caused by this stressful condition. The increases in heart rate and blood pressure observed in spinal toads during exercise may be due to direct mechanical effects of venous return on the heart.

Plasma membrane water permeability of cultured cells and epithelia measured by light microscopy with spatial filtering.

A method was developed to measure the osmotic water permeability (Pf) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10-20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. Pf in control Chinese hamster ovary cells was low (0.0012 cm/s at 23 degrees C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. Pf in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. Pfbl and Pfap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23 degrees C. In intact toad urinary bladder, basolateral Pf was 0.036 cm/s and apical membrane Pf after vasopressin stimulation was 0.025 cm/s at 23 degrees C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types.

Cell volume and plasma membrane osmotic water permeability in epithelial cell layers measured by interferometry

The development of strategies to measure plasma membrane osmotic water permeability (Pf) in epithelial cells has been motivated by the identification of a family of molecular water channels. A general approach utilizing interferometry to measure cell shape and volume was developed and applied to measure Pf in cell layers. The method is based on the cell volume dependence of optical path length (OPL) for a light beam passing through the cell. The small changes in OPL were measured by interferometry. A mathematical model was developed to relate the interference signal to cell volume changes for cells of arbitrary shape and size. To validate the model, a Mach-Zehnder interference microscope was used to image OPL in an Madin Darby Canine Kidney (MDCK) cell layer and to reconstruct the three-dimensional cell shape (OPL resolution < lambda/25). As predicted by the model, a doubling of cell volume resulted in a change in OPL that was proportional to the difference in refractive indices between water and the extracellular medium. The time course of relative cell volume in response to an osmotic gradient was computed from serial interference images. To measure cell volume without microscopy and image analysis, a Mach-Zehnder interferometer was constructed in which one of two interfering laser beams passed through a flow chamber containing the cell layer. The interference signal in response to an osmotic gradient was analyzed to quantify the time course of relative cell volume. The calculated MDCK cell plasma membrane Pf of 6.1 x 10(-4) cm/s at 24 degrees C agreed with that obtained by interference microscopy and by a total internal reflection fluorescence method. Interferometry was also applied to measure the apical plasma membrane water permeability of intact toad urinary bladder; Pf increased fivefold after forskolin stimulation to 0.04 cm/s at 23 degrees C. These results establish and validate the application of interferometry to quantify cell volume and osmotic water permeability in cell layers.

Nature of caveolae in the endothelial cells of toad urinary bladders

Intact toad urinary hemibladder sacs have been in use for many years as a membrane model for experimental studies of transepithelial aqueous transfer under osmotic gradient. The toad bladder tissue is composed of several cell types. Recently, we reported the presence of a large concentration of caveolae in the granular epithelial cells of toad urinary bladders. Caveolae are microstructures which arise by invagination of the plasma membrane on the cytoplasmic side of the cell. Currently, we report the presence of caveolae in endothelial cells of toad urinary bladder.Urinary hemibladder sacs were excised from doubly-pithed toads, Bufo marinus, fixed using 1% glutaraldehyde in PIPES, postfixed in 1% osmium tetroxide, dehydrated and embedded in epon blocks. Ultrathin sections were exposed to uranyl acetate and lead citrate for transmission electron microscopy. In some cases, the bladder sacs were tied up at the ends of glass tubes and equilibrated in aerated Ringer's solution for 30 min prior to experimental procedures.

Calponin phosphorylation in vitro and in intact muscle.

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.

Ionic components of the electrical response of chromaffin cells from the toad ( Caudiverbera caudiverbera) adrenal gland

1. Ultra fine tip muelectrodes (300 MOhm) were used to study the electrical properties of the chromaffin cell membrane in situ in the intact toad adrenal gland. 2. In the presence of physiologic [K +] o(2 mM) the resting membrane potential ( V m) was − 53 ± 3.2 mV. V m, depended on [K +] o as predicted by the constant field equation with P Na P K of 0.16 3. A small fraction (20%) of the impaled cells exhibited spontaneous electrical activity, though in all the cells examined, the injection of depolarizing current pulses elicited repetitive spikes. 4. Our measurements of the chromaffin cell input resistance (326 ± 35 MOhm) is substantially smaller than the values reported for bovine isolated chromaffin cells, suggesting that the toad adrenal chromaffin cells might be electrically coupled. 5. Tetraethylammonium (TEA) increased the amplitude and duration of spikes, probably inhibiting outward K + current. In the presence of tetrodotoxin (TTX) action potentials were abolished, although they reappeared if TEA was added, suggesting the participation of both Na + and Ca 2+ currents in the genesis of spikes. 6. As expected, acetylcholine (ACh) and nicotine depolarized the cells, though they did not always elicit electrical activity. 7. Muscarine (10–100 μM) had no effect on both V m and on the depolarization induced by ACh or nicotine. Since muscarine inhibits catecholamine (CA) secretion induced by ACh and nicotine, we concluded that the inhibition of CA release by muscarine in the toad probably occurs at a level other than the membrane.

Activity of 11 beta-hydroxysteroid dehydrogenase in toad bladder: effects of 11-dehydrocorticosterone.

11 beta-Hydroxysteroid dehydrogenase (11 beta-OHSD) transforms circulating glucocorticoids to their "biologically inert" 11-dehydro derivatives. Isoforms of 11 beta-OHSD with different cofactor requirements and biochemical properties [Michaelis constant (Km) and maximal velocity (Vmax)] exist in the kidney. Since epithelial cells derived from the toad bladder also contain this enzyme, we wished to further characterize its properties in prepared cell homogenates. 11 beta-OHSD from toad bladder demonstrated a clear preference for NAD+ over NADP+ as a cofactor similar to that observed in renal cortical collecting duct (CCD) cells. Furthermore, 11 beta-OHSD had a rapid onset of action. The apparent Km for corticosterone was 16.3 x 10(-8) M, a value comparable to that observed for enzyme from CCD, and a Vmax of 4.8 x 10(-12) mol.mg protein-1.min-1. The end product, 11-dehydrocorticosterone (compound A), influenced enzyme activity; it increased 11 beta-OHSD activity at corticosterone concentrations below the apparent Km for the enzyme and inhibited 11 beta-OHSD activity at corticosterone concentrations above the Km for the enzyme. The inhibitory effects of compound A appeared noncompetitive with an apparent equilibrium constant (Ki) of 2.8 x 10(-7) M. Consistent with its inhibitory action on 11 beta-OHSD, compound A (10(-6) M) enhanced the short-circuit current response to corticosterone (10(-7) M) in the intact toad bladder (experimental 2.03 +/- 0.33 vs. control 1.40 +/- 0.17 times above baseline; n = 7, P < 0.01). Thus 11 beta-OHSD in toad bladder resembles the isoform found in CCD, and compound A may be biologically important as a regulator of 11 beta-OHSD.

The determination of total cGMP levels in rod outer segments from intact toad photoreceptors in response to light superimposed on background and to consecutive flashes: a second light flash accelerates the dark recovery rate of cGMP levels in control media, but not in Na(+)-free, low Ca2+ medium.

In previous experiments we established that a light flash reduced cGMP levels of toad rod outer segments within the transduction time interval, but that recovery of the dark level of cGMP occurred more slowly than reported electrophysiological recovery of membrane potential. We now report that a second light flash accelerates the recovery rate of total cGMP following an initial flash, but that this acceleration is blocked in a medium which is both sodium and calcium deficient. We also noted that calcium deficiency only elevated cGMP levels when sodium was present. For other experiments, we recorded ERG or aspartate isolated PIII responses from eyecups or retinas mounted on our quick-freeze apparatus, the light stimuli originating from the double light-bench of the latter. Whereas background illumination depressed cGMP, no detectable further cGMP loss accompanied the electrical response to a flash superimposed on the background.

HCO3- transport in the toad lens epithelium is mediated by an electronegative Na(+)-dependent symport

The pH-sensitive cell-entrapable dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to continuously monitor epithelial intracellular pH (pHi) of intact toad lenses, enabling a description of a HCO3- transport mechanism that contributes to pHi homeostasis of this organ. In physiological medium, pH 7.40, the steady-state pHi was 7.48 +/- 0.03 (SE; n = 93). Induction of cell depolarization by either elevation of [K+] to 50 mM, addition of 0.2 mM quinidine, a K(+)-channel blocker, or addition of 0.1 mM Li+ ionophore that equalizes Na+ and K+ permeabilities elicited pHi increases (delta pHi = 0.18 +/- 0.02; P less than 0.0005; n = 13, for K+). These increases could be blocked or reverted by DIDS and were not affected by amiloride. Removal of Na+ induced an amiloride-insensitive acidification. pHi recovery seen upon Na+ reintroduction in the presence of amiloride was inhibited by DIDS. Despite the effects of DIDS on induced pHi changes, the agent did not affect control pHi. Elevation of medium HCO3- (pH to 7.7) produced a pHi increase followed by a spontaneous reversal. This increase was both DIDS and Na+ sensitive. pHi was not affected in any condition by removal (or addition) of Cl-, unless the lens was pretreated with the artificial Cl(-)-HCO3- exchanger tributyltin. Collectively, these results suggest that the primary mechanism for HCO3- movement across the lens epithelial membrane is an electronegative Na+ cotransporter and that this system is near equilibrium under normal physiological conditions.

Effects of acetylcholine and substance P on electrical activity of intact toad gastric muscles

By using isolated gastric muscle cells of Bufo marinus, others have studied the mechanism of action of muscarinic agonists and substance P (SP). To compare responses of isolated cells with those of intact muscles, we have studied the effects of acetylcholine (ACh) and SP on membrane potentials of circular muscle cells in strips of intact muscle from the toad gastric corpus region. These cells had average resting potentials of -69 +/- 0.7 mV. Membrane potential rhythmically depolarized, producing slow waves at an average frequency of 1/min and average amplitude of 25 +/- 2.2 mV. The major effect of ACh (10(-7) to 10(-4) M) was chronotropic; the frequency of slow waves was increased by 88 +/- 11% by 10(-6) M ACh. The amplitudes and rates of rise of slow waves were decreased by ACh. SP had effects similar to ACh; its major effect was chronotropic. The data suggest that ACh and SP primarily affect the pacemaker mechanism in gastric muscles. Since rhythmicity is apparently not expressed in isolated gastric myocytes, it is possible that this effect of these agonists may have been missed in studies of dispersed cells. Our data suggest that the excitatory effects of ACh and SP on contractions may be due to summation of Ca2+ signals, a partial tetanus-like effect.

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder.

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.

Propranolol increases the biosynthesis of phosphatidic acid, phosphatidylinositol and phosphatidylserine in the toad retina: Studies in the entire retina and subcellular fractions

1. Intact toad retinas incubated for short periods of time with [2- 3H]glycerol were subject to subcellular fractionation. 2. The composition and labeling of glycerolipids were studied in the following subcellular fractions: rod outer segment (ROS), nuclear-photoreceptor inner segment synaptic body (P 1, synaptosomal-mitochondrial (P 2), microsomal and cytosolic. 3. It was concluded that the biosynthetic de novo route was followed by [2- 3H]glycerol in the toad retina since radioactivity was located solely in the glycerol backbone of lipids and phosphatidic acid specific activity was the highest. 4. Propranolol produces an increase in the biosynthesis of acidic phospholipids and inhibition in the biosynthesis of zwitterionic lipids in the entire toad retina. The effect was mainly located in microsomes and in the soluble fraction during the first minutes of incubation, being spread afterwards to other fractions. 5. These data are consistent with the view that enzymes of the biosynthesis of glycerolipids are modified in the retinal endoplasmic reticulum by propranolol, providing a useful tool to evaluate the regulation of the pathway.

Epithelial organization and hormone sensitivity of toad urinary bladder cells in culture.

Two continuous cell lines (TB-M and TB-6c) derived from epithelial cells of the toad urinary bladder form epithelia in culture that manifest hormone-sensitive transepithelial transport. Development of transepithelial electrical resistance (R) and transport rate (ISC) are dependent on time and density of cells seeded, but steady-state ISC and R are characteristic for each cell line and independent of seeding density. Some responses of intact toad bladder are preserved in culture, whereas others are altered or absent. Neither cell line responds to vasopressin. Analogues of cAMP increase sodium transport and urea permeability in both cell lines but do not affect water permeability. The intramembrane particle aggregates associated with the vasopressin- and cAMP-induced increase in water permeability of the intact bladder could not be detected in the cell lines. Aldosterone increases sodium transport in both cell lines, and the time course and concentration dependence of the response to aldosterone are similar to those of the intact bladder. The relative effect of a series of steroids on ISC reveals corticosterone to be a more potent mineralocorticoid in cultured cells than in the intact bladder.

Cell-free translation of RNA isolated from aldosterone-treated toad bladder mucosal cells

The stimulation of sodium transport by aldosterone in target tissues requires the synthesis of both mRNA and proteins. Aldosterone-induced mRNA and proteins have been demonstrated in toad urinary bladder and rat kidney. We have isolated total RNA and poly(A)-containing RNA from hormone-treated and untreated toad bladder mucosal cells for translation in a rabbit reticulocyte lysate system. Aldosterone-induced proteins synthesized in this system have physical properties similar to those of aldosterone-induced proteins synthesized in the intact toad bladder.

Isolation of epithelial cells from toad bladder.

The epithelial cells of the toad bladder have been isolated by brief exposure to ethylenediaminetetraacetic acid followed by treatment with collagenase, DNAse, and the application of shearing forces. This approach eliminates the need for scraping of the mucosal surface and reduces mechanical damage during harvesting of the epithelium. The method yields intact, isolated epithelial cells and few clumps. The three major types of epithelial cells described in the intact toad bladder were present in the final preparation. The cellular contents of nucleic acids and proteins (in pg/cell) were: DNA 22.5 +/- 1.1; RNA, 12.9 +/- 0.6; and protein, 192 +/- 9. The isolated cells possess rates of oxygen consumption and amino acid incorporation higher than those of epithelial sheets obtained by collagenase treatment and scraping of the intact bladder. However, the production of cyclic nucleotides in response to stimulation by vasopressin and carbachol is comparable in both preparations.

Temperature coefficients for the oxidative metabolic responses to electrical stimulation in cerebral cortex.

Temperature coefficients of both cat and toad brain have been calculated for the active metabolic state induced by electrical stimulation. Values are higher than most of the values previously reported for "rest" metabolism, whether calculated from Arrhenius plots or from linear graphs. Relative rates of oxidative metabolism were obtained by measuring the time course of the transient changes in NADH fluorescence and cytochrome aa3 absorption by reflectance techniques directly from the surface of the exposed cat cerebral cortex in vivo and from the isolated intact toad brain mounted in a cuvet. These findings demonstrate that such optical methods accurately record metabolic processes.

Inhibition of electrogenic sodium transport across toad urinary bladder by the mycotoxin patulin

The effects of the mycotoxin patulin (4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one) on short circuited intact toad bladder and on Na+-K+, activated ATPase were examined in an attempt to elucidate the relationship between toxin, the Na+-K+ ATPase enzyme system and associated active sodium transport. Patulin inhibited transbladder short circuit current and Na+-K+ ATPase from isolated bladder preprations. The effect was exponentially dependent on time. A significantly slower rate of inhibition was achieved within 15-30 min. The results are compatible with the assumption that Na+-K+ ATPase is associated with the pump mechanism since patulin inhibited enzyme activity and concomitantly reduced the rate of electrogenic Na+ transport. A significant correlation suggested a cause-effect relationship.

Role of karyoplasm in the emergence of capacity of egg cytoplasm to induce DNA synthesis in transplanted sperm nuclei

The behaviour of sperm nuclei was studied both in the cytoplasm of intact toad oocytes undergoing maturation and the cytoplasm of oocytes matured without germinal vesicles. The behaviour of the nuclei of pronase-treated sperm injected in the mature egg cytoplasm was shown to be exactly similar to that of the sperm nucleus after fertilization, i.e. they swelled, synthesized DNA, and divided. No changes in such sperm nuclei could be detected in the cytoplasm of the oocytes matured without germinal vesicles.

Regulation of phosphorylation of a specific protein in toad-bladder membrane by antidiuretic hormone and cyclic AMP, and its possible relationship to membrane permeability changes.

Phosphorylation of a specific protein was decreased in intact toad bladders by exposure to either antidiuretic hormone or monobutyryl cyclic AMP. The decrease in phosphorylation caused by these agents preceded the change in electrical potential difference (an indicator of the rate of sodium ion transport) observed in response to the same compounds. The addition of cyclic AMP to homogenates of toad bladder led to a decrease in phosphorylation of the same, or a similar, protein. In subcellular fractionation studies, the effect of cyclic AMP on the phosphorylation of this protein was observed in those fractions rich in membrane fragments, but not in the nuclear or cell-sap fractions. These and other results are compatible with the possibility that the regulation by vasopressin and cyclic AMP of sodium and/or water transport in toad bladder may be mediated through regulation of the phosphorylation of this specific protein.

Oxidative and glycolytic recovery metabolism in muscle.

The average degree of reduction of mitochondrial NAD has been measured in the intact toad sartorius by a fluorometric technique. It has been shown that cytoplasmic NADH does not interfere materially with these measurements. The percentage reduction of this respiratory coenzyme has been determined in a number of physiological steady states which are well correlated with fluorometrically determined levels of NADH in suspensions of mitochondria from the hind leg musculature of the toad. In addition, these findings are closely comparable to similar, spectrophotometric measurements on mitochondria from other sources. In the presence of an adequate O(2) level a single twitch produces a decrease in fluorescence from the resting steady state which is followed by a slow return to the base line condition. This cycle indicates the intensity and the time course of the oxidative recovery metabolism. The area under this curve is directly related to the number of twitches up to three or four. Greater activity produces a curtailment of oxidative recovery due to glycolysis. In the presence of iodoacetate the linear relation holds for five to seven twitches. At still higher levels of activity a curtailment of the change in NAD level sets in, probably due to the removal of AMP by catabolic reactions.