Showing papers on "Toad published in 1983"

Apical sodium uptake in toad kidney epithelial cell line A6

Abstract: The characteristics of the apical entry pathway for sodium into the cultured toad kidney epithelial cell line A6 are studied. Unidirectional apical sodium fluxes were determined by measuring the uptake of 22Na into confluent A6 epithelia growing in filter-bottomed cups. Apical sodium uptake was found to be a saturable function of sodium concentration with a Michaelis constant of 18 mM and a maximum velocity of 2.5 nmol X min-1 X cm-2. Amiloride competitively inhibits this sodium entry pathway with an inhibitor dissociation constant of 5 X 10(-8) M. Incubation of the epithelium with 10(-7) M aldosterone leads to a threefold increase in apical sodium uptake after 4 h. Both the aldosterone-stimulated and base-line sodium fluxes are completely inhibited by 10(-4) M amiloride. The similarity of these results to those from other tissues such as toad bladder and frog skin indicate that the A6 cells provide a useful model system for studying the apical entry pathway for sodium in tight epithelia.

Regulatory Role of Intracellular Calcium Ions in Epithelial Na Transport

Abstract: Whereas several early studies indicated that extracellular calcium levels can influence the rate of sodium movement across amphibian epithelia (15, 16), only during the last decade has it become apparent that cytosolic calcium ions are potentially important regulators of transepithelial ion and water transport. The first suggestive evidence that intracellular calcium ions play such a role was obtained in studies with quinidine in the urinary bladder of the toad (46). Quinidine, an agent presumed to increase the level of cytosolic free calcium ions (4, 29, 10,22), was found to inhibit vasopressin­ dependent water permeability and net sodium transport across the isolated toad bladder (46,50). Subsequently, Erlij & Grinstein (18, 25) reported that a reduction in the sodium concentration of the medium bathing the inner surface of the isolated frog skin inhibits the rate of active transepithelial sodium transport in a calcium-dependent manner. Their interpretation of this finding was based on the concept that a Na-Ca exchange mechanism operates across the basolateral border of epithelial cells, as in excitable cells, as originally proposed by Blaustein (7). Wiesmann et al (56) employed the ionophore A23187 to explore the regulatory role of intracellular calcium in the toad bladder; these investigations first described and characterized the

Actin filaments and vasopressin-stimulated water flow in toad urinary bladder.

Abstract: Vasopressin increases the water permeability of the apical membrane of the granular epithelial cells of the toad urinary bladder. Cytochalasin B inhibits this action of the hormone, indicating that microfilaments may play a role in the water permeability response. We have extended previous functional studies with cytochalasin B and have demonstrated that dihydrocytochalasin B, a more specific inhibitor of actin filament elongation, similarly diminishes the hydrosmotic response to vasopressin. Biochemical studies of isolated epithelial cells indicate that an actin-like protein accounts for about 10% of the soluble protein of the epithelium. Morphological studies of whole toad bladders incubated with heavy meromyosin conclusively demonstrate that actin is a component of the epithelial cells and that actin-containing filaments are associated with both plasma membranes and cytoplasmic organelle membranes. Taken together, these findings provide strong, albeit indirect, evidence that actin microfilaments play a functional role in the hormone-induced increase in water permeability in the toad urinary bladder.

Microelectrode study of K+ accumulation by tight epithelia: I. Baseline values of split frog skin and toad urinary bladder.

Abstract: Toad bladder and split frog skin were impaled with fine-tipped single- and double-barrelled K+-selective microelectrodes. In order to circumvent membrane damage induced by impaling toad bladder, a null point method was developed, involving elevations of mucosal potassium concentration. The results suggest that intracellular potassium activity of short-circuited toad bladder is approximately 82mm, twice as large as earlier estimates. Far more stable and rigorously defined intracellular measurements were recorded from short-circuited split frog skins. The intracellular positions of the micropipette and microelectrode tips were verified by transient hyperpolarizations of the membrane potential with mucosal amiloride or by transient depolarizations with serosal barium or strophanthidin. Simultaneous impalement of distant cells with separate micropipettes demonstrated that both the baseline membrane potentials and the responses to depolarizing agents were similar, further documenting that frog skin is a functional syncytium. Measurements with double-barrelled microelectrodes and simultaneous single-barrelled microelectrodes and reference micropipettes suggest that the intracellular potassium activity is about 104mm, lower than previously reported. Taken together with measurements of intracellular potassium concentration, this datum suggests that potassium is uniformly distributed within the epithelial cells.

45Ca fluxes in isolated toad bladder epithelial cells: effects of agents which alter water or sodium transport.

Abstract: Vasopressin enhances osmotic water flow and sodium transport across the toad urinary bladder by mechanisms involving cyclic AMP and calcium. It is believed that changes in intracellular calcium concentration or in its binding to membranes may in part mediate the effects of vasopressin. In addition, several agents which alter the response of the toad bladder to vasopressin may also act by altering cellular calcium metabolism. The effects of vasopressin and several agents which modify its effects in the toad bladder were studied on 45Ca fluxes in isolated epithelial cells from the toad bladder. Compartmental analysis of 45Ca exchange revealed three components. Vasopressin reduced the amount of calcium in the most rapidly exchanging pool from 1.67 +/- 0.20 to 0.86 +/- 0.12 nmol/mg of protein (P less than .025) and the most slowly exchanging pool from 2.72 +/- 0.26 to 1.90 +/- 0.34 nmol/mg of protein (P less than .001), while not affecting the intermediate pool. Theophylline, which mimics the natriferic and hydroosmotic effects of vasopressin, also mimicked the effects on 45Ca exchange by vasopressin. Exogenous cyclic AMP and the prostaglandin endoperoxide analog U46619, which mimic the hydroosmotic effect of vasopressin, also reduced the amount of calcium in the most slowly exchanging pool. Prostaglandin E1, which inhibits the hydroosmotic effect of vasopressin at the concentrations used increased the size of the most slowly exchanging pool. These studies suggest that prostaglandins and other agents which alter the effect of vasopressin in the isolated toad bladder may elicit their effects in part by influencing the calcium concentration at some critical site.

Peptide-containing nerves in the urinary bladder of the toad, Bufo marinus.

Abstract: The distributions of peptide-containing nerves in the urinary bladder of the toad, Bufo marinus, were studied by means of fluorescence immunohistochemistry of whole-mount preparations. The bundles of smooth muscle in the bladder are well supplied by varicose nerve fibres displaying somatostatin-like immunoreactivity; these fibres probably arise from intrinsic perikarya. The urinary bladder also has a well-developed plexus of nerves containing substance P-like immunoreactive material; these elements probably represent sensory nerves of extrinsic origin. Nerve fibres showing immunoreactivity to vasoactive intestinal polypeptide (VIP) or enkephalin are rare within the urinary bladder of the toad. It is considered unlikely that any of these peptides directly mediates the hyoscine-resistant excitatory response of the smooth muscle to nerve stimulation in the toad bladder.

Irreversible Stimulation of Hydroosmotic Response in Toad Bladder by Photoaffinity Labeling with [Phe2,Phe-(p-N3)3] Vasopressin*

Abstract: The photoreactive analogs of vasopressin, [Phe2,Phe-(p-N3)3]AVP (3a) and [Phe-(p-N3)2]AVP (2a), and the chemically reactive analogs of vasopressin, [Phe2,Phe-(p-NHCOCH2Br)3]AVP (3b) and [Phe-(p-NHCOCH2Br)2]AVP (2c), have been tested in the toad bladder for irreversible stimulation or inhibition of the water permeability response. Analog 3a was found to be an agonist with an ED50 of 4.5 X 10(-7) M and to induce a maximal osmotic water flow across bladders equivalent to 74% of that observed with the parent hormone (AVP). Photolysis of this analog in Ringer's fluid resulted in a decrease in its biological activity, with a half-time of 7 min. However, UV irradiation of the analog in the presence of toad bladders triggered an irreversible increase in the permeability of the bladders to water. Under optimal conditions of irradiation, water permeability remained at about 60% of maximum for more than 3 h after washout of analog 3a. The addition of AVP raised the permeability of these bladders to 100%. Analog 3a did not cause irreversible stimulation without photolysis, nor did this analog induce its characteristic effect when added to the mucosal solution. Compound 2a was found to be a potent antagonist of AVP. This inhibitory action of 2a was readily reversed in both the presence and absence of UV irradiation. Compound 3b was also found to be a reversible inhibitor of AVP. Compound 2c was found to be inactive as agonist or antagonist. These studies suggest that analog 3a binds covalently at or near the toad bladder hydroosmotic receptors, resulting in a persistent increase in permeability to water of the bladder wall.

Effects of frog-skin angiotensin II in amphibians.

Abstract: The role of frog-skin angiotensin II (AII) in amphibia was studied by comparing the sodium and water permeability effects of three angiotensins (AII): frog skin (Ala-Pro-Gly-[Ile3, Val5]-Ang II), human [( Asp1, Ile5]-AII), and Japanese goosefish [( Asn1-Val5]-AII). Frog-skin AII increased the short-circuit current (SCC) significantly after it was added to the dermal side of the isolated skin of the South American frogs, Leptodactylus chaquensis and ocellatus, and the toad, Bufo arenarum, in concentrations of 10(-6) M. In frogs, the effect was significant at 15 minutes and reached 45% over control after 2 1/2 hours. The effect cannot be achieved with concentrations lower than 10(-7) M. Since amiloride (10(-4) M) blocked the SCC response, and absence of chloride in the bathing fluid did not, the effect is probably dependent on sodium transport. Human AII (10(-6) M) produced a similar response in summer frogs that had been treated with 0.1% NaCl for 14 days. Goosefish AII was ineffective at similar concentrations, and none of the angiotensins modified SCC in the toad bladder. Hydrosmotic effects could be achieved with the three angiotensins, the response being dependent on seasonal and species factors but always considerably lower than that of the neurohypophyseal peptides. Vascular reactivity of the isolated frog hindlimbs was compared by dose-response curves. Potency ratios on a molar basis against frog-skin AII was 1.136 for human AII and 1.193 for goosefish AII. The results show that the effects of the angiotensins differ in both the response of SCC to frog-skin angiotensin and its higher vascular effects.

A comparison between field habits and contractile performance of frog and toad sartorius muscle

Abstract: The contractile characteristics of the sartorius muscle isolated from the frog,Rana pipiens, and the toad,Bufo americanus, are compared at 25°C and pH 8.0. Frog sartorius muscles have faster shortening velocities and greater mechanical power at low loads, while toad sartorius muscles develop greater tetanic tension (Fig. 1). During the rising phase of an isometric tetanus, the latent period and time to half-maximum tension are shorter and the rate of tension development is faster in frog sartorius muscles than in toad sartorius muscles (Table 1). There is no difference between the two species during relaxation, i.e., the half-relaxation time after an isometric tetanus is quite similar (Table 1). It is concluded that most of the contractile characteristics of frog and toad sartorius muscles correlate well with the habits, jumping ability, burrowing activities and endurance to fatigue of the two species in the field.

Analysis ofHormonal Regulation ofSodium Transport in Toad Bladder

Abstract: �Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value . This decrease was accompanied by aproportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations . In contrast, the paracellular resistance was unaffected by trypsinization . Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin . Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin

Effects of prostacyclin on short-circuit current and water flow in the toad urinary bladder.

Abstract: The effects of prostaglandins of the E series on sodium and water transport have been studied extensively. PGE2 has been shown to inhibit the increase in osmotic water flow produced by vasopressin and to stimulate short-circuit current (SCC) in the toad bladder. On the other hand, the effects of prostacyclin (PGI2), an arachidonic acid product, on sodium and water transport have not been extensively evaluated. The present studies describe the effects of PGI2 on basal and vasopressin-stimulated osmotic water flow and on SCC in the urinary bladder of the toad. Studies were performed in the absence or presence of indomethacin. PGI2 in the absence of indomethacin had no effect on basal or vasopressin-stimulated osmotic water flow. When indomethacin was present, thereby eliminating intrinsic prostaglandin biosynthesis, PGI2 inhibited basal but not vasopressin-stimulated osmotic water flow. PGI2 increased SCC in the presence or absence of indomethacin. 6-keto PGF1 alpha, the stable metabolite of PGI2, had no effect on SCC. PGI2 stimulated cAMP production in isolated toad bladder epithelial cells. 2',5'-Dideoxyadenosine, an inhibitor of cAMP production, blocked the increase in SCC produced by PGI2, suggesting that the effects of this compound on SCC are mediated via cAMP.

Endogenous Glycoside-Like Substances

Abstract: Publisher Summary This chapter explores the biological effects, chemical nature, source of production, and potential physiologic implications of endogenous glycoside-like compound For these studies extracts of bovine hypothalamus was screened for sodium transport inhibitory activity The basic biological assay in this early work was the isolated toad bladder preparation To explore the possibility that the unknown substance had other ouabain-like features, its interaction with ouabain in frog urinary bladder by the method of Mills and Ernst is demonstrated in the chapter It is concluded that endogenous glycoside-like compound material reversibly inhibits active sodium transport in anuran membranes It directly inhibits renal Na,K-adenosine triphosphate (ATP)ase from a mammalian source, and its ouabain-like activity is further manifested by its ability to inhibit ouabain binding to its cellular receptor in frog urinary bladder and guinea-pig brain microsomes Frog urinary bladder and rabbit renal Na,K–ATPase have been found to be significantly more sensitive to the effects of the compound than toad urinary bladder and enzyme prepared in an identical fashion from rat renal tissue, respectively

Sulfhydryl reagents affect Na+ uptake into toad bladder membrane vesicles.

Abstract: The effect of sulfhydryl reagents on the Na+ permeability mechanisms of toad urinary bladder vesicles was examined. The reagents 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), iodosobenzoate, and ethylenimine were able to decrease amiloride-inhibited sodium uptake into vesicles when used at low concentrations. When used at higher concentrations these reagents were able to induce large increases in vesicle Na+ permeability that were not sensitive to amiloride. The reagentp-chloro-mercuribenzene sulfonate was able to induce such leaks even at low concentrations. The reagent N-ethylmaleimide was incapable of substantially affecting vesicle Na+ transport in any way. All of the effects observed could be reversed by removing the reagents from the solution surrounding the vesicles. Our results help explain the varied actions of sulfhydryl reagents on intact epithelial tissue.

Active transport of calcium across the urinary bladder and colon of the toad Bufo marinus

Abstract: Unidirectional fluxes of calcium were measured (in vitro) across the urinary bladder and colon of the toad Bufo marinus in the absence of electrochemical gradients. A net calcium flux was observed in each tissue, but the polarity differed; it occurred from the mucosal (luminal) to the serosal side of the colon and from the serosal to mucosal (urinary) side of the bladder. The active transport in each tissue appeared to involve different mechanisms; that across the colon exhibited a sodium dependence, possibly involving a sodium-calcium exchange, but this was not seen in the urinary bladder. The net flux in the latter was, however, abolished by metabolic inhibitors, possibly reflecting a role of a calcium-adenosine triphosphatase mechanism. The results are discussed in relation to the calcium metabolism of this amphibian.

Ultrastructure of toad (Bufo paracnemius) mast cells. Their alteration by compound 48/80.

Abstract: The fine structure of toad mast cells and their alteration by compound 48/80 is described. The specific cytoplasmic granules, which stain metachromatically with toluidine blue, seem to be of one type only, notwithstanding wide differences in their appearance. They are composed of an electron-dense, peripheral, lamellar component, and a matrix showing a particulate material embedded in a homogenous ground substance. The alterations induced by compound 48/80 were essentially restricted to mast cell granules. Eventually these disappeared leaving vacuoles filled with a flocculent material which was also found outside the cell. Solubilization of granule contents preceded secretion. Stimulated mast cells seemed to be morphologically intact, even those cells that showed very intense degranulation.

Comparative study on the effects of thyroxine on protein, RNA and DNA contents of liver of different vertebrates at different stages of life.

Abstract: The relative responsiveness of different vertebrates (mammals, amphibia, and fish) at different stages of life to thyroxine (T4) with respect to protein and nucleic acids contents of liver has been studied. The control growing rat, toad, and Lata fish showed a gradual rise in the protein content of liver with the advancement of age. The rat liver RNA reached a maximum level at the 15th d (immature stage) of life and this level was maintained in 30 (juvenile) and 60th d (adult) of life. In the toad, no significant difference in liver RNA was observed with age. Fish liver, however, showed more RNA in juvenile stage than that in immature stage; no such difference was observed in between juvenile and adult stages of life. In normal growing rat, the liver DNA was found to be reduced in juvenile stage from that of immature stage. But in adult stage, the level of DNA was more or less at the same level as that of immature stage of the animals. Fish liver DNA did not exhibit any change with age. But in the toad, the progress of the stages of life was associated with the enhancement of liver DNA. Administration of T4 for 5 consecutive d caused an increase in protein, RNA and DNA contents of liver of rat, toad and Lata fish of different age groups excepting liver DNA in adult toad and fish. The dose of 1 microgram of T4 per g produced maximum effects in these animals. The T4-induced percentage increase in the amount of liver DNA was maximum in immature stage of life; this was followed by an increase in RNA and then by protein. In juvenile stage of these animals, RNA shows maximum increase followed by DNA and/or protein; and in adult stage, the rate of percentage increase in liver RNA was maximum followed by protein and DNA. The dose-response relationship between rat, toad, and Lata fish after T4 treatment (1 microgram/g) revealed that the poikilothermic vertebrates (toad and Lata fish) were more responsive than homoiotherm (rat) so far as the T4-induced increase in liver protein, RNA, and DNA are concerned.

Limb regeneration in the narrow-mouthed toad, Gastrophryne carolinensis, (Amphibia; Anura; Microhylidae)

Abstract: This paper reports the response of the narrow-mouthed toad, Gastrophryne carolinensis, to forelimb amputation. The amputation wound was closed by epidermal migration within 24 h. Within the 1st wee...