Ion-chromatographic study of the possible absorption of copper and zinc by the skin of Rana pipiens.

Frogs are known to obtain some of their nutrients (e.g., glucose and sodium) through their skin. However, no studies have been made of the possible absorption of transition metals, which exist in most river water at low-ppb (w/w) levels. Therefore, this research was undertaken to evaluate the use of ion chromatography for such an investigation. Solutions of copper and zinc (20 ppb in each) were chosen for use in a small-scale screening study. Ten live frogs were each placed in individual baths for approximately 50 h. Of interest were the net changes in the concentrations of the metals. These differences were the result of any absorption and/or excretion processes that took place. A Dionex IonPac CS5 column was used to analyze this simulated river water, both before and after frogs had been placed in the solution. Included in this paper are: (1) methodology and calculation formulas; (2) precautions needed to ensure sample integrity; (3) statistical analyses, which indicated that ion chromatography is an accurate, precise technique for quantifying Cu and Zn in these samples; and (4) screening-study results, which were used to test the null hypothesis that frogs do not absorb copper and zinc either onto or through their skin.

Ammonia transport by the urinary bladder of Bufo marinus.

All experiments were performed in vitro on toad bladders. Bladder sacs from acidotic toads produced a concentration gradient across the bladder with both [NH3] and [NH4] higher in the mucosal media. By varying the pH of the serosal media, paired sacs from normal toads were incubated with similar [NH3] in the serosal media but a 75 fold difference in [NH4] of the serosal media of the pairs. The hemibladders with the higher [NH4] had a 2.4 fold greater excretory flux than the paired sac. Both serosal to mucosal and mucosal to serosal fluxes were determined in normal bladders between chambers at various ammonium concentration gradients. The plot of mucosal to serosal flux against concentration produced a curve compatible with both carrier mediated and diffusion transport; the plot of serosal to mucosal flux produced a straight line with flux increasing when the ammonium concentration was increased. The serosal to mucosal transepithelial flux was augmented by making the serosal side of the bladder 50 mV positive. Although NH3 diffusion may occur, it cannot be the primary method of ammonia transport in the toad bladder.

Histological changes in the skin of Rana pipiens produced by either KCl loading or fasting.

The present study was performed to determine if either potassium loading or fasting results in histological changes in the skin. Rana pipiens were loaded with KCl and skin biopsies obtained (Group I). These biopsies were compared with biopsies from NaCl loaded frogs (Group II). In blind studies of microscopic sections, 13 of 17 biopsies of a mixture of I and II were correctly diagnosed by one observer and similarly, 14 of 17 of Group I and II were correctly diagnosed by a second observer (P = 0.0245 and 0.0063, respectively). The characteristics used to distinguish skins from KCl treated frogs versus controls treatment included: (i) an abundance of large euchromatin cells on or near the surface; (ii) changes in the basal cell layer with elongation and rotation of the nuclei; (iii) lighter cells in the spinosal layers; and (iv) sometimes the skin became thicker. The water-soluble nondialyzable material of the frog skin was extracted, and we found that it increased by 4.4 times following KCl loading (P < 0.05). However, the protein fraction was not increased by loading the frog for 3 days with NaHCO3. We conclude that potassium loading results in characteristic histological changes in the skin and that this is probably related to the ability of the skin to excrete potassium. In addition, a comparison of biopsies of skin from fed frogs with samples from frogs fasted for 40 to 49 days showed a change in the thickness of the skin. Skins of fed frogs averaged 57.0 +/- 1.4 mu thick compared with fasted, 39.9 +/- 2.7 mu (P < 0.001).

Stimulation of HCO3- excretion in frog skin by urinary and adrenal cortical extracts.

1. There is a substance extractable from human urine which stimulates frog skin to excrete bicarbonate. The response to this extract is delayed, taking 4-9 hr, depending upon the dosage. 2. The amount of this substance varies with the bicarbonate intake of the urine donor. 3. This is a reasonable dose-response curve to the extract. 4. A substance with the same solubility characteristics, and which stimulates bicarbonate excretion with a similar time response is present in adrenal cortical extracts.

Histological changes in the skin of Rana pipiens produced by metabolic alkalosis.

The frog skin has been shown to excrete various electrolytes, the rates can be altered by varying metabolic conditions. The present study was performed to determine if metabolic alkalosis results in histological changes in the skin that are characteristic of this state. Rana pipiens were loaded with NaHCO3 and skin biopsies obtained (I). These biopsies were compared with biopsies from either control, unloaded frogs (II), or from NaCl loaded (III) frogs. In blind studies of microscopic sections, 13 of 15 biopsies of a mixture of I and II were correctly diagnosed, and similarly, 18 of 20 of I and III were correctly diagnosed (P = 0.0037, and 0.0002, respectively). The changes due to NaHCO3 treatment included; (1) an abundance of large euchromatin cells on or near the surface; (2) changes in the basal cell layer with elongation and rotation of the nuclei; (3) lighter cells in the spinosal layer; and, (4) sometimes the skin became thicker. We conclude that metabolic alkalosis results in characteristic histological changes in the skin, and that this is probably related to the ability of the skin to excrete bicarbonate.

Atrial natriuretic factor inhibits vasotocin-induced water reabsorption in the toad urinary bladder.

Peptides recently isolated from atrial extracts possess potent natriuretic and diuretic activities, which are thought to be due to hemodynamic actions, such as increased glomerular filtration or altered medullary blood flow. A direct tubular site of action cannot be ruled out; therefore we have examined the effect of one of these peptides, atriopeptin III on vasotocin-induced water absorption in the toad urinary bladder. Our results indicate that equimolar doses (10(-12) to 10(-11) M) of atriopeptin III can significantly inhibit vasotocin-induced water reabsorption in vitro and suggest a physiologic role for the cardiac peptides to alter water reabsorption directly at the level of the tubules or collecting ducts, independent of any hemodynamic effects they might also exert in vivo.

Increased Na+ excretion by the skin of Rana pipiens after NaCl loading.

In vivo the frog skin excretes sodium and the sodium excretion is increased in response to a NaCl load. The sodium excretion can be demonstrated in vitro, and the rate of excretion is greater in skin from NaCl-loaded animals than from control, non-loaded animals. Unidirectional 22Na flux experiments on paired frog skins, as well as 22Na and 24Na bidirectional flux experiments measured in vitro, confirm the above finding that net sodium excretion occurs in response to the NaCl load.

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine.

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH+4 and/or H+ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH4Cl-induced acidosis were prepared. These extracts were tested for their effect on NH+4 excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH+4 excretion in the toad urinary bladder. We found no effect on H+ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH+4. The factor could act as a permease in the bladder cell or as a stimulator of an NH+4 transport system.

Excretion of HCO3- by the urinary bladder of Bufo marinus in metabolic alkalosis.

We studied the role of the urinary bladder of Bufo marinus in the excretion of bicarbonate into the urine. The toads were in metabolic alkalosis, produced by administering 120 mM NaHCO3 by stomach tube or by soaking the toads in 120 mM NaHCO3 solution for 48 to 72 hr. In vitro 10 cannulated whole bladders from toads in alkalosis transported bicarbonate from the serosal to mucosal medium. The average gradient created by this transport was 5.7 meq/1. In 15 whole bladders from toads in metabolic acidosis studied under identical conditions, there was no transport of bicarbonate into mucosal medium.