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Role of sodium on H+ excretion in the integument of the leopard frog Rana pipiens
Camp. Biochem. Physiol. Vol. 9lA, No. Printed in Great Britain
I. pp. 53-59,
0300-9629/88 $3.00 + 0.00 \C 1988 Pergamon Press plc
1988
ROLE OF SODIUM ON H+ EXCRETION IN THE INTEGUMENT OF THE LEOPARD FROG RANA PIPIENS Department
RAY PAGE, LOY W. FRAZIER* and THOMAS YORIO of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107. USA Telephone:
(8 17) 735-2056
(Received 24 November
1987)
The northern leopard frog, Rana pipiens, pipiens, in contrast to the southern leopard frog, Rana pipiens, berlundieri, did not demonstrate any significant H+ excretion across its integument even during a challenge of chronic metabolic acidosis. Likewise, no increase in the number of H+ secreting mitochondria-rich cells were observed in the northern frogs. 2. Under normal acid-base conditions in the southern frogs, H+ excretion was found to be dependent on mucosal sodium concentrations, whereas during chronic metabolic acidosis, H+ excretion was independent of mucosal sodium concentrations, but was amiloride sensitive. 3. High salinity adapted southern frogs, under normal and acidotic conditions, had enhanced H+ excretion rates as compared to the control non-salt adapted frogs. 4. Blood analyses demonstrated that significant acid-base changes were the result of systemic acidosis and not due to salt adaptations. Blood Na+ and K+ concentrations were also efficiently maintained during salt adaptations or chronic metabolic acidosis. 5. The results suggest that H+ excretion in epithelia can be influenced by the sodium transport state of the cell and the systemic acid-base profile. Models are proposed explaining these relationships. Abstract-l.
INTRODUCTION
The main objective of the current study was to further characterize H+ excretion in the skin of the frog Rana pipiens and to determine its relationship to sodium under normal, acidotic conditions and salt adaptations. A comparison of H+ excretion of northern vs southern grass frogs (Rana pipiens) is also presented.
In amphibians, the ability to regulate systemic pH is dependent, in part on its capacity to excrete protons across its integument (Emilio and Menano, 1975; Ehrenfeld and Garcia-Romeu, 1977; Frazier, 1986) and urinary bladder (Frazier and Vanatta, 1973; Frazier, 1974, 1985). Such characteristics can be found among many different sub-species of amphibia (Machen and Erlij, 1975; Benos, 1981; Ehrenfeld et al., 1985) including Rana pipiens (Vanatta and Frazier, 198 1). Similar mechanisms for systemic regulation of pH can also be found in the mammalian nephron (Breyer and Jacobson, 1987). In response to a metabolic acidosis, the skin of the frog increases its capacity to excrete hydrogen ions (Vanatta and Frazier, 1981) and this is accompanied by an increase in the number of mitochondria-rich (MR) cells located in the frog’s active epidermal layer (Page and Frazier, 1987). This latter response is thought to be associated with the location of active proton excretion (Al-Awqati and Schwartz, 1986). Recent evidence has suggested that Na+/H+ exchange may play an important role in H+ secretion and intracellular pH regulation in many cells (Aickin and Thomas, 1977; Deitmer and Ellis, 1980; Grinstein and Rothstein, 1986) including frog skin epithelium (Ehrenfeld et al., 1985, 1987). It has been demonstrated that H+ excretion in the skin of the southern grass frog, Rana pipiens, is amiloride sensitive, both under normal acid-base conditions and in the presence of metabolic acidosis (Frazier, 1986), and in Rana esculenta a basolateral amiloridesensitive Na+/H+ ion exchanger has been described (Ehrenfeld et al., 1987). *Department of Physiology, Dallas, TX 75226, USA
Baylor
College
MATERIALS AND
METHODS
Comparison of northern and southern frogs The frogs used in this study were Rana pipiens of both southern and northern origin and were supplied by Carolina Biological Company (Burlington, NC). An additional group of southern frogs were obtained from Southern Biologicals (McKenzie, TN). The animals were fed and kept in terraria at 23°C. Chronic metabolic acidosis was induced by injecting 120mM NH,CI (O.O25ml/g body weight) into the dorsal lymph sac 3 times/day for 2 days. Normal and acidotic frogs were sacrificed by double pithing, and the ventral abdominal skins were removed. These skins were mounted as sheets between two Ussing-type lucite chambers. Two different sets of chamber sizes were used: one set of chambers had a volume of 2 ml with an exposed area of skin of 1.98cm’. and the other set of chambers was 5 ml in volume with an exposed area of 3.14 cm2. Previous H+ excretion comparisons between the chamber types showed no statistical difference. The mucosal and serosal surfaces of the skins were bathed in frog Ringers solution consisting of (in mM): NaCI, 114.5; KCI, 3.0; CaCI,, 0.9; and sodium phosphate (dibasic), 1.5. The final pH was 7.25-7.35. The mounted skins were allowed to equilibrate in Ringer’s for I5 min, the chambers were then drained and fresh Ringer’s solution was added. Mucosal H+ flux of the isolated skins was measured for 120 min at room temperature with the serosal bathing fluid being bubbled with air. The determination of H+ excretion and the histological preparation and the identification of the tissues were as previously described (Page and Frazier, 1987). In all experiments, the H+ excretion was calculated from a change in the
of Dentistry,
53
RAY PAGE et al.
54
pH and the concentration of phosphate buffer in the mucosal and serosal solutions. A Fisher accumet Model 142pH meter was used for all pH determinations. Skins from the northern frogs were histologically prepared for cell counts. Coded slides of random sections were used, and cells were counted from both normal and acidotic frogs. Instruments Systemic pH in the northern frogs was determined from whole blood obtained by ventricular puncture and aspiration with a syringe immediately after removal of the skins. Blood pH was determined by the aforementioned pH meter. Blood analyses in subsequent experiments were performed by an automated instrument, Stat Profile 1. This instrument directly measures PO,, pCO,, pH, and hematocrit, as well as electrolytes, including K+, Na+ and Ca*+. From these measured variables several other variables were calculated including 0, content and bicarbonate. The effect of low sodium concentrations and amiloride on H + excretion Ventral skins from the southern variety frogs were used in the following experiment and mounted in the modified Ussing chambers with an exposed area of I .98 cm’. To determine the effect of sodium on H+ excretion, the concentration of sodium in the Ringer’s solution was adjusted so that five solutions had the following NaCl concentrations (in mM): 114, 80, 40, 20 and 2. An equimolar amount of choline chloride replaced the NaCl in the solutions. The final pH was adjusted to 7.15, and all solutions had an osmolality of 248-254 mOsm. The choline chloride adjusted Ringer’s solution was placed on the mucosal side and normal Ringer’s solution was placed on the serosal side. The tissues were allowed to flux for 120min. A second group of frogs received amiloride (IO-‘M) (Merck Institute of Therapeutic Research, West Point, PA) in the mucosal bath. This concentration has been shown by Yorio and Bentley (1976) to be adequate to block Nat transport across the skin. Both the mucosal and serosal baths contained normal frog Ringer’s (I I4 mM NaCI), with amiloride administered to the mucosal bath. Again the skins were allowed to flux and H+ excretion was determined. The effects of high salinity adaptation on H+ excretion Southern variety frogs were placed into three groups: (1) control, (2) normal high salinity adapting (HSA), and (3) acidotic high salinity adapting. The controls were placed in I mM NaCl water for 5 days, while the normal HSA frogs
were placed in 60 mM NaCl water for 5 days. The acidotic HSA frogs were also kept in 60 mM NaCl water for 5 days and in addition, on the last 2 days of adapting, metabolic acidosis was induced as previously described. The water for each of the groups was changed twice daily and at that time the animals were weighed to determine any changes in weight. On the fifth day, the animals were sacrificed and H + excretion by the skin was determined. Blood was collected from these frogs and analyzed for acid--base. ionic and metabolic characteristics. Statistics Statistical analyses were performed utilizing the statistical programs from Tallarido and Murray (1987) adapted for the IBM XT. Appropriate statistical analyses were applied to each group of data.
RESULTS Comparison of’ H+ excretion and morphological changes in the skin IJ~ northern and southern j?ogs
We have previously shown that during a challenge of chronic metabolic acidosis the southern variety of the leopard frog, Ram pipiens, compensates by increasing H+ excretion in the skin. Additionally, there is an increase in the number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis (Page and Frazier, 1987). Figure I demonstrates that following the challenge of a 48 hr metabolic acidosis the intact skin of the northern variety of Rana pipiens did not significantly increase its Hi excretion, but the skin of the southern variety frog produces a highly significant increase in H+ excretion in acidosis (Independent t-test, P > 0.2 and P < 0.005, respectively). The mean blood pH of the northern frogs, however. decreased significantly from 7. I3 k 0. I I in the normal frogs (n = I I) to 6.66 & 0.12 in the acidotic frogs (n = 10) (Independent t-test, P < 0.001). Table I indicates that the net H+ flux is directional from serosal to mucosal in both the normal and acidotic states, and that in normal skins the serosal bathing fluid alkalinizes, while during the acidotic condition the serosal bathing fluid acidifies.
2.00, p(O.001
T
p10.2
5 ‘J f : + I
0.75 0.50 0.25 0.00 northern Mucosol
southern bathing
fluid
Fig. I. Comparison of H+ excretion in the skin of northern and southern frogs under normal and acidotic conditions. Each group represents the mean k S.E. of ten skins. Acidosis was produced by NH,CI injections into the dorsal lymph sac three times daily for 48 hr.
Na+/H+ Table
I.
Mucosal
and
serosal
H + excretion
southern
in
relationship
northern
and
55
in frog skin
Table 2. Changes
in cell population
Hydrogen MUCOsal
I
Northern
Ratio
(12)
0.64 + 0.14”
(I 2)
0.73 * 0. I2
Southern
variety
A. Normal
(IO)
0.60 + 0.10
9. Acidotic
(IO)
1.60+0.12
‘As
means i
P value
-0.05
kO.19
0.24+0.10
state
and
Normal
< 0.002b < 0.002
Mitochondria-
B. Acidosis
rich
basal I.0
0.013
(98.8%)
(1.23%) 0.014b
1.0
(1.38%)
(98.6%) -0.15
kO.07
0.10 * 0.05
<0.002 co.002
aRepresents Number
a total
of 2200
in parenthesis
cells counted
is the percentage
from
Paragon
slides.
cell type of the total number
of cells counted.
S.E.
“15 the probability
Acid-base A.
9. Acidotic
of cell types”
Granular
variety
A. Normal II
Serosal
frog skin in response
acidosis
ion excretion
(nmole/cm2/min) Grow
in northern
to metabolic
frees
that
the values are different.
bP > 0.05.
The skins of both the normal and acidotic northern frogs were histologically prepared for identification of cell types and cell counts. Table 2 describes changes in the cell population of the skin in response to metabolic acidosis. The granular + basal : MR cell ratio and percentage of cell types in the northern skins show no significant increase in the number of MR cells in response to chronic metabolic acidosis (Independent t-test, P > 0.05). The effect of low sodium concentration and amiloride on H+ excretion In the following experiment, mucosal sodium concentration was varied and H+ flux measured under normal and acidotic conditions of southern frogs. There was a significant difference in the mean H+ excretion at various mucosal Na+ concentrations in the normal acid-base state (Fig. 2a) (P < 0.05, one way parametric ANOVA, F = 3.5, n = 48). However, zero order kinetics was observed for the H+ excretion of frogs in metabolic acidosis (Fig. 2b), as there were no significant differences between the mean H+ excretion at various mucosal Na+ concentrations (one way ANOVA, P > 0.10, F = 1.45, n = 48). Lmear regression analysis of these data and their respective serosal H+ fluxes showed no 1: 1 mucosal acidification to serosal alkalinization relationship. In the normal acid-base state, the mean H+ excretion by amiloride (IO-‘M) exposed skins was not significantly different from that of the control portion
Na+
concentration
Not
statistically
significant
by the
independent
I-test.
of the skins (Fig. 3) (one-tailed paired t-test, 0.10 > P > 0.05). However in metabolic acidosis, the administration of amiloride significantly decreased from that of the nonthe mean H+ excretion amiloride treated skin (Fig. 3) (one-tailed paired t-test, P < 0.001). Likewise, no significant difference in mean H+ excretion was found between the control skins and low mucosal sodium skins in normal frogs (Independent f-test, one-tailed 0.50 > P > 0.25), whereas, in the acidotic frogs, a highly significant decrease in mean H+ excretion was found in the low mucosal sodium skins as compared to controls (Fig. 3) (Independent t-test, one-tailed, P < 0.005). Effects of &h
salinity adaptation on H+ excretion
To characterize further the relationship between sodium transport and H+ flux, frogs were sodium adapted for 5 days in 60mM NaCl in the presence and absence of metabolic acidosis (present last 2 days). The mean H+ excretion of the 5 day 60mM NaCl adapted frogs in both the normal and acidotic state was significantly different from the control group bathed in 1 mM NaCl and in the normal acid-base state (Fig. 4) (Dunnetts t-test, P < 0.05 and P < 0.01, respectively). As seen in Fig. 5, the percentage change in body weight during high salinity adaptation increased in the frogs that were adapting to the 60 mM NaCI, while the control frogs bathed in the 1 mM NaCl showed a slight decrease in body weight. It was further found that the slopes
(meq/L)
Fig. 2. Effects of different sodium concentrations on H+ excretion in the southern frog skin under normal (left panel) and acidotic conditions. Under normal acid-base conditions, H+ excretion is dependent on mucosal sodium concentrations, whereas, in the presence of acidosis, H+ excretion is independent of mucosal sodium concentrations. Each point represents the mean f S.E. of eight skins.
RAY PAGEet al.
56
h
3.5
1.40
g
1.20
2
1.00
z E.
0.80
s ‘Z E :
0.60
+I
0.40 0.20 0.00 normal
acidotic Metabolic state
Fig. 3. The effects of amiloride and low mucosal sodium concentrations on H + excretion in normal and acidotic frogs. Each group represents the mean + S.E. of eight paired frog skins. Amiloride (1 x IO-’ M) was added to the mucosal bathing fluid.
0.60 Q ‘E 2
0.70
E 0 > 5 .SE
0.60
*E zi t c 2
0.30
ONomWt~ Iuotab&addo8b =Nnmld*
0.50 0.40
0.20 0.10 0.00
60mM
60mM
1mM
NaCl concentration Fig. 4. The effects of high salinity adaptation (60 mM) on H’ excretion in frog skins under normal and acidotic conditions. Each group was statistically compared to the 1mM normal group (mean + SE. of eight skins per group).
-54 0
25
50 Time (houn
75
100
1 !5
during adaptation)
Fig. 5. Percentage weight change of southern frogs under normal and high salinity adapting conditions. Each group of animals were weighed twice daily at time of water change. The means of each group are presented.
51
Na+/H+ relationship in frog skin Table 3. Blood acid-base, Blood Analyses PH PC@ (mm Hg) Na+ (mmol/L) K + (mmol/L) Ca*+ (mmol/L) BE-ECF” (mmol/L) HCO, (mmol/L)
Group Normal 7.4 * 21 * III * 3.3 f I .o i -1.9il.4 13 *
ionic and metabolic
I
0.03 1.0 1.3 0.3 0.02 0.9
characteristics
Group 2 Acid 7.2 f 0.04 15i I.1 Ill f 1.3 3.4 * 0.3 I.1 kO.03 -22 + 1.3 6.4 k 0.8
of acidotic
Group 3 HSA-normal 7.6 k 20 + I IO f 2.5 f 0.9 + -4.2 k l8k
0.07 2.6 3.4 0.6 0.05 2.2 I.3
As mans k SE. HSA-High Salimty Adapted; SNK-Newman-Keuls Multiple Range Test. “Base excessxxtracellular fluid. bF(95%) = 2.92, n = 39. All comoarisons between eroutx I and 2 that demonstrate ’ significantly different groups’are at P < 0.01. -
relating percentage body weight change were not significantly different between the 60 mM NaCl adapting frogs, but were different from the 1 mM NaCl control group (t-test for parallelism, t = 0.121, P > 0.5, and t = 5.171, P < 0.001, respectively). Blood analysis Table 3 lists the results of an indepth blood analysis done on frogs from the above salt adapted experiment. The student Newman-Keuls multiple range test demonstrates several comparisons. It was found that a significant difference in pH, Ca*+, BE-ECF (base excessextracellular fluid), and HC03, occurs between: (1) the normal acid-base groups (groups 1 and 3) vs both acidotic groups (groups 2 and 4) (P < 0.01) and, (2) the normal acid-base normal salinity group (group 1) vs the normal acid-base/ 60 mM salt adapted group (group 3) (P < 0.05). Furthermore, no significant difference is found among the two acidotic groups (groups 2 and 4) in any of the parameters. In addition, the Na+ and K+ concentrations were not significantly different among any of the groups (ANOVA, F = 1.36, and F = 2.08, respectively). The pC0, comparison showed significance only between group 1 and group 2 (P < 0.05). DISCUSSION
The cellular mechanisms involved in proton transport by renal tissues in maintaining homeostatic systemic pH has long been questioned and pursued. The microperfusion techniques of Rector et al. (1965) and Malnic et al. (1972) along with the morphological studies of Madson and Tisher (1985) have demonstrated the presence of H+ excretion in the mammalian proximal and distal tubules. These characteristics have also been demonstrated in several amphibian models including the toad urinary bladder (Frazier, 1985), the turtle bladder (Stetson and Steinmetz, 1985) and various species of frogs skin (Benos, 1981; Vanatta and Frazier, 198 1; Lindenger and McDonald, 1986; Ehrenfeld et al., 1987). However, differences among these species in their membrane permeability characteristics (Bentley and Yorio, 1976) and ion transport relationships with H+ excretion (Frazier, 1986; Duranti et al., 1986; Emilio and Menano, 1975) have left many of the cellular mechanisms of proton transport in question. In Rana pipiens, the skin has been shown to be structurally and biochemically heterogenous and the mitochondria-rich (MR) cell is thought to be the
and salt adapted Group 4 HSA-acid 7.0 f I6 f I I8 * 4.4 * I .3 + -27 k 4.3 *
a difference
southern
ANOVA F-valw?
0.04 1.2 0.5 0.5 0.03 0.8 0.1
are
frogs
at P < 0.05,
11.66 4.01 I .36 2.08 7.83 18.84 21.76
all other
SNK Grouping
I 3 24 123 234 I234 1234 I 3 24 I 3 24 I 3 24
combinations
of
mediator of Hi transport (Farquhar and Palade, 1965; Rosen and Friedley, 1973). It has been shown in both the toad and turtle urinary bladder (Frazier, 1978; Al-Awqati and Schwartz, 1986) and in the skin of the southern variety of Rana pipiens (Page and Frazier, 1987) that an increase in the number of MR cells as well as an increase in the rate of H+ excretion in response to a challenge of chronic metabolic acidosis occurs. However, in the present study it was found that the skin of the northern variety of Rana pipiens was unable to increase H+ excretion in response to metabolic acidosis, and likewise, no evidence of an increase in MR cell number was observed. Recently, Wheeler and Arruda (1987) have shown that an increase in proton excretion by turtle urinary bladders, under acidosis was associated with a corresponding increase in MR cells and intracellular acidic vesicles. These data offer additional support that the MR cell may be the mediator of proton excretion. In the current study, net H+ flux was found to be directional from serosa to mucosa in both normal and acidotic animals. In addition, it was found that in the normal skins the serosal bathing fluid alkalinizes, while during metabolic acidosis the serosal solution acidifies. This phenomenon has previously been shown in the intact skin (Page and Frazier, 1987) and supports the model of Ehrenfeld et al. (1987) which suggests a basolateral H+ secretory mechanism which is quiescent in normal conditions but stimulated in metabolic acidosis. This allows cellular pH to be maintained within physiological limits by “dumping” protons systemically to be eliminated by the kidney. It was further observed that under normal acidbase conditions, H+ fluxes were found to be dependent on mucosal sodium concentrations. Conversely, in metabolic acidosis, zero order kinetics was observed for the H+ excretion rate. These results suggest that under normal conditions there is a sodium dependency of H+ excretion which is saturable at 20-30 mM mucosal sodium, whereas at higher rates of H+ flux, under metabolic acidosis, this is not observed. This is similar to what has been reported for H+ fluxes in the skin of the frog, Rana esculenta (Ehrenfeld and Garcia-Romeu, 1977). This sodium dependency difference was particularly noticeable in the low mucosal sodium (2 mM) H+ flux data. Amiloride, a known inhibitor of passive mucosal Na+ reabsorption, was found to inhibit H+ excretion only in the acidotic frogs. These results agree with
RAY
58
PAGE et al.
previous observations of Frazier (1985) on the amiloride-sensitive H+ flux in toad bladders in the presence of chronic acidosis. It appears, therefore, that a decrease in sodium uptake across the apical membrane results in a significant decrease in H+ excretion during acidotic states. It has been proposed by Leaf et al. (1959), that as much as 50% of endogenous CO, is generated from ATP production used for driving the basolateral Nat-K+ pumps, and that CO1 is also a substrate for carbonic anhydrase in the production of H+ for excretion. Therefore, amiloride, by blocking apical Na+ entry, produced a 40% decrease in H+ excretion, probably through an inhibition in transcellular active sodium transport and a concomitant decrease in CO, generation. Furthermore, Hi excretion was markedly increased in acidosis, partly due to the decrease in cell pH from the increased uptake of systemic COZ, and partly from the presence of active sodium transport (Ehrenfeld et al., 1987). It was interesting to note in the present study, that under low sodium conditions (2mM), only a slight decrease in H+ flux was observed during metabolic acidosis, perhaps reflecting the presence of residual transcellular sodium transport and CO1 production sufficient in maintaining an elevated H+ flux. Additional information concerning the sodium dependence of H’ excretion was obtained by measuring H+ excretion in normal and acidotic animals which were salt adapted. It was found that in normal frogs, which were high salinity adapted (HSA) for 5 days, there was an increase in H+ excretion over those of the normal 1 mM salt adapted frogs. This suggests that the apical Na + gradient has an influence on H+ excretion and the pH gradient. The increased H+ flux may be the result of an increase in metabolic CO1 production resulting from energy utilization in driving the basolateral sodium transport system. An increase in Na+ uptake was evidenced by an increase in weight gain by the HSA animals in comparison
to the normal animals, perhaps as a result of an increased fluid absorption. Blood acid-base profiles in normal HSA animals were also consistent with an increase in H+ excretion, as blood bicarbonate concentrations were elevated. Although no change in blood sodium concentrations were observed, this may reflect an increase in blood volume from an enhanced water reabsorption or a consequence of efficient sodium removal by the kidney. The HSA animals in metabolic acidosis also showed an increase in weight, but had a significant decrease in blood pH and HCO,, reflecting the state of acidosis in the animal. The H+ excretion rates of these animals were similar to that of the HSA normal animals, although blood pH and HCO, concentrations were markedly different between normal HSA vs HSA animals in acidosis. The blood acidbase profiles of the HSA acidotic animals were not different from the non-salt adapted acidotic animals. This suggests that the acid-base changes are primarily a result of systemic acidosis and not due to the salt adaptation. In addition, the increase in H + excretion by normal HSA animals may therefore be a function of increased sodium uptake and transport, possibly through an increased CO2 production. These data suggest that the apical Na+ gradient may influence H+ excretion, perhaps as proposed by Frazier (1985) through energy dependent mechanisms of active sodium reabsorption. Alternatively, at sodium concentrations below 30mM, below sodium transport saturation, there appears to be a Nat/H+ antiporter that is driven by the sodium and pH gradients. Under acidosis, H’ excretion may in part function through this antiporter. but it may also proceed through Na’ independent processes linked to the metabolic state of the cell. Figures 6(A) and (B) are a modification of a model previously proposed by Frazier (1985) for the toad bladder and depicts these alternate mechanisms.
NORMAL
ACIOOSIS
Mucosal
Serosal
Mucosal
> Na+--___.__
Nat--__
--__
pHi
A.
Nat K+
--*H+ ----Na+
B.
Fig.6. Proposed model of
H + excretion characteristics and the role of sodium in (A) the normal state, and (B) in chronic metabolic acidosis (see text).
metabolic
Na+/H+
relationship
In Fig. 6(A), normal H+ excretion is depicted as an electrogenic apical pump which is dependent on the intracellular H+ concentration produced from the enzymatic conversion of CO2 to bicarbonate. The intracellular CO, concentration is, in turn, primarily dependent on the rate of passive apical Na+ influx which is actively removed basolaterally. Thus, energy utilization
for the active
Na+/K+
pump
plays
a major
role in cellular CO, production. Under normal conditions the intracellular pH is efficiently maintained and the Na+/H+ exchanger is quiescent and serosal alkalinization is observed. Under these conditions H+ excretion occurs primarily across the apical membrane. When the animal is challenged with chronic metabolic acidosis, H+ excretion is no longer primarily dependent on apical Nat influx (Fig. 6B), and the main stimulation of H+ excretion is via the increase in cellular CO, from the induced systemic acidosis. The increased CO, masks the Na + dependency of H + excretion, but, in response to a decreased pH, the Na+/H+ exchanger becomes activated. This results in both a serosal acidification and in an increase in intracellular sodium which can stimulate CO* production via the basolateral Na+/K+ pump. Such a model is consistent with the present observations. It is also evident that additional studies are needed to further describe these mechanisms and to elucidate some of the mediators that may regulate these processes. A~,know/edgemenrs-This research was supported in part from a grant from the American Heart Association, Texas Affiliate (TY) and a BRSG grant from TCOM (TY). Ray Page was supported by an AOA/Burroughs Wellcome Osteooathic Research Fellowship . (F87-07). The authors would like to thank Drs L. X. Oakford and A. J. Mia for the assistance with quantification of the MR cells by light microscopy.
REFERENCES Aickin C. C. and Thomas R. C. (1977) An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers, J. Physiol. Lond. 273, 295-3 16. Al-Awqati Q. and Schwartz J. H. (1986) Plasticity of spithelial polarity. Renal Physiol. 9, 1 IO. Benos D. J. (1981) Acidification and sodium entry in frog skin epithelium. B&hem. Biophys. Acta 647, 40-48. Bentley P. J. and Yorio T. (1976) The passive permeability of the skin of anuran Amphibia, a comparison of frogs (Rana pipiens) and toads (Et& marinus). J. Physiol., Lond. 261, 603-615. Brever M. D. and Jacobson H. R. (1987) Mechanisms and Am. J. regulation of renal H+ and HCO, transport. Yephrol. 7, 150-161. Deitmer J. W. and Ellis D. (1980) Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkenje fibers. J. Physiol., Land. 304, 471&488. Duranti E., Ehrenfeld J. and Harvey B. J. (1986) Acid secretion through the Rana esculenra skin: Involvement of an anion-exchange mechanism at the basolateral membrane. J. Physiol, Lond. 378, 195-21 I. Ehrenfeld J. and Garcia-Romeu F. (1977) Active hydrogen excretion and sodium absorption through isolated frog skin. Am. J. Physiol. 233, F46F54.
in frog skin
59
Ehrenfeld J., Garcia-Romeu F. and Harvey B. J. (1985) Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport. J. Physiol., Land. 359, 331-355. Ehrenfeld J., Cragoe E. J. and Harvey B. J. (1987) Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues. P&get-s Arch 409, 200-207. Emilio M. G. and Menano H. P. (1975) The excretion of hydrogen ion by the isolated amphibian skin. Effects of antidiuretic hormone and amiloride. Biochem. Biophys. Acta 382, 344352. Farquhar M. G. and Palade G. E. (1965) Cell junctions in amphibian skin. J. cell Biol. 26, 263-291,
Frazier L. W. (1974) Interrelationship of H+ excretion and Na+ reabsorption in the toad urinary bladder. J. Membrane Biol. 19, 267-276. Frazier L. W. (1978) Cellular changes in the toad urinary bladder in response to metabolic acidosis. J. Membrane Biol. 40, 165-172. Frazier L. W. (1985) Characteristics of proton excretion in normal and acidotic toad urinary bladder. Biochem. Biophys. Acta 817, 75-84. Frazier L. W. (1986) Acidification of the bathing fluid by the skin of Rana pipiens in metabolic acidosis. Camp. Biochem. Physiol. 85A, 57-61. Frazier L. W. and Vanatta J. C. (1973) Characteristics of H+ and NH: excretion by the urinary bladder of the toad. Biochem. Biophys. Acta 311, 98-108. Grinstein S. and Rothstein A. (1986) Mechanisms of regulation of the Na+/H+ exchanger. J. Membrane Biol. 90, 112. Leaf A., Page L. B. and Anderson J. (1959) Respiration and active sodium transport of isolated toad bladder. J. biol. Chem. 234, 1625-1629. Lindinger M. 1. and McDonald D. G. (1986) Cutaneous and renal responses to intravascular infusions of HCI and NH,Cl in the bullfrog (Rana catesbiuna). Camp. Biochem. Physiol. 84A(l), 113-122. Machen T. and Erlij D. (1975) Some features of hydrogen (ion) secretion by the frog skin. Biochem. Biophys. Acra 406, 120&130. Madsen K. M. and Tisher C. C. (1985) Structure-function relationships in H+ secreting epithelia. Fed. Proc. 44, 2704-2709. Malnic G.. deMello Aires M. and Giebisch G. (1972) Micropuncture study of renal tubular hydrogen ion transport in the rat. Am. J. Physiol. 222, 147-158. Page R. D. and Frazier L. W. (1987) Morphological changes in the skin of Rana pipiens in response to metabolic acidosis. Proc. Sot. exp. biol. Med. 184, 416422. Rector F. C., Carter W. W. and Seldin D. W. (1965) The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney. J. clin. Incest. 44, 278-290. Rosen S. and Friedley N. J. (1973) Carbonic anhydrase activity in Rana pipiens skin: biochemical and histochemical analysis. Histochemie 36, l-4. Stetson D. L. and Steinmetz P. R. (1985) A and B types of carbonic anhydraserich cells in turtle bladder. Am. J. Physiol. 249, F553-F565. Tallarido R. J. and Murray R. B. (1987) Manual ofPharmacological Culculufions. Second edition. Springer-Verlag, New York. Vanatta J. C. and Frazier L. W. (1981) The epithelium of Rana pipiens excretes H + and NH: in acidosis and HCO, in alkalosis. Camp. Biochem. Physiol. 68A, 51 I-513. Wheeler R. P. and Arruda J. A. (1987) Adaptation to metabolic acidosis by turtle urinary bladder. Am. J. Physiol. 252, F256266. Yorio T. and Bentley P. J. (1976) Effects of ethanol on the permeability of frog skin. J. Pharmac. exp. Ther. 197, 340-351.
I. pp. 53-59,
0300-9629/88 $3.00 + 0.00 \C 1988 Pergamon Press plc
1988
ROLE OF SODIUM ON H+ EXCRETION IN THE INTEGUMENT OF THE LEOPARD FROG RANA PIPIENS Department
RAY PAGE, LOY W. FRAZIER* and THOMAS YORIO of Pharmacology, Texas College of Osteopathic Medicine, Fort Worth, TX 76107. USA Telephone:
(8 17) 735-2056
(Received 24 November
1987)
The northern leopard frog, Rana pipiens, pipiens, in contrast to the southern leopard frog, Rana pipiens, berlundieri, did not demonstrate any significant H+ excretion across its integument even during a challenge of chronic metabolic acidosis. Likewise, no increase in the number of H+ secreting mitochondria-rich cells were observed in the northern frogs. 2. Under normal acid-base conditions in the southern frogs, H+ excretion was found to be dependent on mucosal sodium concentrations, whereas during chronic metabolic acidosis, H+ excretion was independent of mucosal sodium concentrations, but was amiloride sensitive. 3. High salinity adapted southern frogs, under normal and acidotic conditions, had enhanced H+ excretion rates as compared to the control non-salt adapted frogs. 4. Blood analyses demonstrated that significant acid-base changes were the result of systemic acidosis and not due to salt adaptations. Blood Na+ and K+ concentrations were also efficiently maintained during salt adaptations or chronic metabolic acidosis. 5. The results suggest that H+ excretion in epithelia can be influenced by the sodium transport state of the cell and the systemic acid-base profile. Models are proposed explaining these relationships. Abstract-l.
INTRODUCTION
The main objective of the current study was to further characterize H+ excretion in the skin of the frog Rana pipiens and to determine its relationship to sodium under normal, acidotic conditions and salt adaptations. A comparison of H+ excretion of northern vs southern grass frogs (Rana pipiens) is also presented.
In amphibians, the ability to regulate systemic pH is dependent, in part on its capacity to excrete protons across its integument (Emilio and Menano, 1975; Ehrenfeld and Garcia-Romeu, 1977; Frazier, 1986) and urinary bladder (Frazier and Vanatta, 1973; Frazier, 1974, 1985). Such characteristics can be found among many different sub-species of amphibia (Machen and Erlij, 1975; Benos, 1981; Ehrenfeld et al., 1985) including Rana pipiens (Vanatta and Frazier, 198 1). Similar mechanisms for systemic regulation of pH can also be found in the mammalian nephron (Breyer and Jacobson, 1987). In response to a metabolic acidosis, the skin of the frog increases its capacity to excrete hydrogen ions (Vanatta and Frazier, 1981) and this is accompanied by an increase in the number of mitochondria-rich (MR) cells located in the frog’s active epidermal layer (Page and Frazier, 1987). This latter response is thought to be associated with the location of active proton excretion (Al-Awqati and Schwartz, 1986). Recent evidence has suggested that Na+/H+ exchange may play an important role in H+ secretion and intracellular pH regulation in many cells (Aickin and Thomas, 1977; Deitmer and Ellis, 1980; Grinstein and Rothstein, 1986) including frog skin epithelium (Ehrenfeld et al., 1985, 1987). It has been demonstrated that H+ excretion in the skin of the southern grass frog, Rana pipiens, is amiloride sensitive, both under normal acid-base conditions and in the presence of metabolic acidosis (Frazier, 1986), and in Rana esculenta a basolateral amiloridesensitive Na+/H+ ion exchanger has been described (Ehrenfeld et al., 1987). *Department of Physiology, Dallas, TX 75226, USA
Baylor
College
MATERIALS AND
METHODS
Comparison of northern and southern frogs The frogs used in this study were Rana pipiens of both southern and northern origin and were supplied by Carolina Biological Company (Burlington, NC). An additional group of southern frogs were obtained from Southern Biologicals (McKenzie, TN). The animals were fed and kept in terraria at 23°C. Chronic metabolic acidosis was induced by injecting 120mM NH,CI (O.O25ml/g body weight) into the dorsal lymph sac 3 times/day for 2 days. Normal and acidotic frogs were sacrificed by double pithing, and the ventral abdominal skins were removed. These skins were mounted as sheets between two Ussing-type lucite chambers. Two different sets of chamber sizes were used: one set of chambers had a volume of 2 ml with an exposed area of skin of 1.98cm’. and the other set of chambers was 5 ml in volume with an exposed area of 3.14 cm2. Previous H+ excretion comparisons between the chamber types showed no statistical difference. The mucosal and serosal surfaces of the skins were bathed in frog Ringers solution consisting of (in mM): NaCI, 114.5; KCI, 3.0; CaCI,, 0.9; and sodium phosphate (dibasic), 1.5. The final pH was 7.25-7.35. The mounted skins were allowed to equilibrate in Ringer’s for I5 min, the chambers were then drained and fresh Ringer’s solution was added. Mucosal H+ flux of the isolated skins was measured for 120 min at room temperature with the serosal bathing fluid being bubbled with air. The determination of H+ excretion and the histological preparation and the identification of the tissues were as previously described (Page and Frazier, 1987). In all experiments, the H+ excretion was calculated from a change in the
of Dentistry,
53
RAY PAGE et al.
54
pH and the concentration of phosphate buffer in the mucosal and serosal solutions. A Fisher accumet Model 142pH meter was used for all pH determinations. Skins from the northern frogs were histologically prepared for cell counts. Coded slides of random sections were used, and cells were counted from both normal and acidotic frogs. Instruments Systemic pH in the northern frogs was determined from whole blood obtained by ventricular puncture and aspiration with a syringe immediately after removal of the skins. Blood pH was determined by the aforementioned pH meter. Blood analyses in subsequent experiments were performed by an automated instrument, Stat Profile 1. This instrument directly measures PO,, pCO,, pH, and hematocrit, as well as electrolytes, including K+, Na+ and Ca*+. From these measured variables several other variables were calculated including 0, content and bicarbonate. The effect of low sodium concentrations and amiloride on H + excretion Ventral skins from the southern variety frogs were used in the following experiment and mounted in the modified Ussing chambers with an exposed area of I .98 cm’. To determine the effect of sodium on H+ excretion, the concentration of sodium in the Ringer’s solution was adjusted so that five solutions had the following NaCl concentrations (in mM): 114, 80, 40, 20 and 2. An equimolar amount of choline chloride replaced the NaCl in the solutions. The final pH was adjusted to 7.15, and all solutions had an osmolality of 248-254 mOsm. The choline chloride adjusted Ringer’s solution was placed on the mucosal side and normal Ringer’s solution was placed on the serosal side. The tissues were allowed to flux for 120min. A second group of frogs received amiloride (IO-‘M) (Merck Institute of Therapeutic Research, West Point, PA) in the mucosal bath. This concentration has been shown by Yorio and Bentley (1976) to be adequate to block Nat transport across the skin. Both the mucosal and serosal baths contained normal frog Ringer’s (I I4 mM NaCI), with amiloride administered to the mucosal bath. Again the skins were allowed to flux and H+ excretion was determined. The effects of high salinity adaptation on H+ excretion Southern variety frogs were placed into three groups: (1) control, (2) normal high salinity adapting (HSA), and (3) acidotic high salinity adapting. The controls were placed in I mM NaCl water for 5 days, while the normal HSA frogs
were placed in 60 mM NaCl water for 5 days. The acidotic HSA frogs were also kept in 60 mM NaCl water for 5 days and in addition, on the last 2 days of adapting, metabolic acidosis was induced as previously described. The water for each of the groups was changed twice daily and at that time the animals were weighed to determine any changes in weight. On the fifth day, the animals were sacrificed and H + excretion by the skin was determined. Blood was collected from these frogs and analyzed for acid--base. ionic and metabolic characteristics. Statistics Statistical analyses were performed utilizing the statistical programs from Tallarido and Murray (1987) adapted for the IBM XT. Appropriate statistical analyses were applied to each group of data.
RESULTS Comparison of’ H+ excretion and morphological changes in the skin IJ~ northern and southern j?ogs
We have previously shown that during a challenge of chronic metabolic acidosis the southern variety of the leopard frog, Ram pipiens, compensates by increasing H+ excretion in the skin. Additionally, there is an increase in the number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis (Page and Frazier, 1987). Figure I demonstrates that following the challenge of a 48 hr metabolic acidosis the intact skin of the northern variety of Rana pipiens did not significantly increase its Hi excretion, but the skin of the southern variety frog produces a highly significant increase in H+ excretion in acidosis (Independent t-test, P > 0.2 and P < 0.005, respectively). The mean blood pH of the northern frogs, however. decreased significantly from 7. I3 k 0. I I in the normal frogs (n = I I) to 6.66 & 0.12 in the acidotic frogs (n = 10) (Independent t-test, P < 0.001). Table I indicates that the net H+ flux is directional from serosal to mucosal in both the normal and acidotic states, and that in normal skins the serosal bathing fluid alkalinizes, while during the acidotic condition the serosal bathing fluid acidifies.
2.00, p(O.001
T
p10.2
5 ‘J f : + I
0.75 0.50 0.25 0.00 northern Mucosol
southern bathing
fluid
Fig. I. Comparison of H+ excretion in the skin of northern and southern frogs under normal and acidotic conditions. Each group represents the mean k S.E. of ten skins. Acidosis was produced by NH,CI injections into the dorsal lymph sac three times daily for 48 hr.
Na+/H+ Table
I.
Mucosal
and
serosal
H + excretion
southern
in
relationship
northern
and
55
in frog skin
Table 2. Changes
in cell population
Hydrogen MUCOsal
I
Northern
Ratio
(12)
0.64 + 0.14”
(I 2)
0.73 * 0. I2
Southern
variety
A. Normal
(IO)
0.60 + 0.10
9. Acidotic
(IO)
1.60+0.12
‘As
means i
P value
-0.05
kO.19
0.24+0.10
state
and
Normal
< 0.002b < 0.002
Mitochondria-
B. Acidosis
rich
basal I.0
0.013
(98.8%)
(1.23%) 0.014b
1.0
(1.38%)
(98.6%) -0.15
kO.07
0.10 * 0.05
<0.002 co.002
aRepresents Number
a total
of 2200
in parenthesis
cells counted
is the percentage
from
Paragon
slides.
cell type of the total number
of cells counted.
S.E.
“15 the probability
Acid-base A.
9. Acidotic
of cell types”
Granular
variety
A. Normal II
Serosal
frog skin in response
acidosis
ion excretion
(nmole/cm2/min) Grow
in northern
to metabolic
frees
that
the values are different.
bP > 0.05.
The skins of both the normal and acidotic northern frogs were histologically prepared for identification of cell types and cell counts. Table 2 describes changes in the cell population of the skin in response to metabolic acidosis. The granular + basal : MR cell ratio and percentage of cell types in the northern skins show no significant increase in the number of MR cells in response to chronic metabolic acidosis (Independent t-test, P > 0.05). The effect of low sodium concentration and amiloride on H+ excretion In the following experiment, mucosal sodium concentration was varied and H+ flux measured under normal and acidotic conditions of southern frogs. There was a significant difference in the mean H+ excretion at various mucosal Na+ concentrations in the normal acid-base state (Fig. 2a) (P < 0.05, one way parametric ANOVA, F = 3.5, n = 48). However, zero order kinetics was observed for the H+ excretion of frogs in metabolic acidosis (Fig. 2b), as there were no significant differences between the mean H+ excretion at various mucosal Na+ concentrations (one way ANOVA, P > 0.10, F = 1.45, n = 48). Lmear regression analysis of these data and their respective serosal H+ fluxes showed no 1: 1 mucosal acidification to serosal alkalinization relationship. In the normal acid-base state, the mean H+ excretion by amiloride (IO-‘M) exposed skins was not significantly different from that of the control portion
Na+
concentration
Not
statistically
significant
by the
independent
I-test.
of the skins (Fig. 3) (one-tailed paired t-test, 0.10 > P > 0.05). However in metabolic acidosis, the administration of amiloride significantly decreased from that of the nonthe mean H+ excretion amiloride treated skin (Fig. 3) (one-tailed paired t-test, P < 0.001). Likewise, no significant difference in mean H+ excretion was found between the control skins and low mucosal sodium skins in normal frogs (Independent f-test, one-tailed 0.50 > P > 0.25), whereas, in the acidotic frogs, a highly significant decrease in mean H+ excretion was found in the low mucosal sodium skins as compared to controls (Fig. 3) (Independent t-test, one-tailed, P < 0.005). Effects of &h
salinity adaptation on H+ excretion
To characterize further the relationship between sodium transport and H+ flux, frogs were sodium adapted for 5 days in 60mM NaCl in the presence and absence of metabolic acidosis (present last 2 days). The mean H+ excretion of the 5 day 60mM NaCl adapted frogs in both the normal and acidotic state was significantly different from the control group bathed in 1 mM NaCl and in the normal acid-base state (Fig. 4) (Dunnetts t-test, P < 0.05 and P < 0.01, respectively). As seen in Fig. 5, the percentage change in body weight during high salinity adaptation increased in the frogs that were adapting to the 60 mM NaCI, while the control frogs bathed in the 1 mM NaCl showed a slight decrease in body weight. It was further found that the slopes
(meq/L)
Fig. 2. Effects of different sodium concentrations on H+ excretion in the southern frog skin under normal (left panel) and acidotic conditions. Under normal acid-base conditions, H+ excretion is dependent on mucosal sodium concentrations, whereas, in the presence of acidosis, H+ excretion is independent of mucosal sodium concentrations. Each point represents the mean f S.E. of eight skins.
RAY PAGEet al.
56
h
3.5
1.40
g
1.20
2
1.00
z E.
0.80
s ‘Z E :
0.60
+I
0.40 0.20 0.00 normal
acidotic Metabolic state
Fig. 3. The effects of amiloride and low mucosal sodium concentrations on H + excretion in normal and acidotic frogs. Each group represents the mean + S.E. of eight paired frog skins. Amiloride (1 x IO-’ M) was added to the mucosal bathing fluid.
0.60 Q ‘E 2
0.70
E 0 > 5 .SE
0.60
*E zi t c 2
0.30
ONomWt~ Iuotab&addo8b =Nnmld*
0.50 0.40
0.20 0.10 0.00
60mM
60mM
1mM
NaCl concentration Fig. 4. The effects of high salinity adaptation (60 mM) on H’ excretion in frog skins under normal and acidotic conditions. Each group was statistically compared to the 1mM normal group (mean + SE. of eight skins per group).
-54 0
25
50 Time (houn
75
100
1 !5
during adaptation)
Fig. 5. Percentage weight change of southern frogs under normal and high salinity adapting conditions. Each group of animals were weighed twice daily at time of water change. The means of each group are presented.
51
Na+/H+ relationship in frog skin Table 3. Blood acid-base, Blood Analyses PH PC@ (mm Hg) Na+ (mmol/L) K + (mmol/L) Ca*+ (mmol/L) BE-ECF” (mmol/L) HCO, (mmol/L)
Group Normal 7.4 * 21 * III * 3.3 f I .o i -1.9il.4 13 *
ionic and metabolic
I
0.03 1.0 1.3 0.3 0.02 0.9
characteristics
Group 2 Acid 7.2 f 0.04 15i I.1 Ill f 1.3 3.4 * 0.3 I.1 kO.03 -22 + 1.3 6.4 k 0.8
of acidotic
Group 3 HSA-normal 7.6 k 20 + I IO f 2.5 f 0.9 + -4.2 k l8k
0.07 2.6 3.4 0.6 0.05 2.2 I.3
As mans k SE. HSA-High Salimty Adapted; SNK-Newman-Keuls Multiple Range Test. “Base excessxxtracellular fluid. bF(95%) = 2.92, n = 39. All comoarisons between eroutx I and 2 that demonstrate ’ significantly different groups’are at P < 0.01. -
relating percentage body weight change were not significantly different between the 60 mM NaCl adapting frogs, but were different from the 1 mM NaCl control group (t-test for parallelism, t = 0.121, P > 0.5, and t = 5.171, P < 0.001, respectively). Blood analysis Table 3 lists the results of an indepth blood analysis done on frogs from the above salt adapted experiment. The student Newman-Keuls multiple range test demonstrates several comparisons. It was found that a significant difference in pH, Ca*+, BE-ECF (base excessextracellular fluid), and HC03, occurs between: (1) the normal acid-base groups (groups 1 and 3) vs both acidotic groups (groups 2 and 4) (P < 0.01) and, (2) the normal acid-base normal salinity group (group 1) vs the normal acid-base/ 60 mM salt adapted group (group 3) (P < 0.05). Furthermore, no significant difference is found among the two acidotic groups (groups 2 and 4) in any of the parameters. In addition, the Na+ and K+ concentrations were not significantly different among any of the groups (ANOVA, F = 1.36, and F = 2.08, respectively). The pC0, comparison showed significance only between group 1 and group 2 (P < 0.05). DISCUSSION
The cellular mechanisms involved in proton transport by renal tissues in maintaining homeostatic systemic pH has long been questioned and pursued. The microperfusion techniques of Rector et al. (1965) and Malnic et al. (1972) along with the morphological studies of Madson and Tisher (1985) have demonstrated the presence of H+ excretion in the mammalian proximal and distal tubules. These characteristics have also been demonstrated in several amphibian models including the toad urinary bladder (Frazier, 1985), the turtle bladder (Stetson and Steinmetz, 1985) and various species of frogs skin (Benos, 1981; Vanatta and Frazier, 198 1; Lindenger and McDonald, 1986; Ehrenfeld et al., 1987). However, differences among these species in their membrane permeability characteristics (Bentley and Yorio, 1976) and ion transport relationships with H+ excretion (Frazier, 1986; Duranti et al., 1986; Emilio and Menano, 1975) have left many of the cellular mechanisms of proton transport in question. In Rana pipiens, the skin has been shown to be structurally and biochemically heterogenous and the mitochondria-rich (MR) cell is thought to be the
and salt adapted Group 4 HSA-acid 7.0 f I6 f I I8 * 4.4 * I .3 + -27 k 4.3 *
a difference
southern
ANOVA F-valw?
0.04 1.2 0.5 0.5 0.03 0.8 0.1
are
frogs
at P < 0.05,
11.66 4.01 I .36 2.08 7.83 18.84 21.76
all other
SNK Grouping
I 3 24 123 234 I234 1234 I 3 24 I 3 24 I 3 24
combinations
of
mediator of Hi transport (Farquhar and Palade, 1965; Rosen and Friedley, 1973). It has been shown in both the toad and turtle urinary bladder (Frazier, 1978; Al-Awqati and Schwartz, 1986) and in the skin of the southern variety of Rana pipiens (Page and Frazier, 1987) that an increase in the number of MR cells as well as an increase in the rate of H+ excretion in response to a challenge of chronic metabolic acidosis occurs. However, in the present study it was found that the skin of the northern variety of Rana pipiens was unable to increase H+ excretion in response to metabolic acidosis, and likewise, no evidence of an increase in MR cell number was observed. Recently, Wheeler and Arruda (1987) have shown that an increase in proton excretion by turtle urinary bladders, under acidosis was associated with a corresponding increase in MR cells and intracellular acidic vesicles. These data offer additional support that the MR cell may be the mediator of proton excretion. In the current study, net H+ flux was found to be directional from serosa to mucosa in both normal and acidotic animals. In addition, it was found that in the normal skins the serosal bathing fluid alkalinizes, while during metabolic acidosis the serosal solution acidifies. This phenomenon has previously been shown in the intact skin (Page and Frazier, 1987) and supports the model of Ehrenfeld et al. (1987) which suggests a basolateral H+ secretory mechanism which is quiescent in normal conditions but stimulated in metabolic acidosis. This allows cellular pH to be maintained within physiological limits by “dumping” protons systemically to be eliminated by the kidney. It was further observed that under normal acidbase conditions, H+ fluxes were found to be dependent on mucosal sodium concentrations. Conversely, in metabolic acidosis, zero order kinetics was observed for the H+ excretion rate. These results suggest that under normal conditions there is a sodium dependency of H+ excretion which is saturable at 20-30 mM mucosal sodium, whereas at higher rates of H+ flux, under metabolic acidosis, this is not observed. This is similar to what has been reported for H+ fluxes in the skin of the frog, Rana esculenta (Ehrenfeld and Garcia-Romeu, 1977). This sodium dependency difference was particularly noticeable in the low mucosal sodium (2 mM) H+ flux data. Amiloride, a known inhibitor of passive mucosal Na+ reabsorption, was found to inhibit H+ excretion only in the acidotic frogs. These results agree with
RAY
58
PAGE et al.
previous observations of Frazier (1985) on the amiloride-sensitive H+ flux in toad bladders in the presence of chronic acidosis. It appears, therefore, that a decrease in sodium uptake across the apical membrane results in a significant decrease in H+ excretion during acidotic states. It has been proposed by Leaf et al. (1959), that as much as 50% of endogenous CO, is generated from ATP production used for driving the basolateral Nat-K+ pumps, and that CO1 is also a substrate for carbonic anhydrase in the production of H+ for excretion. Therefore, amiloride, by blocking apical Na+ entry, produced a 40% decrease in H+ excretion, probably through an inhibition in transcellular active sodium transport and a concomitant decrease in CO, generation. Furthermore, Hi excretion was markedly increased in acidosis, partly due to the decrease in cell pH from the increased uptake of systemic COZ, and partly from the presence of active sodium transport (Ehrenfeld et al., 1987). It was interesting to note in the present study, that under low sodium conditions (2mM), only a slight decrease in H+ flux was observed during metabolic acidosis, perhaps reflecting the presence of residual transcellular sodium transport and CO1 production sufficient in maintaining an elevated H+ flux. Additional information concerning the sodium dependence of H’ excretion was obtained by measuring H+ excretion in normal and acidotic animals which were salt adapted. It was found that in normal frogs, which were high salinity adapted (HSA) for 5 days, there was an increase in H+ excretion over those of the normal 1 mM salt adapted frogs. This suggests that the apical Na + gradient has an influence on H+ excretion and the pH gradient. The increased H+ flux may be the result of an increase in metabolic CO1 production resulting from energy utilization in driving the basolateral sodium transport system. An increase in Na+ uptake was evidenced by an increase in weight gain by the HSA animals in comparison
to the normal animals, perhaps as a result of an increased fluid absorption. Blood acid-base profiles in normal HSA animals were also consistent with an increase in H+ excretion, as blood bicarbonate concentrations were elevated. Although no change in blood sodium concentrations were observed, this may reflect an increase in blood volume from an enhanced water reabsorption or a consequence of efficient sodium removal by the kidney. The HSA animals in metabolic acidosis also showed an increase in weight, but had a significant decrease in blood pH and HCO,, reflecting the state of acidosis in the animal. The H+ excretion rates of these animals were similar to that of the HSA normal animals, although blood pH and HCO, concentrations were markedly different between normal HSA vs HSA animals in acidosis. The blood acidbase profiles of the HSA acidotic animals were not different from the non-salt adapted acidotic animals. This suggests that the acid-base changes are primarily a result of systemic acidosis and not due to the salt adaptation. In addition, the increase in H + excretion by normal HSA animals may therefore be a function of increased sodium uptake and transport, possibly through an increased CO2 production. These data suggest that the apical Na+ gradient may influence H+ excretion, perhaps as proposed by Frazier (1985) through energy dependent mechanisms of active sodium reabsorption. Alternatively, at sodium concentrations below 30mM, below sodium transport saturation, there appears to be a Nat/H+ antiporter that is driven by the sodium and pH gradients. Under acidosis, H’ excretion may in part function through this antiporter. but it may also proceed through Na’ independent processes linked to the metabolic state of the cell. Figures 6(A) and (B) are a modification of a model previously proposed by Frazier (1985) for the toad bladder and depicts these alternate mechanisms.
NORMAL
ACIOOSIS
Mucosal
Serosal
Mucosal
> Na+--___.__
Nat--__
--__
pHi
A.
Nat K+
--*H+ ----Na+
B.
Fig.6. Proposed model of
H + excretion characteristics and the role of sodium in (A) the normal state, and (B) in chronic metabolic acidosis (see text).
metabolic
Na+/H+
relationship
In Fig. 6(A), normal H+ excretion is depicted as an electrogenic apical pump which is dependent on the intracellular H+ concentration produced from the enzymatic conversion of CO2 to bicarbonate. The intracellular CO, concentration is, in turn, primarily dependent on the rate of passive apical Na+ influx which is actively removed basolaterally. Thus, energy utilization
for the active
Na+/K+
pump
plays
a major
role in cellular CO, production. Under normal conditions the intracellular pH is efficiently maintained and the Na+/H+ exchanger is quiescent and serosal alkalinization is observed. Under these conditions H+ excretion occurs primarily across the apical membrane. When the animal is challenged with chronic metabolic acidosis, H+ excretion is no longer primarily dependent on apical Nat influx (Fig. 6B), and the main stimulation of H+ excretion is via the increase in cellular CO, from the induced systemic acidosis. The increased CO, masks the Na + dependency of H + excretion, but, in response to a decreased pH, the Na+/H+ exchanger becomes activated. This results in both a serosal acidification and in an increase in intracellular sodium which can stimulate CO* production via the basolateral Na+/K+ pump. Such a model is consistent with the present observations. It is also evident that additional studies are needed to further describe these mechanisms and to elucidate some of the mediators that may regulate these processes. A~,know/edgemenrs-This research was supported in part from a grant from the American Heart Association, Texas Affiliate (TY) and a BRSG grant from TCOM (TY). Ray Page was supported by an AOA/Burroughs Wellcome Osteooathic Research Fellowship . (F87-07). The authors would like to thank Drs L. X. Oakford and A. J. Mia for the assistance with quantification of the MR cells by light microscopy.
REFERENCES Aickin C. C. and Thomas R. C. (1977) An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers, J. Physiol. Lond. 273, 295-3 16. Al-Awqati Q. and Schwartz J. H. (1986) Plasticity of spithelial polarity. Renal Physiol. 9, 1 IO. Benos D. J. (1981) Acidification and sodium entry in frog skin epithelium. B&hem. Biophys. Acta 647, 40-48. Bentley P. J. and Yorio T. (1976) The passive permeability of the skin of anuran Amphibia, a comparison of frogs (Rana pipiens) and toads (Et& marinus). J. Physiol., Lond. 261, 603-615. Brever M. D. and Jacobson H. R. (1987) Mechanisms and Am. J. regulation of renal H+ and HCO, transport. Yephrol. 7, 150-161. Deitmer J. W. and Ellis D. (1980) Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkenje fibers. J. Physiol., Land. 304, 471&488. Duranti E., Ehrenfeld J. and Harvey B. J. (1986) Acid secretion through the Rana esculenra skin: Involvement of an anion-exchange mechanism at the basolateral membrane. J. Physiol, Lond. 378, 195-21 I. Ehrenfeld J. and Garcia-Romeu F. (1977) Active hydrogen excretion and sodium absorption through isolated frog skin. Am. J. Physiol. 233, F46F54.
in frog skin
59
Ehrenfeld J., Garcia-Romeu F. and Harvey B. J. (1985) Electrogenic active proton pump in Rana esculenta skin and its role in sodium ion transport. J. Physiol., Land. 359, 331-355. Ehrenfeld J., Cragoe E. J. and Harvey B. J. (1987) Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues. P&get-s Arch 409, 200-207. Emilio M. G. and Menano H. P. (1975) The excretion of hydrogen ion by the isolated amphibian skin. Effects of antidiuretic hormone and amiloride. Biochem. Biophys. Acta 382, 344352. Farquhar M. G. and Palade G. E. (1965) Cell junctions in amphibian skin. J. cell Biol. 26, 263-291,
Frazier L. W. (1974) Interrelationship of H+ excretion and Na+ reabsorption in the toad urinary bladder. J. Membrane Biol. 19, 267-276. Frazier L. W. (1978) Cellular changes in the toad urinary bladder in response to metabolic acidosis. J. Membrane Biol. 40, 165-172. Frazier L. W. (1985) Characteristics of proton excretion in normal and acidotic toad urinary bladder. Biochem. Biophys. Acta 817, 75-84. Frazier L. W. (1986) Acidification of the bathing fluid by the skin of Rana pipiens in metabolic acidosis. Camp. Biochem. Physiol. 85A, 57-61. Frazier L. W. and Vanatta J. C. (1973) Characteristics of H+ and NH: excretion by the urinary bladder of the toad. Biochem. Biophys. Acta 311, 98-108. Grinstein S. and Rothstein A. (1986) Mechanisms of regulation of the Na+/H+ exchanger. J. Membrane Biol. 90, 112. Leaf A., Page L. B. and Anderson J. (1959) Respiration and active sodium transport of isolated toad bladder. J. biol. Chem. 234, 1625-1629. Lindinger M. 1. and McDonald D. G. (1986) Cutaneous and renal responses to intravascular infusions of HCI and NH,Cl in the bullfrog (Rana catesbiuna). Camp. Biochem. Physiol. 84A(l), 113-122. Machen T. and Erlij D. (1975) Some features of hydrogen (ion) secretion by the frog skin. Biochem. Biophys. Acra 406, 120&130. Madsen K. M. and Tisher C. C. (1985) Structure-function relationships in H+ secreting epithelia. Fed. Proc. 44, 2704-2709. Malnic G.. deMello Aires M. and Giebisch G. (1972) Micropuncture study of renal tubular hydrogen ion transport in the rat. Am. J. Physiol. 222, 147-158. Page R. D. and Frazier L. W. (1987) Morphological changes in the skin of Rana pipiens in response to metabolic acidosis. Proc. Sot. exp. biol. Med. 184, 416422. Rector F. C., Carter W. W. and Seldin D. W. (1965) The mechanism of bicarbonate reabsorption in the proximal and distal tubules of the kidney. J. clin. Incest. 44, 278-290. Rosen S. and Friedley N. J. (1973) Carbonic anhydrase activity in Rana pipiens skin: biochemical and histochemical analysis. Histochemie 36, l-4. Stetson D. L. and Steinmetz P. R. (1985) A and B types of carbonic anhydraserich cells in turtle bladder. Am. J. Physiol. 249, F553-F565. Tallarido R. J. and Murray R. B. (1987) Manual ofPharmacological Culculufions. Second edition. Springer-Verlag, New York. Vanatta J. C. and Frazier L. W. (1981) The epithelium of Rana pipiens excretes H + and NH: in acidosis and HCO, in alkalosis. Camp. Biochem. Physiol. 68A, 51 I-513. Wheeler R. P. and Arruda J. A. (1987) Adaptation to metabolic acidosis by turtle urinary bladder. Am. J. Physiol. 252, F256266. Yorio T. and Bentley P. J. (1976) Effects of ethanol on the permeability of frog skin. J. Pharmac. exp. Ther. 197, 340-351.