AMERICAN JOURNAL OF PHYSIOLOGY Vol. 228, No. 5, May 1975. Printed
Effect
of metabolic
renal
action NAMA
MAMA,
HEI
BECK,
PAIK
on
of parathyroid HE1 PAIK
Department of Medicine, Pittsburgh, Pennsylvania
BECK,
acidosis
KIM,
AND
KIM,
hormone
AND
KWANG
Veterans Administration 15240
KWANG
SUP
KIM.
Effect of
metabolic acidosis on renal action of pamthyroid hormone. Am. J . Physid. 1975.~Effects of metabolic acidosis on renal 228(5): 1483-1488. action of parathyroid hormone (PTH) was investigated in rats. Metabolic acidosis was induced by infusing NH&l, and the control animals received the equivalent volume of NaCl. The extracellufar fluid volume was not measurably different between the two groups of animals. The changes of TRP and urinary excretion of cyclic AMP by PTH was significantly inhibited in the acidotic animals, but the phosphaturia induced by an infusion of dibutyryl cyclic AMP was not affected by metabolic acidosis. The increase of cyclic AMP concentration by PTH was significantly less in the renal cortical slices obtained from the acidotic animals than in those obtained from the control animals. The activation of adenylate cyclase by PTH was also significantly inhibited in the acidic media. These results suggest that in metabolic acidosis the renal response to PTH is decreased, and that the decrease is due to the inhibition of the PTH-dependent cyclic AMP system in the kidney at the level of adenylate cyclase in renal cortex. cyclic
AMP;
BONE
IS A RESERVQIR
buffer,
and
in
University
of Pittsburgh
parathyroidectomized mediated through metabolic acidosis METHODS
AND
School
of Medicine,
rats. Since the renal effect of PTH cyclic AMP (2, 4, 1 l), the effects on that system was also investigated.
is of
MATERIALS
In viuo experiments. Sprague-Dawlev rats, weighing 270290 g, were surgically parathyroidectomized to evaluate the renal response to PTH in the absence of endogenous PTH secretion. Four to six days were allowed for the animals to stabilize after the surgery. During this period, the animals were maintained on calcium chloride, 1 g/l 00 ml of drinking water. On the day of experiments the animals were divided into three groups : alkalotic, control, and acidotic. Into each group of animals, 15 ml of solution were injected peritoneally. The solution contained either NaHC03 1.5 mmol, NaCl 1.5 mmol, or NH&l 1.5 mmol, inulin 15 mg, and sodium pentobarbital 7.5 mg. Polyethylene catheters were inserted into the urinary bladders through suprapubic incisions for collection of urine, and into the femoral veins for intravenous infusion. The intravenous infusion was initiated 30 min after the peritoneal injection. The solution contained either 140 mM NaHCOs, 140 mM NaCl, or 140 mM NH&l, with inulin, 0.3 mg/ml; the infusion rate was 70 &min until the end of the experiment. In the first series of experiments, the respective intravenous solutions were infused for 2.5 h, and arterial blood pH and bicarbonate concentrations and the phosphate excretion rates were measured during that period in the absence of PTH injection. The pH and bicarbonate concentrations were stable from 1 to 2.5 h after the initiation of intravenous infusion with the changes of pH less than 0.02 in each group. Therefore, subsequent experiments were performed during that l2.5 h period. In the second seriesof experiments, three 15-min urine specimens were collected and served as the basal values. Then, 10 U of synthetic, amino-terminal, l-34 fragment of bovine PTH, hereafter referred to as PTH, were injected intravenously to each animal, followed by collections of 15min urine specimens. Ten units of PTH per rat was a submaximal dose to induce phosphaturia (7). Blood samples were obtained from the femoral artery immediately prior to PTH injection and 60 min after the injection. The maximal phosphaturic response to PTH was usually seen 30-60 min after PTH injection. Therefore, the phosphaturic response to PTH was measured during that 30 to 60.
phosphaturia
of phosphate
SUP KIM
Hus@al,
metabolic
acidosis there is an increased release of phosphate buffer from the bone (3, 10, 16, 19, 23, 29, 30). It has been postulated that the increased mobilization of phosphate buffer may have, at least in part, a protective role against metabolic acidosis (16, 19). Barzel (3), Wachman and Bernstein (29), and other investigators (3, 10) postulated that parathyroid hormone (PTH) has a role in acid-base balance through the modulation of the release of phosphate buffer from bone (3, 10, 29). Furthermore, Bernstein, Wachman, and Hattner (10) and Wachman and Bernstein (29) postulated that the increased release of phosphate buffer is due to an increased skeletal end-organ response to PTH in metabolic acidosis. In the kidney, PTH also affects acid-base balance (17, 18). According to the data obtained from micropuncture experiments (8, 9, 14, 26), the major fractions of filtered phosphate and bicarbonate are reabsorbed in proximal tubules. PTH inhibits the proximal tubular reabsorption and consequently increases excretions of those ions (I 7). Since it has been suggested that metabolic acidosis alters the end-organ response to PTH in bone (l&29), it is reasonable to speculate that metabolic acidosis also alters the renal end-organ response to PTH. Therefore, the effect of metabolic acidosis on renal response to PTH was investigated in 1483
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1484 min period. The maximal increase of urinary excretion of cyclic AMP was usually seen 15-45 min after PTH injection. Therefore, the increase of urinary excretion of cyclic AMP by PTH was measured during that 15- to 45min period. The extracellular fluid volumes in the control group and in the acidotic animals were compared on the basis of chloride space with the use of Wl, and urinary excretion rates of water and chloride. In the third series of experiments, dibutyryl cyclic AMP was infused into both the control animals and the acidotic animals at the rate of 50 pg/min. The phosphaturic responses to dibutyryl cyclic AMP were then compared between the two groups of animals. In vitro experiments. In another series of experiments the animals were prepared in an identical manner to the above in viva experiments, infusing NaCl into the control group and NH&l into the acidotic animals. One hour and 45 minutes after the initiation of the intravenous infusion, which is the time corresponding to that of PTH injection in vivo, the kidneys were removed rapidly and the cortex was sliced to a thickness of less than 0.5 mm. Each slice, weighing between 30 and 50 mg, was incubated in KrebsRinger-Tris buffer, pH 7.2. The PTH group contained PTH, 10 U/ml. After 10 min incubation at 37”C, the slice was homogenized in 0.5 ml of glass distilled water using a glass tissue grinder, and then the tube was placed in a boiling water bath for 3 min to terminate the enzyme activity in the homogenate. After centrifugation for 15 min at 7,000 X g, cyclic AMP in the supernatant was assayed using Gilman’s method (15) with modifications (6). To evaluate the efiect of pH of the incubation medium, the cortical slices obtained from the control-group animals were incubated in Krebs-Ringer-Tris buffer with two diKerent pH’s, 7.2 and 7.4. Adenylate cyclase of renal cortex was prepared as described by Marcus and Aurbach (20). The cortex of kidney was homogenized in 50 mM Tris, pH 7.4, and centrifuged for 15 min at 2,000 X g. The precipitate was resuspended in 50 mM Tris, pH 7.4, and centrifuged again for 15 min at 2,000 X g. The precipitate after the second centrifugation was resuspended in 5 mM Tris, pH 7.4. The homogenate of renal cortex, hereafter referred to as the enzyme adenylate cyclase, was incubated in the medium containing, in millimoles per liter: MgClz, 1.6; KCl, 25; theophvlline, 10; ATP, 1; Tris, 50; and PTH, 10 U/ml (for the PT’H group) ; total volume, 0.60 ml per tube. The pH’s of the mixtures were adjusted to 7.4 for the control and 7.2 for the acidotic group. After 15 min incubation at 37°C (during this period the enzyme activity was linear with time), the reaction was terminated by placing the tubes in a boiling water bath for 3 min. The aggregate of the boiled enzyme was precipitated by centrifugini for 15 min at 700 X g. Cyclic AMP in the supernatant ias measured using Gilman’s method (15) with modifications (6). The difference in pH of the media, ATP remaining after 15 min incubation, PTH, or other study substances, had no measurable effect on the cyclic AMP assay system. Phosphodiesterase was prepared as described by Cheung (13) by homogenizing renal cortical tissue in glass-distilled water and then centrifugin g the homogenate at 30,000 X g for 30 min. Phosphodiesterase activity was assayed as de-
BECK,
KIM,
KIM
AND
scribed by Thompson and Appleman (28). The precipitate fraction of 30,000 X g centrifugation was not studied because there was a negligible amount of phosphodiesterase activity in the precipitate fraction (7). The proper amount of enzyme preparation from the supernatant fraction, that which hydrolyzes 15-20 % of substrate, was incubated in the medium containing 10L4 M cyclic AMP, a tracer amount of 3H-labeled cyclic I , AMP 1 .6 mM MgC12, and 25 mM KCl. The pH of the mixture; was adjusted to 7.4 for the control group and 7.2 for the acidotic group. After 15 min incubation at 37”C, the reaction was terminated by placing the tubes in a boiling water bath for 3 min. The mixtures were then reincubated for 10 min at 37°C with @ili@ugus hannah venom, 0.1 mg/tube. Then, 2 ml of Dowex AGLX2 anion-exchange resin, ZOO-400 mesh, 1: 3 suspension, were added to each tube, and the tubes were centrifuged at 700 X g for 15 min. The radioactivity in the supernatant was counted by a beta liquid scintillation spectrometer. Synthetic, amino-terminal, l-34 bovine PTH, lot no. 150 12, was obtained from Beckman Instruments, Inc. (Palo Alto) and its activity was rechecked using renal cortical adenylate cyclase as described by Marcus and Aurbach (20). 3H-labeled cyclic AMP, 25 Ci/mmol, was obtained from New England Nuclear Corp. (Boston), AGl -X2 anion exchange resin, 200-400 mesh, from Bio-Rad Laboratories, (Richmond, Calif.), and cyclic AMP, dibutyryl cyclic AMP, and ATP from Sigma Chemical Co. (St. Louis). RESULTS
In vim cxfieriments. The chang-es of arterial blood pH and bicarbonate concentration aft& the infusion of NaHCOs, _nSaCl, or NH&l were significantly different among the three groups of animals, P < 0.01 among the three groups
1. Eflicts of metaboh’c alkalosis and acidosis on rats --- “,“.--I ,__.__.I.._-.-~ -.-~---
TABLE
---
iAcidotic ”-
Alkalotic
-
Rats
P value
I
No. of experiments Intravenous infusion, pmol/min Arterial pH
I ’
Arterial HCQ-, meq/liter Plasma Ca++, mM
/ ’ I
Plasma phosphate, mg/lOO ml GFR, ml/min Filtered load of phosphate, &min i TRP before PTH, yO 1 TRP
after
Changes
PTH,
(/;1
of TRP
by
PTH, %
1
9 NaHC03 9.8 7.65 ztO.04 29.4 +I .OO 0.72 *o -06 6.1 +l.l 2.09 zto.19 128 442 99.11 *0.34 76 -4-2 k4.38 a-22.69 zt4.33
12 NaCl 9.8 7.46 +o .Ol 15.9 +I .4 0.82 hO.07 9.4 ~1.8 1.67 *urn 17 156 zkl6 98.99 ztO.86 76.83 zt4.06 !A- 22.16 +4.45
I
13 NH&l 9.8 7.23 +0.01 6.8 zt2.50 1.11 zto.09 8.7 h1.2 1.66 +o.zo 144 zkl7 99.44 ztO.28 87.97 +0.28 A -11.47 zk2.96
/
;0.05 i>O.O5 I * /
*
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METABOLIC
ACIDOSIS
ON
RENAL
ACTION
OF
1485
PTH
(Table 1). Those values were stable during the period of experiments (l-Z.5 h) and the change of pH was less than 0.02 in each group. The plasma concentration of ionized calcium was significantly higher in the acidotic animal than in the control animals, P < 0.05. However, plasma phosphate concentration, mean glomerular filtration rate (GFR), and filtered load of phosphate were not measurably diflerent among the three groups of animals, P > 0.05 (Table 1). The extracellular fluid volume measured by “%l space and urinary excretion rates of water and chloride were not measurably different between the control and the acidotic groups of animals, P > 0.05 (TabIe Z), and those values were not measurably altered during the experiments, P > 0.05. TubuZar reabsorption rate of phospkute (TRP). In the first series of experiments without PTH injection, there was no measurable change of TRP during the experimental periods of l-Z.5 h after the initiation of the intravenous infusion, and the values remained not measurably dEerent among the three groups of animaIs at the end of the 2.5-h period : 99.21 & 0.38 % in the alkalotic animals, 99.25 & 0.40 in the control animals, and 99.48 s+. 0.35 in the acidotic animals 2.5 h after the initiation of the intravenous infusion, P > 0.05 among the three groups of animals. In the second series of experiments, TRP was not measurably different among the three groups of animals during the basal periods prior to PTH injection (Table 1). But: the decrease of TRP by PTH was significantly less in the acidotic animals than in the control or the alkalotic animals (Table 1). However, the decrease of TRP by PTH was not measurably different between the control and the alkalotic animals, P > 0.05 (Table 1 and Fig. 1). Urinary cyclic AMP excretion rates. During the basal periods prior to PTH injection, the urinary cyclic AMP excretion rate was not measurably diRerent between the acidotic animals, 52.5 + 6.5 pmol/min, and the control animals, 49.6 & 6.2; P > 0.05 (Fig. 2). However, the increase of urinary cyclic AMP excretion rate after the PTH injection was significantly less in the acidotic animals, to 93.8 & 9.4 (A41.3 rk 9.3), th an in the control animals, to 135.6 =t 18.8 (~86.0 h 15.1); P < 0.05. Dibutyryl cyclic AMP infusion. The mean decrease of TRP after the infusion of dibutyryl cyclic AMP was not measTABLE 2. liffecfs of an intrclvenous infusion of NHdCl or NaCI on extracellular Juid volume and urinary excretion of chloride ---- .- _ --~._----_ ^_- -. .---Control
No. of animals studied Intravenous infusion, 100 pl/ min for 3 h Plasma osmolality, mosmol/kg ““Cl space, ml/kg body wt Urine flow rate, pi/kg-min Cl- excretion, peq/kg-min Osmolal clearance, PI/kg-min
_- .-
Rats
Acidotic
5 150 mM 295 336.9 86.2 20.4 317.3
Rats ---... ---
5 NaCl s+ + A zt +
1 4.3 17.2 3.3 16.6
150 mM 295 331.0 106.5 18.5 293.4
NH&l It Ik + * zk
2 8.2 18.4 2.2 24.3
Values are means + SE. After the respective intravenous infusion for 3 h, the average of the subsequent 90-min urine specimen in each rat was calculated. Chloride space was determined 3 h after the intravenous infusion and 90 min after the injection of 36C1 for equilibration.
A cidotic
Alkalotic
7s
basal
PTH
FIG. 1. Changes of rate of tubular phosphate reabsorption (TRP) in control, alkalotic, and acidotic animals. Basal values are averages of three 15-min values, and PTH values are averages of 30- to GO-min values after PTH injection. Each point represents mean + SE of 12-13 experiments.
165
-
1 4
Control
140;
z gm ‘g 115 =.u \z; .-E .: goSE
// // : 1’ /’ //
/’ J,/ ’ /’ ,, ‘I /’ ’ $*. basal
n ,’ -?’
-
Acidotic
PTH
FIG. 2. Changes of urinary cyclic AMP excretion rates in control and acidotic animals. Basal values are average of three 15-min values, and PTH values are averages of 30- to 60-min values after PTH injection. Each point represents mean + SE of basal and PTH values of 1 Z- 13 experiments.
urably different: between the acidotic animals, from 99.70 & 0.05 % in the basal periods to 60.95 & 11.49 (% in 3075 min after the initiation of the infusion, and the control animals, from 99.73 j= 0.04 to 61.52 j= 3.80; P > 0.05. CjcliG AMP concentration in corfz’cal slices. The basal cyclic AMP concentration without PTH was not measurably d’fF 1 erent between the slices obtained from the acidotic animals, 3-42 & 0.30 pmol/mg wet tissue, and those from the control animals, 4.08 & 0.24; P > 0.05. However, the increase of cyclic AMP concentration by PTH, 10 U/ml, was significantly less in the slices obtained from the acidotic animals, 4.77 & 0.32 (Al.35 & 0.20), than in the control group, 6.50 & 0.42 (AZ.42 + 0.31);P < 0.01 (Fig. 3). On the other hand, when the slices obtained from the control-group animals, having the same intracellular pH were incubated in the medium with two dif&rent pH’s, 7.2 vs. 7.4, the increase of cyclic AMP concentration by PTH, 10 U/ml, was not measurably different between the two groups: from 4.08 =t 0.24 to 6.50 =t 0.42 in the group with pH 7.2 vs. from 4.27 & 0.22 to 6.29 & 0.23 in the group with DH 7.4. resDectiveIv; P > 0.05. Adkylate Lyclaie activikk. In the absence of PTH, adenylate cyclase activities were not measurably different between the acidic group (pH 7.2), 2.3 & 0.5 pmol/mg protein-min, and the control group (pH 7.4), 2.3 + 0.2; P >
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BECK,
J
rats //3 Control Acidotic
rats
FIG. 3. Changes of cyclic AMP concentration in renal cortical slices. Acidotic, slices obtained from acidotic animals. Control, slices obtained from control animals, Basal, without PTH. Values are means j= SE of 10 experiments each.
KIM,
AND
KIM
acidosis masks the effect of PTH on urinary bicarbonate excretion (17) and makes the results difficult to interpret. Therefore, the renal response to PTH was measured by tubular reabsorption rate of phosphate. During the baial periods prior to PTH injection, TRP was not measurably different among alkalotic, control, and acidotic animals. These results suggest that metabolic acidosis or alkalosis alone does not affect TRP. However, the decrease of TRP by PTH was significantly less in the acidotic animals than in the other two groups of animals. On the other hand, there was no measurable difference in TRP between the control group and the alkalotic animals. These results suggest that the renal response to PTH is inhibited in metabolic acidosis, but not in alkalosis. Therefore, the effect of metabolic alkalosis on the renal response to PTH was not further investigated. The data in the present experiments do not explain why the renal action of PTH is affected by acidosis, but not by alkalosis. However, it is interesting to note that the discrepancy in the present experiments, viz., phenomenon opposite from that observed in acidosis does not occur in alkalosis, is similar in that the intracellular pH is protected against the extracellular acidosis, but not against the extracellular alkalosis (1) Plasma phosphate concentration, mean GFR, and filtered load of phosphate were not measurably different among the three groups of animals, indicating the lesser decrease of TRP by PTH in the acidotic animals is not due to an alteration of filtered load of phosphate. These findings are consistent with the data of other investigators (22). Expansion of extracellular fluid volume induces phosphaturia unrelated to renal effect of PTH (27). Since the electrolytes in the intravenous solutions are different, i.e., NaCl for the control animals and NH&l for the acidotic animals, the status of extracellular fluid volume might be different between the two groups of animals, and consequently may induce a difference in TRP. However, the extracellular fluid volume, measured by 36Cl space, and that compared by urinary excretion rates of water and chloride was not measurably different between the control group of animals with an infusion of NaCl and the acidotic group with an infusion of NH&l. Furthermore, in the first series of experiments with the same intravenous infusions as the second series of experiments, but without PTH injection, TRP was not measurably different among the three groups of animals, and TRP did not change during the 1 -to 2.5-h experiments after the initiation of the intravenous infusion. These results sqgest that, in the system tested, there was no c measurable difference in extracellular fluid volume between the two groups of animals. Therefore, it is very unlikely that the lesser decrease of TRP by PTH in the acidotic animals is due to the lesser extracellular fluid volume rather than due to the acidosis per se, even though an unmeasurable small difference in extracellular fluid volume between the two groups of animals cannot be excluded. Since the phosphaturic effect of PTH is mediated through the cyclic AMP system, that system is also evaluated in vivo and in vitro. The increase of urinary excretion of cyclic AMP after PTH injection was less in the acidotic animals than in the control animals. These results are consistent with the findings of TRP. These data suggest that the altered l
PTH 10 U/ml
FIG. 4, Changes of renal cortical adenylate cyclase activity by PTH. Each point represents mean =t SE of 10 experiments in control group (pH 7.4) and acidotic group (pH 7.2). Basal indicates group without PTH.
0.05. However, the activation of adenylate cyclase by PTH was significantly less in the acidic group, to 5.3 & 0.5 (A3.0 & 0.5), than in the control group, to 14.3 =k 0.4 (A12.0 =t 0.3); P < 0.01 (Fig. 4). Phosj9hodiesterase activities. The basal activity of high-K, ( 10m4 M) cyclic AMP phosphodiesterase in the acidic group (pH 7.2), 825 Z& 20 pmol hydrolysis per milligram protein per minute, was not measurably different from the control group, 746 =t 39; P > 0.05. PTH also had no measurable effect on the phosphodiesterase activity: 819 I!= 17 in the acidic group (pH 7.2) with PTH, 10 U/ml, and 751 & 13 in the control group (pH 7.4) with PTH; P > 0.05 as compared to the corresponding basal values.
The data in the present experiments demonstrate that metabolic acidosis inhibits the phosphaturic response to PTH. However, in the animals with intact parathyroid glands, the changes of PTH activity in metabolic acidosis could be due to an alteration of either hormonal secretion or the response of the end-organ to the hormone, or a combination of both. Therefore, the effect of metabolic acidosis was evaluated exclusively on renal end-organ response to PTH in parathyroidectomized animals in which the secretory mechanism of PTH had been eliminated. In our present experiments, the change of bicarbonate excretion associated with the induction of alkalosis or
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METABOLIC
AC1DOSIS
ON
RENAL
ACTION
OF
PTH
end-organ response to PTH in metabolic acidosis is at or prior to the PTH-dependent cyclic AMP system in the kidney* The urinary excretion of cyclic AMP can be affected by the change of blood cyclic AMP concentration and consequently the change of g1omerular filtered load of cyclic AMP. Furthermore, urinary cyclic AMP does not exclusively represent PTH-dependent cyclic AMP. There are several other hormones inducing cyclic AMP generation in the kidney, e.g., vasopressin (5), catecholamines (6), prostaglandins (4, 5), and calcitonin (24). Also certain unknown or unmeasured factors associated with the induction of metabolic acidosis may indirectly affect urinary cyclic AMP excretion either through the alteration of efiects of those hormones or through other unknown mechanism. However, if those factors or hormones other than metabolic acidosis or PTH per se affected the urinary excretion of cyclic AMP, that effect should be demonstrable during the basal period in the absence of PTH. But, those basal values in the absence of PTH were not measurably different between the acidotic and the control animals. Furthermore, the increase of urinary excretion of cyclic AMP after PTH injection specifically represents the ‘change of the PTHdependent urinary cyclic AMP, and that increase was significantly less in the acidotic animals than in the control animals. Therefore, the results of urinary cyclic AMP suggest that the PTH-dependent cyclic AMP system in the kidney is inhibited in metabolic acidosis. If the findings of urinary excretion of cyclic AMP were considered alone, the changes of urinary cyclic AMP does not necessarily exclusively reflect the change of the PTHdependent cyclic AMP system in the kidney, and the findings of urinary cyclic AMP alone should be interpreted with caution. Therefore, in the in vitro system, the direct effect of metabolic acidosis on the PTH-dependent cyclic AMP system was further investigated in renal cortical slices in vitro. The cortex but not the medulla of the kidney was used because the PTH-dependent cyclic AMP system is mainly located in the cortex (12). The cortical slices were obtained from the control and the acidotic animals, which had been prepared in an identical manner to those in the above in vivo experiments. In those slices, the basal values in the absence of PTH was not measurablv different between the two groups of slices. But the increase of cyclic AlMP concentration bv PTH, 10 U/ml, in the incubation media, pH 7.2 for both groups, was significantly less in the acidotic group than in the control group. These results are consistent with the findings of urinary excretion of cyclic AMP, and further suggest that metabolic acidosis inhibits the PTH-dependent cyilic AMP system in the kidney. On the other hand, when the slices of the control animals with the same intracellular pH were incubated in the media with two different pH’s, 7.2 and 7.4, both the basal values and the increases bv PTH, 10 U/ml, were not measurably different betwee; the two groups. These latter findings suggest that the change of intracellular milieu associated with metabolic acidosis, but not the simple lowering of extracellular pH, inhibits the PTH-dependent cyclic AMP in the kidney. The lesser cyclic AMP concentration in cortical slices could be due either to the inhibition of cyclic AMP forma-
1487 tion, viz., the inhibition of adenylate cyclase, or to the augmentation of cyclic AMP catabolism, viz., the activation of phosphodiesterase. Therefore, the effect of pH on the PTHdependent cyclic AMP system was further evaluated on the enzyme activities of adenylate cyc1ase and PhosP hodiesterae. The basal activity of adenylate cyclase was not measurably different between the two groups, pH 7.2 vs. 7.4. However, the activation of enzyme by PTH, which specifically represents the change of PTH-dependent adenylate cyclase, was significantly less in the acidic group (pH 7.2) than in the control group (pH 7.4). These results indicate that in metabolic acidosis, the lesser increase of cyclic AMP concentration by PTH in renal cortical slices is due at least in part to the decreased synthesis of cyclic AMP. However, the data in the present experiments do not differentiate whether the inhibitory mechanism of acidosis on PTHdependent adenylate cvclase in renal cortex is at the hormonal receptor or the citalytic unit in the adenylate cyclase complex. Neither PTH nor the change of pH in the media had any measurable effect on cyclic AMP-phosphodiesterase activity of renal cortex in the high-K, system. These results suggest that the lesser increase of cyclic AMP concentration by PTH in metabolic acidosis is not due to the increased catabolism of cvclic AMP. The changes of TRP, the end results of PTH effects in the kidney, can also be affected by the reaction or reactions subsequent to cyclic AMP generation. Therefore, the eflect of metabolic acidosis was evaluated on phosphaturia induced by an infusion of dibutyryl cyclic AIMP as described by Nagata and Rasmussen (25). However, the phosphaturic response to dibutyryl cyclic AMP infusion was not afl’ected by metabolic acidosis. These results suggest that the inhibition of end-organ response to PTH in metabolic acidosis is due to the inhibition of the PTH-dependent cyclic AMP generation, but not due to the alteration of the reactions subsequent to cyclic AMP generation. Plasma concentration of ionized calcium was increased in acidosis and diminished in alkalosis (Table 1). Those findings are comparable to those described by Moore (2 1). Since Beck et al. (7) have reported an inhibition of renal effect of PTH in hypercalcemia, it is reasonable to postulate that the increase of ionized calcium concentration in acidosis may at least in part play a role to mediate the inhibitory effect of acidosis on the renal action of PTH. On the other hand, the in vitro data of adenylate cyclase in the present experiments suggest that the inhibitory efl?ect of acidosis on the PTH-dependent cyclic AMP system in the kidney is the alteration of pH per se rather than med ia ted through the al tera tion of ionized calcium concentrati on, because there was no measurable calcium in the incubation media in both the control group and the acidic group. Those findings suggest that the increase of the concentration of ionized calcium is not the sole mechanism, but that there is probably more than one mechanism that mediates the inhibitory effect of acidosis on the renal action of PTH. Regardless of those inhibitory mechanisms postulated, the data in the present experiments clearly demonstrate that metabolic acidosis inhibits the PTH-dependent cyclic AMP system in the kidney.
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1488 When all the above results are considered together, as a whole, they demonstrate that metabolic acidosis inhibits the renal response to PTH, measured by TRP, through the inhibition of the PTH-dependent cyclic AMP system in the kidney at or prior to the level of adenylate cyclase. The inhibition of renal end-organ response to PTH in metabolic
BECK,
KIM,
acidosi s may serve at least in p.art to protect milieu against meta .bolic acidosis. The authors assistance. Received
for
thank publication
Elinor
M. 20 May
Moody
and
Diane
AND
KIM
the internal
S. Heller
for
their
1974.
REFERENCES 1. iiDLER, S., A. ROY, AND A. S. RELMAN. Intracellular acid-base regulation. XL The interaction between CO2 tension and extracellular bicarbonate in the determination of muscle cell PH. J. Clin. Invest. 44 : 21-30, 1965. 2. AGUS, A. S., J. B. PUSCHETT, D. SENESKY, AND M. GOLDBERG. Mode of action of parathyroid hormone and cyclic adenosine 3’, 5’monophosphate on rerlal tubular phosphate reabsorption in the dog. J. C&n. Invest, 50: 617-626, 1971. 3. BARZEL, U. S. Parathyroid hormone, blood phosphorus, and acidbase metabolism. Lancet 1: 1329-1331, 1971. 4. BECK, N. P., F. R. DERUBERTIS, M. F. MICHELIS, R. D. FUSCO, AND B. B. DAVIS. Effect of prostaglandin El on certain renal actions of parathyroid hormone. J. Clin. Invest. 51: 2352-2358, 1972. 5. BECK, N., T. KANEKO, U. ZOR, J. B. FIELD, AND B. B. DAVIS. Effects of vasopressin and prostaglandin El on the adenyl cycJasecyclic AMP system of the renal medulla of the rat. J. Clin. Invest. 50: 2461-2465, 1971. 6. BECK, N. P., S. w. R EED, V. D. MURDAUGH, AND B. B. DAVIS. Effect of catecholamines and their interaction with other hormones on cyclic 3’ ,5’-adenosine monophosphate of the kidney. J. Clin. Invest. 5 1 : 939-944, 1972. 7. BECK, N., H. SINGH, S. W. REED, AND B. B. DAVIS. Direct inhibitory effect of hypercalcemia on renal actions of parathyroid hormone. J. Glin. Invest. 53 : 717-725, 1974. 8. BENNET, C. M., B. M. BRENNER, AND R. W. BERLINER. Micropuncture study of nephron function in the rhesus monkey. J. Clin. Invest. 47 : 203-216, 1968. 9. BERNSTEIN, B. A., AND J. R. CLAPP. Micropuncture study of bicarbonate reabsorption by the dog nephron. Am. J. Physiol. 214: 251-257, 1968, 10. BERNSTEIN, D. S., A. WACHMAN, AND R. S. HATTNER. Acid base balance in metabolic bone disease. In: Osteoporosis, edited by U. S. Barzel. N ew York: Grune & Stratton, 1970, p. 207-216. 11. CHASE, L. R., AND G. ID. AURBACH. Parathyroid function and the renal excretion of 3’5’-adenylic acid. Proc. iVatZ. Acad. Sk., US 58: 518-525, 1967. 12. CHASE, L. R., AND G. D. AURBACH. Renal adenyl cyclase. Anatomically separate sites for parathyroid hormone and vasopressin. Science 159 : 545-547, 1968. 13. CHEUNG, W* Properties of cyclic 3’5’-nucleotide phosphodiesterase from rat brain. Biochemistry 6 : 1079-1087, 1967. 14. CLAPP, J, R., J. F. WATSON, AND R. MT. BERLINER. Effect of carbonic anhydrase inhibition on proximal tubular bicarbonate reabsorption. Am. J, Physiol. 205 : 693-696, 1963.
15, GILMAN,
A.
G. A protein binding assay for adenosine 3’5’-monoProc. Natl. Acad. Sci., US 67 : 305-312, 1970. 16, GOTO, K. Mineral metabolism in experimental acidosis. J. &oZ. Chem. 36 : 355-376, 1918. 1% HELLMAN, D. E., W, Y. W. Au, AND F. C. BARTTER. Evidence for a direct effect of parathyroid hormone on urinary acidification. Am. J. Physiol. 209: 643-650, 1965. 18. KURTZMAN, N. A., M. L. KARLINSKY, AND D. S. SAGER. Effects of infusion of cyclic AMP and parathyroid hormone on renal bicarbonate reabsorption. Glin. Res. 21: 283, 1973. 19. LEMANN, F., JR., E. J. LENNON, A. II GOODMAN, J. R. LITZOW, AND A. S. RELMAN. The net balance of acid in subjects given large loads of acid or alkali. J. Ciin. Invest. 44 : 507-5 17, 1965. 20. MARCUS, R., AND G. D. AURBACH. Bioassay of parathyroid hormone in vitro with a stable preparation of adenyl cyclase from rat kidney. Endocrinology. 85 : 801-8 10, 1969. 21. MOORE, E. W. Ionized calcium in normal serum, ultrafiltrates, and whole blood determined by ion-exchange electrode. J. CZin. Invest. 49 : 3 18-334, 1970. 22. MOSTELLAR, M. E., AND E. P. TUTTLE, JR. Effects of alkalosis on plasma concentration and urinary excretion of inorganic phosphate in man. J. Clin, Invest. 43 : 138-149, 1964. 2% MULDOWNEY, F. P., R. FREANEY, AND T. MCGEENEY. Renal tubular acidosis and amino-aciduria in osteomalacia of dietary or intestinal origin. Quart. J. Med. 37 : 5 17-539, 1968. 24. MURAD, F., H. B. BREWER, JR., AND M. VAUGHAN. Effect of thyrocalcitonin on cyclic AMP formation by rat kidney and bone. Proc. Natl. Acad. Sci. 65 : 446-453, 1970. 25. NAGATA, N., AND H. RASMUSSEN. Parathyroid hormone and renal cell metabolism. Biochemistry 7 : 3728-3 733, 1968. 26. RECTOR, F. C., JR., H, A. BLOOMER, AND D. MT. SELDIN. Effect of potassium deficiency on the reabsorption of bicarbonate in the proximal tubule of the rat kidney. J. Clin. Invest. 43 : 19764982, 1964. 27. SuKl, W. N., M. M ARTINEZ-MALDONADO, D. ROOSE, AND A. TERRY, Effect of expansion of extracellular fluid volume on renal phosphate handling. J, C&n. best. 48 : 1888-l 894, 1969. 28. THOMPSON, W. J., AND M. M. APPLEMAN. Multiple cyclic nucleotide phosphodiesterases from rat brain. Biochemistry. 10 : 3 1 l-3 16, 1971, 29. WACHMAN, A., AND D. S, BERNSTEIN. Parathyroid hormone in metabolic acidosis : its role in pH homeostasis. C&n. Orthofaed. Rel. Res. 69: 252-263, 1970. 30. YORK, S. E., AND E. R. YENST. Qsteomalacia associated with renal bicarbonate loss. Can. Med. Assoc. J. 94. 1329-133 1, 1966. phosphate.
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Effect
of metabolic
renal
action NAMA
MAMA,
HEI
BECK,
PAIK
on
of parathyroid HE1 PAIK
Department of Medicine, Pittsburgh, Pennsylvania
BECK,
acidosis
KIM,
AND
KIM,
hormone
AND
KWANG
Veterans Administration 15240
KWANG
SUP
KIM.
Effect of
metabolic acidosis on renal action of pamthyroid hormone. Am. J . Physid. 1975.~Effects of metabolic acidosis on renal 228(5): 1483-1488. action of parathyroid hormone (PTH) was investigated in rats. Metabolic acidosis was induced by infusing NH&l, and the control animals received the equivalent volume of NaCl. The extracellufar fluid volume was not measurably different between the two groups of animals. The changes of TRP and urinary excretion of cyclic AMP by PTH was significantly inhibited in the acidotic animals, but the phosphaturia induced by an infusion of dibutyryl cyclic AMP was not affected by metabolic acidosis. The increase of cyclic AMP concentration by PTH was significantly less in the renal cortical slices obtained from the acidotic animals than in those obtained from the control animals. The activation of adenylate cyclase by PTH was also significantly inhibited in the acidic media. These results suggest that in metabolic acidosis the renal response to PTH is decreased, and that the decrease is due to the inhibition of the PTH-dependent cyclic AMP system in the kidney at the level of adenylate cyclase in renal cortex. cyclic
AMP;
BONE
IS A RESERVQIR
buffer,
and
in
University
of Pittsburgh
parathyroidectomized mediated through metabolic acidosis METHODS
AND
School
of Medicine,
rats. Since the renal effect of PTH cyclic AMP (2, 4, 1 l), the effects on that system was also investigated.
is of
MATERIALS
In viuo experiments. Sprague-Dawlev rats, weighing 270290 g, were surgically parathyroidectomized to evaluate the renal response to PTH in the absence of endogenous PTH secretion. Four to six days were allowed for the animals to stabilize after the surgery. During this period, the animals were maintained on calcium chloride, 1 g/l 00 ml of drinking water. On the day of experiments the animals were divided into three groups : alkalotic, control, and acidotic. Into each group of animals, 15 ml of solution were injected peritoneally. The solution contained either NaHC03 1.5 mmol, NaCl 1.5 mmol, or NH&l 1.5 mmol, inulin 15 mg, and sodium pentobarbital 7.5 mg. Polyethylene catheters were inserted into the urinary bladders through suprapubic incisions for collection of urine, and into the femoral veins for intravenous infusion. The intravenous infusion was initiated 30 min after the peritoneal injection. The solution contained either 140 mM NaHCOs, 140 mM NaCl, or 140 mM NH&l, with inulin, 0.3 mg/ml; the infusion rate was 70 &min until the end of the experiment. In the first series of experiments, the respective intravenous solutions were infused for 2.5 h, and arterial blood pH and bicarbonate concentrations and the phosphate excretion rates were measured during that period in the absence of PTH injection. The pH and bicarbonate concentrations were stable from 1 to 2.5 h after the initiation of intravenous infusion with the changes of pH less than 0.02 in each group. Therefore, subsequent experiments were performed during that l2.5 h period. In the second seriesof experiments, three 15-min urine specimens were collected and served as the basal values. Then, 10 U of synthetic, amino-terminal, l-34 fragment of bovine PTH, hereafter referred to as PTH, were injected intravenously to each animal, followed by collections of 15min urine specimens. Ten units of PTH per rat was a submaximal dose to induce phosphaturia (7). Blood samples were obtained from the femoral artery immediately prior to PTH injection and 60 min after the injection. The maximal phosphaturic response to PTH was usually seen 30-60 min after PTH injection. Therefore, the phosphaturic response to PTH was measured during that 30 to 60.
phosphaturia
of phosphate
SUP KIM
Hus@al,
metabolic
acidosis there is an increased release of phosphate buffer from the bone (3, 10, 16, 19, 23, 29, 30). It has been postulated that the increased mobilization of phosphate buffer may have, at least in part, a protective role against metabolic acidosis (16, 19). Barzel (3), Wachman and Bernstein (29), and other investigators (3, 10) postulated that parathyroid hormone (PTH) has a role in acid-base balance through the modulation of the release of phosphate buffer from bone (3, 10, 29). Furthermore, Bernstein, Wachman, and Hattner (10) and Wachman and Bernstein (29) postulated that the increased release of phosphate buffer is due to an increased skeletal end-organ response to PTH in metabolic acidosis. In the kidney, PTH also affects acid-base balance (17, 18). According to the data obtained from micropuncture experiments (8, 9, 14, 26), the major fractions of filtered phosphate and bicarbonate are reabsorbed in proximal tubules. PTH inhibits the proximal tubular reabsorption and consequently increases excretions of those ions (I 7). Since it has been suggested that metabolic acidosis alters the end-organ response to PTH in bone (l&29), it is reasonable to speculate that metabolic acidosis also alters the renal end-organ response to PTH. Therefore, the effect of metabolic acidosis on renal response to PTH was investigated in 1483
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1484 min period. The maximal increase of urinary excretion of cyclic AMP was usually seen 15-45 min after PTH injection. Therefore, the increase of urinary excretion of cyclic AMP by PTH was measured during that 15- to 45min period. The extracellular fluid volumes in the control group and in the acidotic animals were compared on the basis of chloride space with the use of Wl, and urinary excretion rates of water and chloride. In the third series of experiments, dibutyryl cyclic AMP was infused into both the control animals and the acidotic animals at the rate of 50 pg/min. The phosphaturic responses to dibutyryl cyclic AMP were then compared between the two groups of animals. In vitro experiments. In another series of experiments the animals were prepared in an identical manner to the above in viva experiments, infusing NaCl into the control group and NH&l into the acidotic animals. One hour and 45 minutes after the initiation of the intravenous infusion, which is the time corresponding to that of PTH injection in vivo, the kidneys were removed rapidly and the cortex was sliced to a thickness of less than 0.5 mm. Each slice, weighing between 30 and 50 mg, was incubated in KrebsRinger-Tris buffer, pH 7.2. The PTH group contained PTH, 10 U/ml. After 10 min incubation at 37”C, the slice was homogenized in 0.5 ml of glass distilled water using a glass tissue grinder, and then the tube was placed in a boiling water bath for 3 min to terminate the enzyme activity in the homogenate. After centrifugation for 15 min at 7,000 X g, cyclic AMP in the supernatant was assayed using Gilman’s method (15) with modifications (6). To evaluate the efiect of pH of the incubation medium, the cortical slices obtained from the control-group animals were incubated in Krebs-Ringer-Tris buffer with two diKerent pH’s, 7.2 and 7.4. Adenylate cyclase of renal cortex was prepared as described by Marcus and Aurbach (20). The cortex of kidney was homogenized in 50 mM Tris, pH 7.4, and centrifuged for 15 min at 2,000 X g. The precipitate was resuspended in 50 mM Tris, pH 7.4, and centrifuged again for 15 min at 2,000 X g. The precipitate after the second centrifugation was resuspended in 5 mM Tris, pH 7.4. The homogenate of renal cortex, hereafter referred to as the enzyme adenylate cyclase, was incubated in the medium containing, in millimoles per liter: MgClz, 1.6; KCl, 25; theophvlline, 10; ATP, 1; Tris, 50; and PTH, 10 U/ml (for the PT’H group) ; total volume, 0.60 ml per tube. The pH’s of the mixtures were adjusted to 7.4 for the control and 7.2 for the acidotic group. After 15 min incubation at 37°C (during this period the enzyme activity was linear with time), the reaction was terminated by placing the tubes in a boiling water bath for 3 min. The aggregate of the boiled enzyme was precipitated by centrifugini for 15 min at 700 X g. Cyclic AMP in the supernatant ias measured using Gilman’s method (15) with modifications (6). The difference in pH of the media, ATP remaining after 15 min incubation, PTH, or other study substances, had no measurable effect on the cyclic AMP assay system. Phosphodiesterase was prepared as described by Cheung (13) by homogenizing renal cortical tissue in glass-distilled water and then centrifugin g the homogenate at 30,000 X g for 30 min. Phosphodiesterase activity was assayed as de-
BECK,
KIM,
KIM
AND
scribed by Thompson and Appleman (28). The precipitate fraction of 30,000 X g centrifugation was not studied because there was a negligible amount of phosphodiesterase activity in the precipitate fraction (7). The proper amount of enzyme preparation from the supernatant fraction, that which hydrolyzes 15-20 % of substrate, was incubated in the medium containing 10L4 M cyclic AMP, a tracer amount of 3H-labeled cyclic I , AMP 1 .6 mM MgC12, and 25 mM KCl. The pH of the mixture; was adjusted to 7.4 for the control group and 7.2 for the acidotic group. After 15 min incubation at 37”C, the reaction was terminated by placing the tubes in a boiling water bath for 3 min. The mixtures were then reincubated for 10 min at 37°C with @ili@ugus hannah venom, 0.1 mg/tube. Then, 2 ml of Dowex AGLX2 anion-exchange resin, ZOO-400 mesh, 1: 3 suspension, were added to each tube, and the tubes were centrifuged at 700 X g for 15 min. The radioactivity in the supernatant was counted by a beta liquid scintillation spectrometer. Synthetic, amino-terminal, l-34 bovine PTH, lot no. 150 12, was obtained from Beckman Instruments, Inc. (Palo Alto) and its activity was rechecked using renal cortical adenylate cyclase as described by Marcus and Aurbach (20). 3H-labeled cyclic AMP, 25 Ci/mmol, was obtained from New England Nuclear Corp. (Boston), AGl -X2 anion exchange resin, 200-400 mesh, from Bio-Rad Laboratories, (Richmond, Calif.), and cyclic AMP, dibutyryl cyclic AMP, and ATP from Sigma Chemical Co. (St. Louis). RESULTS
In vim cxfieriments. The chang-es of arterial blood pH and bicarbonate concentration aft& the infusion of NaHCOs, _nSaCl, or NH&l were significantly different among the three groups of animals, P < 0.01 among the three groups
1. Eflicts of metaboh’c alkalosis and acidosis on rats --- “,“.--I ,__.__.I.._-.-~ -.-~---
TABLE
---
iAcidotic ”-
Alkalotic
-
Rats
P value
I
No. of experiments Intravenous infusion, pmol/min Arterial pH
I ’
Arterial HCQ-, meq/liter Plasma Ca++, mM
/ ’ I
Plasma phosphate, mg/lOO ml GFR, ml/min Filtered load of phosphate, &min i TRP before PTH, yO 1 TRP
after
Changes
PTH,
(/;1
of TRP
by
PTH, %
1
9 NaHC03 9.8 7.65 ztO.04 29.4 +I .OO 0.72 *o -06 6.1 +l.l 2.09 zto.19 128 442 99.11 *0.34 76 -4-2 k4.38 a-22.69 zt4.33
12 NaCl 9.8 7.46 +o .Ol 15.9 +I .4 0.82 hO.07 9.4 ~1.8 1.67 *urn 17 156 zkl6 98.99 ztO.86 76.83 zt4.06 !A- 22.16 +4.45
I
13 NH&l 9.8 7.23 +0.01 6.8 zt2.50 1.11 zto.09 8.7 h1.2 1.66 +o.zo 144 zkl7 99.44 ztO.28 87.97 +0.28 A -11.47 zk2.96
/
;0.05 i>O.O5 I * /
*
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
METABOLIC
ACIDOSIS
ON
RENAL
ACTION
OF
1485
PTH
(Table 1). Those values were stable during the period of experiments (l-Z.5 h) and the change of pH was less than 0.02 in each group. The plasma concentration of ionized calcium was significantly higher in the acidotic animal than in the control animals, P < 0.05. However, plasma phosphate concentration, mean glomerular filtration rate (GFR), and filtered load of phosphate were not measurably diflerent among the three groups of animals, P > 0.05 (Table 1). The extracellular fluid volume measured by “%l space and urinary excretion rates of water and chloride were not measurably different between the control and the acidotic groups of animals, P > 0.05 (TabIe Z), and those values were not measurably altered during the experiments, P > 0.05. TubuZar reabsorption rate of phospkute (TRP). In the first series of experiments without PTH injection, there was no measurable change of TRP during the experimental periods of l-Z.5 h after the initiation of the intravenous infusion, and the values remained not measurably dEerent among the three groups of animaIs at the end of the 2.5-h period : 99.21 & 0.38 % in the alkalotic animals, 99.25 & 0.40 in the control animals, and 99.48 s+. 0.35 in the acidotic animals 2.5 h after the initiation of the intravenous infusion, P > 0.05 among the three groups of animals. In the second series of experiments, TRP was not measurably different among the three groups of animals during the basal periods prior to PTH injection (Table 1). But: the decrease of TRP by PTH was significantly less in the acidotic animals than in the control or the alkalotic animals (Table 1). However, the decrease of TRP by PTH was not measurably different between the control and the alkalotic animals, P > 0.05 (Table 1 and Fig. 1). Urinary cyclic AMP excretion rates. During the basal periods prior to PTH injection, the urinary cyclic AMP excretion rate was not measurably diRerent between the acidotic animals, 52.5 + 6.5 pmol/min, and the control animals, 49.6 & 6.2; P > 0.05 (Fig. 2). However, the increase of urinary cyclic AMP excretion rate after the PTH injection was significantly less in the acidotic animals, to 93.8 & 9.4 (A41.3 rk 9.3), th an in the control animals, to 135.6 =t 18.8 (~86.0 h 15.1); P < 0.05. Dibutyryl cyclic AMP infusion. The mean decrease of TRP after the infusion of dibutyryl cyclic AMP was not measTABLE 2. liffecfs of an intrclvenous infusion of NHdCl or NaCI on extracellular Juid volume and urinary excretion of chloride ---- .- _ --~._----_ ^_- -. .---Control
No. of animals studied Intravenous infusion, 100 pl/ min for 3 h Plasma osmolality, mosmol/kg ““Cl space, ml/kg body wt Urine flow rate, pi/kg-min Cl- excretion, peq/kg-min Osmolal clearance, PI/kg-min
_- .-
Rats
Acidotic
5 150 mM 295 336.9 86.2 20.4 317.3
Rats ---... ---
5 NaCl s+ + A zt +
1 4.3 17.2 3.3 16.6
150 mM 295 331.0 106.5 18.5 293.4
NH&l It Ik + * zk
2 8.2 18.4 2.2 24.3
Values are means + SE. After the respective intravenous infusion for 3 h, the average of the subsequent 90-min urine specimen in each rat was calculated. Chloride space was determined 3 h after the intravenous infusion and 90 min after the injection of 36C1 for equilibration.
A cidotic
Alkalotic
7s
basal
PTH
FIG. 1. Changes of rate of tubular phosphate reabsorption (TRP) in control, alkalotic, and acidotic animals. Basal values are averages of three 15-min values, and PTH values are averages of 30- to GO-min values after PTH injection. Each point represents mean + SE of 12-13 experiments.
165
-
1 4
Control
140;
z gm ‘g 115 =.u \z; .-E .: goSE
// // : 1’ /’ //
/’ J,/ ’ /’ ,, ‘I /’ ’ $*. basal
n ,’ -?’
-
Acidotic
PTH
FIG. 2. Changes of urinary cyclic AMP excretion rates in control and acidotic animals. Basal values are average of three 15-min values, and PTH values are averages of 30- to 60-min values after PTH injection. Each point represents mean + SE of basal and PTH values of 1 Z- 13 experiments.
urably different: between the acidotic animals, from 99.70 & 0.05 % in the basal periods to 60.95 & 11.49 (% in 3075 min after the initiation of the infusion, and the control animals, from 99.73 j= 0.04 to 61.52 j= 3.80; P > 0.05. CjcliG AMP concentration in corfz’cal slices. The basal cyclic AMP concentration without PTH was not measurably d’fF 1 erent between the slices obtained from the acidotic animals, 3-42 & 0.30 pmol/mg wet tissue, and those from the control animals, 4.08 & 0.24; P > 0.05. However, the increase of cyclic AMP concentration by PTH, 10 U/ml, was significantly less in the slices obtained from the acidotic animals, 4.77 & 0.32 (Al.35 & 0.20), than in the control group, 6.50 & 0.42 (AZ.42 + 0.31);P < 0.01 (Fig. 3). On the other hand, when the slices obtained from the control-group animals, having the same intracellular pH were incubated in the medium with two dif&rent pH’s, 7.2 vs. 7.4, the increase of cyclic AMP concentration by PTH, 10 U/ml, was not measurably different between the two groups: from 4.08 =t 0.24 to 6.50 =t 0.42 in the group with pH 7.2 vs. from 4.27 & 0.22 to 6.29 & 0.23 in the group with DH 7.4. resDectiveIv; P > 0.05. Adkylate Lyclaie activikk. In the absence of PTH, adenylate cyclase activities were not measurably different between the acidic group (pH 7.2), 2.3 & 0.5 pmol/mg protein-min, and the control group (pH 7.4), 2.3 + 0.2; P >
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BECK,
J
rats //3 Control Acidotic
rats
FIG. 3. Changes of cyclic AMP concentration in renal cortical slices. Acidotic, slices obtained from acidotic animals. Control, slices obtained from control animals, Basal, without PTH. Values are means j= SE of 10 experiments each.
KIM,
AND
KIM
acidosis masks the effect of PTH on urinary bicarbonate excretion (17) and makes the results difficult to interpret. Therefore, the renal response to PTH was measured by tubular reabsorption rate of phosphate. During the baial periods prior to PTH injection, TRP was not measurably different among alkalotic, control, and acidotic animals. These results suggest that metabolic acidosis or alkalosis alone does not affect TRP. However, the decrease of TRP by PTH was significantly less in the acidotic animals than in the other two groups of animals. On the other hand, there was no measurable difference in TRP between the control group and the alkalotic animals. These results suggest that the renal response to PTH is inhibited in metabolic acidosis, but not in alkalosis. Therefore, the effect of metabolic alkalosis on the renal response to PTH was not further investigated. The data in the present experiments do not explain why the renal action of PTH is affected by acidosis, but not by alkalosis. However, it is interesting to note that the discrepancy in the present experiments, viz., phenomenon opposite from that observed in acidosis does not occur in alkalosis, is similar in that the intracellular pH is protected against the extracellular acidosis, but not against the extracellular alkalosis (1) Plasma phosphate concentration, mean GFR, and filtered load of phosphate were not measurably different among the three groups of animals, indicating the lesser decrease of TRP by PTH in the acidotic animals is not due to an alteration of filtered load of phosphate. These findings are consistent with the data of other investigators (22). Expansion of extracellular fluid volume induces phosphaturia unrelated to renal effect of PTH (27). Since the electrolytes in the intravenous solutions are different, i.e., NaCl for the control animals and NH&l for the acidotic animals, the status of extracellular fluid volume might be different between the two groups of animals, and consequently may induce a difference in TRP. However, the extracellular fluid volume, measured by 36Cl space, and that compared by urinary excretion rates of water and chloride was not measurably different between the control group of animals with an infusion of NaCl and the acidotic group with an infusion of NH&l. Furthermore, in the first series of experiments with the same intravenous infusions as the second series of experiments, but without PTH injection, TRP was not measurably different among the three groups of animals, and TRP did not change during the 1 -to 2.5-h experiments after the initiation of the intravenous infusion. These results sqgest that, in the system tested, there was no c measurable difference in extracellular fluid volume between the two groups of animals. Therefore, it is very unlikely that the lesser decrease of TRP by PTH in the acidotic animals is due to the lesser extracellular fluid volume rather than due to the acidosis per se, even though an unmeasurable small difference in extracellular fluid volume between the two groups of animals cannot be excluded. Since the phosphaturic effect of PTH is mediated through the cyclic AMP system, that system is also evaluated in vivo and in vitro. The increase of urinary excretion of cyclic AMP after PTH injection was less in the acidotic animals than in the control animals. These results are consistent with the findings of TRP. These data suggest that the altered l
PTH 10 U/ml
FIG. 4, Changes of renal cortical adenylate cyclase activity by PTH. Each point represents mean =t SE of 10 experiments in control group (pH 7.4) and acidotic group (pH 7.2). Basal indicates group without PTH.
0.05. However, the activation of adenylate cyclase by PTH was significantly less in the acidic group, to 5.3 & 0.5 (A3.0 & 0.5), than in the control group, to 14.3 =k 0.4 (A12.0 =t 0.3); P < 0.01 (Fig. 4). Phosj9hodiesterase activities. The basal activity of high-K, ( 10m4 M) cyclic AMP phosphodiesterase in the acidic group (pH 7.2), 825 Z& 20 pmol hydrolysis per milligram protein per minute, was not measurably different from the control group, 746 =t 39; P > 0.05. PTH also had no measurable effect on the phosphodiesterase activity: 819 I!= 17 in the acidic group (pH 7.2) with PTH, 10 U/ml, and 751 & 13 in the control group (pH 7.4) with PTH; P > 0.05 as compared to the corresponding basal values.
The data in the present experiments demonstrate that metabolic acidosis inhibits the phosphaturic response to PTH. However, in the animals with intact parathyroid glands, the changes of PTH activity in metabolic acidosis could be due to an alteration of either hormonal secretion or the response of the end-organ to the hormone, or a combination of both. Therefore, the effect of metabolic acidosis was evaluated exclusively on renal end-organ response to PTH in parathyroidectomized animals in which the secretory mechanism of PTH had been eliminated. In our present experiments, the change of bicarbonate excretion associated with the induction of alkalosis or
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 19, 2019.
METABOLIC
AC1DOSIS
ON
RENAL
ACTION
OF
PTH
end-organ response to PTH in metabolic acidosis is at or prior to the PTH-dependent cyclic AMP system in the kidney* The urinary excretion of cyclic AMP can be affected by the change of blood cyclic AMP concentration and consequently the change of g1omerular filtered load of cyclic AMP. Furthermore, urinary cyclic AMP does not exclusively represent PTH-dependent cyclic AMP. There are several other hormones inducing cyclic AMP generation in the kidney, e.g., vasopressin (5), catecholamines (6), prostaglandins (4, 5), and calcitonin (24). Also certain unknown or unmeasured factors associated with the induction of metabolic acidosis may indirectly affect urinary cyclic AMP excretion either through the alteration of efiects of those hormones or through other unknown mechanism. However, if those factors or hormones other than metabolic acidosis or PTH per se affected the urinary excretion of cyclic AMP, that effect should be demonstrable during the basal period in the absence of PTH. But, those basal values in the absence of PTH were not measurably different between the acidotic and the control animals. Furthermore, the increase of urinary excretion of cyclic AMP after PTH injection specifically represents the ‘change of the PTHdependent urinary cyclic AMP, and that increase was significantly less in the acidotic animals than in the control animals. Therefore, the results of urinary cyclic AMP suggest that the PTH-dependent cyclic AMP system in the kidney is inhibited in metabolic acidosis. If the findings of urinary excretion of cyclic AMP were considered alone, the changes of urinary cyclic AMP does not necessarily exclusively reflect the change of the PTHdependent cyclic AMP system in the kidney, and the findings of urinary cyclic AMP alone should be interpreted with caution. Therefore, in the in vitro system, the direct effect of metabolic acidosis on the PTH-dependent cyclic AMP system was further investigated in renal cortical slices in vitro. The cortex but not the medulla of the kidney was used because the PTH-dependent cyclic AMP system is mainly located in the cortex (12). The cortical slices were obtained from the control and the acidotic animals, which had been prepared in an identical manner to those in the above in vivo experiments. In those slices, the basal values in the absence of PTH was not measurablv different between the two groups of slices. But the increase of cyclic AlMP concentration bv PTH, 10 U/ml, in the incubation media, pH 7.2 for both groups, was significantly less in the acidotic group than in the control group. These results are consistent with the findings of urinary excretion of cyclic AMP, and further suggest that metabolic acidosis inhibits the PTH-dependent cyilic AMP system in the kidney. On the other hand, when the slices of the control animals with the same intracellular pH were incubated in the media with two different pH’s, 7.2 and 7.4, both the basal values and the increases bv PTH, 10 U/ml, were not measurably different betwee; the two groups. These latter findings suggest that the change of intracellular milieu associated with metabolic acidosis, but not the simple lowering of extracellular pH, inhibits the PTH-dependent cyclic AMP in the kidney. The lesser cyclic AMP concentration in cortical slices could be due either to the inhibition of cyclic AMP forma-
1487 tion, viz., the inhibition of adenylate cyclase, or to the augmentation of cyclic AMP catabolism, viz., the activation of phosphodiesterase. Therefore, the effect of pH on the PTHdependent cyclic AMP system was further evaluated on the enzyme activities of adenylate cyc1ase and PhosP hodiesterae. The basal activity of adenylate cyclase was not measurably different between the two groups, pH 7.2 vs. 7.4. However, the activation of enzyme by PTH, which specifically represents the change of PTH-dependent adenylate cyclase, was significantly less in the acidic group (pH 7.2) than in the control group (pH 7.4). These results indicate that in metabolic acidosis, the lesser increase of cyclic AMP concentration by PTH in renal cortical slices is due at least in part to the decreased synthesis of cyclic AMP. However, the data in the present experiments do not differentiate whether the inhibitory mechanism of acidosis on PTHdependent adenylate cvclase in renal cortex is at the hormonal receptor or the citalytic unit in the adenylate cyclase complex. Neither PTH nor the change of pH in the media had any measurable effect on cyclic AMP-phosphodiesterase activity of renal cortex in the high-K, system. These results suggest that the lesser increase of cyclic AMP concentration by PTH in metabolic acidosis is not due to the increased catabolism of cvclic AMP. The changes of TRP, the end results of PTH effects in the kidney, can also be affected by the reaction or reactions subsequent to cyclic AMP generation. Therefore, the eflect of metabolic acidosis was evaluated on phosphaturia induced by an infusion of dibutyryl cyclic AIMP as described by Nagata and Rasmussen (25). However, the phosphaturic response to dibutyryl cyclic AMP infusion was not afl’ected by metabolic acidosis. These results suggest that the inhibition of end-organ response to PTH in metabolic acidosis is due to the inhibition of the PTH-dependent cyclic AMP generation, but not due to the alteration of the reactions subsequent to cyclic AMP generation. Plasma concentration of ionized calcium was increased in acidosis and diminished in alkalosis (Table 1). Those findings are comparable to those described by Moore (2 1). Since Beck et al. (7) have reported an inhibition of renal effect of PTH in hypercalcemia, it is reasonable to postulate that the increase of ionized calcium concentration in acidosis may at least in part play a role to mediate the inhibitory effect of acidosis on the renal action of PTH. On the other hand, the in vitro data of adenylate cyclase in the present experiments suggest that the inhibitory efl?ect of acidosis on the PTH-dependent cyclic AMP system in the kidney is the alteration of pH per se rather than med ia ted through the al tera tion of ionized calcium concentrati on, because there was no measurable calcium in the incubation media in both the control group and the acidic group. Those findings suggest that the increase of the concentration of ionized calcium is not the sole mechanism, but that there is probably more than one mechanism that mediates the inhibitory effect of acidosis on the renal action of PTH. Regardless of those inhibitory mechanisms postulated, the data in the present experiments clearly demonstrate that metabolic acidosis inhibits the PTH-dependent cyclic AMP system in the kidney.
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1488 When all the above results are considered together, as a whole, they demonstrate that metabolic acidosis inhibits the renal response to PTH, measured by TRP, through the inhibition of the PTH-dependent cyclic AMP system in the kidney at or prior to the level of adenylate cyclase. The inhibition of renal end-organ response to PTH in metabolic
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