Pfli.igers Archiv
Pfltigers Arch. 379, 37- 41 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Influence of Calcium and Ionophore 23187 on Tubular Phosphate Reabsorption*' * * Hans Oberleithner, Florian Lang, Rainer Greger, and Hans Sporer Institut f~ir Physiologie, Universitfit Innsbruck, Fritz-Pregl Stral3e 3, A-6010 Innsbruck, Austria
Abstract. In previous studies it has been demonstrated
Key words: Thyroparathyroidectomy -
that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading. Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect. Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 retool/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 rain. After 18 rain a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused iubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12 % of the microinfused phosphate load within 24 rain. In another series of experiments, the calcium specific ionophore A 23187 - a substance which is known to increase intracellular calcium - was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15 % after 24 rain. In contrast, when calcium chloridefree as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7 %). F r o m these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.
transport calcium.
* This study was supported by Dr. LegerlotzStiftung ** Parts of this study were presented at the fall meeting of the ,,Nephrologische Gesellschaft" in Bonn, 1977and at the spring meeting of the ,,Deutsche Physiologische Gesellschaft" in G6ttingen, 1978
-
Ionophore A23187
-
Phosphate Intracellular
Introduction It has been demonstrated that a constant decline of maximal reabsorptive rate of phosphate occurs during continuous phosphate loading [2, 5]. Recently it was shown that in thyroparathyroidectomized (TPTX) and phosphate-loaded rats severe hypocalcemia is responsible for the decline in renal phosphate reabsorption [15]. Normalization of plasma calcium concentration is followed by an increase of phosphate reabsorption. The question arises whether calcium exerts its effect on renal phosphate reabsorption by an extrarenal or by a direct renal mechanism. The present study was designed 1. to detect a possible direct renal effect of calcium and 2. to evaluate the influence of intracellular calcium on renal phosphate transport using ionophore A 23187, a substance which shifts extracellular calcium into the cell.
Methods Studies were carried out on 22 male Wistar rats (200- 250 g B. W.). The animals were fed a standard altromin chow and had access to tapwater ad libitum. The animals wereanesthetized with Inactin(Byk Gulden, Konstanz, FRG 120 mg/kg B. W. i.p.) and placed on an electrically heated table, to maintain body temperature at 310~ Following tracheostomythe parathyroid glands were destroyed by thermocautery and finally the remainder of the thyroid gland was cauterized. The right jugular vein and the right femoral artery were cannulated and a bladder catheter was inserted for sampling of urine of the contralateral kidney,whereas the ipsilateral urine was collected from a PE50 ureteral catheter. Animals were infused with a phosphate solution (sodium phosphate 100 mmol/1,pH 7.4) at a rate
0031-6768/79/0379/0037/$ 01.00
38
Pflfigers Arch. 379 (1979)
/
+10 --*8 a-§ 6
N
Fig. 1 Microinfusion experiments with superfusion of CaCl2 (solid line) and NaCI (dashed line). Change of fractional reabsorption of 33p [A reabsorption (%)] is plotted versus time after beginning of microinfusion in proximal convoluted tubules. Shaded bars indicate beginning of superfusion 18 rain after microinfusion was started * Significantly different from zero, n = 13, + SEM
~+2 rY 0 O u3
~-2 Ill rY
-6 -8-IO
of 0.2 ml/min, kg B. W. Three hours after TPTX the animals were prepared for micropuncture using standard techniques [11]. When plasma calcium concentration had dropped below 1.30mmol/1, a proximal tubular segment was indentified by neutral lissamine green dye injected systemically (0.03 ml, 5g/l, i.v.). Thereafter this unblocked proximal tubule was microinfused using a microinfusion pump [20] at 10 nl/min for 42 min. The microinfused fluid consisted of isotonic NaCI solution to which 33p phosphate (2 mmol/1 and 3H inulin (120 mg/1) were added (33p phosphate at a specific activity of 10 mCi/mmol was obtained from New England Nuclear, Boston, Mass. and 3H Inulin at a specific activity of 400 mCi/g from Amersham, Radiochemical Centre, Buckinghamshire, GB). After 18 min ofmicroinfusion 10gl of a calcium chloride solution (CaClz5 mmol/1, NaC1 138 mmol/1) was delivered from a glass capillary onto the microinfused segment over the following 24 min. In controls the same amount of CaC1z was given systemically and 10 ~tl of a sodium chloride solution (NaCI 145 mmol/l) were applied superficially (superfused). In a second series of experiments, proximal tu bular segments were superfused with the calcium specific ionophore A 231871 (100 mg/1, 10 gl) which was initially dissolved in absolute ethanole (10 mg/ml). This solution was dissolved in a CaCI 2 containing solution (CaC12 0.5 mmol/1, NaC1 145 mmol/l) in order to obtain an ionophore concentration of 100 mg/1, In corresponding control experiments the same solution without the ionophore was used. Urine from the ipsilateral kidney was sampled every 6 min and from the contralateral kidney every 12 min throughout the experiment. Blood samples (300 gl) were taken from the femoral artery catheter 180 min and 360 rain after TPTX and analyzed for plasma phosphate, acid base status and plasma calcium concentration. A~alytical Procedures. Plasma phosphate concentration ([Pi]p~) was measured colorimetrically using the molybdate method described by Chen [3]. Plasma calcium concentration ([Ca]p0 was measured by flamephotometry (Type Eppendorf, Hamburg, FRG). Plasma pH (pH00 and plasma carbon dioxide tension (Pco2pl) of the arterial blood were determined at 310~ utilizing a blood microsystem and a glass electrode unit (BMS2MK2, BEU1, Radiometer Copenhagen, DK). a Kindly provided by Eli Lilly S_ A., CH.
Plasma bicarbonate concentration ([HCO;-]) was computed using the Henderson-Hasselbalch equation. The 3H and 3~p activities in the urine were measured by liquid scintillation counting. The scintillation fluid consisted of Readysolv HP scintillation fluid (Beckmann Instruments, Wien, Austria). Fractional reabsorption of 33p phosphate was calculated using the formula: @33p i --e33pe). 3Hin f
Fractional 33p reabsorption = 1 (e3Hi--g3X-[e) - 33pin f
In this formula/;33p i and 833pe, and g3H i and e3H~ represent the sum of 33p activity and 3H activity in ipsi- and contralateral urine respectively. 3Hinf/33Pin f is the tracer ratio in the microinfusate. Only tubules were considered for further evaluation where the sum of 3Hc was less than 10 ~ of the sum of 3Hi. All data are expressed as mean values -L-_standard error of the mean (SEM). Statistica/significance of difference was evaluated by the paired Student's t-test. If not otherwise indicated, P
Pfltigers Arch. 379, 37- 41 (1979)
EuropeanJournal of Physiology 9 by Springer-Verlag 1979
Influence of Calcium and Ionophore 23187 on Tubular Phosphate Reabsorption*' * * Hans Oberleithner, Florian Lang, Rainer Greger, and Hans Sporer Institut f~ir Physiologie, Universitfit Innsbruck, Fritz-Pregl Stral3e 3, A-6010 Innsbruck, Austria
Abstract. In previous studies it has been demonstrated
Key words: Thyroparathyroidectomy -
that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading. Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect. Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 retool/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 rain. After 18 rain a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused iubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12 % of the microinfused phosphate load within 24 rain. In another series of experiments, the calcium specific ionophore A 23187 - a substance which is known to increase intracellular calcium - was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15 % after 24 rain. In contrast, when calcium chloridefree as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7 %). F r o m these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.
transport calcium.
* This study was supported by Dr. LegerlotzStiftung ** Parts of this study were presented at the fall meeting of the ,,Nephrologische Gesellschaft" in Bonn, 1977and at the spring meeting of the ,,Deutsche Physiologische Gesellschaft" in G6ttingen, 1978
-
Ionophore A23187
-
Phosphate Intracellular
Introduction It has been demonstrated that a constant decline of maximal reabsorptive rate of phosphate occurs during continuous phosphate loading [2, 5]. Recently it was shown that in thyroparathyroidectomized (TPTX) and phosphate-loaded rats severe hypocalcemia is responsible for the decline in renal phosphate reabsorption [15]. Normalization of plasma calcium concentration is followed by an increase of phosphate reabsorption. The question arises whether calcium exerts its effect on renal phosphate reabsorption by an extrarenal or by a direct renal mechanism. The present study was designed 1. to detect a possible direct renal effect of calcium and 2. to evaluate the influence of intracellular calcium on renal phosphate transport using ionophore A 23187, a substance which shifts extracellular calcium into the cell.
Methods Studies were carried out on 22 male Wistar rats (200- 250 g B. W.). The animals were fed a standard altromin chow and had access to tapwater ad libitum. The animals wereanesthetized with Inactin(Byk Gulden, Konstanz, FRG 120 mg/kg B. W. i.p.) and placed on an electrically heated table, to maintain body temperature at 310~ Following tracheostomythe parathyroid glands were destroyed by thermocautery and finally the remainder of the thyroid gland was cauterized. The right jugular vein and the right femoral artery were cannulated and a bladder catheter was inserted for sampling of urine of the contralateral kidney,whereas the ipsilateral urine was collected from a PE50 ureteral catheter. Animals were infused with a phosphate solution (sodium phosphate 100 mmol/1,pH 7.4) at a rate
0031-6768/79/0379/0037/$ 01.00
38
Pflfigers Arch. 379 (1979)
/
+10 --*8 a-§ 6
N
Fig. 1 Microinfusion experiments with superfusion of CaCl2 (solid line) and NaCI (dashed line). Change of fractional reabsorption of 33p [A reabsorption (%)] is plotted versus time after beginning of microinfusion in proximal convoluted tubules. Shaded bars indicate beginning of superfusion 18 rain after microinfusion was started * Significantly different from zero, n = 13, + SEM
~+2 rY 0 O u3
~-2 Ill rY
-6 -8-IO
of 0.2 ml/min, kg B. W. Three hours after TPTX the animals were prepared for micropuncture using standard techniques [11]. When plasma calcium concentration had dropped below 1.30mmol/1, a proximal tubular segment was indentified by neutral lissamine green dye injected systemically (0.03 ml, 5g/l, i.v.). Thereafter this unblocked proximal tubule was microinfused using a microinfusion pump [20] at 10 nl/min for 42 min. The microinfused fluid consisted of isotonic NaCI solution to which 33p phosphate (2 mmol/1 and 3H inulin (120 mg/1) were added (33p phosphate at a specific activity of 10 mCi/mmol was obtained from New England Nuclear, Boston, Mass. and 3H Inulin at a specific activity of 400 mCi/g from Amersham, Radiochemical Centre, Buckinghamshire, GB). After 18 min ofmicroinfusion 10gl of a calcium chloride solution (CaClz5 mmol/1, NaC1 138 mmol/1) was delivered from a glass capillary onto the microinfused segment over the following 24 min. In controls the same amount of CaC1z was given systemically and 10 ~tl of a sodium chloride solution (NaCI 145 mmol/l) were applied superficially (superfused). In a second series of experiments, proximal tu bular segments were superfused with the calcium specific ionophore A 231871 (100 mg/1, 10 gl) which was initially dissolved in absolute ethanole (10 mg/ml). This solution was dissolved in a CaCI 2 containing solution (CaC12 0.5 mmol/1, NaC1 145 mmol/l) in order to obtain an ionophore concentration of 100 mg/1, In corresponding control experiments the same solution without the ionophore was used. Urine from the ipsilateral kidney was sampled every 6 min and from the contralateral kidney every 12 min throughout the experiment. Blood samples (300 gl) were taken from the femoral artery catheter 180 min and 360 rain after TPTX and analyzed for plasma phosphate, acid base status and plasma calcium concentration. A~alytical Procedures. Plasma phosphate concentration ([Pi]p~) was measured colorimetrically using the molybdate method described by Chen [3]. Plasma calcium concentration ([Ca]p0 was measured by flamephotometry (Type Eppendorf, Hamburg, FRG). Plasma pH (pH00 and plasma carbon dioxide tension (Pco2pl) of the arterial blood were determined at 310~ utilizing a blood microsystem and a glass electrode unit (BMS2MK2, BEU1, Radiometer Copenhagen, DK). a Kindly provided by Eli Lilly S_ A., CH.
Plasma bicarbonate concentration ([HCO;-]) was computed using the Henderson-Hasselbalch equation. The 3H and 3~p activities in the urine were measured by liquid scintillation counting. The scintillation fluid consisted of Readysolv HP scintillation fluid (Beckmann Instruments, Wien, Austria). Fractional reabsorption of 33p phosphate was calculated using the formula: @33p i --e33pe). 3Hin f
Fractional 33p reabsorption = 1 (e3Hi--g3X-[e) - 33pin f
In this formula/;33p i and 833pe, and g3H i and e3H~ represent the sum of 33p activity and 3H activity in ipsi- and contralateral urine respectively. 3Hinf/33Pin f is the tracer ratio in the microinfusate. Only tubules were considered for further evaluation where the sum of 3Hc was less than 10 ~ of the sum of 3Hi. All data are expressed as mean values -L-_standard error of the mean (SEM). Statistica/significance of difference was evaluated by the paired Student's t-test. If not otherwise indicated, P
