Effect of ionophore RO Z-2985 on the efflux of calcium from the rat nephron HARRY 0. SENEKJIAN, THOMAS F. KNIGHT, ANN Renal Section, Department of Internal Medicine, Veterans and Baylor College of Medicine, Houston, Texas 77211
SENEKJIAN, HARRY O., THOMAS F. KNIGHT, ANN INCE, AND EDWARD J. WEINMAN. Effect of ionophore RO 2-2985 on the efflux of calcium from the rat nephron. Am. J. Physiol. 235(4): F381-F384, 1978 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 4(4): F381-F384, 1978. -The effect of the ionophore RO 2-2985 on the efflux of calcium from the renal tubule was studied employing the in vivo microinjection technique. Microinjection solutions contained either RO 2-2985 (E) or its diluent (C). Following microinjections into the early proximal tubule, urinary Wa recoveries averaged 10.1 t 1.9 (C) and 3.5 t 1.4% (E) (P < 0.005>, while recoveries averaged 32.3 t 6.9 (C) and 24.9 t 6.5% (E) (P < 0.05) following microinjections into the late proximal tubule. To determine if the decreased recovery of calcium was a specific effect, the effect of RO 2-2985 on the efIlux of sodium, phosphate, and 3-0methyl-n-glucose was examined. Compared to controls, RO 22985 did not affect the urinary recoveries of 22Na, [:S2P]orthophosphoric or 3-@methyl-n-[ 14C]glucose. acid, These studies demonstrate that RO 2-2985 enhances the efflux of calcium microinjected into the proximal portions of the rat nephron. tubular
permeability;
microinjection
IONOPHORES ARE A CLASS OF compounds which function as exogenous carriers of electrolytes across (10, 12, 14). This effect derives biologic membranes from the fact that these compounds form lipid-soluble complexes with charged electrolytes, thereby facilitating the transfer of the electrolytes. Several classes of ionophores have been identified and have been grouped based upon their structure and specificity for given cations (10, 14). One of these compounds, RO Z-2985 (also coded X-537A, Hoffmann-LaRoche, Nutley, N.J.), has been shown to complex with and enhance the transport of several cations as well as a number of biogenic amines (10-12, 14). The ability of RO 2-2985 to transport calcium across lipid membranes has been employed in a number of in vitro biologic systems. RO 2-2985 can produce muscle contractions and increase resting tension in skeletal, cardiac, and smooth muscle (5), induce release of adenine nucleotides and serotonin from platelets (6), and stimulate vasopressin release from the isolated rat neurohypophysis (7, 8). These effects have been attributed to ionophore-induced shifts of calcium across cell membranes and within intracellular compartments. Despite the use of RO 2-2985 in a variety of in vitro THE
INCE, AND Administration
EDWARD J. WEINMAN Hospital,
studies, only limited data are available on the effects of this agent on the renal transport of electrolytes. Since the systemic administration of these agents may alter renal function by virtue of changes in blood pressure or renal blood flow, or cause alterations in circulating regulatory hormones such as parathyroid hormone or vasopressin, it might be difficult to determine if ionophores directly affect the renal transport of electrolytes and nonelectrolytes (9, 11, 13). The current studies, therefore, were designed to examine the direct effects of RO 2-2985 on renal transport by employing the intratubular microinjection technique in the rat. METHODS
Male Sprague-Dawley rats weighing 200-450 g were used in all experiments. Anesthesia was induced by pentobarbital sodium, 50 mg/kg body wt, injected intraperitoneally. After a tracheostomy, both jugular veins and the urinary bladder were catheterized. The left kidney was decapsulated and prepared for micropuncture as previously described (16). The left ureter was cannulated with PE-50 tubing near the renal pelvis to permit separate urine collections from each kidney. Estimated surgical losses of fluid were replaced with a volume of isotonic saline equal to 1% of body wt. A solution of 5% mannitol in isotonic saline was infused intravenously throughout the experiment at a rate of 22 ml/h to ensure high urine flow rates. The microinjection solutions were prepared from isotonic saline to which 3-o -methyl-n-glucose (2.58 mM), calcium as CaC1, (2 mM), or phosphate as Na,HPO, (0.6 mM) was added. To the corresponding microinjection solutions were added: 3-@methyl-n-[methyl-14C]glucose (50 &i/ ml) (New England Nuclear, Boston), [45Ca]calcium chloride (67 &i/ml) (Amersham/Searle, Arlington Heights, Ill.), [32P]orthophosphoric acid (30 &i/ml) (New England Nuclear) or [22Na]sodium chloride (67 &i/ml) (Amershamlsearle). [methoxy-3H]Inulin (70 &i/ml) (New England Nuclear) was added to all microinjection solutions. The ionophore RO 2-2985, dissolved in dimethyl sulfoxide, was added to the microinjection solutions to a final concentration of 1.7 x lOwe M (experimental solution). Control microinjection solutions were prepared by adding an equal amount of the ionophore diluent to the microinjection solutions. Triplicate droplets of the test solutions of approximately 20 nanoliters each were prepared, one of which F381
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F382
SENEKJIAN,
was used as the microinjectate, and the other two were counted directly for total radioactivity. Microinjections were performed into either early or late portions of the proximal convoluted tubule or into the distal tubule over a 90- to 120-s interval. The rate of injection was controlled so that it was less than the tubular flow rate. The microinjections were performed under direct observation, and samples were discarded in the presence of dilatation of the tubule, obvious leakage of the injectate, retrograde flow, or obstruction to flow. Proximal tubules were localized by the injection of small droplets of oil into the tubular lumen and were considered to be an early portion of the proximal tubule if the oil droplet reappeared on the surface of the kidney in two or more successive loops. The injection site was considered to be a late portion of the proximal tubule if the oil droplet disappeared immediately below the surface of the kidney. Prior microdissection studies from this laboratory (16) have demonstrated that these procedures correspond to the initial third and distal third of the proximal convoluted tubule, respectively. Distal convoluted tubules were localized following the intravenous infusion of lissamine green dye. Following each microinjection, five 1-min and one 5-min urine collections were obtained from the left kidney. Urine was simultaneously collected from the right kidney over the same time interval. In any given animal, the recovery of only a single test isotope was examined. Microinjections were performed into the same nephron segments in each animal but previously unpunctured nephrons were used for each individual microinjection. Microinjections with control or ionophore-containing microinjectates vvere alternated. Individual microinjections having greater than 1% of the microinjected inulin appearing in the contralateral right kidney were assumed to have had undetected leakage at the microinjection site and were discarded from further analysis. This occurred in less than 5% of the microinjections and occurred with equal frequency with both the control and experimental microinjection solutions. Inulin recoveries were greater than 93% in all studies. In none of the studies were significant amounts of the test isotope recovered from the contralateral kidney. No corrections were required for delayed excretion. The results, therefore, are expressed as total recoveries and are calculated from the formula LHe
recovery
(%) =
KNIGHT,
INCE,
AND
WEINMAN
dpm,/dpm [:‘H]inulin in urine dpm,/dpm [:sH]inulin in injectate
x loo
where dpm are the disintegrations per minute of the test isotope x and of [:‘H]inulin. The radioactivity of urine and droplet solutions was determined in Biofluor (New England Nuclear) in a Packard Tri-Carb liquid scintillation counter (Packard Instrument Co., Downers Grove, Ill.), with appropriate corrections for quench and crossover counts. The recoveries of isotopes using control or ionophore-containing solutions were averaged for each animal and the results expressed as the mean of means t SE. Statistical significance was determined by the t test for paired data. RESULTS
The experiments were designed so that a given animal received alternate microinjections with solutions containing either the ionophore or the diluent alone. The results of control and experimental microinjections performed in each animal were averaged so that each animal served as its own control. The results of the calcium microinjections in each individual animal are shown in Fig. 1 and are summarized in Table 1. Following microinjections into the early proximal tubule, calcium recoveries averaged 10.1 t 1.9% with the control solution. The addition of the ionophore to the microinjection solution resulted in significantly lower rates of recovery in the urine of 3.5 t 1.4% (P < 0.005), indicating enhanced calcium efflux. Following microinjections into the late portions of the proximal convoluted tubule, recoveries averaged 32.3 t 6.9% with control solutions compared to 24.9 t 6.5% with the solution containing ionophores (P < 0.05). After microinjections into the distal convoluted tubule, calcium recoveries averaged 86.2 t 3.3 and 80.4 t 7.4% with control and ionophore solutions, respectively (P = NS). In order to examine the specificity of the effects of RO 2-2985 for calcium, additional studies were performed to examine its effects on the efflux of sodium, phosphate, and on the carrier-mediated transport of 3O-methyl-n-glucose. In these studies, microinjections were performed only into the early portion of the proximal convoluted tubule, and the results are summarized in Table 2. Sodium recoveries were 6.1 t 1.1% Pmdmd
Tubule loo
farly
Pmxlmal
Tubule
ejo- c
90 ZI B a
a
FIG. 1. Effect of ionophore RO 2-2985 on urinary recovery of calcium following microinjection into early proximal, late proximal, and distal tubules. Each point is mean of urinary recovery of calcium
for each animal tions.
00 !
following
control
(0 or experimental
(E) microinjec-
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 28, 2018. Copyright © 1978 the American Physiological Society. All rights reserved.
IONOPHORE
TABLE
following
RO 2-2985 AND
RENAL
HANDLING
1. urinary recoveries (%) of calcium intratubular microinjection
Microinjection
Site
Early proximal h = 9) Late proximal (n = 5)
tubule tubule
Distal convoluted (n = 6)
tubule
Control
3.5 * 1.4
SENEKJIAN, HARRY O., THOMAS F. KNIGHT, ANN INCE, AND EDWARD J. WEINMAN. Effect of ionophore RO 2-2985 on the efflux of calcium from the rat nephron. Am. J. Physiol. 235(4): F381-F384, 1978 or Am. J. Physiol.: Renal Fluid Electrolyte Physiol. 4(4): F381-F384, 1978. -The effect of the ionophore RO 2-2985 on the efflux of calcium from the renal tubule was studied employing the in vivo microinjection technique. Microinjection solutions contained either RO 2-2985 (E) or its diluent (C). Following microinjections into the early proximal tubule, urinary Wa recoveries averaged 10.1 t 1.9 (C) and 3.5 t 1.4% (E) (P < 0.005>, while recoveries averaged 32.3 t 6.9 (C) and 24.9 t 6.5% (E) (P < 0.05) following microinjections into the late proximal tubule. To determine if the decreased recovery of calcium was a specific effect, the effect of RO 2-2985 on the efIlux of sodium, phosphate, and 3-0methyl-n-glucose was examined. Compared to controls, RO 22985 did not affect the urinary recoveries of 22Na, [:S2P]orthophosphoric or 3-@methyl-n-[ 14C]glucose. acid, These studies demonstrate that RO 2-2985 enhances the efflux of calcium microinjected into the proximal portions of the rat nephron. tubular
permeability;
microinjection
IONOPHORES ARE A CLASS OF compounds which function as exogenous carriers of electrolytes across (10, 12, 14). This effect derives biologic membranes from the fact that these compounds form lipid-soluble complexes with charged electrolytes, thereby facilitating the transfer of the electrolytes. Several classes of ionophores have been identified and have been grouped based upon their structure and specificity for given cations (10, 14). One of these compounds, RO Z-2985 (also coded X-537A, Hoffmann-LaRoche, Nutley, N.J.), has been shown to complex with and enhance the transport of several cations as well as a number of biogenic amines (10-12, 14). The ability of RO 2-2985 to transport calcium across lipid membranes has been employed in a number of in vitro biologic systems. RO 2-2985 can produce muscle contractions and increase resting tension in skeletal, cardiac, and smooth muscle (5), induce release of adenine nucleotides and serotonin from platelets (6), and stimulate vasopressin release from the isolated rat neurohypophysis (7, 8). These effects have been attributed to ionophore-induced shifts of calcium across cell membranes and within intracellular compartments. Despite the use of RO 2-2985 in a variety of in vitro THE
INCE, AND Administration
EDWARD J. WEINMAN Hospital,
studies, only limited data are available on the effects of this agent on the renal transport of electrolytes. Since the systemic administration of these agents may alter renal function by virtue of changes in blood pressure or renal blood flow, or cause alterations in circulating regulatory hormones such as parathyroid hormone or vasopressin, it might be difficult to determine if ionophores directly affect the renal transport of electrolytes and nonelectrolytes (9, 11, 13). The current studies, therefore, were designed to examine the direct effects of RO 2-2985 on renal transport by employing the intratubular microinjection technique in the rat. METHODS
Male Sprague-Dawley rats weighing 200-450 g were used in all experiments. Anesthesia was induced by pentobarbital sodium, 50 mg/kg body wt, injected intraperitoneally. After a tracheostomy, both jugular veins and the urinary bladder were catheterized. The left kidney was decapsulated and prepared for micropuncture as previously described (16). The left ureter was cannulated with PE-50 tubing near the renal pelvis to permit separate urine collections from each kidney. Estimated surgical losses of fluid were replaced with a volume of isotonic saline equal to 1% of body wt. A solution of 5% mannitol in isotonic saline was infused intravenously throughout the experiment at a rate of 22 ml/h to ensure high urine flow rates. The microinjection solutions were prepared from isotonic saline to which 3-o -methyl-n-glucose (2.58 mM), calcium as CaC1, (2 mM), or phosphate as Na,HPO, (0.6 mM) was added. To the corresponding microinjection solutions were added: 3-@methyl-n-[methyl-14C]glucose (50 &i/ ml) (New England Nuclear, Boston), [45Ca]calcium chloride (67 &i/ml) (Amersham/Searle, Arlington Heights, Ill.), [32P]orthophosphoric acid (30 &i/ml) (New England Nuclear) or [22Na]sodium chloride (67 &i/ml) (Amershamlsearle). [methoxy-3H]Inulin (70 &i/ml) (New England Nuclear) was added to all microinjection solutions. The ionophore RO 2-2985, dissolved in dimethyl sulfoxide, was added to the microinjection solutions to a final concentration of 1.7 x lOwe M (experimental solution). Control microinjection solutions were prepared by adding an equal amount of the ionophore diluent to the microinjection solutions. Triplicate droplets of the test solutions of approximately 20 nanoliters each were prepared, one of which F381
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 28, 2018. Copyright © 1978 the American Physiological Society. All rights reserved.
F382
SENEKJIAN,
was used as the microinjectate, and the other two were counted directly for total radioactivity. Microinjections were performed into either early or late portions of the proximal convoluted tubule or into the distal tubule over a 90- to 120-s interval. The rate of injection was controlled so that it was less than the tubular flow rate. The microinjections were performed under direct observation, and samples were discarded in the presence of dilatation of the tubule, obvious leakage of the injectate, retrograde flow, or obstruction to flow. Proximal tubules were localized by the injection of small droplets of oil into the tubular lumen and were considered to be an early portion of the proximal tubule if the oil droplet reappeared on the surface of the kidney in two or more successive loops. The injection site was considered to be a late portion of the proximal tubule if the oil droplet disappeared immediately below the surface of the kidney. Prior microdissection studies from this laboratory (16) have demonstrated that these procedures correspond to the initial third and distal third of the proximal convoluted tubule, respectively. Distal convoluted tubules were localized following the intravenous infusion of lissamine green dye. Following each microinjection, five 1-min and one 5-min urine collections were obtained from the left kidney. Urine was simultaneously collected from the right kidney over the same time interval. In any given animal, the recovery of only a single test isotope was examined. Microinjections were performed into the same nephron segments in each animal but previously unpunctured nephrons were used for each individual microinjection. Microinjections with control or ionophore-containing microinjectates vvere alternated. Individual microinjections having greater than 1% of the microinjected inulin appearing in the contralateral right kidney were assumed to have had undetected leakage at the microinjection site and were discarded from further analysis. This occurred in less than 5% of the microinjections and occurred with equal frequency with both the control and experimental microinjection solutions. Inulin recoveries were greater than 93% in all studies. In none of the studies were significant amounts of the test isotope recovered from the contralateral kidney. No corrections were required for delayed excretion. The results, therefore, are expressed as total recoveries and are calculated from the formula LHe
recovery
(%) =
KNIGHT,
INCE,
AND
WEINMAN
dpm,/dpm [:‘H]inulin in urine dpm,/dpm [:sH]inulin in injectate
x loo
where dpm are the disintegrations per minute of the test isotope x and of [:‘H]inulin. The radioactivity of urine and droplet solutions was determined in Biofluor (New England Nuclear) in a Packard Tri-Carb liquid scintillation counter (Packard Instrument Co., Downers Grove, Ill.), with appropriate corrections for quench and crossover counts. The recoveries of isotopes using control or ionophore-containing solutions were averaged for each animal and the results expressed as the mean of means t SE. Statistical significance was determined by the t test for paired data. RESULTS
The experiments were designed so that a given animal received alternate microinjections with solutions containing either the ionophore or the diluent alone. The results of control and experimental microinjections performed in each animal were averaged so that each animal served as its own control. The results of the calcium microinjections in each individual animal are shown in Fig. 1 and are summarized in Table 1. Following microinjections into the early proximal tubule, calcium recoveries averaged 10.1 t 1.9% with the control solution. The addition of the ionophore to the microinjection solution resulted in significantly lower rates of recovery in the urine of 3.5 t 1.4% (P < 0.005), indicating enhanced calcium efflux. Following microinjections into the late portions of the proximal convoluted tubule, recoveries averaged 32.3 t 6.9% with control solutions compared to 24.9 t 6.5% with the solution containing ionophores (P < 0.05). After microinjections into the distal convoluted tubule, calcium recoveries averaged 86.2 t 3.3 and 80.4 t 7.4% with control and ionophore solutions, respectively (P = NS). In order to examine the specificity of the effects of RO 2-2985 for calcium, additional studies were performed to examine its effects on the efflux of sodium, phosphate, and on the carrier-mediated transport of 3O-methyl-n-glucose. In these studies, microinjections were performed only into the early portion of the proximal convoluted tubule, and the results are summarized in Table 2. Sodium recoveries were 6.1 t 1.1% Pmdmd
Tubule loo
farly
Pmxlmal
Tubule
ejo- c
90 ZI B a
a
FIG. 1. Effect of ionophore RO 2-2985 on urinary recovery of calcium following microinjection into early proximal, late proximal, and distal tubules. Each point is mean of urinary recovery of calcium
for each animal tions.
00 !
following
control
(0 or experimental
(E) microinjec-
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on November 28, 2018. Copyright © 1978 the American Physiological Society. All rights reserved.
IONOPHORE
TABLE
following
RO 2-2985 AND
RENAL
HANDLING
1. urinary recoveries (%) of calcium intratubular microinjection
Microinjection
Site
Early proximal h = 9) Late proximal (n = 5)
tubule tubule
Distal convoluted (n = 6)
tubule
Control
3.5 * 1.4
