669
J. Physiol. (1975), 252, pp. 669-679 With 2 text-figures Printed in Great Britain
SENSITIVE OSMOMETER FUNCTION OF
JUXTAGLOMERULAR CELLS IN VITRO BY 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER* From the University Institute for Experimental Medicine,t 71 N0rre Alle', Copenhagen, Denmark
(Received 21 March 1975) SUMMARY
1. The rate of renin release from viable juxtaglomerular cells was studied during prolonged superfusion of isolated rat renal glomeruli with Ringer solutions of differing osmolarities. 2. Reduction in osmolarity from 305 to 285 m-osmole/l. by lowering sucrose concentration caused renin release rate to double. A rise in osmolarity of 30 m-osmole/l. by raising sucrose concentration halved release rate. 3. The response to osmolarity was graded. During the first 30 min following a 20 m-osmole/l. decrease in osmolarity, 1*57 + 0.22 % (s.E. of mean) of cellular renin content was released; three times this amount was released with a decrease of 50 m-osmole/l. The effect persisted at lower release rates for 60-90 min. 4. The juxtaglomerular cells were four to five times more sensitive to changes in osmolarity through sucrose than sodium chloride concentration. Changes in potassium chloride concentration (7-57 mM) had little effect. 5. Sodium chloride had no direct ionic effect on renin release outside its osmotic properties. 6. The findings support a previous proposal that the rate of renin release in vitro relates directly to the volume of the juxtaglomerular cell. The hypothesis is developed that a similar mechanism may underlie renin secretion in vivo. INTRODUCTION
Recently, we developed a technique that permits the study of renin release from superfused, metabolically active juxtaglomerular cells prepared free from neural, vascular and tubular influences (Blendstrup, Visiting Scientist, from the University of Melbourne, Australia. t Jens Bing, Director. *
670 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER Leyssac, Poulsen & Skinner, 1975). Prepared in this way, juxtaglomerular cells release renin at physiological rates over many hours. Because renin release was increased by reduction in temperature and by cyanideiodoacetate, it was concluded that retention of stored renin by the cell requires energy and that renin release may be controlled physiologically by mechanisms which regulate the volume of the juxtaglomerular cell. The present work tests and supports this proposal. The juxtaglomerular cells are revealed to be sensitive osmometers which release renin in response to changes in superfusate sucrose, sodium and potassium concentrations in a manner consistent with the anticipated reflexion coefficients of these solutes at the membranes of viable cells. METHODS
Batches of 400 microscopically selected rat renal glomeruli (five batches per experiment in fifteen experiments) were prepared by the magnetic iron-oxide method of Cook & Pickering (1959) and superfused in five polyethylene lines with CO2bicarbonate or phosphate Ringer solutions for periods up to 5 hr as previously described (Blendstrup et al. 1975). The glomeruli with contained juxtaglomerular cells were held in the superfusion lines by a magnetic field and were superfused at a rate of 140 ,sl./30 min from one of two infusion machines. This arrangement permitted changes of superfusate composition without disturbance to the glomeruli, a point of some importance since renin release was increased by movement (Blendstrup et al. 1975). The dead space time of the system was 10 min. Superfusate was collected over 30 min periods and renin was assayed in 25 /A. volumes by radioimmunoassay of generated angiotensin I during incubations with homologous renin substrate. Renin content of the batches of glomeruli was assayed at the end of the experiment after extraction by freezing and thawing. Renin is expressed in terms of the Goldblatt hog unit (M.R.C., Holly Hill, London). Descriptions of all these techniques with investigations of their limitations have been published elsewhere (Blendstrup et al. 1975). The composition of the solution in m-mole/l. in which the glomeruli were prepared and initially superfused was: NaCl, 91 0; NaHCO3 17-5; KCl, 7 0; CaCl2, 2-0; MgSO4, 12; NaH2PO4, 1-2; glucose, 11 0; sucrose, 50 0. The solution was saturated with 5% CO2 and 95% 02. The pH was 7 4. This sodium concentration (11O mM) is slightly lower than physiological but sucrose (50 mM) was added to raise osmolarity to the normal level (305 m-osmole/l.). These solute concentrations were used to prevent swelling of the cells during preparation of the glomeruli. Compared with solutions containing 135 mm sodium and 305 m-osmole/l., it has been shown that the content of renin in the juxtaglomerular cell is not altered but that renin release rate is lower with the lower sodium-higher sucrose concentration (Blendstrup et al. 1975). Osmolarity was adjusted as indicated in results by altering the concentrations of sucrose, sodium or potassium. Direct estimation of osmolarity was not performed and ideal osmolarity was calculated from molar solute concentrations. When altering solute concentrations the assumption was made that sucrose would be reflected to a greater extent than either sodium or potassium at the juxtaglomerular cell membrane. Consequently the colligative properties of the solutes were not immediately relevant and of more importance was the presumed ability of individual solutes to influence water movement across the juxtaglomerular cell membrane.
OSMOMETER FUNCTION OF JUXTAGLOMERULAR CELLS 671 RESULTS
Fig. 1 shows for a particular experiment, the manner in which all experiments were performed and also illustrates the important findings. The first superfusion line was used as a control and demonstrates the previously documented fall in release with time (Blendstrup et al. 1975). The total amount released during 4-5 hr in this control line was less than 3 % of the amount initially present. The glomeruli in other lines were subjected to changes in superfusate sodium and sucrose concentrations at times indicated by the vertical lines. By conducting the experiment in this way, controls were obtained both within and between the different experiments. In the second superfusion line, on lowering sucrose concentration so as to reduce osmolarity by 25 m-osmole/l. (from 305 to 280) without change in sodium concentration, renin release was increased 160 % above the immediate preceding level. This increase persisted at lesser levels for 90 min up to the time when the original superfusate was re-introduced, whereupon release was suppressed by 70 % from the last value at 280 m-osmole/l. Following two control periods the lower osmolarity superfusate was again introduced and there was a 133 % increase in release from the now lower level of release. Comparison must be made with the top histogram which shows the expected fall in release in the absence of any change in superfusate composition. The quantity of renin released during the first 30 min period following introduction of the hypotonic superfusate was 2-35 % of the initial content. In the immediate preceding control period, 0-92 % of content was released. The third superfusion line was a repeat of this sequence but with a fall in osmolar concentration by 50 m-osmole/l. Again the effect was not sustained, but more renin was released at both the first (+ 550 %) and second (+ 330 %) introduction of the solution. The response to osmolarity was therefore graded, and reproducible but not sustained. The fourth superfusion line shows the effect of raising the sodium concentration from 110 to 135 mm at unchanged osmolarity. This was accomplished by reducing the sucrose concentration by 50 mm and increasing sodium chloride by 25 mm. Renin release increased on both presentations of the solution (+ 208 and + 150 %). Release was however less than that provoked by an osmolarity reduction of 50 m-osmole/l. in the absence of a change in sodium chloride (i.e. reduced sucrose concentration as in the third superfusion line). Superfusion Line 5 reveals that the release seen in Line 4 is related to the requirement of removing sucrose in order to maintain osmolarity constant; when osmolarity was allowed to rise together with sodium chloride concentration, renin release was not provoked. Indeed in Line 5, release (+ 250 %) only occurred on changing from the 135 mm sodium,
672 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER Content 2 1101~10 110 2 3051110 3051 3051 3051 (4uunits) Final Initial 1 1150
4 110 110 3 1 305K
280
154
110 110 305 280
._
@1 0
140 153
0
-s0
8 110 110 305 7
E
6 S 4 3 2 I
f" 0
IS-
0 0
0 ._
CD
0 0
255
110 110 305 255
-60
A
0 4J
75
4 110 135 110 135 305 305 305 305 3 ISO
2
2 110305
J. Physiol. (1975), 252, pp. 669-679 With 2 text-figures Printed in Great Britain
SENSITIVE OSMOMETER FUNCTION OF
JUXTAGLOMERULAR CELLS IN VITRO BY 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER* From the University Institute for Experimental Medicine,t 71 N0rre Alle', Copenhagen, Denmark
(Received 21 March 1975) SUMMARY
1. The rate of renin release from viable juxtaglomerular cells was studied during prolonged superfusion of isolated rat renal glomeruli with Ringer solutions of differing osmolarities. 2. Reduction in osmolarity from 305 to 285 m-osmole/l. by lowering sucrose concentration caused renin release rate to double. A rise in osmolarity of 30 m-osmole/l. by raising sucrose concentration halved release rate. 3. The response to osmolarity was graded. During the first 30 min following a 20 m-osmole/l. decrease in osmolarity, 1*57 + 0.22 % (s.E. of mean) of cellular renin content was released; three times this amount was released with a decrease of 50 m-osmole/l. The effect persisted at lower release rates for 60-90 min. 4. The juxtaglomerular cells were four to five times more sensitive to changes in osmolarity through sucrose than sodium chloride concentration. Changes in potassium chloride concentration (7-57 mM) had little effect. 5. Sodium chloride had no direct ionic effect on renin release outside its osmotic properties. 6. The findings support a previous proposal that the rate of renin release in vitro relates directly to the volume of the juxtaglomerular cell. The hypothesis is developed that a similar mechanism may underlie renin secretion in vivo. INTRODUCTION
Recently, we developed a technique that permits the study of renin release from superfused, metabolically active juxtaglomerular cells prepared free from neural, vascular and tubular influences (Blendstrup, Visiting Scientist, from the University of Melbourne, Australia. t Jens Bing, Director. *
670 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER Leyssac, Poulsen & Skinner, 1975). Prepared in this way, juxtaglomerular cells release renin at physiological rates over many hours. Because renin release was increased by reduction in temperature and by cyanideiodoacetate, it was concluded that retention of stored renin by the cell requires energy and that renin release may be controlled physiologically by mechanisms which regulate the volume of the juxtaglomerular cell. The present work tests and supports this proposal. The juxtaglomerular cells are revealed to be sensitive osmometers which release renin in response to changes in superfusate sucrose, sodium and potassium concentrations in a manner consistent with the anticipated reflexion coefficients of these solutes at the membranes of viable cells. METHODS
Batches of 400 microscopically selected rat renal glomeruli (five batches per experiment in fifteen experiments) were prepared by the magnetic iron-oxide method of Cook & Pickering (1959) and superfused in five polyethylene lines with CO2bicarbonate or phosphate Ringer solutions for periods up to 5 hr as previously described (Blendstrup et al. 1975). The glomeruli with contained juxtaglomerular cells were held in the superfusion lines by a magnetic field and were superfused at a rate of 140 ,sl./30 min from one of two infusion machines. This arrangement permitted changes of superfusate composition without disturbance to the glomeruli, a point of some importance since renin release was increased by movement (Blendstrup et al. 1975). The dead space time of the system was 10 min. Superfusate was collected over 30 min periods and renin was assayed in 25 /A. volumes by radioimmunoassay of generated angiotensin I during incubations with homologous renin substrate. Renin content of the batches of glomeruli was assayed at the end of the experiment after extraction by freezing and thawing. Renin is expressed in terms of the Goldblatt hog unit (M.R.C., Holly Hill, London). Descriptions of all these techniques with investigations of their limitations have been published elsewhere (Blendstrup et al. 1975). The composition of the solution in m-mole/l. in which the glomeruli were prepared and initially superfused was: NaCl, 91 0; NaHCO3 17-5; KCl, 7 0; CaCl2, 2-0; MgSO4, 12; NaH2PO4, 1-2; glucose, 11 0; sucrose, 50 0. The solution was saturated with 5% CO2 and 95% 02. The pH was 7 4. This sodium concentration (11O mM) is slightly lower than physiological but sucrose (50 mM) was added to raise osmolarity to the normal level (305 m-osmole/l.). These solute concentrations were used to prevent swelling of the cells during preparation of the glomeruli. Compared with solutions containing 135 mm sodium and 305 m-osmole/l., it has been shown that the content of renin in the juxtaglomerular cell is not altered but that renin release rate is lower with the lower sodium-higher sucrose concentration (Blendstrup et al. 1975). Osmolarity was adjusted as indicated in results by altering the concentrations of sucrose, sodium or potassium. Direct estimation of osmolarity was not performed and ideal osmolarity was calculated from molar solute concentrations. When altering solute concentrations the assumption was made that sucrose would be reflected to a greater extent than either sodium or potassium at the juxtaglomerular cell membrane. Consequently the colligative properties of the solutes were not immediately relevant and of more importance was the presumed ability of individual solutes to influence water movement across the juxtaglomerular cell membrane.
OSMOMETER FUNCTION OF JUXTAGLOMERULAR CELLS 671 RESULTS
Fig. 1 shows for a particular experiment, the manner in which all experiments were performed and also illustrates the important findings. The first superfusion line was used as a control and demonstrates the previously documented fall in release with time (Blendstrup et al. 1975). The total amount released during 4-5 hr in this control line was less than 3 % of the amount initially present. The glomeruli in other lines were subjected to changes in superfusate sodium and sucrose concentrations at times indicated by the vertical lines. By conducting the experiment in this way, controls were obtained both within and between the different experiments. In the second superfusion line, on lowering sucrose concentration so as to reduce osmolarity by 25 m-osmole/l. (from 305 to 280) without change in sodium concentration, renin release was increased 160 % above the immediate preceding level. This increase persisted at lesser levels for 90 min up to the time when the original superfusate was re-introduced, whereupon release was suppressed by 70 % from the last value at 280 m-osmole/l. Following two control periods the lower osmolarity superfusate was again introduced and there was a 133 % increase in release from the now lower level of release. Comparison must be made with the top histogram which shows the expected fall in release in the absence of any change in superfusate composition. The quantity of renin released during the first 30 min period following introduction of the hypotonic superfusate was 2-35 % of the initial content. In the immediate preceding control period, 0-92 % of content was released. The third superfusion line was a repeat of this sequence but with a fall in osmolar concentration by 50 m-osmole/l. Again the effect was not sustained, but more renin was released at both the first (+ 550 %) and second (+ 330 %) introduction of the solution. The response to osmolarity was therefore graded, and reproducible but not sustained. The fourth superfusion line shows the effect of raising the sodium concentration from 110 to 135 mm at unchanged osmolarity. This was accomplished by reducing the sucrose concentration by 50 mm and increasing sodium chloride by 25 mm. Renin release increased on both presentations of the solution (+ 208 and + 150 %). Release was however less than that provoked by an osmolarity reduction of 50 m-osmole/l. in the absence of a change in sodium chloride (i.e. reduced sucrose concentration as in the third superfusion line). Superfusion Line 5 reveals that the release seen in Line 4 is related to the requirement of removing sucrose in order to maintain osmolarity constant; when osmolarity was allowed to rise together with sodium chloride concentration, renin release was not provoked. Indeed in Line 5, release (+ 250 %) only occurred on changing from the 135 mm sodium,
672 0. FREDERIKSEN, P. P. LEYSSAC AND S. L. SKINNER Content 2 1101~10 110 2 3051110 3051 3051 3051 (4uunits) Final Initial 1 1150
4 110 110 3 1 305K
280
154
110 110 305 280
._
@1 0
140 153
0
-s0
8 110 110 305 7
E
6 S 4 3 2 I
f" 0
IS-
0 0
0 ._
CD
0 0
255
110 110 305 255
-60
A
0 4J
75
4 110 135 110 135 305 305 305 305 3 ISO
2
2 110305
