J. Phyeiol. (1977), 273, pp. 745-764 With 6 text-ftgure8 Printed in Great Britain

745

STUDIES ON THE MECHANISM OF RENIN RELEASE FROM ISOLATED SUPERFUSED RAT GLOMERULI: EFFECTS OF CALCIUM, CALCIUM IONOPHORE AND LANTHANUM

BY L. BAUMBACH AND P. P. LEYSSAC From the Univer8ity Institute for Experimental Medicine, 71 Norre Alle, 2100 Copenhagen 0, Denmark

(Received 2 May 1977) SUMMARY

1. The effects of external medium calcium concentration, the ionophore A23187 and lanthanum on the rate of renin release in vitro were studied with particular emphasis on results obtained from isolated superfused glomeruli of rat kidneys. 2. The response to reduction in superfusate calcium concentration from 2 mm was a graded and reversible increase in the rate of renin release. An increase in release was detectable at 02 mm calcium; a threefold increase was found 36 mm after a change from 2 mm calcium to calcium-free superfusate. A similar relative increase in release resulted from reductions from 0-1 mm to zero calcium, but the absolute amounts of renin released were greater in this latter series. Renin release from kidney cortical slices similarly increased in response to calcium-free incubation medium. 3. The effects of A23,87 on renin release were modest. Changing from 2 mm calcium during control periods to calcium-free Ringer with A23187 added caused an attenuated and more delayed increase in release than the change to calcium-free Ringer without ionophore. This difference in response was abolished when glomeruli were superfused with 0.1 mM calcium during the preceding 1 hr control period. There was no significant difference in renin release from glomeruli exposed to calcium-free EGTARinger with and without A23187 in the 2 mm calcium series; in the 0.1 mm calcium series the increase in release following a shift to calcium-free EGTA-containing superfusate with A23187 added was significantly greater than in the absence of the ionophore. 4. Addition of lanthanum (1 or 0 05 mM) to calcium-containing as well as calcium-free superfusate resulted in a significant depression of renin Some of the present data were presented in preliminary form at the VIth Int. Nephrol. Congress, Florence, 1975. Abstracts free communications no. 518.

L. BAUMBACH AND P. P. LEYSSAC release. Subsequent removal of the lanthanum did not restore the rate of release unless EGTA was added; in the latter case a massive increase in renin release occurred resulting in a marked depletion of the remaining renin content of the glomeruli. 5. It is concluded that calcium influences renin release by a direct action on the juxtaglomerular cells. The data support the previous suggestion that basal renin release is a function of active, calcium-dependent cell volume regulation - swelling causing an increase in the release; and further suggest that membrane-bound calcium has a direct effect on the cell membrane permeability to renin. 6. The results exclude that calcium-stimulated exocytosis is responsible for basal renin release from the juxtaglomerular cells adhering to isolated glomeruli. 746

INTRODUCTION

Calcium stimulated exocytosis has been established as a secretary mechanism for export of a variety of enzymes, hormones and transmitter substances used by endocrine as well as exocrine secretary cells. Following its first suggestion based on evidence obtained from adrenal chromaffin cells (Douglas & Rubin, 1961) and salivary exocrine secretion (Douglas & Poisner, 1963) such a mechanism has been demonstrated in an increasing number of secretary systems leading to the concept that calcium-activated exocytosis is the key to stimulus-secretion coupling generally (Douglas, 1974). Renin is a proteolytic enzyme synthetized by and stored in the granules of the juxtaglomerular cells which are located in the media of the glomerular end mainly of the afferent arteriole. Renin is secreted presumably into the vascular lumen, to a large extent at least, in response to a variety of stimuli both from the macula densa and the vascular side of the system, including stimuli from the sympathetic nervous system (cf. review by Davis & Freeman, 1976). It might therefore be anticipated that calciumstimulated exocytosis is the mechanism of secretion used also by the juxtaglomerular cells. Results obtained from kidney cortical slices in vitro on the effect of calcium on renin release have been conflicting; some investigators have reported a stimulating effect of calcium (e.g. Michelakis 1971; Yamamoto, Iwao, Abe & Morimoto, 1974), while others found no effect (Aoi, Wade, Rosner & Weinberger 1974; Saruta & Matsuki, 1975). The reason(s) for these conflicting results remain unsolved, but differences in pre-incubation conditions, medium composition, slice thickness, etc. may well influence the results of kidney cortex slice experiments. In contrast to the disagreement between these in vitro observations, results obtained in studies on

Ca AND La ON RENIN RELEASE 747 renin release from isolated perfused rat kidneys by Peart and his colleagues (Vandongen & Peart, 1974a, b; Logan, Tenyi, Quesada, Peart, Breathnach & Martin, 1975) and. from in vivo studies in dogs (Kotchen, Mauli, Luke, Rees & Flamenbaum, 1974; Watkins, Davis, Lohmeier & Freeman, 1976) are consistent in the finding that elevated calcium concentration inhibits basal (resting) renin release, while calcium-free perfusion elevates basal release. In addition there is agreement that calcium-free perfusion fluid does not abolish or even attenuate isoprenaline-stimulated renin release from isolated perfused whole kidneys of either rat or rabbit (Vandongen & Peart, 1974b; Viskoper, Rosenfeld, Maxwell, DeLima, Lupu & Rosenfeld, 1976), while on the other hand calcium is required for the inhibitory action of angiotensin on isoprenaline-stimulated release (Vandongen & Peart, 1974 b). These results and the demonstration ofreduced renin release during intrarenal calcium-infusion in dogs with non-filtering kidneys (Watkins et al. 1976) suggest, first, a vascular site of action of calcium, possibly a direct effect on the juxtaglomerular cells; and secondly a mechanism of secretion distinct from exocytosis. However, indirect effects cannot be excluded in studies on intact kidneys. Previous studies have indicated that a preparation of viable isolated, superfused glomeruli is a useful system for studies on the cellular mechanisms of renin release from juxtaglomerular cells. This preparation is also suitable for answering questions about the direct or indirect nature of action on the juxtaglomerular cells of various known stimuli, because of the absence of influences from either tubular, neural or vascular smoothmuscle tissue elements and extra-renal factors (Blendstrup, Leyssac, Poulsen & Skinner, 1975; Frederiksen, Leyssac & Skinner, 1975; Baumbach, Leyssac & Skinner, 1976; Morris, Nixon & Johnston, 1976). In the present investigation we have utilized the advantages of such a preparation of isolated glomeruli in an attempt to establish whether or not the effect of calcium observed in isolated perfused kidneys and in kidneys in vivo is a direct effect on the juxtaglomerular cells; and, further, to define more precisely the possible mechanisms of the calcium action in these cells. The results, which are in perfect agreement with those obtained in intact kidneys, indicate that calcium has a direct action on the juxtaglomerular cells influencing the release of renin by a mechanism entirely different from exocytosis. METHODS

I8Olae glomeruli Batches of 300, 400 or 600 microscopically selected rat glomeruli (five batches per rat) were prepared by the magnetic iron oxide technique of Cook & Pickering (1959) and superfused with Ringer solutions in five polyethylene tubings as previously described in detail (Blendstrup et al. 1975). The glomeruli with the attached end of

748

L. BAUMBACH AND P. P. LEYSSAC

the afferent vessel containing some of the juxtaglomerular cells were held in the superfusion lines by a magnetic field (magnetic flux 0-0030 Wb) during superfusion at a rate of 10 ul./min from one of two or three constant perfusion pumps. This arrangement permitted abrupt changes in the composition of the superfusate during an experiment without detectable disturbances to the glomeruli, and allowed each line to serve as its own control. In addition one of the five lines usually served as a control line throughout the entire experiment by maintaining medium composition constant when changing from one perfusion pump to another. In most experiments glomeruli were prepared in, and superfused during control periods with, a bicarbonate-Ringer, the composition of which was (m-mole/l.): NaCl, 101-0; NaHCO., 17-5: KC1, 7-0; CaCl2, 2-0; MgSO4, 1-2; NaH2PO4, 1-2; glucose, 11-0; and sucrose, 34-0; giving an ideal calculated osmolality of 305 mosmole/kg. The Ringer was adjusted to and maintained at pH 7-4 by bubbling with 5 % CO2 and 95 % 02 at 37 0C. In the A23187 series the ionophore, dissolved in 96 % ethanol, was added to calcium-free bicarbonate-Ringer to give a final concentration of 17 x 10-6 M-A2.7 and 1 % (vol./vol.) of ethanol. In these experiments the control superfusate also contained 1 % (vol./vol.) of ethanol. In the lanthanum series the control medium was a Tris-Ringer of the following composition (m-mole/l.): NaCl, 120-0; KC1, 7-0; C0Cl2, 2-0; MgCl2, 1P2; Tris, 1-0; glucose, 11 0; and sucrose, 34-0; giving an osmolality of 305 m-osmolejkg. The experimental superfusate in these series contained in addition 1-0 mm-LaCl3, except for a few experiments in which a LaCl3 concentration of 0-05 mm was used. Tris-Ringers were adjusted to pH 7-4 and aerated with 100% 02. When ethylene-glycol-2-(-2 aminoethyl)-tetracetic acid (EGTA) was added to the calcium-free superfusion buffers a final concentration of 0-5 mx-EGTA was used and no corrections were made for the resulting change in total osmolality. In all other cases, when the ionic composition of the Ringer was altered, the total sodium concentration was maintained at 120-0 mm and the osmolality kept constant by appropriate adjustments of the sucrose concentration. Molal solute concentrations were checked by direct measurements of the osmolality by the cryoscopic method of Ramsay & Brown (1955); the measured osmolality agreed within 5 m-osmole/kg with that calculated. Superfusate was collected over 12 min periods and renin assayed in 25 ul. volumes by radioimmunoassay of generated angiotensin- 1, using the 'trapping' technique of Poulsen & J0rgensen (1974). The remaining renin content of the batches was assayed at the end of the experiment after extraction by freezing and thawing three times (Blendstrup et al. 1975). Renin is expressed in terms of G}oldblatt hog units (M.R.C., Holly Hill, London). Detection limit of the assay was 10-7 Goldblatt units contained in 25 #1. In a few experiments of each series a millipore filter (0-5 um) was inserted between the polythylene tubing containing the glomeruli retained by the magnetic field and the collection tube in order to test whether or not loosened juxtaglomerular cells or single glomeruli were responsible for the increased renin concentration measured in superfusate collected during calcium-free conditions. The interposition of the filters did not influence the results. All experiments were performed at a temperature of 30 'C. When glomeruli were exposed to changes in superfusate composition these changes were introduced after approximately 60 min of superfusion with the control buffer, and the moment of change referred to as zero time.

Ca AND La ON RENIN RELEASE

749

Kidney cortical 81wie8 For preparation of cortical slices both kidneys were rapidly removed from amytalanaesthetized rats (300-400 g body weight) and immediately placed in ice-cold control bicarbonate-Ringer solution. Two cortex slices, about 0-2-0-4 mm in thickness, were cut by hand with a razor blade from each kidney, and each slice was placed in an incubation flask containing 500 ,ul. of control bicarbonate-Ringer and kept at 0 0C until incubation which started within 20 min from removal of the kidneys. The experiments included four consecutive 30 min incubation periods. Calciumfree Ringer solution was introduced in the third period, and control Ringer reintroduced again in the fourth period. At the end of each 30 min period the supernatant was removed as completely as possible and replaced by 500 jl. of fresh Ringer solution. The supernatant was stored at -20 TC until assay. Slices were incubated at 37 'C during gentle shaking of the incubation flasks kept submerged in a thermostatically controlled water bath. At the end of the experiment the renin content remaining in each slice was assayed after extraction by freezing and thawing 3 times. The composition of the bicarbonate-Ringer solutions used for the cortical slice experiments was identical to that described above for superfusion of isolated glomeruli. The changes in renin release from cortical slices are expressed relative to the value obtained in the last incubation period preceding the change in medium calcium concentration (i.e. during the second period of incubation). The absolute values of supernatant renin content are corrected for differences in initial renin content of the slices due to differences in size and/or thickness. The initial renin content of each slice was calculated as the sum of the total amount of renin released during the experiment and that remaining in the tissue slice at the end of the experiment. Sktietiail analysis For the statistical analysis, the absolute value of renin release in the last control period preceding the zero time was used as the control value for the individual experiment and was ascribed the value of 100 %. Significance of changes in renin release were estimated by Student's t test. RESULTS

As previously observed (Baumbach et al. 1976) basal (or resting) renin release from isolated glomeruli was gradually but slowly decreasing spontaneously over a period of more than 2 hr during superfusion with control bicarbonate Ringer. Some differences in Ringer composition used for superfusion in the control periods between the various series were inevitable in the present study. Thus, for the calcium series the normal control bicarbonate-Ringer was used. For the ionophore series the control bicarbonate-Ringer contained 1 % (vol./vol.) of ethanol, while for the lanthanum series the control superfusate was a Tris-Ringer containing 2 mm calcium. Preliminary control experiments revealed no detectable differences either in absolute rates of release or in the pattern of release with time between glomeruli superfused with these buffer media; nor were any changes in renin release observed after changing from one of these superfusates to the other during an experiment.

L. BAUMBACH AND P. P. LEYSSAC

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The effect of lowering calcium concentration (a) Isolated glomeruli. The effects of graded reduction in superfusate calcium concentration on renin release from isolated glomeruli are given in Fig. 1. Shifting from the control Ringer containing 2 mm calcium 600

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Time (min) Fig. 1. Effects on renin release from isolated glomeruli in response to changes in the calcium concentration of bicarbonate-Ringer. Control Ringer contained 2 mm calcium. Experimental superfusate contained calcium ions: 0-3 mM (@, n = 8); 0-2 mm (A, n = 7); 0-1 mm (0 n = 10); 0 mm (a, n = 9). The dashed line indicates the decrease in basal release with time in control experiments (calcium 2 mm, n = 19). Bars indicate + s.E. of means.

resulted in a stimulation of renin release in a concentration-dependent pattern. Thus, reduction of the calcium concentration to 0-3 mm did not significantly change the release (90 % at 36 min; P > 0.1) although there

751 Ca AND La ON RENIN RELEASE was a tendency to attenuate the decrease in release with time observed in the continued presence of 2 mm calcium. Changing the calcium concentration down to 0*2 mm caused a small increase in release (to 133 % at 36 min), a difference on the borderline of significance from that recorded after 36 min of superfusion with 2 mm calcium in control experiments (dashed line in Fig. 1). A substantial elevation in renin release resulted from reduction in external calcium concentration to 0 1 mm (to 195 % at 36 min; P < 0-001), and even more renin was released when calcium was omitted from the medium (267 % at 36 min; P < 0.005). No significant additional increase in renin release was seen after adding EGTA (0.5 mM) to the medium (308 % at 36 min). (b) Cortical slices. The conflicting results reported in the literature between the effects on renin release of changing extracellular calcium concentration in studies on cortical slices and intact kidneys contrasted with the agreement between the effects of such changes recorded in intact kidneys and in the present series on isolated glomeruli. This motivated a few experiments on conventionally prepared cortical slices, chilled in the pre-incubation period, in order to test whether or not a significant difference could be detected in the response of slices and isolated glomeruli incubated and superfused, respectively, with identical Ringer solutions. The data given in Fig. 2 show an increase in renin release with time from slices incubated in control bicarbonate-Ringer (Fig. 2, hatched columns), contrasting to the decrease in release with time from superfused isolated glomeruli. The apparent difference in release in the first control period (A) between the control and experimental series was not significant (P > 0.1). However, subsequent incubation of slices in calcium-free bicarbonateRinger caused a marked increase in release significantly larger than that seen during continued incubation in control Ringer (P < 0.025). Thus, the response of cortical slices to removal of extracellular calcium was qualitatively the same as that observed in isolated glomeruli under similar conditions. Therefore, studies on kidney slices were not carried on to any further extent in the present investigation.

Effects of the divalent cation ionophore A23187 (a) 2 mm calcium in control periods. Shifting from the control bicarbonate Ringer to a calcium-free superfusate containing 17 x 106 M-A23187 (Fig. 3, open triangles) resulted in a delayed and very modest increase in renin release (104% at 36 min, P < 0-025), significantly different from the marked response to calcium-free medium without the ionophore (cf. Fig. 1). However, subsequent removal ofthe ionophore in the continued absence of calcium in the Ringer (Fig. 3, filled squares) caused a marked additional increase similar in magnitude and steepness to that observed going from

752 L. BAUMBACH AND P. P. LEYSSAC control to calcium-free buffer (cf. Fig. 1). Re-introducing the control Ringer (2 mm calcium) following superfusion with calcium-free Ringer containing A23187 resulted in a rapid decline in the release, which reached control levels within 36 min (Fig. 3, filled circles). Addition of the ionoI

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Fig. 2. Renin release from cortical slices incubated in bicarbonate-Ringer. Slices were incubated with 2 mm calcium in all periods in control experiments (hatched columns; n = 7). In the experimental series (open columns; n = 9) slices were exposed to calcium-free Ringer in period C. Renin release is given as per cent of values obtained in the second control period (B). Bars indicate + s.E. of mean.

phore to calcium-free buffer containing 0 5 mM-EGTA (Fig. 3, filled triangles) gave a prompt and steep increase in release (614 % at 36 min, P < 0.001), possibly slightly larger than that resulting from addition of EGTA alone (Fig. 3, open circles). However, the scatter was so large that

753 Ca AND La ON RENIN RELEASE the difference in release between these two latter series was insignificant (P > 0.1). (b) 0-1 mm calcium in control period Fig. 4 shows the effects of calcium removal and addition of the ionophore A28187 following superfusion with 0-1 mm calcium Ringer in the control periods. The absolute value of the control resting release was about 6 times higher than that measured in 2 MM-Ca2+ 1

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Fig. 3. Effects of An1 on renin release from isolated glomeruli superfused in control periods with bicarbonate-Ringer containing 2 mM calcium. In one series superfusate composition was changed (at zero time) to calciumfree Ringer containing A2,187 (V, n = 10). The arrow indicates a subsequent change, either to calcium-free Ringer without A3,187 (*, n = 3); or back to control Ringer (2 mm calcium) without A3,1 (O, n = 7). (0) represents data resulting from changing to calcium-free superfusate with 06 m EGTA in the absence of A.,,, (n = 21), to be compared with those obtained from calcium-free superfusion with EGTA and A231.7 (A, n = 3). The dashed line indicates the decrease in basal release with time in control experiments. Bars indicate + s.E. of mean.

754 L. BAUMBACH AND P. P. LEYSSAC control superfusate (2 mm calcium) (14.6 x 10 Goldblatt u./30 min. 100 glomeruli, and 2-4 x 10- Goldblatt u./30 min. 100 glomeruli, respectively). The additional relative increase in release obtained following the shift to calcium-free medium was only modest, 174% at 36 min (Fig. 4, open 800

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Fig. 4. Effects on renin release from isolated glomeruli superfused in control periods with bicarbonate-Ringer containing 0-1 mM calcium. At zero time superfusate composition was changed to calcium-free Ringer without ionophore (V, n = 3); or to calcium-free Ringer with A23187 (*, n = 3); or to calcium-free Ringer with 0-5 mM-EGTA but without A2,U7 (0O n = 6); or to calcium-free Ringer with both EGTA and A23187 (A, n = 6). Bars indicate + S.E. of mean.

Ca AND La ON RENIN RELEASE 755 triangles), as compared to the change in release elicited by shifting from 2 mm calcium to the calcium-free buffer (cf. Fig. 1); but the absolute amounts of rein released were larger. The presence of the ionophore A28187 during the calcium-free superfusion did not significantly affect this change in release (Fig. 4, filled squares). EGTA in the calcium-free medium caused a similar relative increase in renin release as that seen after exposure to 2 mm calcium in the control periods (360 % at 36 min, P < 0*001); the further addition of the ionophore to the EGTA-containing calcium-free superfusate resulted on an average in a larger increase in release. However, again the scatter was large, but the difference in mean values of data collected with and without the presence of the ionophore was statistically significant in this series (P < 0.05). Before terminating the individual experiments of the two ionophore series and their control series Ca2+-containing control Ringer was always re-introduced for a recovery period of 36 min at least, but after various durations of exposure to the calcium-free superfusates. For the sake of clarity in presentation of the data given in Figs. 3 and 4, the recovery data were excluded. Re-introduction of control medium consistently depressed renin release after a 12 min period of delay. Thereafter release of renin rapidly declined towards the control level; and there was no detectable difference in the rate of decline in release during recovery between glomeruli exposed to A23187 in the experimental period as compared to those superfused with calcium-free Ringer without the ionophore. The effect of lanthanum The data presented in Fig. 5 show, firstly, that removal of calcium from Tris-buffered superfusate caused the same stimulation of renin release as that obtained in bicarbonate-Ringer (Fig. 5, filled triangles); secondly, that addition of lanthanum chloride to the calcium-free superfusate (Fig. 5, open circles) depressed release below control levels measured during continued superfusion with 2 mm calcium (Fig. 5, dashed line); after 24 min of superfusion with lanthanum-containing calcium-free Ringer renin release had decreased to 59 % of the control value, significantly below the value of 89 % obtained in control experiments at that time (P < 0*001). In most ofthese experiments (n = 6) a lanthanum concentration of 1 0 mm was used, but in a few experiments (n = 3) the lanthanum concentration was reduced to 0 05 mM. The data obtained in these latter experiments were indistinguishable from those obtained with the higher concentration; therefore these data were pooled for presentation in Fig. 5. A similar inhibitory effect of lanthanum on renin release was observed in a few experiments in which 1 mM lanthanum was added to the calciumcontaining control Tris-Ringer.

L. BAUMBACH AND P. P. LEYSSAC Fig. 6. shows the effect on renin release of removing lanthanum from the calcium-free superfusate after 24 min of exposure to this trivalent metal ion. It is apparent that renin release did not increase following this change (Fig. 6, filled circles); rather release continued unchanged at the very low 756

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Time (min) Fig. 5. Effect of lanthanum on renin release from isolated glomeruli superfused with Tris-Ringer containing 2 mM calcium in control periods. The superfusate was changed to calcium-free Tris-Ringer at zero time, either without addition of lanthanum chloride (A, n = 9); or with 1 mm lanthanum (O. n = 9). The dashed line indicates the decrease in basal release with time in control experiments (2 min calcium). Bars indicate + s.E. of mean.

level, significantly below that measured during superfusion with 2 mM calcium. However, the lanthanum effect was not irreversible, as demonstrated by the tremendous increase in release following the removal of lanthanum and simultaneous addition of 0.5 mM-EGTA to the calcium-free superfusate (Fig. 6, open circles). A consequence of this release was a severe

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Ca AND La ON RENIN RELEASE7757 reduction of the renin content remaining in the glomeruli after 60 min of superfusion with this medium. Preliminary control experiments had indi10000 7500

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Fig. 6. Effects on renin release from isolated glomeruli following removal of lanthanum from the medium. After 24 min of superfusion with calcium. free Tris-Ringer containing 1 mm lanthanum chloride superfusion was continued in calcium-free Ringer in the absence of lanthanum ions either without EGTA (@, n = 9), or with 0 5 mM-EGTA (O. n = 6). Bars indicate + s.E. of mean.

cated that such massive release of rein could not be ascribed to the effect of EGTA in Tris-Ringer alone, since changing superfusate from the control Tris-Ringer to a calcium-free Tris-Ringer with EGTA caused a similar

L. BAUMBACH AND P. P. LEYSSAC 758 increase in renin release as that obtained with bicarbonate-Ringer, far less than the release provoked by lanthanum removal and addition of EGTA.

Magnesium In nineteen experiments the magnesium concentration of bicarbonate Ringer was reduced from 1*2 mm in the control periods to 0-12 or 0 mM in the experimental periods. No other change in medium composition was made in this series. Neither the reduction in magnesium concentration nor superfusion with magnesium-free Ringer had any consistent or significant effect on rein release. DISCUSSION

The present results confirm the previous observations that renin release from kidney cortical slices in vitro increases with time during incubation in control Ringer solution (e.g. Yamamoto et al. 1974; Corsini, Crosslan & Bailie, 1974). In this respect the response of cortical slices to the in vitro conditions is opposite to that of isolated glomeruli (Baumbach et al. 1976; Morris et al. 1976). We consider this increase in renin release from the slices as an indication of functional deterioration of the slice preparation, because increases in renin release from isolated glomeruli in vitro occur as a result of metabolic poisoning (Blendstrup et al. 1975), low temperature (Baumbach et al. 1976), and conditions favouring cell swelling (Frederiksen et al. 1975). Nevertheless, in the present study renin release from cortical slices increased even more during incubation in calcium-free Ringer in accordance with the results obtained from isolated glomeruli, but in contrast to previous findings in kidney slice studies (Michelakis, 1971). The reason for this discrepancy remains unexplained. However, the present data indicate that incubation of cortical slices and superfusion of isolated glomeruli with media of identical composition may yield qualitatively similar results, suggesting that medium composition might be an essential factor. Satisfied so far with this result and taking into account the above criticism of the slice preparation we preferred to complete the present study utilizing the preparation of isolated glomeruli. The perfect agreement between the present in vitro results and results obtained from intact kidneys, both isolated perfused and under in vivo conditions, confirms the usefulness of results obtained from isolated superfused glomeruli, and strengthens the possibility that results from this preparation may have physiological relevance. Thus, in perfect agreement with the response of intact kidneys (see Introduction) the renin release from isolated glomeruli increased substantially when exposed to calcium-free superfusion fluid; a reduction of the superfusate calcium concentration to only 0-2 mm was effective in

759 Ca AND La ON RENIN RELEASE augmenting the release, indicating a high sensitivity ofthe juxtaglomerular cells to the external calcium concentration under these conditions. Such high sensitivity to external calcium strongly suggests a high calcium permeability of the juxtaglomerular cell membrane. The effect of calcium appeared to be specific, since moderate changes in the concentration, or even removal of magnesium from the superfusate were without detectable effects on renin release. The major effect of the divalent cation ionophores is supposed to be facilitation of the transmembrane calcium fluxes. Therefore, it would a prior be expected that the effect of these ionophores would be limited, if detectable at all, in systems characterized by a high calcium permeability. The present data do not contradict this anticipation. However, it was surprising to us that the renin response of glomeruli exposed to calcium-free superfusate containing the ionophore A23187 following preceding exposure to 2 mm calcium was delayed and considerably attenuated as compared to the effect ofsolely removing calcium from the medium. A likely explanation to this apparently paradoxical effect of the ionophore might be an ionophore action intracellularly in addition to its facilitation of the calcium fluxes across the cell membrane. A23187 is very efficient in releasing calcium accumulated and stored in mitochondria of the neurohypophysis (Robinson, Russell & Thorn, 1976), and the mitochondrial calcium pool of kidney and liver cells is about 100-300 times greater than the estimated cytosolic pool. Thus, the release of a small fraction of the mitochondrial calcium pool, e.g. by A23187, would produce a large increase in the intracellular concentration of free calcium ions, as emphasized by Rasmussen, Jensen, Lake & Goodman (1976); or counteract the decrease in cytosolic calcium concentration otherwise resulting from calcium-free external media. Support for this explanation may be found in some of the present results. First, subsequent removal of the ionophore in the continued absence of calcium in the superfusate did provoke a release of renin quantitatively similar to that seen in other experiments following a shift from calcium-containing (2 mM) to calcium-free superfusate. Secondly, the presence of EGTA in the media abolished this difference in response to calcium-free superfusion fluid with and without A23187 in the 2 mm calcium series, probably because EGTA more effectively removes the fraction of free intracellular calcium ions. Finally, preceding exposure to 0.1 mm calcium for about 60 min in the control period was followed by equal increases in renin release during the subsequent calcium-free (and EGTA-free) superfusion with or without the addition of A23187. This fact would fit the above interpretation, if it is accepted that the low external calcium concentration might have caused partial depletion of the intracellular calcium pool before the exposure to A23187'

L. BAUMBACH AND P. P. LEYSSAC 760 Partial depletion of intracellular calcium stores, e.g. following such superfusion with a low calcium concentration of 0 1 mm, might be a more favourable situation for disclosing an effect of A23187 on the calcium fluxes across the cell membrane, because of the reduced contribution to the cytosolic calcium concentration from mitochondrial stores. In fact the increase in renin release, possibly reflecting a decrease in intracellular calcium concentration, was found to be significantly greater following a challenge with calcium-free EGTA-Ringer in the presence of the ionophore than in its absence in the 0 I mm calcium series. In contrast there was no significant contribution of the ionophore to renin release from glomeruli precedingly superfused with 2 mm calcium-Ringer. This result suggests an action of the ionophore A23157 both on the cell membrane and intracellularly, and suggests an effect of intracellular free calcium ions on renin release. Further, the significance of difference between data collected with and without A23187 in the 0-1 mM calcium series implies that the reflexion coefficient of the juxtaglomerular cell membrane to calcium is well above zero in our preparation, even though the permeability to this ion is relatively high. In summary, the present data permit the following conclusions. Low calcium concentrations or calcium-free perfusion fluid stimulates rein release. The results establish that the calcium effect on renin release is due to a direct action on the juxtaglomerular cells. The fact that reduction rather than elevation of external, and most likely also intracellular, calcium concentration stimulates the release reveals that a mechanism other than exocytosis can be operating in the export of enzyme (protein) even from cell systems, in which the protein is stored in granular form. To which extent such an alternative mechanism may apply to cells other than the juxtaglomerular cells will be a matter for future research. Previous studies in our laboratory have led to the working hypothesis that renin release from isolated glomeruli is a function of the actively regulated cell volume, i.e. swelling causes increased release (Frederiksen et al. 1975; Baumbach et al. 1976). Calcium (and ATP) plays a critical role in cell volume regulation of, e.g., tubule - and smooth muscle cells (Rorive & Kleinzeller, 1972; Rorive, Nielsen & Kleinzeller, 1972; Rangachari, Daniel & Paton, 1973); thus, the marked cellular swelling caused by ATP in the absence of calcium was prevented by addition of calcium (2-5 mM) to the incubation medium (Rorive & Kleinzeller, 1972; Rorive et al. 1972). The finding of elevated renin release at low calcium concentrations and the marked loss of renin from glomeruli superfused with calcium-free Ringer therefore provides additional support to our previous suggestion. According to our working hypothesis, at least basal renin release is a passive process. The rate of release should, therefore, depend both upon

Ca AND La ON RENIN RELEASE 761 the concentration difference of renin between the cytosol and the extracellular fluid, and, more importantly perhaps, upon the cell membrane permeability, the latter depending on cell volume, among other things. Consequently the role of calcium in the mechanism of renin release could be (1) an effect of calcium as a second messenger in the control of cell volume, e.g. analogous to that of calcium in muscle contraction; and/or (2) a direct effect of membrane-bound calcium on the permeability. The present findings on the effect of lanthanum on renin release, which are in complete agreement with those published from studies on isolated perfused kidneys by Logan et al. (1975), seem to favour the latter possibility. If the lanthanum effect was due solely, or predominantly, to blockade of transmembrane calcium fluxes as suggested by Peart and his colleagues (Logan et al. 1975) one would expect, first, a stimulation rather than inhibition of renin release when lanthanum is added in the presence of 2 mm calcium, because the blockade of calcium fluxes would tendto prevent the gradient-favoured passive influx permitting a more efficient regulation (removal) of the intracellular free calcium ions. Secondly, one would predict an inhibition, or even abolishment of the stimulating effect on release by calcium-free extracellular medium, because of blockade of the calcium outflux. But one would not anticipate the observed substantial drop in release following exposure to lanthanum both in the presence and absence of calcium in the external medium. This drop in renin release is more likely a result of calcium displacement from the cell membrane by lanthanum, which itself mimics the calcium effect only with much higher efficiency. The critical assumption underlying this interpretation is that lanthanum does not penetrate through the cell membrane into the intracellular compartments. This assumption is based on the previous failure to detect any lanthanum intracellularly by electron microscopic examination (Logan et al. 1975); thus it depends, of course, on the limits of electron microscopic detection. If eventually it may be demonstrated that sufficient amounts of lanthanum do penetrate into the cells, then our interpretation would be that lanthanum, like calcium, influences the cell volume regulation causing shrinkage, and thereby a decrease in cell membrane permeability to renin. The above interpretation leaves open the possibility that, besides a direct effect of calcium on the cell membrane permeability to renin (and possibly to other small proteins as well), the intracellular concentration of free calcium ions may act as a messenger in the active control of cell volume and thereby influence the release of renin. The results of the ionophore series, discussed above, would seem to support that such an additional action of calcium actually may influence renin release, although this evidence is not more than indirect and suggestive. In this context it is interesting that calcium is required both for the smooth muscle contractor 28

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762 L. BAUMBACH AND P. P. LEYSSAC action and the inhibitory action of angiotensin-II on isoprenaline-stimulated renin release from isolated perfused kidneys (Vandongen & Peart, 1974b). In conclusion our previous and present observations support the suggestion that renin stored in granules of the juxtaglomerular cells may be released into the cytoplasm, from which activated (and perhaps also inactive) renin molecules are passively transferred somehow across the cell membrane into the surrounding fluid space. The previous findings of increased loss of renin at low temperatures both from isolated juxtaglomerular granules (Hong & Poisner, 1976) and from the intact juxtaglomerular cells of isolated glomeruli (Baumbach et al. 1976) are consistent with this suggestion. Consequently changes in renin release by such a mechanism would depend on the rate of release from the granules into the cytosol and on the permeability of the cell membrane to renin. The membrane permeability, in turn, seems to be influenced by the extracellular calcium concentration by a direct action of membrane-bound calcium as well as indirectly via changes in membrane tension secondary to calciumdependent cell volume regulation. Similarly, changes in membrane tension caused by vascular distension and vascular smooth muscle contraction should also influence renin release according to this concept, consistent with experimental results from intact kidneys (cf. e.g. Davis & Freeman, 1975; Vandongen, Greenwood, Strang, Poesse & Birkenhdger, 1976). The data seem to exclude that calcium stimulated exocytosis is the mechanism responsible for basal renin release from the juxtaglomerular cells adhering to isolated glomeruli. The present work was supported by grants from the Danish Medical Research Council, the Danish Foundation for the Advancement of Medical Science, and by grants from Ebba Celinder's Legacy and Miss P. A. Brandt's Legacy to Dr Leyssac, and by grants from Johan and Hanne Weimann's Legacy and the Danish Heart Association to Dr Baumbach. The skilful technical assistance of Anni Salomonsson, Pia Svaneborg and Conni Temdrup is acknowledged. REFERENCES Aoi, W., WADE, M. B., ROSNER, D. R. & WEINBERGER, M. H. (1974). Renin release by rat kidney slices in vitro: effects of cations and catecholamines. Am. J. Physiol. 227, 630-634. BAUMBACH, L., LEYSSAC, P. P. & SKINNER, S. L. (1976). Studies on renin release from isolated superfused glomeruli: effects of temperature, urea, ouabain and ethacrynic acid. J. Physiol. 258, 243-259. BLENDsTRUp, K., LEYSSAC, P. P., POULSEN, K. & SKINNER, S. L. (1975). Characteristics of renin release from isolated superfused glomeruli in vitro. J. Physiol. 246, 653- 672. COOK, W. F. & PICKERING, G. W. (1959). The location of renin in rabbit kidney. J. Phypiol. 149, 526-536.

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CoRsmNI, W. A., CROSSLAN, K. L. & BAILIE, M. D. (1974). Renin secretion by rat kidney slices in vitro. Proc. Soc. exp. Biol. Med. 145, 403-406. DAVIS, J. 0. & FREEMAN, R. H. (1976). Mechanisms regulating renin release. Phy8iol. Rev. 56, 1-56. DouGLAs, W. W. (1974). Exocytosis and the exocytosis-vesiculation sequence: with special reference to neurohypophysis, chromaffin and mast cells, calcium and calcium ionophores. In Secretory Mechanism8 of Exocrine Glands, ed. THORN, N. A. & PETERSEN, 0. H., pp. 116-129. Copenhagen: Munksgaard. DOuGLAs, W. W. & POISNER, A. M. (1963). The influence of calcium on the secretary response of the submaxillary gland to acetylcholine or to noradrenaline. J. Physiol. 165, 528-541. DouGLAS, W. W. & RUBIN, R. P. (1961). The role of calcium in the secretary response of the adrenal medulla to acetylcholine. J. Physiol. 159, 40-57. FREDERIKSEN, O., LEYSSAC, P. P. & SKINNER, S. L. (1975). Sensitive osmometer function of juxtaglomerular cells in vitro. J. Phy8iol. 252, 669-679. HONG, J.-S. & POISNER, A. M. (1976). Properties of renin granules isolated from rat kidney. Molec. cell. Endocr. 5, 331-337. KOTCHEN, T. A., MAULI, I. I., LuKE, R., REES, D. & FLAMENBAuM, W. (1974). Effect of acute and chronic calcium administration on plasma renin. J. din. Invest. 54, 1279-1286. LOGAN, A. G., TENYi, I., QUESADA, T., PEART, W. S., BREATENACH, A. S. & MARTIN, B. G. H. (1975). Blockade of renin release by lanthanum. Clin. Sci. 48, 31-328. MICHEALS, A. M. (1971). The effect of sodium and calcium on renin release in vitro. Proc. Soc. exp. Biol. Med. 137, 833-836. Momus, B. J., NIXON, R. L. & JOHNSTON, C. I. (1976). Release of renin from glomeruli isolated from rat kidney. Clin. exp. Pharm. Physiol. 3, 37-47. POULSEN, K. & JORGENSEN, J. (1974). An easy radioimmunological microassay of renin activity, concentration and substrate in human and animal plasma and tissues based on angiotensin-I trapping by antibody. J. clin. Endocr. Metab. 39, 764-773. RAMSAY, J. A. & BROWN, R. H. (1955). Simplified apparatus and procedure for freezing point determinations upon small volumes of fluid. J. 86Cent. Inmtrum. 32, 372-375. RANGACHARI, P. K., DANIEL, E. E. & PATON, D. M. (1973). Regulation of cellular volume in rat myometrium. Biochim. biophy8. Acta 274, 297-308. RASMUSSEN, H., JENSEN, P., LARE, W. & GOODMAN, D. B. P. (1976). Calcium ion as second messenger. Clin. Endocr. suppl. 11-278. ROBINSON, I. C. A., RUSSELL, J. T. & THORN, N. A. (1976). Calcium and stimulussecretion coupling in the neurohypophysis. V. The effects of the Ca2+ ionophores A23187 and X537A on vasopressin release and 45Ca2+ efflux: interactions with sodium and a verapamil analogue (D600). Acta Endocr., (Copenh. 83, 3-49. RORIVE, G. & KLEiNZELTLTER, A. (1972). The effect of ATP and Ca2+ on the cell volume in isolated kidney tubules. Biochim. biophy8. Acta 274, 226-239. RORIVE, G., NIELSEN, R. & KLEINZELLER, A. (1972). Effect of pH on the water and electrolyte content of renal cells. Biochim. biophy8. Acta 266, 376-396. SARUTA, T. & MATsUEi, S. (1975). The effects of cyclic AMP, theophylline, angiotensin II and electrolytes upon renin release from rat kidney slices. Endocr. jap. 22, 137-140. VANDONGEN, R. & PEART, W. S. (1974a). The inhibition of renin secretion by alphaadrenergic stimulation in the isolated rat kidney. Clin. Sci. 47, 471-479. VANDONGEN, R. & PEART, W. S. (1974b). Calcium dependence of the inhibitory effect of angiotensin on renin secretion in the isolated perfused kidney of the rat. Br. J. Pharmac. 50, 125-129. 28-2

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VANDONGEN, R., GREENWOOD, D. M., STRANG, K. D., POESSE, M. H. & BIRKENHAGER, W. H. (1976). Effects of vaso-active substances on renin secretion in the isolated rat kidney. In Interference with Mechanism8 in Hypertension, Proc. of a workshop. Dept. Int. Med. Zuiderziekenhuis, ed. BIRKENEAGER, W. H. & VANDONGEEN, R. pp. 5-17, Rotterdam. VISKOPER, R. J., ROSENFELD, S., MAxwiEi., M. H., DELmIA, J., LuPu, A. N. & ROSENFELD, J. B. (1976). Effect of Ca2+ binding by EGTA on renin release in the isolated perfused rabbit kidney. Proc. Soc. exp. Bil. Med. 152, 415-418. WATKIS, B. E., DAVIS, J. O., LOHMEIER, T. E. & FREEMAN, R. H. (1976). Intrarenal site of action of calcium on renin secretion in dogs. Circulation Re8. 39, 847-853. YAM"OTO, K., IwAo, H., ABE, Y. & MORIMOTO, S. (1974). Effect of Ca on renin release in vitro and in vivo. Jap. Circ. J. 38, 1127-1131.