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Receptors for bradykinin and prostaglandin E coupled to Ca2+ signalling in rat cortical collecting duct
Cell Calcium (1997) 22(4), 269-275 Q Pearson Professional Ltd 1997
Research
Receptors for bradykinin and prostaglandin E, coupled to Ca*+ signalling in rat cortical collecting duct leva Ankorina-Stark, Sabine Haxelmans, Eberhard Schlatter Experimentelle
Nephrologie,
Medizinische
Poliklinik,
Westfalische
Wilhelms-Universitat,
Munster,
Germany
In freshly isolated rat cortical collecting ducts (CCD) we measured intracellular Ca2+ activity ([Ca*+],) with the Fura- method. Bradykinin (BK) induced a transient and biphasic increase in [Caz+li. This increase was concentration dependent and was half maximal at a concentration of 15 nM. The B, receptor antagonist HOE 140 (100 nM, n = 6) completely abolished BK (100 nM) induced increase in [Ca*+],. The B, receptor agonist des-Argg-bradykinin (100 nM, n = 4) had no effect on [Ca*+],. In the absence of extracellular Ca *+, the maximal increase in [Ca2+], induced by BK was diminished and the secondary plateau phase was completely abolished. Prostaglandin E, (PGE,) elevated [Ca*+], also concentration-dependently and biphasically. A half maximal effect was reached with 1 nM PGE,. The secondary plateau phase was absent when extracellular Ca2+ was removed. Sulprostone (100 nM, n = 6) mimicked the PGE, (100 nM) induced increase in [Ca*+],. The effect of BK (100 nM) on [Ca*+], was not inhibited by the cyclooxygenase inhibitor indomethacin (10 PM, n = 5). Dopamine (1 PM, n = 4) did not significantly alter [Ca2+li. BK and PGE, regulate [Ca*+], in the rat CCD via release of Ca*+ from intracellular Ca*+ stores as well as via Ca*+ influx from extracellular space. BK directly modulates [Ca*+], through B, receptors. EP, receptors are most likely to be responsible for the PGE, induced increase in [Ca*+],. Summary
INTRODUCTION
Recently we reported that arginine-vasopressin, oxytocin and adrenalin modulate intracellular Ca2+ activity ([Ca*+]j in rat cortical collecting duct (CCD) through V, and B receptors [ 11.In the present study, we continued to examine other agonists and their receptors, possibly regulating [Ca*+], in rat CCD. Kinins participate in the regulation of salt and water transport [2,3] and were
Received
28 May 1997
Revised 7 August 1997 Accepted 14 August 1997 Correspondence to: E. Schlatter, Westfalische Wilhelms-Universitat, Medizinische Poliklinik, Experimentelle Nephrologie, Domagkstrasse 48149 Mtinster, Germany. Tel: +49 251 83 56991; Fax: +49 251 83 56973 E-mail: schlateauni-muenster.de
3a, D-
shown to increase [Ca2+li in rabbit papillary collecting tubule cells [4]. Bradykinin binding sites have been localised in the kidney and highest binding was observed in the CCD [5]. In rat medullary collecting duct, B, receptor blockade enhances Cl- and water absorption indicating that kinins can regulate tubular transport [6]. Stoos et al [7] reported that BK inhibits transport processes in the mouse M-l cell line. An action of BK on the transport was synergistic with the effects of atrial natriuretic factor in these cells. Prostaglandin E, (PGE,) is a potent regulator of renal hemodynamics and salt, as well as water transport, in the kidney [8,9]. The collecting duct is an important target site for the PGE, mediated inhibition of water and Na+ transport in rabbit kidney [&lo]. It was shown that in rabbit CCD different PGE, receptors are coupled to distinct transport processes [I 11. EP, receptors stimulate CAMP production, whereas EP, receptors increase [Ca2+li and therefore, inhibit Na+ reabsorption. EP, receptors are
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coupled to inhibitory G proteins. Also the presence of luminal EP, receptors which stimulate CAMP generation in rabbit CCD was reported [ 121. The present study was undertaken to examine the effects of BK and PGE, on [Caz+], in the rat CCD and to characterise the receptors involved. We further examined the effects of increases in [Ca*+], by these agonists on membrane voltage (VA to elucidate a possible role of [Caz+], in regulation of ion conductances and thus, electrolyte transport.
CCDs were isolated from kidneys of female Wistar rats (Charles River Wiga, Sulzfeld, Germany) which had a body weight of 100-200 g using the enzymatic method described before in detail [ 131. With this method, short CCD segments or cell clusters were obtained. These clusters were held by a glass pipette in the perfusion chamber allowing access of the perfusion solution to both the basolateral as well as the luminal surface of the CCD cells. CCDs were obtained from animals fed a normal diet (1324, Altromin, Lage, Germany). In some experiments, we used animals which received a low-Na+ diet (C 1036, Altromin; Na+ content 137 mg/kg) for lo-20 days to stimulate electrolyte transport in the CCD. Under these conditions, a possible modulation of transport processes by [Ca2+li would probably be more pronounced and, thus, could be seen more easily in functional studies. Since there were no differences in the basal [Ca2+], nor in the effects of bradykinin on [Ca2+li between CCDs obtained from rats fed either a normal or a low Na+ diet, all data were pooled.
Ham’s F- 12 medium (Gibco BRL, Eggenstein, Germany) at room temperature in the dark. Loaded CCDs were equilibrated for 15 min while superfused with control solution (see below) at 37°C before starting the experiments. Cells were excited at 340, 360 and 380 run with a xenon-quartz lamp (XBO 75W, Zeiss) using a filter wheel (Physiologisches Institut, Universitat Freiburg, Germany) rotating at 10 Hz. Furaemission was registered at 500-530 nm from about 5 cells using an iris diaphragm and a photon counting tube (Hamamatsu H 3460-04, Herrsching, Germany). The ratio of emissions after excitation at 340/380 nm (FR) was calculated and averaged with a time resolution of 1 Hz. The emission at 360 nm ([Ca*+], independent wavelength) for Fura- was used to identify cell volume changes or artefacts, e.g. air bubbles in the bath [15]. The signal noise and autofluorescence, which did not exceed 5% of the Fura- fluorescence, was measured before loading the cells and subtracted from the original data for each experiment. Experiments were controlled and data were analysed with an AT-486 computer system and specific software (II. Frobe, Universitat Freiburg, Germany). The calibration of [Caz+], was attempted at the end of each experiment by incubation of the cells with the CaZ+ ionophore ionomycin (1 @I, Sigma) in the presence (1.3 mM) and absence of extracellular Ca2+ (buffered with 5 mM EGTA) according to standard methods using a dissociation constant of Fura- for CaZ+of 224 nM [16]. Since calibration was not successful in all experiments, summaries are given as fluorescence ratios (FR) and original traces as calculated Ca2+values. Effects of removal of extracellular Caz+, sulprostone, des-Argg-bradykinin or HOE 140 on [Ca2+], were always tested with the respective control effects in the same tubule.
[Ca’+], measurements
Chemicals
Isolated CCD segments were transferred to a perfusion chamber and fixed with a glass pipette. Experiments with isolated CCD segments were performed in a constantly perfused bath (the exchange rate was 0.3 Hz at 37”C), mounted on an inverted microscope (Axiovert 135, Zeiss, Jena, Germany). The standard bath solution contained in mM: NaCl 145, K,HPO, 1.6, KH,PO, 0.4, Ca-gluconate 1.3, MgCl, 1 and D-glucose 5; pH was adjusted to 74. All standard chemicals used were obtained in highest purity available from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany). [Caz+], of CCD segments was measured with the Ca2+ sensitive dye Fura- as described previously [14,15]. CCD segments were incubated with 5 pM of Furaacetoxymethyl ester (Fura-Z/AM), dissolved with 0.1 g/l Pluronic F-127 (Bad Soden, Germany) for 50 + 2 min in
Bradykinin, des-Arg9-bradykinin, prostaglandin E,, dopamine (3-hydroxytyramine) and indomethacin were purchased from Sigma. Thapsigargin was purchased from Calbiochem (Bad Soden, Germany). Nalador@ (sulprostone) was obtained from Schering AG (Berlin, Germany). HOE 140 was a gift from Hoechst Company (Frankfurt am Main, Germany). All agonists were dissolved in control solution as a stock solution and kept frozen in aliquots. Experimental solutions were prepared freshly every day and were kept at 4°C until use.
Isolated tubule preparation
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Membrane voltages (VA of principal cells of rat CCD (identified by their response to 1 ~.tIvlamiloride) were obtained using the slow whole-cell patch-clamp 0 Pearson
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Bradykinin and PGE, receptors
technique that has been described in detail before [17]. Patch clamp electrodes were filled with a solution containing (in mM): K+-gluconate 95, KC1 30, NaH,PO, 1.2, Na,HPO, 4.8, D-glucose 5, Ca2+-gluconate 0.73, EGTA 1, M&l, 1.03, ATP 1, pH was adjusted to 72, to which 100 mg/l nystatin was added. The input resistance of these pipettes was 6-12 MQ. An Ag/AgCl wire served as reference electrode. Data were sampled using a patchclamp amplifier (EPC-9, HEKA Elektronik, Lambrecht, Germany) and recorded using a pen recorder (Rikadenki, Freiburg, Germany).
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Statistical analysis
Data are given as originals or mean values z!zSEM (n), 1z refers to the number of experiments. Paired or nonpaired two sided &test (where appropriate) was used to test for statistically significant differences. P I 0.05 was set as significance level.
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RESULTS
In CCDs from 45 rats receiving normal diet, basal Furafluorescence ratio (FR) was 1.11 + 0.02, n = 66, corresponding to a [Ca”‘], of 169 + 14 nM. In CCDs from 79 animals fed a low Na+ diet FR and [Ca2+], were 1.06 + 0.02 and 145 + 13 (n = 106), respectively. FR and calculated [Ca2+li was not significantly different between these two sets of experiments. Effects of bradykinin on [Ca’+],
BK increased [Caz+], in a biphasic manner with a short initial peak and a secondary plateau phase (Fig. 1). In 12 paired experiments, we explored the effects of BK (100 WI) in the absence of extracellular Ca2+ on FR. The removal of extracellular Ca2+ reduced the BK induced peak increase in FR significantly by 0.08 + 0.01 and abolished the secondary plateau phase completely (Fig. 1). To examine whether stimulated transport in CCDs from animals under low Na+ diet alters the response in [Caz+], the effects of bradykinin (BK) on FR between CCDs from these two groups were compared. BK (100 r&l) increased FRby0.18+0.03(n=21)and0.23+0.03(n=18)inCCDs from animals receiving normal or low Na+ diet, respectively. Since these data were not significantly different all data were pooled. All further experiments were done in CCDs from rats on normal diet. Halfmaximal peak increase in [Ca’+], was reached at 15 nM and the maximal effect of BK was reached at 100 nM (Fig. 2). To elucidate which BK receptor is involved in the’ [Ca2+], modulation, the B, receptor agonist des-Argg-bradvkinin (100 nM, 1~.= 4) &as tested. IFdid not sign&x& alter [Ca*+], (Fig. 2). The B, receptor antagonist Hoe 140 (100 0 Pearson
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5 min Fig. 1 Original recordings of the effects of bradykinin on [Ca2+], of rat CCD segments. Upper trace: effects of bradykinin (BK, 100 nM) on [Ca’+], in the presence and absence of extracellular Caz+ in the same tubule. In this example, BK induced a fast peak increase followed by a plateau phase. Note that the secondary plateau phase is absent after Ca2+ removal. The interruption of the trace corresponds to 12 min. Lower trace: effect of repetitive additions of distinct concentrations of BK in short time intervals on [Cap+], of a rat CCD segment.
nM, 11 = 6) completely abolished the BK (100 nM) induced increase in [Ca2+], (Fig. 2). After removal of HOE 140, BK did not induce an increase in [Ca2+liany more for at least 1 h due to the known low reversibility of this drug. Repetitive addition of BK under control conditions, however, increased [Ca2+], several times with only minor desensitisation (Fig. 1). In the presence of the cyclooxygenase inhibitor indomethacin (10 @l, n = 5), BK (100 nM) induced increase in [Ca’+], was 72 + 15 % of that in the absence of indomethacin, which is not significantly different. Effect of prostaglandin
E, on [Ca2+],
PGE, increased [Ca2+], with a large peak and long lasting secondary plateau phase. This effect was fully reversible Cell Calcium
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Fig. 2 Concentration response curve for the effect of bradykinin (BK) and absence of effects of the B, receptor agonist des-Arg*bradykinin and the B, receptor antagonist HOE 140 (each 100 nM) on Furafluorescence ratio (FR) of rat CCD segments. The changes in FR reflect the initial peak increase of FR. Mean values i SEM, numbers in brackets refer to the number of experiments performed for the different concentrations.
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and no desensitisation was seen (Fig. 3). PGE, induced increase in [Caz+liwas concentration dependent (EC,, = 1 r-&I) and reached its maximum with 10 nM PGE, (Fig. 4). In the absence of extracellular Caz+,PGE, evoked a peak in [Caz+li without a secondary plateau (n = 3, data not shown). Sulprostone (100 r&I, n = 6), an EP, and EP, receptor agonist, mimicked the effect of PGE, on [Ca2+], (Figs 3 & 4). It increased FR by 0.20 f 0.03 comparable to the increase in FR by 0.22 _+ 0.03 induced by 100 nM PGE,.
In rat CCD, dopamine was shown to inhibit Na+ and water permeability [ 181 and to stimulate PGE, production in inner medullary collecting duct cells of the rat, probably via [Ca’+], [ 191. In rat CCD, dopamine (1 I.&I, n = 4) had no significant effect on [Caz+],(data not shown). Table
Effects
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Amiloride (1 W) Change
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Fig. 3 Original recording of repetitive additions of distinct concentrations of prostaglandin E, (PGE,) on [Ca’+]! of a rat CCD segment (top trace). Note the PGE, induced biphasrc increases in [Ca’+], with initial peak and secondary plateau phase and no apparent desensitisation. Original recording of the effects of the prostaglandin E, (PGE,) receptor agonist sulprostone and PGE, (each 100 nM) on [Ca’+], of a rat CCD segment (bottom trace).
No effects of BK and PGE, on membrane voltage (V,)
To examine possible effects of increases in [Ca:+], induced by BK or PGE, on electrogenic transport in rat CCD V, was measured. Vm under resting conditions was -82 f 2 mV (rt = 9). Amiloride (1 l.t.M)induced a significant hyperpolarisation of Vm by 3 -t 1 mV (n = 9, Table) in these cells identifying them as principal cells. Neither 1 pM BK nor 0.1 @I PGE, altered Vm significantly (Table). Neither elevation of [Ca2+],with the Ca2+ionophore ionomycin (0.1
cells
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Fig. 4 Concentration response curve for the effects of prostaglandin E, (PGE,) and the effect of the PGE, receptor agonist sulprostone (100 nM) on the Furafluorescence ratio (FR) of rat CCD segments. The changes in FR reflect the initial peak increase of FR. Data are presented as mean values * SEM. The numbers in brackets refer to the number of experiments performed for the different concentrations.
ltIvI, n = 6) nor with the cellular Ca2+-ATPaseinhibitor thapsigargin (1 @I, n = 6) had any effect on V,,, (Table). Arginine vasopressin, an agonist stimulating electrogenic transport of rat CCD via CAMP activation [ZO], depolarised Vm also in this set of experiments. Figure 5 depicts an example for the effects of amiloride, BK, PGE, and arginine vasopressin on V,. DISCUSSION
Ca2+ signalling is involved in the regulation of transport processes in collecting duct (for review, see [Z 11).The role of different hormones and autakoids in the regulation of water and salt absorption through Ca2+signalling is well established in rabbit collecting duct, but far less is known about a possible modulation of electrolyte transport by Ca2+ in the rat CCD. As opposed to rabbit CCD, in rat CCD elevation of [Ca2+], by ionomycin or thapsigargin does not alter Na+ transport or H,O permeability [22]. This indicates differences in the signalling pathway involved in the regulation of transport between these species. So far, for rat CCD only few studies have been undertaken to demonstrate the presence of receptors coupled to phospholipase C and [Ca2+li. Recently we reported that [Ca2+liin rat CCD is modulated by arginine 0 Pearson
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Fig. 5 Original slow-whole-cell recordings of the membrane voltage (Vm) of principal cells of rat CCD segments. Application of amiloride (1 uM) hyperpolarised V, by 4 mV. Prostaglandin E, (PGE,, 100 nM) and bradykinin (BK, 1 PM) had no significant effect on Vm (top trace). Application of arginine vasopressin (AVP, 1 PM) depolarised V,,, reversibly by 11 mV (bottom trace).
vasopressin, oxytocin and adrenalin via specific receptors [l], comparable to the situation in rabbit CCD. In the current study, we demonstrate that bradykinin (BK) and prostaglandin E, (PGE,) also increase [Ca2+], of rat CCD, again comparable to rabbit CCD. In rat inner medullary collecting duct, B, receptor blockade with the antagonist HOE 140 enhanced Cl- and water reabsorption stressing the role of kinins in the regulation of renal transport also in rat kidney [6]. BK was shown to modulate [Ca2+],in a number of renal cells. Shayman et al [4] reported increases in [Ca’+], after BK stimulation in papillary collecting tubules of the rabbit. In canine distal MDCK cells, BK elevated [Ca2+], and IP, production [23]. BK acts via two types of receptors: B, and B, receptors. Activation of B, receptors leads to stimulation of the phospholipase C activity resulting in formation of inositol phosphates and diacylglycerol and thus, elevation of [Ca*+],. The B, receptor and its signal transduction is less explored 1241. BK induced an instantaneous and biphasic increase in [Ca2+], in our study in rat CCD indicating a stimulation of the release from intracellular stores and Ca2+ influx across the plasma membrane. The specific B, receptor antagonist HOE 140 completely inhibited BK induced increase in [Ca2+],, while the B, receptor agonist had no significant Cell Calcium
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effect on [Ca2+li. Therefore, we propose that, in rat CCD, BK modulates [Ca2+],through B, receptors. In contrast to these results in rat inner medullary collecting duct, BK failed to elevate [Caz+li [25]. In the medullary thick ascending limb of the rat NaCl transport is inhibited by BK via increases in [Ca2+],.This increase is also mediated through B, receptors [26]. BK action in the kidney is often coupled to PGE, production as BK is shown to stimulate renal PGE, synthesis [27,28]. In MDCK cells, BK induced elevation of [Ca’+], was only partially regulated by PKC, whereas PGE,-induced [Caz+], increase was completely blocked by PKC [23]. The authors suggested that the BK-induced signal transduction is different to the PGE,-induced signal transduction pathway. There are also differences in the sources of the increases in [Ca2+],. In MDCK cells,’ BK as well as PGE, induced increase in [Ca2+], was independent of extracellular Ca2+ [23]. However, in our study a biphasic increase in [Caz+], with an initial peak and a secondary plateau phase, dependent on extracellular CaZ+, was seen for both BK and PGE,. These differences could be due to different intracellular signalling in these cells. Also the peak increases in [CaZ+], induced by BK or PGE,, in the absence of extracellular Ca2+ was significantly smaller compared to control conditions. This indicates a dependence of the Ca2+ store refilling on extracellular Caz+. A partial desensitisation of the receptors may also be responsible for this difference. Our data indicate that in rat CCD BK effects on [Caz+], are independent of PGE, synthesis as the increase in [Ca’+], by BK occurs within seconds and cannot be significantly affected by inhibition of the cyclooxygenase pathway. Hebert et al [29] showed that PGE, increases [Ca*+], in rabbit CCD. In agreement with this finding, we observed an increase in [Ca2+li in rat CCD by PGE,. Four EP receptors (for Eprostanoid) have been cloned: EP,, EP,, EP, and EP, [30]. PGE, activates three different EP receptors in the CCD and these receptors have distinct signalling pathways (for reviews see [31,32]). EP, receptors are shown to inhibit both Na+ and water permeability in rabbit CCD [29,33]. These receptors are also highly expressed in human collecting duct [34]. We observed a similar increase in [Ca2+li with sulprostone, which is an EP, and EP, specific receptor agonist, as with PGE,. As stimulation of the EP, receptor increases [Ca2+], but EP, receptors are coupled to a Gi protein [3 11,we can conclude that this increase in [Ca2+], is mediated via EP, receptors. Increases in [Ca2+], with sulprostone were also shown in rabbit CCD [l 11. Dopamine, which binds to D, like receptors in rat CCD [18], failed to modulate [Ca2+], in our study, indicating that this agonist does not act via [Ca2+li in rat CCD. In rabbit CCD, elevation of [Ca2+], by BK or PGE, decreases Na+ and water transport [29,35]. Neither BK nor PGE, influenced Na+ and water transport in rat CCD Cell Calcium (1997) 22(4), 269-275
136,371. The presence of receptors for both agonists in rat CCD and the number of data showing regulation of ion channels of rat CCD by [Ca2+], prompted us to examine possible effects of agonist induced [Ca”+], increases on electrogenic transport by measuring V,. An inhibition of Na+ channels via an increase in [Ca2+li, probably by an indirect mechanism, was observed [38]. Wang and Giebisch [39] and our group [40,41] showed different modulation of luminal and basolateral membrane K+ channels of rat CCD by [Ca2+li. Neither BK or PGE,, nor elevation of [Ca2+], with ionomycin or thapsigargin had any effect on V,,, even when high concentrations of these agonists were used. This confirms the absence of any effect on Na+ and water transport of rat CCD by elevating [Ca2+liwith thapsigargin or ionomycin [22]. The current study shows, that BK and PGE, are potential and independent regulators of [Ca2+],also in rat CCD. BK increases [Ca’+], via specific B, receptors and PGE, modulates [Ca2+], through EP, receptors. Elevation of [Ca’+], in CCD of the rat by these agonists does not alter electrogenic transport. Thus, the role of stimulation of these specific receptors coupled to Ca2+ signalling in the rat CCD remains to be elucidated. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the excellent technical assistance of MS D. Rehder and I. Kleta. This work was supported by the Deutsche Forschungsgemeinschaft Schl 277/2-5 and a grant from the MaxPlanck-Gesellschaft and the von Humboldt Foundation. This work is part of the PhD thesis of I. Ankorina-Stark.
1. Ankorina-Stark I., Haxelmans S.,S&latter E. Functional evidence for the regulation of cytosolic CaZ+activity via V,*receptors and j3-adrenoceptors in rat CCD. Cell Calcium 1997; 21: 163-171. 2. Tomita K., Ujiie K., Maeda Y., Iino Y., Yoshiyama N., Shiigai T. Effects of kinin on electrolytes transport and regulation of kininase activity in distal nephron segments of the rat. / Clin Invest 1986; 7’7: 136-141. 3. Tomita K., Ujiie K., Maeda Y., Iino Y., Yoshiyama N., Shiigai T. Effects of kinin on electrolytes transport and regulation of kininase activity in distal nephron segments of the rat. Adv Exp Med B&1989; 247A: 97- 104. 4. Shayman J.A., Hruska K.A., Morrison A.R. Bradykinin stimulates increased intracellular calcium in papillary collecting tubules of the rabbit. Biochem Biophys Res Commun 1986; 134: 299-304. 5. Tomita K., Pisano JJ. Binding of [3H]-bradykinin in isolated nephron segments of the rabbit. Am J Physioll984; 246: F732-F737. 6. Mukai H., Fitzgibbon W.R., Bozeman C., Margolius H.S., Ploth D.W. Bradykinin B, receptor antagonist increases chloride and water absorption in rat medullary collecting duct. Am J Physol 1996; 271: R352-R360. 0 Pearson Professional Ltd 1997
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7 Stoos B.A., Carretero O.A., Garvin J.L. ANF and bradykinin synergistically inhibit transport in M- 1 cortical collecting duct cell line. AmJPhysioll992; 263: Fl-F6. 8. Bonvalet J-P.,Pradelles P., Farman N. Segmental synthesis and actions of prostaglandins along the nephron. Am J Physiol 1987; 253: F377-F387 9. Mene P., Simonson M.S., Dunn M.J. Physiology of the mesangial cell. Physiol Rev 1989; 69: 1347-1423. 10. Breyer M.D., Ando Y. Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu Rev Physiol 1994; 56: 71 l-739. 11. Hebert R.L. Cellular signalling of PGE, and its selective receptor analogue sulprostone in rabbit cortical collecting duct. ProskzgZandins Leukot Essent Fatty Acids 1994; 51: 147-155. 12. Sakairi Y., Jacobson H.R., Noland Breyer M.D. Luminal prostaglandin E receptors regulate salt and water transport in rabbit cortical collecting duct. Am J Physiol 1995; 269: F257-F265. 13. Schlatter E., Frobe II., Greger R. Ion conductances of isolated cortical collecting duct cells. @tigers Avck 1992; 421: 381-387. 14. Schlatter E., Haxelmans S.,Ankorina I., Kleta R. Regulation of Na+/H+ exchange by diadenosine polyphosphates, angiotensin II and vasopressin in rat cortical collecting duct. J Am Sot NephrolI995; 6: 1223-1229. 15. Nitschke R., Frobe U., Greger R. Antidiuretic hormone acts via V, receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb. @tigers Arch 199 1; 417: 622-632. 16. Grynkiewicz G., Poenie M., Tsien R.Y. A new class of Ca*+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260; 3440-3450. 17 Horn R., Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 1988; 92: 145-159. 18. Sun D., Schafer J.A. Dopamine inhibits AVI-dependent Na+ transport and water permeability in rat CCD via a D,-like receptor. Am JPhysiol 1996; 271; F391-F400. 19. Huo T., Healy D.P.Prostaglandin E, production in rat IMCD cells. I. Stimulation by dopamine. AmJPhysioll991; 261: F647-F654. 20. Schlatter E., Schafer J.A. Electrophysiological studies in principal cells of rat cortical collecting tubules. Pfltigers Arch 1987; 409: 81-92. 2 1. Breyer M.D. Regulation of water and salt transport in collecting duct through calcium-dependent signaling mechanisms. Am] PhysioZl991; 260: Fl-Fll. 22. Rouch A.L., Chen L., Kudo L.H. et al. Intracellular Ca2+ and PKC activation do not inhibit Na+ and water transport in rat CCD. Am JPhysioll993; 265: F569-F577. 23. Aboolian A., Vander Molen M., Nord E.P. Differential effects of phorbol esters on PGE, and bradykinin-induced elevation of [CaZ+]~inMDCKcells.AmJPhysio~1989;256:F1135-F1143. 24. Farmer S.G. Biochemical and molecular pharmacology of kinin receptors. Annu Rev Pharmacol Toxicol 1992; 32: 51 l-536.
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25. Star R.A., Nonoguchi H., Knepper M.A. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct. / CZin Invest 1988; 81: 1879-1888. 26. Grider J., Falcone J., Kilpatrick E., Ott C., Jackson B. Effect of bradykinin on NaCl transport in the medullary thick ascending limb of the rat. EurJPhumtaco~ 1995; 287: 101-104. 27 McGiff J.C., Itskovitz H.D., Terragno A., Wong P.Y. Modulation and mediation of the action of the renal kallikrein-kinin system. Fed Proc 1976; 35: 175-l 80. 28. Grenier F.C., Rollins T.E., Smith W.L. Kinin-induced prostaglandin synthesis by renal papillary collecting tubule cells in culture. AmJPhysiol1981; 241: F94-F104. 29. Hebert R.L.,Jacobson H.R., Breyer M.D. Prostaglandin Ez inhibits sodium transport in rabbit cortical collecting duct by increasing intracellular calcium. J clin Invest 199 1; 87: 1992-l 998. 30. Coleman R.A., Smith W.L., Narumiya S. Classification of prostan oid receptors: properties, distribution, and structure of the receptors and their subtypes. Phurmacol Rev 1994; 46: 205-229. 31. Hebert R.L., Breyer R.D., Jacobson H.R., Breyer M.D. Functional and molecular aspects of prostaglandin E receptors in the cortical collecting duct, Can JPhysiol Pharmacoll995; 73: 172-179. 32. Breyer M.D., Jacobson H.R., Breyer R.M. Functional and molecular aspects of renal prostaglandin receptors. J Am Sot Nephroll996; 7: S-l% 33. Hebert R.T.,Jacobson H.R., Breyer M.D. PGE, inhibits AVIproduced water flow in cortical collecting ducts by protein kinase C activation. Am J Physiol 1990; 259: F3 18-F325. 34. Breyer M.D., Davis L.,Jacobson H.R., Breyer R.D. Differential localization of prostaglandin E receptor subtypes in human kidney. Am J Physioll996; 270: F9 12-F9 18. 35. Schuster V.L. Mechanism of bradykinin, ADH, and CAMP interaction in rabbit cortical collecting duct. Am J Physiol 1985; 249: F645-F653. 36. Rouch A.J., Chen L., Troutman S.L., Schaefer J. Na+ transport in isolated rat CCD: effects of bradykinin, ANP, clonidine, and hydrochlorthiazide. Am J Physiol 199 1; 260: F86-F95. 37 Chen L., Reif M.C., Schafer J.A. Clonidine and PGE, have different effects on Na+ and water transport in rat and rabbit CCD. Am JPhysioll991; 261: F126-F136. 38. Silver R.B., Frindt G., Windhager E.E.,Palmer L.G. Feedback regulation of Na channels in rat CCT I. Effects of inhibition of Na pump. Am JPhysioZ 1993; 264: F557-F564. 39. Wang W., Giebisch G. Dual modulation of renal ATP-sensitive K+ channel by protein kinases A and C. Proc Nat1 Acad Sci USA 1991;88:9722-9725. 40. Hirsch J., S&latter E. K+ channels in the basolateral membrane of rat cortical collecting duct. lyttigers Arch 1993; 424: 470-472 4 1. Hirsch J., Leipziger J., Frobe U., S&latter E. Regulation and possible physiological role of the Ca2+-dependent K+ channel of cortical collecting ducts of the rat. pfliigets Arch 1993; 422: 492-49s.
Cell Calcium (1997) 22(4), 269-275
Research
Receptors for bradykinin and prostaglandin E, coupled to Ca*+ signalling in rat cortical collecting duct leva Ankorina-Stark, Sabine Haxelmans, Eberhard Schlatter Experimentelle
Nephrologie,
Medizinische
Poliklinik,
Westfalische
Wilhelms-Universitat,
Munster,
Germany
In freshly isolated rat cortical collecting ducts (CCD) we measured intracellular Ca2+ activity ([Ca*+],) with the Fura- method. Bradykinin (BK) induced a transient and biphasic increase in [Caz+li. This increase was concentration dependent and was half maximal at a concentration of 15 nM. The B, receptor antagonist HOE 140 (100 nM, n = 6) completely abolished BK (100 nM) induced increase in [Ca*+],. The B, receptor agonist des-Argg-bradykinin (100 nM, n = 4) had no effect on [Ca*+],. In the absence of extracellular Ca *+, the maximal increase in [Ca2+], induced by BK was diminished and the secondary plateau phase was completely abolished. Prostaglandin E, (PGE,) elevated [Ca*+], also concentration-dependently and biphasically. A half maximal effect was reached with 1 nM PGE,. The secondary plateau phase was absent when extracellular Ca2+ was removed. Sulprostone (100 nM, n = 6) mimicked the PGE, (100 nM) induced increase in [Ca*+],. The effect of BK (100 nM) on [Ca*+], was not inhibited by the cyclooxygenase inhibitor indomethacin (10 PM, n = 5). Dopamine (1 PM, n = 4) did not significantly alter [Ca2+li. BK and PGE, regulate [Ca*+], in the rat CCD via release of Ca*+ from intracellular Ca*+ stores as well as via Ca*+ influx from extracellular space. BK directly modulates [Ca*+], through B, receptors. EP, receptors are most likely to be responsible for the PGE, induced increase in [Ca*+],. Summary
INTRODUCTION
Recently we reported that arginine-vasopressin, oxytocin and adrenalin modulate intracellular Ca2+ activity ([Ca*+]j in rat cortical collecting duct (CCD) through V, and B receptors [ 11.In the present study, we continued to examine other agonists and their receptors, possibly regulating [Ca*+], in rat CCD. Kinins participate in the regulation of salt and water transport [2,3] and were
Received
28 May 1997
Revised 7 August 1997 Accepted 14 August 1997 Correspondence to: E. Schlatter, Westfalische Wilhelms-Universitat, Medizinische Poliklinik, Experimentelle Nephrologie, Domagkstrasse 48149 Mtinster, Germany. Tel: +49 251 83 56991; Fax: +49 251 83 56973 E-mail: schlateauni-muenster.de
3a, D-
shown to increase [Ca2+li in rabbit papillary collecting tubule cells [4]. Bradykinin binding sites have been localised in the kidney and highest binding was observed in the CCD [5]. In rat medullary collecting duct, B, receptor blockade enhances Cl- and water absorption indicating that kinins can regulate tubular transport [6]. Stoos et al [7] reported that BK inhibits transport processes in the mouse M-l cell line. An action of BK on the transport was synergistic with the effects of atrial natriuretic factor in these cells. Prostaglandin E, (PGE,) is a potent regulator of renal hemodynamics and salt, as well as water transport, in the kidney [8,9]. The collecting duct is an important target site for the PGE, mediated inhibition of water and Na+ transport in rabbit kidney [&lo]. It was shown that in rabbit CCD different PGE, receptors are coupled to distinct transport processes [I 11. EP, receptors stimulate CAMP production, whereas EP, receptors increase [Ca2+li and therefore, inhibit Na+ reabsorption. EP, receptors are
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coupled to inhibitory G proteins. Also the presence of luminal EP, receptors which stimulate CAMP generation in rabbit CCD was reported [ 121. The present study was undertaken to examine the effects of BK and PGE, on [Caz+], in the rat CCD and to characterise the receptors involved. We further examined the effects of increases in [Ca*+], by these agonists on membrane voltage (VA to elucidate a possible role of [Caz+], in regulation of ion conductances and thus, electrolyte transport.
CCDs were isolated from kidneys of female Wistar rats (Charles River Wiga, Sulzfeld, Germany) which had a body weight of 100-200 g using the enzymatic method described before in detail [ 131. With this method, short CCD segments or cell clusters were obtained. These clusters were held by a glass pipette in the perfusion chamber allowing access of the perfusion solution to both the basolateral as well as the luminal surface of the CCD cells. CCDs were obtained from animals fed a normal diet (1324, Altromin, Lage, Germany). In some experiments, we used animals which received a low-Na+ diet (C 1036, Altromin; Na+ content 137 mg/kg) for lo-20 days to stimulate electrolyte transport in the CCD. Under these conditions, a possible modulation of transport processes by [Ca2+li would probably be more pronounced and, thus, could be seen more easily in functional studies. Since there were no differences in the basal [Ca2+], nor in the effects of bradykinin on [Ca2+li between CCDs obtained from rats fed either a normal or a low Na+ diet, all data were pooled.
Ham’s F- 12 medium (Gibco BRL, Eggenstein, Germany) at room temperature in the dark. Loaded CCDs were equilibrated for 15 min while superfused with control solution (see below) at 37°C before starting the experiments. Cells were excited at 340, 360 and 380 run with a xenon-quartz lamp (XBO 75W, Zeiss) using a filter wheel (Physiologisches Institut, Universitat Freiburg, Germany) rotating at 10 Hz. Furaemission was registered at 500-530 nm from about 5 cells using an iris diaphragm and a photon counting tube (Hamamatsu H 3460-04, Herrsching, Germany). The ratio of emissions after excitation at 340/380 nm (FR) was calculated and averaged with a time resolution of 1 Hz. The emission at 360 nm ([Ca*+], independent wavelength) for Fura- was used to identify cell volume changes or artefacts, e.g. air bubbles in the bath [15]. The signal noise and autofluorescence, which did not exceed 5% of the Fura- fluorescence, was measured before loading the cells and subtracted from the original data for each experiment. Experiments were controlled and data were analysed with an AT-486 computer system and specific software (II. Frobe, Universitat Freiburg, Germany). The calibration of [Caz+], was attempted at the end of each experiment by incubation of the cells with the CaZ+ ionophore ionomycin (1 @I, Sigma) in the presence (1.3 mM) and absence of extracellular Ca2+ (buffered with 5 mM EGTA) according to standard methods using a dissociation constant of Fura- for CaZ+of 224 nM [16]. Since calibration was not successful in all experiments, summaries are given as fluorescence ratios (FR) and original traces as calculated Ca2+values. Effects of removal of extracellular Caz+, sulprostone, des-Argg-bradykinin or HOE 140 on [Ca2+], were always tested with the respective control effects in the same tubule.
[Ca’+], measurements
Chemicals
Isolated CCD segments were transferred to a perfusion chamber and fixed with a glass pipette. Experiments with isolated CCD segments were performed in a constantly perfused bath (the exchange rate was 0.3 Hz at 37”C), mounted on an inverted microscope (Axiovert 135, Zeiss, Jena, Germany). The standard bath solution contained in mM: NaCl 145, K,HPO, 1.6, KH,PO, 0.4, Ca-gluconate 1.3, MgCl, 1 and D-glucose 5; pH was adjusted to 74. All standard chemicals used were obtained in highest purity available from Sigma (Deisenhofen, Germany) and Merck (Darmstadt, Germany). [Caz+], of CCD segments was measured with the Ca2+ sensitive dye Fura- as described previously [14,15]. CCD segments were incubated with 5 pM of Furaacetoxymethyl ester (Fura-Z/AM), dissolved with 0.1 g/l Pluronic F-127 (Bad Soden, Germany) for 50 + 2 min in
Bradykinin, des-Arg9-bradykinin, prostaglandin E,, dopamine (3-hydroxytyramine) and indomethacin were purchased from Sigma. Thapsigargin was purchased from Calbiochem (Bad Soden, Germany). Nalador@ (sulprostone) was obtained from Schering AG (Berlin, Germany). HOE 140 was a gift from Hoechst Company (Frankfurt am Main, Germany). All agonists were dissolved in control solution as a stock solution and kept frozen in aliquots. Experimental solutions were prepared freshly every day and were kept at 4°C until use.
Isolated tubule preparation
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Membrane voltages (VA of principal cells of rat CCD (identified by their response to 1 ~.tIvlamiloride) were obtained using the slow whole-cell patch-clamp 0 Pearson
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Bradykinin and PGE, receptors
technique that has been described in detail before [17]. Patch clamp electrodes were filled with a solution containing (in mM): K+-gluconate 95, KC1 30, NaH,PO, 1.2, Na,HPO, 4.8, D-glucose 5, Ca2+-gluconate 0.73, EGTA 1, M&l, 1.03, ATP 1, pH was adjusted to 72, to which 100 mg/l nystatin was added. The input resistance of these pipettes was 6-12 MQ. An Ag/AgCl wire served as reference electrode. Data were sampled using a patchclamp amplifier (EPC-9, HEKA Elektronik, Lambrecht, Germany) and recorded using a pen recorder (Rikadenki, Freiburg, Germany).
500 -
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IO _I
Statistical analysis
Data are given as originals or mean values z!zSEM (n), 1z refers to the number of experiments. Paired or nonpaired two sided &test (where appropriate) was used to test for statistically significant differences. P I 0.05 was set as significance level.
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RESULTS
In CCDs from 45 rats receiving normal diet, basal Furafluorescence ratio (FR) was 1.11 + 0.02, n = 66, corresponding to a [Ca”‘], of 169 + 14 nM. In CCDs from 79 animals fed a low Na+ diet FR and [Ca2+], were 1.06 + 0.02 and 145 + 13 (n = 106), respectively. FR and calculated [Ca2+li was not significantly different between these two sets of experiments. Effects of bradykinin on [Ca’+],
BK increased [Caz+], in a biphasic manner with a short initial peak and a secondary plateau phase (Fig. 1). In 12 paired experiments, we explored the effects of BK (100 WI) in the absence of extracellular Ca2+ on FR. The removal of extracellular Ca2+ reduced the BK induced peak increase in FR significantly by 0.08 + 0.01 and abolished the secondary plateau phase completely (Fig. 1). To examine whether stimulated transport in CCDs from animals under low Na+ diet alters the response in [Caz+], the effects of bradykinin (BK) on FR between CCDs from these two groups were compared. BK (100 r&l) increased FRby0.18+0.03(n=21)and0.23+0.03(n=18)inCCDs from animals receiving normal or low Na+ diet, respectively. Since these data were not significantly different all data were pooled. All further experiments were done in CCDs from rats on normal diet. Halfmaximal peak increase in [Ca’+], was reached at 15 nM and the maximal effect of BK was reached at 100 nM (Fig. 2). To elucidate which BK receptor is involved in the’ [Ca2+], modulation, the B, receptor agonist des-Argg-bradvkinin (100 nM, 1~.= 4) &as tested. IFdid not sign&x& alter [Ca*+], (Fig. 2). The B, receptor antagonist Hoe 140 (100 0 Pearson
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100 BK (10 r&l)
BK (100 nM)
BK (1 IJM)
5 min Fig. 1 Original recordings of the effects of bradykinin on [Ca2+], of rat CCD segments. Upper trace: effects of bradykinin (BK, 100 nM) on [Ca’+], in the presence and absence of extracellular Caz+ in the same tubule. In this example, BK induced a fast peak increase followed by a plateau phase. Note that the secondary plateau phase is absent after Ca2+ removal. The interruption of the trace corresponds to 12 min. Lower trace: effect of repetitive additions of distinct concentrations of BK in short time intervals on [Cap+], of a rat CCD segment.
nM, 11 = 6) completely abolished the BK (100 nM) induced increase in [Ca2+], (Fig. 2). After removal of HOE 140, BK did not induce an increase in [Ca2+liany more for at least 1 h due to the known low reversibility of this drug. Repetitive addition of BK under control conditions, however, increased [Ca2+], several times with only minor desensitisation (Fig. 1). In the presence of the cyclooxygenase inhibitor indomethacin (10 @l, n = 5), BK (100 nM) induced increase in [Ca’+], was 72 + 15 % of that in the absence of indomethacin, which is not significantly different. Effect of prostaglandin
E, on [Ca2+],
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T
(Ill
IOnM -
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Fig. 2 Concentration response curve for the effect of bradykinin (BK) and absence of effects of the B, receptor agonist des-Arg*bradykinin and the B, receptor antagonist HOE 140 (each 100 nM) on Furafluorescence ratio (FR) of rat CCD segments. The changes in FR reflect the initial peak increase of FR. Mean values i SEM, numbers in brackets refer to the number of experiments performed for the different concentrations.
sulprostone 5 min
and no desensitisation was seen (Fig. 3). PGE, induced increase in [Caz+liwas concentration dependent (EC,, = 1 r-&I) and reached its maximum with 10 nM PGE, (Fig. 4). In the absence of extracellular Caz+,PGE, evoked a peak in [Caz+li without a secondary plateau (n = 3, data not shown). Sulprostone (100 r&I, n = 6), an EP, and EP, receptor agonist, mimicked the effect of PGE, on [Ca2+], (Figs 3 & 4). It increased FR by 0.20 f 0.03 comparable to the increase in FR by 0.22 _+ 0.03 induced by 100 nM PGE,.
In rat CCD, dopamine was shown to inhibit Na+ and water permeability [ 181 and to stimulate PGE, production in inner medullary collecting duct cells of the rat, probably via [Ca’+], [ 191. In rat CCD, dopamine (1 I.&I, n = 4) had no significant effect on [Caz+],(data not shown). Table
Effects
of amiloride
and increases
Amiloride (1 W) Change
in Vm
*Significant
Cell Calcium
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22(4),
in [Ca*+], on Vm of principal
PGE,
Fig. 3 Original recording of repetitive additions of distinct concentrations of prostaglandin E, (PGE,) on [Ca’+]! of a rat CCD segment (top trace). Note the PGE, induced biphasrc increases in [Ca’+], with initial peak and secondary plateau phase and no apparent desensitisation. Original recording of the effects of the prostaglandin E, (PGE,) receptor agonist sulprostone and PGE, (each 100 nM) on [Ca’+], of a rat CCD segment (bottom trace).
No effects of BK and PGE, on membrane voltage (V,)
To examine possible effects of increases in [Ca:+], induced by BK or PGE, on electrogenic transport in rat CCD V, was measured. Vm under resting conditions was -82 f 2 mV (rt = 9). Amiloride (1 l.t.M)induced a significant hyperpolarisation of Vm by 3 -t 1 mV (n = 9, Table) in these cells identifying them as principal cells. Neither 1 pM BK nor 0.1 @I PGE, altered Vm significantly (Table). Neither elevation of [Ca2+],with the Ca2+ionophore ionomycin (0.1
cells
of rat CCD lonomycin (0.1 PM)
(l$, o*o 7
Thapsigargin (1 PM)
iii
1 *o 6
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Bradykinin and PGE, receptors
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a0 -1i)oh4e-r-
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i /
,.i
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,/_,,i
i
AVP
Fig. 4 Concentration response curve for the effects of prostaglandin E, (PGE,) and the effect of the PGE, receptor agonist sulprostone (100 nM) on the Furafluorescence ratio (FR) of rat CCD segments. The changes in FR reflect the initial peak increase of FR. Data are presented as mean values * SEM. The numbers in brackets refer to the number of experiments performed for the different concentrations.
ltIvI, n = 6) nor with the cellular Ca2+-ATPaseinhibitor thapsigargin (1 @I, n = 6) had any effect on V,,, (Table). Arginine vasopressin, an agonist stimulating electrogenic transport of rat CCD via CAMP activation [ZO], depolarised Vm also in this set of experiments. Figure 5 depicts an example for the effects of amiloride, BK, PGE, and arginine vasopressin on V,. DISCUSSION
Ca2+ signalling is involved in the regulation of transport processes in collecting duct (for review, see [Z 11).The role of different hormones and autakoids in the regulation of water and salt absorption through Ca2+signalling is well established in rabbit collecting duct, but far less is known about a possible modulation of electrolyte transport by Ca2+ in the rat CCD. As opposed to rabbit CCD, in rat CCD elevation of [Ca2+], by ionomycin or thapsigargin does not alter Na+ transport or H,O permeability [22]. This indicates differences in the signalling pathway involved in the regulation of transport between these species. So far, for rat CCD only few studies have been undertaken to demonstrate the presence of receptors coupled to phospholipase C and [Ca2+li. Recently we reported that [Ca2+liin rat CCD is modulated by arginine 0 Pearson
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Fig. 5 Original slow-whole-cell recordings of the membrane voltage (Vm) of principal cells of rat CCD segments. Application of amiloride (1 uM) hyperpolarised V, by 4 mV. Prostaglandin E, (PGE,, 100 nM) and bradykinin (BK, 1 PM) had no significant effect on Vm (top trace). Application of arginine vasopressin (AVP, 1 PM) depolarised V,,, reversibly by 11 mV (bottom trace).
vasopressin, oxytocin and adrenalin via specific receptors [l], comparable to the situation in rabbit CCD. In the current study, we demonstrate that bradykinin (BK) and prostaglandin E, (PGE,) also increase [Ca2+], of rat CCD, again comparable to rabbit CCD. In rat inner medullary collecting duct, B, receptor blockade with the antagonist HOE 140 enhanced Cl- and water reabsorption stressing the role of kinins in the regulation of renal transport also in rat kidney [6]. BK was shown to modulate [Ca2+],in a number of renal cells. Shayman et al [4] reported increases in [Ca’+], after BK stimulation in papillary collecting tubules of the rabbit. In canine distal MDCK cells, BK elevated [Ca2+], and IP, production [23]. BK acts via two types of receptors: B, and B, receptors. Activation of B, receptors leads to stimulation of the phospholipase C activity resulting in formation of inositol phosphates and diacylglycerol and thus, elevation of [Ca*+],. The B, receptor and its signal transduction is less explored 1241. BK induced an instantaneous and biphasic increase in [Ca2+], in our study in rat CCD indicating a stimulation of the release from intracellular stores and Ca2+ influx across the plasma membrane. The specific B, receptor antagonist HOE 140 completely inhibited BK induced increase in [Ca2+],, while the B, receptor agonist had no significant Cell Calcium
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effect on [Ca2+li. Therefore, we propose that, in rat CCD, BK modulates [Ca2+],through B, receptors. In contrast to these results in rat inner medullary collecting duct, BK failed to elevate [Caz+li [25]. In the medullary thick ascending limb of the rat NaCl transport is inhibited by BK via increases in [Ca2+],.This increase is also mediated through B, receptors [26]. BK action in the kidney is often coupled to PGE, production as BK is shown to stimulate renal PGE, synthesis [27,28]. In MDCK cells, BK induced elevation of [Ca’+], was only partially regulated by PKC, whereas PGE,-induced [Caz+], increase was completely blocked by PKC [23]. The authors suggested that the BK-induced signal transduction is different to the PGE,-induced signal transduction pathway. There are also differences in the sources of the increases in [Ca2+],. In MDCK cells,’ BK as well as PGE, induced increase in [Ca2+], was independent of extracellular Ca2+ [23]. However, in our study a biphasic increase in [Caz+], with an initial peak and a secondary plateau phase, dependent on extracellular CaZ+, was seen for both BK and PGE,. These differences could be due to different intracellular signalling in these cells. Also the peak increases in [CaZ+], induced by BK or PGE,, in the absence of extracellular Ca2+ was significantly smaller compared to control conditions. This indicates a dependence of the Ca2+ store refilling on extracellular Caz+. A partial desensitisation of the receptors may also be responsible for this difference. Our data indicate that in rat CCD BK effects on [Caz+], are independent of PGE, synthesis as the increase in [Ca’+], by BK occurs within seconds and cannot be significantly affected by inhibition of the cyclooxygenase pathway. Hebert et al [29] showed that PGE, increases [Ca*+], in rabbit CCD. In agreement with this finding, we observed an increase in [Ca2+li in rat CCD by PGE,. Four EP receptors (for Eprostanoid) have been cloned: EP,, EP,, EP, and EP, [30]. PGE, activates three different EP receptors in the CCD and these receptors have distinct signalling pathways (for reviews see [31,32]). EP, receptors are shown to inhibit both Na+ and water permeability in rabbit CCD [29,33]. These receptors are also highly expressed in human collecting duct [34]. We observed a similar increase in [Ca2+li with sulprostone, which is an EP, and EP, specific receptor agonist, as with PGE,. As stimulation of the EP, receptor increases [Ca2+], but EP, receptors are coupled to a Gi protein [3 11,we can conclude that this increase in [Ca2+], is mediated via EP, receptors. Increases in [Ca2+], with sulprostone were also shown in rabbit CCD [l 11. Dopamine, which binds to D, like receptors in rat CCD [18], failed to modulate [Ca2+], in our study, indicating that this agonist does not act via [Ca2+li in rat CCD. In rabbit CCD, elevation of [Ca2+], by BK or PGE, decreases Na+ and water transport [29,35]. Neither BK nor PGE, influenced Na+ and water transport in rat CCD Cell Calcium (1997) 22(4), 269-275
136,371. The presence of receptors for both agonists in rat CCD and the number of data showing regulation of ion channels of rat CCD by [Ca2+], prompted us to examine possible effects of agonist induced [Ca”+], increases on electrogenic transport by measuring V,. An inhibition of Na+ channels via an increase in [Ca2+li, probably by an indirect mechanism, was observed [38]. Wang and Giebisch [39] and our group [40,41] showed different modulation of luminal and basolateral membrane K+ channels of rat CCD by [Ca2+li. Neither BK or PGE,, nor elevation of [Ca2+], with ionomycin or thapsigargin had any effect on V,,, even when high concentrations of these agonists were used. This confirms the absence of any effect on Na+ and water transport of rat CCD by elevating [Ca2+liwith thapsigargin or ionomycin [22]. The current study shows, that BK and PGE, are potential and independent regulators of [Ca2+],also in rat CCD. BK increases [Ca’+], via specific B, receptors and PGE, modulates [Ca2+], through EP, receptors. Elevation of [Ca’+], in CCD of the rat by these agonists does not alter electrogenic transport. Thus, the role of stimulation of these specific receptors coupled to Ca2+ signalling in the rat CCD remains to be elucidated. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the excellent technical assistance of MS D. Rehder and I. Kleta. This work was supported by the Deutsche Forschungsgemeinschaft Schl 277/2-5 and a grant from the MaxPlanck-Gesellschaft and the von Humboldt Foundation. This work is part of the PhD thesis of I. Ankorina-Stark.
1. Ankorina-Stark I., Haxelmans S.,S&latter E. Functional evidence for the regulation of cytosolic CaZ+activity via V,*receptors and j3-adrenoceptors in rat CCD. Cell Calcium 1997; 21: 163-171. 2. Tomita K., Ujiie K., Maeda Y., Iino Y., Yoshiyama N., Shiigai T. Effects of kinin on electrolytes transport and regulation of kininase activity in distal nephron segments of the rat. / Clin Invest 1986; 7’7: 136-141. 3. Tomita K., Ujiie K., Maeda Y., Iino Y., Yoshiyama N., Shiigai T. Effects of kinin on electrolytes transport and regulation of kininase activity in distal nephron segments of the rat. Adv Exp Med B&1989; 247A: 97- 104. 4. Shayman J.A., Hruska K.A., Morrison A.R. Bradykinin stimulates increased intracellular calcium in papillary collecting tubules of the rabbit. Biochem Biophys Res Commun 1986; 134: 299-304. 5. Tomita K., Pisano JJ. Binding of [3H]-bradykinin in isolated nephron segments of the rabbit. Am J Physioll984; 246: F732-F737. 6. Mukai H., Fitzgibbon W.R., Bozeman C., Margolius H.S., Ploth D.W. Bradykinin B, receptor antagonist increases chloride and water absorption in rat medullary collecting duct. Am J Physol 1996; 271: R352-R360. 0 Pearson Professional Ltd 1997
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