Osmoregulation in the renal papilla: Membranes, messengers and molecules

Kidney International, VoL 49 (1996), pp. 1686—1689

Osmoregulation in the renal papilla: Membranes, messengers and molecules ROLF K.H. KINNE, STEFAN H. BOESE, EVAMARIA KINNE-SAFFRAN, BIRGIT RUHFUS, HANNA TINEL, and FRANK WEHNER Ma.x-Planck-Institut für molekulare Physiologie, Abteilung Epitheiphysiologie, Dortmund, Germany

Osmoregulation in the renal papilla: Membranes, messengers and molecules. This contribution summarizes recent progress in the understanding of the molecular basis of the release of organic osmolytes that occurs when inner medullary cells are confronted with a drop in osmolar-

ity in their environment. For sorbitol release across the basolateral membrane an increase in intracellular calcium seems to be the prominent signal, initiated by G-protein activation, followed by phosphatidyicholine phospholipase activation and generation of arachidonic acid. The increase in betaine permeability is also G-protein dependent but calcium indepen-

dent, and is restricted to the basal-lateral cell face. Myo-inositol and glycerophosphorylcholine efflux are calcium and G-protein independent and occur both across the apical and basolateral membrane, although to a different extent. Taurine release is also calcium and G-protein independent; a swelling-activated anion channel at the basolateral membrane represents the major efflux pathway.

show, as demonstrated in Figure 1, that at least in IMCD cells the

calcium dependence is a quite unique phenomenon for the stimulation of sorbitol efflux during hypotonic shock. In the meantime, it has been shown by Mooren and Kinne [13] and by

Tinel, Wehner and Sauer [141 that during hypotonic shock intracellular calcium is transiently elevated in IMCD cells, and that this increase apparently involves an initial calcium release from intracellular stores followed by a rapid Ca2 influx from the extracellular medium. Accordingly, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamine)-octylester), which inhibits the hypotonicity-induced Ca2 increase [15, 16], also inhibits the hypotonicity-evoked sorbitol efflux from IMCD cells. As expected other "calcium-independent" organic osmolyte fluxes are not affected by this experimental maneuver (Fig. 2).

Route to the elevation of intracellular calcium This contribution summarizes some of the progress made during the last several years in understanding the molecular Signal transduction pathways that have been partially revealed mechanisms underlying the release of organic osmolytes from for the regulation of (in particular) ion permeabilities of plasma renal medullary cells. The term 'molecular' is used on the one membranes have often involved the activation of one or several hand to refer to those molecules that are elements in the signal G-proteins [17]. We, therefore, recently investigated whether

transduction pathway by which the signal "cell swelling" is trans- G-proteins might be involved in (initiating) the cascade of reacduced into an increase in the permeability of the plasma mem- tions culminating in the release of intracellular calcium [15, 16]. branes to organic osmolytes. On the other hand, 'molecular' refers Indeed, it could be demonstrated that pretreatment of IMCD to those mechanisms that actually mediated the rapid efflux of cells in primary culture with pertussis toxin decreased the hypoorganic osmolytes across the plasma membrane. tonicity-evoked calcium response, indicating that G1,,, proteins are In a very broad sense the current interest in organic osmolytes activated when cells swell. In the same course mastoparan that in mammals [reviewed in 1—4] is a consequence of studies in the mimicks the activation of G-proteins evokes a transient rise of field of marine biology, in particular algae, bacteria and fish [5—7]. intracellular calcium in IMCD cells not subjected to a change in Therefore, the approach of comparative physiology advocated so extracellular osmolality. The calcium movements altered by perstrongly by Homer W. Smith has come to fruition [8]. tussis toxin and mastoparan occur apparently in a compartment of the IMCD cells that is relevant for the regulation of sorbitol efflux. There is an inhibition of the hypotonicity-induced sorbitol efflux Molecules transducing the signal after pretreatment of IMCD cells with pertussis toxin, and a Extra- and intracellular calcium release under isotonic conditions when the cells are exposed to In 1990 Bevan, Theill and Kinne [9] pointed to the importance mastoparan [18].

of the presence of extracellular calcium for the hypotonicityevoked release of sorbitol from inner medullary collecting duct (IMCD) cells from rat papilla. We have recently extended these studies to the other organic osmolytes present in IMCD cells [Ruhfus B, Kinne RKH, unpublished results; 101. These studies

One of the consequences of G-protein activation is the alteration of the activity of membrane bound enzymes, some of them acting on membrane components such as phospholipids. In the

attempt to trace the path for triggering intracellular calcium release, the phospholipases C are of particular importance. Indeed, it could be demonstrated by Tinel et al that phospholipase C inhibitors reduce the hypotonicity-induced calcium response [15, 16]. In the majority of cases this finding would be interpreted

© 1996 by the International Society of Nephrology

to indicate that a phospholipase acting on phosphatidylinositol 1686

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Kinne et al: Organic osmolytes in renal medulla

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Isotonicity Hypotonic shock

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Fig. 3. Effects of arachidonic acid (10 jsM) on isotonic and hypotonicityactivated sorhitol effiux from isolated IMCD cells. Values are means of the

5

efflux in % of total content/lO minutes

se, N 4. Symbols are: ()

control; (•) arachidonic acid (10 jrM). Organic osmolytes were quantified

0

GPC Betaine

Myo-inositol Sorbitol

by HPLC (for further information see legend of Fig. 1). Taurine

Fig. 1. Dependence of hypotonicity-activated organic osmolyte effiux from freshly isolated rat renal IMCD cells on extracellular calcium. Symbols are: calcium; (•) without calcium. Values are means of efflux in % of total SE, taurine efflux is given in % efflux of total content/lO minutes

()

radioactivity/10 minutes SE, N 5 for each experimental condition. All values are corrected for release of lactate dehydrogenase (LDH). Isolated IMCD cells (according to Stokes et al [11]) were incubated for 10 minutes at 37°C followed by centrifugation for one minute to separate supernatants and pellets. Organic osmolytes were quantified in supernatants and

pellets using HPLC according to Wolff et al [12]. Taurine efflux was measured in supernatants and pellets after preloading the cells with 3H-taurine.

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phospholipids. This pathway seems to be also involved predominantly in IMCD cells. Addition of arachidonic acid to IMCD cells elicits changes in intracellular calcium under isotonic conditions

that are otherwise only observed when the osmolality of the extracellular medium is reduced. Again these changes in calcium occur in the compartment determining sorbitol permeability of the membrane as evidenced in Figure 3.

Further studies demonstrated a multiplicity of actions that arachidonic acid (and other polyunsaturated long-chain fatty acids) exert on various pathways for organic osmolytes. Such phenomena have also been observed in other cells during volume regulation, but cannot be discussed in this contribution in more detail [19, 201. It also remains an open question whether arachi-

donic acid itself or one of its metabolites are responsible for initiating calcium release. It should be noted in this context that extracellular ATP via purinergic receptors leads also to a dramatic increase of intracellular calcium associated with an increase in 1P3 [15, 16, 21], apparently activating 1P1-sensitive calcium stores. Thus, at least two different intracellular calcium stores must exist that are closely coupled to different cellular responses, as shown recently for other epithelial cells [221. Once calcium is released several events ensue. As proposed in

T

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earlier studies, fusion of membrane reserve vesicles with the

5

0 Myo-inositol

I*

Sorbitol

GPC

Betaine

Fig. 2. Effects of TMB-8 on the hypotonicity-activated organic osmolyte efflux from freshly isolated IMCD cells. Symbols arc: () control; (•) TMB-8 (100 rM). Values are means of the efflux in % of total content/lU minutes SE, N = 3 for each experimental condition. Organic osmolytes were quantified by HPLC (for further information see legend of Fig. 1).

(P1) would be involved. In IMCD cells, however, no change in 1P3

plasma membrane at the basal-lateral cell pole occurs [23]. This fusion process inserts sorbitol transporters into the membrane that, in addition, might be (or might have been) activated by a calcium calmodulin-dependent phosphorylation process [241. Molecules mediating the efflux The biochemical nature of those molecules mediating the efflux

of organic osmolytes is still rather ill defined. There appears, however, to emerge a general agreement that in their biophysical and kinetic properties they bear more resemblance to channels rather than carriers. The initial evidence for this assumption is the

apparent lack of saturation kinetics. This has been found for In some signal transduction pathways phosphatidylcholine sorbitol efflux via the "permease" in rabbit kidney papillary (PC), rather than P1, is the substrate of the activated phospho- epithelial cells [25], and for efflux pathways in glial cells [26] and lipase C. One important intermediate of this pathway is arachi- in bovine collecting duct cells [Kinne-Saifran E, K.inne RKH, donic acid derived from the C2 position of the phosphatidic acid unpublished results]. Similarly taurine and myo-inositol efflux (or diacylglycerol-3-phosphate) that forms the backbone of the from skate red blood cells is not saturable [see 7]. The strongest

could be detected during the hypotonic shock [15, 16].

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Kinne et al: Organic osmolytes in renal medulla

Table 1. Hypotonically-activated organic osmolyte release in rate IMCD cells Osmolyte

Sorbitol

Ca2 dependent G protein dependent Ca2 release from intracellular stores (triggered by arachidonic acid) Ca2 influx through plasma membrane channel

hyposmotic

A

Signal transduction

11iiiiiiiiIU

Target systems reserve vesicles basolateral sorbitol efflux pathway (specific for sorbitol)

Ca2 independent

apical and basolateral efflux pathway(s)? apical and Glycerophosphorylcholine Ca2 independent G protein independent basolateral efflux pathway(s)? Betaine basolateral efflux Ca2 independent G protein dependent pathway Taurine basolateral efflux Ca2 independent G protein independent pathway shared with other anions Myo-inositol

G protein independent

500 pA 2 mm

apical efflux pathway

B

I'll hO

[29]. A representative recording for the activation of the taurine conductance is shown in Figure 4. Currently it is, however, not clear whether taurine can pass the channel also as a zwitterion. In addition, there appear to exist channels which allow the passage of a variety of chemically not related organic osmolytes such as amino acids (taurine), polyols (myo-inositol or sorbitol) and

methylamines (such as betaine) [7, 26]. In efflux studies on isolated rat IMCD cells both, efflux pathways specific for only one organic osmolyte (sorbitol) and pathways with a transport activity

125 pA

2 mm

for several osmolytes could be detected. The discrimination of transport pathways is thereby based on the signal transduction pathways identified thus far for the regulation of these pathways, their sensitivitiy to inhibitors, and their cellular polarity. These data are compiled in Table 1.

Fig. 4. Time course of activation of the anion conductance in rat IMCD cells

under symmetrical chloride or taurine conditions. Activation of anion conductance was under symmetrical CsC1 (A) (140 mmol/liter solution at pH 7.2 in the pipette and at pH 7.4 in the superfusate) and under symmetrical taurine conditions (B) (300 mmol/liter in pipette and bath at pH 7.8; 31 mmol/liter negatively charged). Outside osmolality was reduced from 600 to 500 mOsm/liter at the time point indicated (representative experiments; for further details see Boese et al [29]).

Concluding remarks This brief review has concentrated on work done in the authors'

laboratory. It became clear that IMCD cells possess a wide repertoire to regulate organic osmolyte release and to fine tune their response to the changing tides of urinary and/or interstitial osmolarity. Several signal transduction pathways—one of which has been detailed in this contribution— converge on various efflux

evidence for the existence of channels through which organic pathways for organic osmolytes. A pathway for taurine efflux osmolytes are transported has been provided by electrophysiolog- common to a variety of cells has also been described above. ical studies. In an increasing number of cells, it has been observed that exposure of cells to hypotonicity activates an anion channel that is characterized by a broad substrate specificity that includes not only inorganic anions but also organic osmolytes. The prop-

Whether there are indeed several pathways for organic osmolytes

is currently a matter of dispute. If there is only one pathway,

according to the studies presented above, it would have to change its selectivity according to the different signals transmitted to it. erties of these channels are reviewed in more detail in the Currently we prefer the view of a multiplicity of signal transduccontribution by Jackson and Strange [271. In this context it has to tion pathways, each pathway having only one efflux mediator as its be mentioned that also rat papillary collecting duct cells show an target. Such an assumption does not exclude, or it rather requires, activation of an anion conductance when exposed to a hypotonic cross talk of the signal transduction pathways as well as the efflux shock [28]. This outwardly rectifying anion channel also carries mediators. Thus a network of communications exists that remains the taurine anion as demonstrated in whole cell patch experiments to be unravelled.

Kinne et a!: Organic osmolytes in renal medulla

Acknowledgments The fruitful discussion and support by Kevin Strange is gratefully acknowledged. Special thanks go also to Daniela Magdefessel for her skillful secretarial assistance.

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15. TINEL H: Kalziumfreisetzung aus intrazellulhren Speichern und Kalziumeinstrom während der hypoosmotischen Volumenregulation papillärer Sammelrohrzellen der Rattenniere: Eine fluorometrische Analyse der Signaltransduktion. PhD Thesis, Ruhr-Universität Bochum, 1995

16. TINEL H, WEHNER F, KINNE RKH: Signal transduction during regu-

Reprint requests to Prof Dr. Rolf KH. Kinne, Mwc-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Postfach 10 26 64, 44026 Dortmund, Germany.

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