Cholinergic stimulation in isolated rat glomeruli PIERRE MENETON, FRANCOIS MOREL,

of phosphoinositide

MAY BLOCH-FAURE, GILLES AND RABARY M. RAJERISON

GUILLON,

metabolism DANIELLE

CHABARDES,

Laboratoire de Physiologie Cellulaire, Unit& de Recherche Associke 219 au Centre National de la Recherche Scientifique (CNRS), Collkge de France, 75231 Paris Cedex 05; and CNRS-Institut National de la Sant6 et de la Recherche Mkdicale de Pharmacologic-Endocrinologie, 34094 Montpellier Cedex 02, France Meneton, Pierre, May Bloch-Faure, Gilles Guillon, Danielle Chabardbs, Frangois Morel, and Rabary M. Rajerison. Cholinergic stimulation of phosphoinositide metabolism in isolated rat glomeruli. Am. J. Physiol. 262 (Renal

Fluid Electrolyte physiol. 31): F256-F266, 1992.-Cholinergic effects on kidney function have been observedin somemammals but the intrarenal localization and the cellular mechanismsof these effects are poorly defined to date. The aim of this work was to study the effects of carbachol on phosphoinositide metabolism in freshly isolated rat glomeruli labeled with myo- [3H]inositol. Carbachol rapidly and markedly stimulates phosphoinositide metabolism with a 50% effective concentration of 3 PM. The enormousmagnitude of the response is enlightened by the use of 10 mM lithium, which provokes in the presenceof the agonist a large accumulation of inositol phosphatesand a correspondingdepletion of cellular free inositol. The responseis inhibited by 85% by pirenzepine, is pertussis toxin insensitive, and shows no desensitization at maximum doseof carbachol up to 40 min of stimulation. rat kidney; isolated glomeruli; muscarinic agonist; inositol phosphates;phosphoinositides

has not been clearly demonstrated to date, several studies indicate that cholinergic agonists act on the kidney. The anatomic localization of muscarinic receptors has been reported in rat and dog kidneys (8, 28,4O) as well as the regulation of some renal functions by cholinergic agents, e.g., acetylcholine infusion into dog or rat kidney produces renal vasodilatation, diuresis, natriuresis (1, 12, 16-18, 28, 30, 34, 36, 41), and a decrease in glomerular ultrafiltration coefficient (&) (2)) and carbachol inhibits sodium and bicarbonate reabsorption in the rat proximal convoluted tubule (37). Biochemical effects of muscarinic agonist have also been described in specific renal sites, i.e., hydrolysis of phosphoinositides in rabbit inner medullary collecting duct cells (23), rise of cytosolic calcium in rat outer medullary collecting duct and in isolated rat glomeruli (2O,21), and increase in guanosine 3’,5’-cyclic monophosphate (cGMP) cell content of isolated rat glomeruli (32, 35). We show here that carbachol stimulates phosphoinositide metabolism in isolated rat glomeruli; this effect has been also found in glomeruli of some other mammalian species (desert rat, cat, rabbit). These results are consistent with a role of acetylcholine in glomerular function in vivo. They also support the idea that freshly isolated glomeruli may constitute a valuable preparation in which to study the regulation of phosphoinositide signaling pathway in noncultured cells. ALTHOUGH RENAL PARASYMPATHETIC innervation

F256

036306127/92 $2.00 Copyright0 1992

MATERIALS AND METHODS Chemicals and composition of assay solution. Collagenase (CLS II, 151 U/mg) was purchasedfrom Worthington (Freehold, NJ); myo-[3H]inositol (-100 Ci/mmol) and 3H-radiolabeled D-myo-inositol mono-, bis-, tris-, and tetrakisphosphate [Ins( l)P, Ins(1,4)Pz, Ins( l,4,5)P3, and Ins( 1,3,4,5)P4, respectively] were from Amersham France (Les Ulis, France); Dowex resin AGl-X8 (200-400 mesh, formate form) was from BioRad laboratories (Richmond, CA); Partisil lo-SAX column was from Whatman (Springfield Mill, UK); all other chemicalswere from Merck Sharp & Dohme ResearchLaboratories (Rahway, NJ) or Sigma Chemical (St. Louis, MO). Except where specified,the solution usedfor isolation, myo[3H]inositol labeling, rinsing, and incubation of glomeruli contained (in mM) 120 NaCl, 5 KCl, 0.8 MgSO,, 0.33 Na2HP04, 0.44 KH2P04, 1 MgC12, 1.5 CaC12,4 NaHC03, 5 glucose, 1 pyruvate, 2 lactate, and 20 N-2-hydroxyethylpiperazine-N’-2ethanesulfonic acid (HEPES), pH 7.4. Solution also included 3% dextran (wt/vol) and 0.1% bovine serum albumin (BSA) (wt/vol). Isolation of glomeruli. For sievedglomeruli, experiments were carried out on either male Wistar or Sprague-Dawleyrats (150250 g body wt) that were fed a standard laboratory diet and had free accessto tap water until experiments. Under pentobarbital sodiumanesthesia(Nembutal, 50 mg/kg body wt), the left kidney wasperfusedvia the renal artery with 5 ml solution to remove blood cells from glomerular capillaries. At room temperature, the renal cortex was chopped finely and then filtered with extensive washing through nylon nets of decreasing size, i.e., 150, 106, and 75 pm. Glomeruli were retained on the 75 pm net and resuspendedin a few milliliters of solution. For dissectedglomeruli another method of isolation wasused; the perfusion wasperformed with a solution containing 300 U/ ml of collagenaseto start the digestion of the extracellular matrix. Thin pyramids, cut from the renal cortex, were incubated for 10 min at 30°C in aerated solution containing 150U/ ml of collagenaseand then rinsed in collagenase-freesolution before freehand dissectionof glomeruli. It should be mentioned that the sieving method results in collection of many more glomeruli that are more responsiveto carbachol than the dissection method (see RESULTS). The method of isolation is indicated for each experiment in the legends of Figs. l-10. Following isolation by either method, glomeruli used for experiments were selected at 4°C under stereomicroscopicobservation to be uniform in size and free of microvasculature and tubular fragments. Glomeruli with and without Bowman’scapsulewere not separatedasno difference was found in the responsivenessof the two types of glomeruli to carbachol (results not shown). Labeling of glomeruli with myo-rH]inositoZ. With regard to the small number of glomeruli usedper determination (usually lo), these were labeled in a solution containing a high radioactive concentration of myo- [3H]inositol(w300 &i/ml). Labeling was performed for various time periods ranging from 2 to the AmericanPhysiological Society

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CARBACHOL

STIMULATES

IP AND

PI METABOLISM

12 h (but usually 2 h) in a humid atmosphere at 37”C, and all glomeruli (typically -1,000) were in only 60 ~1 solution to avoid problems of oxygenation. After this labeling period, glomeruli were extensively rinsed under stereomicroscopic observation at room temperature as follows: they were aspirated with generally less than 5 ~1 of labeling solution using a polyethylene catheter fixed on a Hamilton syringe, and transferred into 500 ~1 of myo-[3H]inositol-free solution. Such a transfer was repeated four more times. Thus the initial radioactivity present with glomeruli was theoretically diluted by a factor of =lO”. Indeed, in these conditions, we did not detect any radioactivity in 2 ~1 of the last rinsing solution. Incubation of glomeruli with agonist. After removal of extracellular myo- [3H]inositol by washing, glomeruli (10, unless specified)were transferred with 2 ~1of the last rinsing solution into glasstubes containing 48 ~1of lithium-free solution. The incubation at 37°C was initiated by addition of 50 ~1 solution containing carbachol. When used, lithium (10 mM final) was present in this solution, becausewe have noted that this ion was effective in lessthan 1.5 min of incubation with glomeruli (see RESULTS).

Reactions were terminated by adding either 940 ~1 chloroform:methanol (1:2, vol:vol) and 200 ~1 of 5 mM EDTA for measurementof total [3H]inositol phosphatesor 100 ~1 of 1.7 M perchloric acid (PCA) for measurementof free inositol, total [3H]phosphoinositides,and the different types of [3H]inositol phosphatesby anion exchangechromatography. For separation of [3H]inositol phosphate isomersby high-performance liquid chromatography (HPLC), the reaction was stopped with 1 ml of a phenol-saturated solution containing 5 mM KH2PO+ 10 mM EDTA, pH 6.3, 0.2 pg each of myo-inositol, mannitol, and phytic acid, and 0.1 M each of unlabeled Ins(l)P, Ins(l,4)Pz, Ins( 1,3,4)P3,Ins( 1,4,5)P3,and Ins( 1,3,4,5)P4as carriers. Measurements of [3HJinositol phosphates nositides by anion exchange chromatography.

and [3H]phosphoi-

After termination of reactions by chloroform:methanol method (3), 310 ~1 chloroform and 310 ~1water were addedagain to samplesthat were then vortexed and centrifuged at 3,000g for 5 min to separate the phases.For each sample 1 ml of the upper hydrophilic phasewas recovered and diluted in 3 ml of 0.003 M HEPESNaOH-buffered water (pH 7.4) before application to columns (seebelow). Phosphoinositideswere not determined when this method wasused.All extraction stepswere carried out at 4°C. After termination of reactions by PCA method (5,15), 20 ~1 BSA 20 mg/ml were addedto samples,and these were centrifuged at 150 g for 5 min. Supernatants that contained free inositol and inositol phosphateswere neutralized and buffered to neutral pH with 0.75 M KOH-0.075 M HEPES in the presenceof universal pH indicator. After 5-min centrifugation at 4,200 g, precipitated potassium perchlorate was discarded and the supernatant was diluted to 4 ml with 0.003M HEPESNaOH-buffered water (pH 7.4) and applied to columns that contained 0.25 g of the Dowex resin AGl-X8 (200-400 mesh, formate form). On the basisof control experimentswith labeled inositol phosphatestandards,free inositol waseluted with 8 ml of 0.003 M HEPES-buffered water, glycerophosphoinositol with 8 ml of 0.03 M ammoniumformate, InsP with 8 ml of 0.18 M ammonium formate, InsPs with 8 ml of 0.4 M ammonium formate-0.1 M formic acid and InsP3 + InsP4 with 8 ml of 1 M ammonium formate-0.1 M formic acid. For measurementsof total inositol phosphates(chloroform:methanol or PCA methods), these were eluted with 8 ml of 1 M ammonium formate0.1 M formic acid after elution of free inositol and glycerophosphoinositolas describedabove. The PCA-precipitated pellets that contained phosphoinositideswere resuspendedwith 4 ml chloroform-methanol-10 M HCl (200:100:1, vol/vol/vol). These suspensionswere mixed with 1.1 ml of 0.1 M HCl and 2.6 ml chloroform and centrifuged for 5 min at 2,500 g to

IN

ISOLATED

GLOMERULI

F257

separate the phases. The lower hydrophobic phase was recovered and dried at 65°C in counting vials to determine radioactivity in total phosphoinositides. We checkedthat addition of carriers, i.e., 0.2 PM final each of unlabeled myo-inositol and inositol phosphates[Ins( l,4)P2, Ins( l,3,4)P3, Ins( l,4,5)P3, and Ins( 1,3,4,5)P,J did not modify recoveriesof the labeledcompoundsthroughout extraction and separationprocedures.Recoveriesof free inositol/inositol phosphates and phosphoinositideswere ~90% with PCA method. With chloroform:methanol method, inositol phosphate recovery was =75%. The radioactivity contained in each eluate (8 ml) and phospholipid extract was counted (Beckman LS 7500 or Kontron Betamatic V) after addition of 10 ml scintillation liquid (ACS II, Amersham) for 10 or 20 min after experiments. The radioactivity wascorrected for variable quenching due to the different elution solutions by meansof quench curves obtained with each solution and was thereby converted into disintegrations per minute (dpm). Separation of [3H]inositol phosphate isomers by HPLC. A Partisil lOSAX (25 x 0.46 cm) anion exchange column was used as previously reported (27, 39). After injection of the samplethe column was washedwith distilled water for 10 min to remove any unbound 3H-labeled material. Elution of the different inositol phosphateisomerswasachievedby increasing the ammoniumformate concentration (adjustedto pH 3.7 with orthophosphoric acid) from 0 to 3 M (as shown in Fig. 9). The flow rate was kept constant at 1.1 ml/min, and fractions were taken every 0.5 min. Scintillation liquid (3.5 ml) and distilled water (0.55 ml) were added in each fraction to count radioactivity (dpm) with the samecorrections as describedabove. Mass measurement of cellular free inositol. The polyol was measuredby a method that couplesthe NADH-forming reaction of inositol dehydrogenasefrom Enterobacter aerogenes to the sensitive Fe3’ bathophenantrolindisulfonic acid detection systemusing phenazine methosulfatefor electron transfer (10). Each determination was performed on 100 ~1 of neutralized extracts in a total volume of 420 ~1 containing the suitable concentrations of reagents(9). To avoid glucoseinterference in the assay, glucoseand dextran were omitted in all solutions during isolation, labeling, washing,and incubation of glomeruli. RESULTS

Turnover rate of [3H]inositol in nonstimulated glomeruli. The free inositol contained in sieved control glomer-

uli (labeled with myo-[3H]inositol for 2 h, washed, and then incubated for 30 or 10 min with 10 mM lithium in the absence of agonist) was measured chemically as indicated under MATERIALS AND METHODS. The amount found was 0.83 pmol/glomerulus (average value of 2 experiments), a value almost identical to that previously reported (31) for rat glomerulus. This content would correspond to a concentration of ~1.5 mM, assuming a volume of zO.5 nl/glomerulus. The turnover rate of cellular free inositol with extracellular inositol on the one hand, and with inositol contained in phosphoinositides on the other hand, was evaluated by performing kinetic experiments with myo-[3H]inositol in sieved glomeruli. In washed glomeruli that were incubated for various times in myo-[3H]inositol solution of high specific activity, the total radioactivity (free inositol plus total phosphoinositide plus total inositol phosphate radioactivities) increased almost linearly as a function of time up to 12 h as shown on Fig. 1. In this experiment, myo- [3H]inositol influx [calculated from

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F258

CARBACHOL z 2 ,.i!

STIMULATES

IP AND PI METABOLISM

IN ISOLATED

GLOMERULI

Table

1. Time-course of myo-[3H]inositol equilibration between cellular free inositol, total phosphoinositide, and total inositol phosphate pools

7000060000-

m 0

2

50000-

4 z 40000.)r :5 30000G z! f 20000L

Time, h Experiment

[3H]I, %

0

2

4

labeling

6

8

10

12

time (hours)

Fig. 1. Time course of glomerular uptake of myo-[3H]inositol. Sieved glomeruli were incubated in presence of myo-[3H]inositol (150 Ci/ mmol) at 37°C during various times indicated on abscissa. After each labeling time, extracellular myo- [ 3H] inositol was removed by washing, and total radioactivity in glomeruli was determined; dpm, disintegrations per minute. Each point is mean * SE of 5 determinations. This incorporation of [3H]inbsitol in glomerular cells follows the straight line y = -1,813 + 5,504t; r = 0.99.

the slope of the regression line relating total radioactivity in glomeruli with time (r = 0.99) and from the specific radioactivity of the extracellular myo- [ 3H] inositol (150 Ci/mmol)] corresponded to 0.016 pmol al0 glomeruli-’ h-l. When related to the amount of free inositol contained in glomeruli (8.3 pmol/lO glomeruli), this influx would represent only 0.19% of intracellular pool per hour. The estimation of how fast the specific activity of [3H]inositol incorporated into total phosphoinositide pool tends to approach that of the precursor, the intracellular free [3H]inositol, can be evaluated in the following way. If during the labeling period glomeruli are assumed to remain under steady-state conditions, then the fraction of the total radioactivity of glomeruli that is present in phosphoinositides should increase with time as the [3H]inositol specific activity in free inositol and phosphoinositide pools progressively equilibrates. The data of Table 1 (experiment A) and Fig. 2A concerning the radioactivities contained in free inositol, total phosphoinositides, and total inositol phosphates after the different labeling times are expressed as percent of the total radioactivity of the corresponding samples. As for free inositol and total phosphoinositides, control values measured after an additional lo-min incubation of the samples with 10 mM lithium were not different from those measured at the end of the labeling period (basal values), these values were pooled in Table 1. It is clear that the percentage of radioactivity present in total phosphoinositides increased, whereas that of free inositol decreased, as a function of labeling time. Such variations probably resulted from an equilibration of specific radioactivity between phosphoinositide and free inositol pools, which could be linearized as a function of time as shown on Fig. 2A. In fact the radioactivity fraction present in total phosphoinositides ( [3H]ZPIt, in %) increased exponentially towards the equilibrium ( [ 3H] ZPI,,) during l

%

[3H]ZIP,

%

Mean Total 3H Radioactivity, dpm/lO glomeruli

A

2 5 7 12 Experiment 0

[3H]ZPI,

82.2zk1.3 75.1k0.7 69.3ckO.4 67.3zkl.O

15.6k1.3 24.0zk0.7 28.5k0.8 30.820.9

2.1kO.4 0.9kO.2 2.2k0.4 1.8kO.4

8,694&827 22,404&1,279 35,364+1,995 60,798&2,860

B

78.821.0 19.8kO.8 1.4k0.2 0.5 72.OkO.B 26.2k0.8 1.7kO.l 1 68.3t1.2 29.921.0 1.9kO.2 2.2kO.l 1.5 68.0k1.7 29.8t1.6 2.OkO.l 2 66.020.8 32.OkO.7 62.Ok2.0 35.7k2.0 2.3k0.2 2.5 61.Okl.l 36.8zkl.O 2.2k0.3 3 Values are in % of total radioactivity recovered in each sample and A, labeling are means k SE of 10 determinations. In experiment duration with myo-[3H]inositol varied from 2 to 12 h. In experiment B, glomeruli were labeled for 2 h and distributed after washing into tubes containing a myo-[3H]inositol-free solution where they were kept for increasing times (O-3 h) before measurement of radioactivities in cellular free inositol ( [3H] I), total phosphoinositides ( [3H] ZPI), and total inositol phosphates ( [3H] ZIP). For both experimental conditions, measurements were also performed after a further 10 min in the presence of 10 mM lithium, and no difference was noticed with values obtained without this lo-min incubation with lithium; therefore these B was 7,052 values were pooled. Mean total radioactivity in experiment k 158 (n = 70).

the 12-h period of labeling, y = In

according to the equation

( [3H] ZPI,, - [3H] ZPI,) ([3H]zPIeq - [3H]t;lPI,,o)

= -kt

(I)

where [3H]ZPI, = 0 at time zero ( [3H] ZPI& and -k = 27.5%/h (r = 0.99), with [3H]ZPIeq = 32%. In the additional experiment depicted on Table 1 (experiment B ) and Fig. 2B, all glomeruli were first labeled for 2 h, then washed and incubated in the absence of myo-[3H]inositol. Every 30 min, samples were analyzed for free inositol, phosphoinositide, and inositol phosphate 3H content (basal values). Other samples were incubated for 10 min more in the presence of 10 mM lithium (control values) before such measurements. As previously, for each incubation time no difference was observed between basal and control values, and these were therefore pooled. Though total sample radioactivity remained constant in this experiment with washed prelabeled glomeruli, it is obvious from Table 1 (experiment B) and Fig. 2B that phosphoinositide radioactivity increased with the time at the expense of free inositol radioactivity. The percentage of total radioactivity contained in phosphoinositides increased again exponentially toward equilibrium according to Eq. 1, with [3H] ZPI,, = 45%, [3H]ZPIt=o = 21.2%, and -k = 34%/h (r = 0.98).

Phosphoinositide turnover rate may be calculated from the slope (12)and the size of phosphoinositide ( [3H] ZPI,,) and free inositol ( [3H] ZI,,) pools (in %), according to Eq. 2, where F is the mass flux of inositol entering or leaving the phosphoinositide pool per unit of time under steady-

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CARBACHOL

STIMULATES

IP AND PI METABOLISM

IN ISOLATED

F259

GLOMERULI

Table

2. Phosphoinositide turnover in isolated glomeruli Total

-k, %/h

-1

[3H]21eq,

%

[3H]ZPIeq,

%

‘;;;;;.F;e”

9 %/h

Y -2

-3

;

i!

f,

ii

lb

1;

Table 3. Radioactivities in cellular inositol-containing pools as a function of the labeling time: effect of a further incubation with carbachol in the presence of lithium

time (hours)

B

OQ

Experiment A 27.5 66 32 18.5 Experiment B 34.0 53 45 18.4 Kinetic analysis of data obtained in experiments A and B (see Table 1 and Fig. 2), corresponding to radioactivity measurements during and after glomerular labeling, respectively. As obtained from the slope of the regression lines (Fig. 2), lz is the sum of phosphoinositide and free inositol turnover rates. [3H] ZPI,,, % of radioactivity present at equilibrium in total phosphoinositides obtained from Eq. 1 as described in the text. [3H]ZIeq, % of radioactivity present at equilibrium in free inositol obtained by difference between 98% (see Table 1) and [3H] ZPI,, values.

Labeling Duration, h

y

Free Inositol

ZPI

ZIP

2 42.2H.2 14.3kO.8 43.5t0.9 5 34.3t0.7 15.1t1.4 50.6k1.6 7 34.3zkl.O 19.5t1.0 46.2t1.6 12 34.622.4 18.1kO.6 47.3t2.8 Values are in % of total radioactivity and are means 2 SE of 5 determinations. Compare with experiment A in Table 1. After various times of labeling with myo-[3H]inositol, sieved glomeruli were washed and incubated for 10 min in the presence of 10 mM lithium and 10B4 M carbachol before measurement of radioactivities in cellular free inositol, total phosphoinositides (ZPI), and total inositol phosphates (ZIP).

- 0.6

- 0.8

-1

0

1

3

2

time (hods)

greater than the rate of cellular free inositol turnover from extracellular inositol (=0.19%/h) under our experimental conditions. data of Table 1. According

Fig. 2. Linearization of phosphoinositide to &. 1 discussed in text, relationships obtained are as follows. A: y = 0.079 - 0.275t (r = 0.99) for experiment A where t is labeling time of glomeruli. B: y = 0.026 - 0.340t (r = 0.98) for experiment B where t represents time during which glomeruli are kept in a myo-[3H]-inositolfree solution after a 2-h labeling time.

state conditions PI turnover

rate =

[3H]PI

eq (2) --

k[3H]

ZIeq

[3H] ZPIeq + [3H] ZIeq Table 2 shows that the relative size of free inositol and phosphoinositide pools were not the same in the two experiments. However, kinetic analysis of the data leads to a similar value in the two experiments when the flux of inositol between the two pools is expressed as percent of the phosphoinositide pool size (18.5%/h). In contrast, when the flux is expressed as percent of the free inositol pool size, the results are 9 and 16%/h for experiments A and B, respectively. This suggests that, from one experiment to another, the free inositol content of glomeruli varied rather than the phosphoinositide content. The above experiments demonstrate that the rate of phosphoinositide turnover from intracellular free inositol is =18%/h in sieved glomeruli, i.e., a value -100-fold

Effects of carbachol on phosphoinositide metabolism in isolated glomeruli. The comparison of Table 1 (experiment A) and Table 3 data shows that, in the presence of

10 mM lithium, a lo-min stimulation with 10m4 M carbachol increased the fraction of the total radioactivity in inositol phosphates and decreased those in free inositol and phosphoinositides. Note that these fractions remained the same whatever the labeling duration. Similarly, for 2-h labeled glomeruli the radioactivity of inositol phosphates measured after 10 min in the presence of lOa M carbachol and 10 mM lithium did not change throughout at least a 3-h subsequent storage in a myo[ 3H]inositol-free solution (Fig. 3), although specific radioactivities in free inositol and phosphoinositide pools progressively equilibrated as during the labeling periods (see Table 1). Thus, in the presence of 10 mM lithium, the increase of inositol phosphate radioactivity induced by a IO-min stimulation with 10B4 M carbachol seems to be independent of the state of isotopic equilibration between phosphoinositide and free inositol pools. Glomeruli were labeled for 2 h in most of the following experiments. The sensitivity of the method is illustrated in Fig. 4. At the end of labeling, radioactivities in free inositol, phosphoinositides, and inositol phosphates were proportional to the number of glomeruli used (Fig. 4A). After

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F260

CARBACHOL

g 5000 tl E ,o 0"

4000

c E 6 E

3000-

3 5 r g

2000-

2 P "0 z

1000

l

IP AND PI METABOLISM

IN ISOLATED

6000

GLOMERULI

A 1

f

-

T

T f

carbachol

W4

M

-

.-E

7 c9 c;1

STIMULATES

0-y 0

r5 I I 1

control cl .

” I 2

El .

a 1 3

time (hours)

Fig. 3. Carbachol-induced accumulation of total [3H]inositol phosphates at various times after a 2-h labeling period. After labeling and washing, sieved glomeruli were kept in a myo-[3H]inositol-free solution during various times and then incubated for 10 min in presence of 10 mM lithium without or with 10B4 M carbachol before determination of radioactivity in total (2) inositol phosphates. Each point is mean * SE of 4 determinations. This experiment was repeated twice and produced the same results.

incubation of glomeruli (10 min with 10 mM lithium), the radioactivity associated with total inositol phosphates in control and stimulated conditions (without or with low4 M carbachol) was also proportional to the number of glomeruli (Fig. 4B). These results demonstrate that in the same experiment both the labeling of glomeruli and the stimulation of phosphoinositide metabolism are reproducible from one glomerulus to another. The effect of carbachol is evident in a single glomerulus, indicative of the high sensitivity of the method. Note that this sensitivity can beimproved by increasing the labeling time. We have used 10 glomeruli for each determination in all other experiments. For HPLC experiments, 100 glomeruli were used per determination to allow detection of isomers present in small quantities [Ins(1,4,5)P3 and Ins(1,3,4,5)P4]. Table 4 summarizes all data from experiments using sieved and dissected glomeruli labeled for 2 h. Total inositol phosphates (as % of total radioactivity) were significantly higher in sieved than in dissected glomeruli after lo-min incubation with 10 mM lithium and 10m4 M carbachol. The increase in inositol phosphate radioactivity was associated with a decrease in both free inositol and phosphoinositide radioactivities in sieved glomeruli and only in free inositol radioactivity in dissected glomeruli. The stimulation of phosphoinositide metabolism by carbachol (10 min with 10 mM lithium) was dose dependent (Fig. 5) with a mean 50% effective concentration (EC& of 3.5 t 1.5 (SE) PM (from 4 experiments) in dissected glomeruli. As shown in Fig. 5 the increase of the radioactivity in inositol phosphates for each carbachol dose was accompanied by an equivalent decrease of radioactivity in free inositol. Note that ECsO values obtained with sieved glomeruli were similar (data not shown). Experiments depicted in Table 5 were designed to

F .-s xL

1500

, c inositol

phosphates

mI” -

1000

500

0 6

number

of glomeruli

Fig. 4. Correlation between number of dissected glomeruli and radioactivities in inositol-containing cellular pools. A: after a 2-hour labeling period and washout of extracellular myo-[3H]inositol, radioactivities in cellular free inositol, total (Z) phosphoinositides, and total inositol phosphates were immediately determined. B: glomeruli were incubated for a further 10 min after washing in a myo-[3H]inositol-free solution in presence of 10 mM lithium and without -or with 10m4 M carbachol before determination of radioactivity in total inositol phosphates. Each point is mean t SE of 3 determinations. The same results were obtained in a duplicate experiment.

Table

4. Comparison of sieved and dissected glomeruli Free Inositol

ZPI

HP

Sieved glomeruli Control 75.9t1.5 22.5kl.2 1.6kO.2 Carbachol (10m4M) 40.5k1.6 14.7k1.2 44.822.2 Dissected glomeruli Control 70.1t2.4 27.6k2.1 2.3kO.3 Carbachol (10B4 M) 44.7t2.4 27.8k2.5 27.5k1.2 Values are in % of total radioactivity and are means t SE of 18 and 12 experiments for sieved and dissected glomeruli, respectively. Both types of glomeruli were labeled for 2 h, washed to remove extracellular myo-[3H]inositol, and incubated during 10 min in the presence of 10 mM lithium without or with 10m4M carbachol before determination of radioactivities in cellular free inositol, total phosphoinositides, and total inositol phosphates. Total radioactivity was 7,839 k 765 for sieved glomeruli and 11,022 k 1,761 dpm/lO glomeruli for dissected glomeruli.

verify whether the observed carbachol-induced decrease in free inositol radioactivity corresponded to a decrease in cellular free inositol content. Clearly, 300min incubation of glomeruli with 10 mM lithium and 10e4 M carbachol led to a comparable decrease of ~40% in both radioactivity and mass of free inositol. After 10 min of incubation, a reduction of ~17% in free inositol content was observed. The unusual conditions of these experi-

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CARBACHOL z 3 6E

IP AND PI METABOLISM

0

F261

GLOMERULI

A

~~yl-~

3000

3000

free

r

inositol 2000

2000

* h .Cr .-> : .-0 zL I I c9 -

IN ISOLATED

Z

0 m 2 &

STIMULATES

1000

control 1000

. 0

c inositol

phosphates

1 0

10

-8

10

carbachol

1

I

-6

10

concentration

e z al g

1

-4

10

400

1

300

-

5

-2

(M)

Fig. 5. Dose-dependent changes in total inositol phosphates and cellular free inositol radioactivities. Dissected glomeruli were labeled for 2 h with myo-[3H]inositol, washed, and incubated 10 min in presence of 10 mM lithium with indicated carbachol concentrations before measurement of radioactivities in cellular free inositol and inositol phosphates. Each point is mean t SE of 3 determinations from a single experiment, which is representative of 4.

0 c E

Q E 8 2 B ,o p. 5 .f: B

.

0

I

I

1

R

3 200

.

100

.

o-

Table 5. Cellular mass content of free inositol: effect of carbachol in presence of lithium Cellular Free Inositol, nmol/l,OOO glomeruli

3H Radioactivity, dpm/lO glomeruli Free inositol

ZIP

Control 0.83t0.05 18,005*1,337 4,477+189 Carbachol (10m4M) 0.48+0.03 10,244*1,571 16,954+772 Values are means * SE of 12 determinations. A dextran- and glucose-free solution was used throughout the experiment. Sieved glomeruli were incubated for K30 min in the presence of 10 mM lithium without or with lOa M carbachol. Cellular free inositol content was measured on samples of 1,000 unlabeled glomeruli, whereas radioactivities in cellular free inositol and total inositol phosphates were determined on samples of 10 glomeruli labeled 2 h with myo-[3H]inositol. From another experiment where incubation was carried out for only 10 min in presence of 10 mM lithium, cellular free inositol content was of .0.83 * 0.05 and 0.68 k 0.05 nmol/l,OOO glomeruli (n = 8) in absence or presence of lOa M carbachol, respectively. No radioactivity measurement was performed in this experiment.

ments (absence of glucose, see MATERIALS AND METHboth the higher incorporation of [3H]inositol in glomeruli (38) and probably the lower rate of stimulation of inositol phosphate production due to very high control values. The time course of the accumulation of different types of inositol phosphates during the incubation step with carbachol is shown in Figs. 6 and 7. In the presence of 10 mM lithium (Fig. 6), i.e., where inositol phosphate phosphatases are inhibited (4), lo-* M carbachol increased the radioactivity in InsP3 + InsP* and InsP2 with a maximum at 10 min. InsP-associated radioactivity increased linearly up to 10 min and thereafter tended slowly to a plateau. In the absence of lithium (Fig. 7), the radioactivity associated with all types of inositol phosphates was enhanced as soon as 5 s after the addition of 10m4 M carbachol. This increase reached a maximum at 1 min for InsP3 + InsP4 and InsP2 and at 3 min for InsP. Levels reached by the three types of inositol phosODS) may explain

loo4 M

control 0 0

10

I

0 1

20

30

time (minutes)

Fig. 6. Time course of radioactivities in inositol phosphates in presence of lithium. A: inositol phosphate (InsP). B: inositol 1,4-bisphosphate [Ins( 1,4)Pz]. C: inositol 1,4,5-trisphosphate plus inositol 1,3,4,5tetrakis-phosphate [Ins( 1,4,5)P3 + Ins( 1,3,4,5)P4]. After 2 h of labeling and washing, dissected glomeruli were incubated for various times in presence of 10 mM lithium without or with 10m4M carbachol before measurement of radioactivities in inositol phosphates. Each point is mean t SE of 3 determinations. The same data were obtained in a duplicate experiment.

phates were lower than in the presence of lithium and did not decline significantly down to control values in the continuous presence of carbachol during 10 min and even 30 min (see Fig. 8), indicating the absence of a desensitization phenomenon by the maximum dose of carbachol. Figure 8 also shows that, when 10 mM lithium was added after a 30-min incubation of glomeruli with carbachol in lithium-free solution, the radioactivities associated with the three types of inositol phosphates rapidly and markedly increased, which points out the rapid action of lithium on inositol phosphate phosphatases. In this experiment with sieved glomeruli, the increase of the radioactivity in InsP was linear up to 10 min of stimulation by carbachol as in the experiment shown in

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F262

CARBACHOL

STIMULATES

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4

120

-

90

-

60

-

30

-

.

.

0

~100 B Sur 1

control e

kl

1

T

10 . 1

LA

IP

control

0 0

2

4

6

0

10

time (minutes)

Fig. 7. Time course of radioactivities in inositol phosphates in absence of lithium. Experiment was carried out on dissected glomeruli as described in legend of Fig. 6 except that lithium was absent during incubation. A: InsP. B: Ins(1,4)P2. C: Ins(1,4,5)P3 + Ins(1,3,4,5)P4. Each point is mean k SE of 3 determinations. This experiment was repeated twice and produced the same results.

Fig. 6 in which dissected glomeruli were used. To identify precisely the inositol phosphate isomers in which the radioactivity increased after carbachol stimulation, HPLC anion exchange chromatography was performed on water-soluble extracts from 100 glomeruli that were incubated for 10 s (Fig. 9A) or 10 min (Fig. 9B) with low4 M carbachol in the absence or presence of 10 mM lithium, respectively. [3H]inositol phosphate isomer standards were run in parallel under the same experimental procedure to determine the nature of the six major peaks observed. We assume that peaks B, C, E, and F in Fig. 9 correspond to InsP, Ins(l,4)P2, Ins( l,4,5)P3, and Ins( 1,3,4,5)P4, respectively, because they have the same retention times. Peaks A and D probably represent glycerophosphoinositol and Ins( 1,3,4)P3, respectively, because the retention times correspond to

IN ISOLATED

GLOMERULI

those determined previously (27). As shown on Table 6, a 10-s stimulation with 10m4 M carbachol increased the radioactivity in InsP, Ins( 1,4)P2, and Ins( 1,3,4)P3 with little changes in Ins(1,4,5)P3 and Ins( 1,3,4,5)P4. No significant increase of the radioactivity in glycerophosphoinositol was observed. These data are in good agreement with the Dowex experiments in which we also observed a rapid increase of the radioactivity associated with InsP, InsPB, and InsP3 + InsP4 (Fig. 7). Furthermore, HPLC data clearly indicate that the radioactivity found in the InsPa + InsP4 fraction principally corresponds to Ins( 1,3,4)P3. In conditions of longer stimulation where inositol phosphate phosphatases were inhibited (lo-min incubation in the presence of 10 mM Li+), carbachol increased the radioactivity in InsP, Ins(l,4)Pa, and Ins(1,3,4)P3 but not in Ins(1,4,5)P3 and Ins(1,3,4,5)P4, compared with control values. There was no InsP5 or InsPG detected in these experiments. The specificity of the carbachol effect is supported by the effect of low4 M pirenzepine (a muscarinic antagonist) preincubated 15 min with glomeruli before the agonist stimulation. Pirenzepine inhibited by ~85% the increase of radioactivity in inositol phosphates resulting from lo-min incubation with 10 mM lithium and 10B4 M carbachol (Fig. 10). The effect of pirenzepine was reversible by washing (result not shown). As muscarinic stimulation of phosphoinositide metabolism is known to occur via G protein(s) (26), glomeruli were labeled with myo-[3H]inositol for 6 h in the absence or presence of pertussis toxin. We have observed that the toxin at 0.1 or 1 pg/ml slightly impairs the incorporation of myo-[3H]inositol in cellular free inositol, phosphoinositides, and inositol phosphates during the labeling period (see legend of Table 7). However, the carbachol-induced increase of total inositol phosphate and decrease in cellular free inositol radioactivities during a lo-min incubation in the presence of 10 mM lithium were not modified by pertussis toxin treatment. Note that a 12-h treatment with the toxin gave the same results (data not shown). DISCUSSION

Before addressing the question of the clear stimulation of phosphoinositide metabolism by carbachol in isolated rat glomeruli, it is necessary to analyze some characteristics of this biological material with regard to my~-[~H]inositol labeling. The specificity and some features of the uptake of myo-[3H]inositol by isolated rat glomeruli have been previously reported (38) and indicate the possible assimilation of glomerular free inositol to cellular free inositol after a few hours of labeling of glomeruli with myo- [3H]inositol. The rate of cellular free inositol turnover from extracellular myo- [ 3H] inositol was only 0.19%/ h under our experimental conditions. With such a rate, extracellular solution and cellular pools were still far from isotopic equilibrium even after 12 h of labeling. However, at this time, specific radioactivity in total phosphoinositides represents ~88% of that in cellular free inositol, because the rate of phosphoinositide turnover from cellular free inositol was ~18% h. In most of our experiments, i.e., after 2 h of labeling, specific radio-

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CARBACHOL

STIMULATES

IP AND PI METABOLISM

IN ISOLATED

20

10

incubation

without

30

30

t&no (minutes)

Li*

32

34 incubation

36

F263

Fig. 8. Carbachol-induced changes in radioactivities in inositol phosphates: effect of addition of lithium. After 2 h of myo- [3H] inositol labeling and washing, sieved glomeruli were incubated during various times without or with 10e4 M carbachol before determination of radioactivities in inositol phosphate. A: InsP. B: Ins(1,4)P2. C: Ins(1,4,5)P3 + Ins(1,3,4,5)P4. From 0 to 30 min incubation was performed without lithium (left), whereas 10 mM lithium was present between 30 and 40 min of incubation (r&h@. Note change in ordinate scale between left and right. Each point is mean & SE of 4 determinations. The same results were found in a duplicate experiment.

control

0

GLOMERULI

38

40

with 10 mM Li*

lns(1,4)P2 800

'600

Fig. 9. High-performance liquid chromatography (HPLC) separation of [3H]inositol phosphate isomers produced during carbachol stimulation. Sieved glomeruli were labeled for 2 h with myo- [3H]inositol, washed, and incubated without (thin lines) or with low4 M carbachol (thick lines) for 10 s in absence of lithium (A) or for 10 min in presence of 10 mM lithium (B). [3H]inositol phosphate isomers were separated by means of a discontinuous gradient of ammonium formate, which is shown in two profiles. Arrows show retention time of 4 types of [3H]inositol phosphate standards that were mixed and eluted under same experimental procedure.

Ins( 1,4)P2 InsP

1

B

I

rI

4000

C

10000

time (minutes)

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F264

CARBACHOL

STIMULATES

IP AND PI METABOLISM

IN ISOLATED

GLOMERULI

Table 6. Effect of carbachol on 3H radioactivity in inositol phosphate isomers Peak

A GroPIns

B InsP

C Ins( 1,4)Pz

D

E

Ins( 1,3,4)P3

F

Ins( 1,4,5)P3

Ins( 1,3,4,5)P4

s without Li+ Control 717234 16t4 72tlO 3k2 22k5 1423 Carbachol 773k55 540t42 913t63 199t15 44*5 27k7 10 min with 10 mM Li+ Control 641k168 137k27 77t9 17k2 74kll 13k5 Carbachol 790&72 32,824*5,282 5,090*348 1,157+101 4925 19t3 Values are means t SE and are in dpm/lOO glomeruli. GroPIns, glycerophosphoinositol; for definitions of other abbreviations, see MATERIALS AND METHODS. Five profiles were performed on extracts from labeled sieved glomeruli as described in the legend of Fig. 9 in each of the 4 conditions (10 s without lithium and 10 min with 10 mM lithium in absence or presence of 0.5 mM carbachol). 10

Table

8. Carbachol-induced accumulation of total inositol phosphates in glomeruli from different mammalian species Control

Fig. 10. Effect of pirenzepine on carbachol-induced accumulation of total [3H]inositol phosphates. After 2 h of myo-[3H]inositol labeling and washing, sieved glomeruli were incubated during 10 min in presence of 10 mM lithium without or with 10B4 M carbachol and in presence or absence of 10B4 M pirenzepine before measurement of radioactivity in total inositol phosphates. Each bar is mean k SE of 4 determinations. This experiment was repeated twice and produced the same results.

Table

7. Effect of pertussis toxin on m$o-[3H]inositol labeling of glomeruli and carbachol stimulation of phosphoinositide metabolism Control Untreated

Free inositol ZIP XPI

Treated with 1 Ilglml pertussis toxin

75.OkO.8

76.9k0.9

1.OkO.l 24.0k0.8

1.OkO.l 22.1z!zo.9

10B4 M Carbachol

Untreated

Treated with 1 /%/ml pertussis toxin

33.4k1.7 56.5k1.9 lO.lkO.4

33.8kl.l 55.4t1.3 10.8kO.5

Values are means k SE of 4 determinations from 1 experiment typical of 3 and are in % total radioactivity for each sample. Sieved glomeruli were labeled with myo-[3H]inositol for 6 h in absence or presence of 1 pg/ml pertussis toxin. After washing, glomeruli were incubated during 10 min in presence of 10 mM lithium without or with 10B4 M carbachol before measurement of radioactivities in cellular free inositol, total phosphoinositide, and total inositol phosphates. Total radioactivity was 23,558 k 1,155 and 17,996 k 693 dpm/lO glomeruli (n = 8) in untreated and treated glomeruli, respectively.

activity in total phosphoinositides was only ~30% of that in cellular free inositol. It can be noted that a quasiequilibrium (=99%) would be reached in 15 h between cellular [3H]inositol-containing pools, whereas at the same time specific radioactivity of these pools would be only ~3% of that of extracellular solution. This illustrates the slowness of the entry of myo-[3H]inositol in cells compared to the turnover rate between cellular pools

10m4 M Carbachol

Rat 71.2t8.5 3,388&65 Desert rat 36.227.7 632k50 Rabbit 9.7t2.9 64k7 Cat 22.2t3.1 105t5 Values are means k SE of 4 determinations in 3 different experiments and are in dpm/lO glomeruli. Sieved glomeruli were labeled for 2 h with myo- [3H]inositol, washed, and incubated for 10 min in presence of 10 mM lithium and without or with 10m4 M carbachol before measurement of radioactivity in total inositol phosphates. Rats, rabbits, and cats are laboratory animals, whereas desert rats (Merkes shuwi) are not bred in the laboratory.

as described in many systems (25). It is worth noting that the increase in inositol phosphate radioactivity, due to a lo-min incubation of prelabeled glomeruli with 10 mM lithium and 10m4M carbachol, did not depend on the state of [3H]inositol equilibration between cell free inositol and phosphoinositide pools at which the incubation was performed. This was observed in experiment of Table 3, where labeling duration was varied from 2 to 12 h, and also in that of Fig. 3, where glomeruli labeled for 2 h were washed and then kept for increasing times (O-3 h) in inositol-free solution before incubation with lithium and carbachol. These results can hardly be explained if, during carbachol stimulation, inositol phosphates are only produced from the phosphoinositide pool where the relative specific activity increased with time. They suggest that inositol phosphates also result from hydrolysis of phosphoinositides that are newly resynthetized from cytosolic free inositol pool where the relative specific radioactivity decreased. This hypothesis is supported by the following additional observations: intracellular free inositol radioactivity was always reduced by a lo-min stimulation of glomeruli with lOa M carbachol in the presence of lithium (compare Tables 1 and 3; see Tables 4,5, and 7; see Fig. 5). There was a time-dependent decrease in free inositol radioactivity induced by 10D4M carbachol (decrease significant at 3 min, data not shown). Similarly, there was a dosedependent decrease of free inositol after a lo-min stimulation by carbachol (Fig. 5). In dissected glomeruli, inositol phosphate-associated radioactivity represented ~27% of the total radioactivity after 10 min of stimulation with a maximum dose of

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CARBACHOL

STIMULATES

IP AND PI METABOLISM

carbachol (10e4 M), the increase of inositol phosphate radioactivity and decrease of free inositol radioactivity were equivalent, and no change was observed in phosphoinositides (Table 4). On the other hand, in sieved glomeruli inositol phosphate radioactivity constituted ~45% of the total radioactivity after carbachol stimulation (Table 4), and both free inositol and phosphoinositide radioactivities were reduced. This latter point may be explained probably by a higher rate of carbacholinduced breakdown of phosphoinositides in comparison to the rate of the resynthesis and not by a limiting supply of free inositol as discussed below. The reduction in cellular free inositol mass, after carbachol stimulation in the presence of lithium (Table 5), definitely demonstrates that the agonist induces a resynthesis of phosphoinositides and that the observed changes in free inositol radioactivity reflect mass changes. These results indicate also that at least ~40% of the glomerular free inositol pool is carbachol sensitive, and these support the hypothesis of the cellular inositol depletion to explain the effects of lithium on some tissues (3, 4, 10, 11). On the whole, the above observations suggest that both hydrolysis of phosphoinositides by phosphoinositidase(s) C and their synthesis from free inositol are enhanced by carbachol stimulation. Other aspects of the effects of lithium may also be considered. This cation greatly enhanced the accumulation of inositol phosphates (Figs. 6 and 8). The accumulation of InsP2 and InsPs + InsP4 decreased after ~10 min of stimulation (Fig. 6), indicating the existence of InsPB-, InsPs-, and InsP4-phosphatases, which are not totally inhibited by 10 mM lithium (4). In contrast, InsP accumulated linearly during 10 min in the presence of 10m4 M carbachol (Fig. 6 and 8), and then the accumulation tended to saturate (Fig. 6). These observations show that 1) lithium (10 mM) rapidly enters glomerular cells and completely inhibits InsP-phosphatase (4), 2) the supply of free inositol is not limiting during 10 min of stimulation by a maximum dose of carbachol in both dissected and sieved glomeruli, and 3) the saturation of !InsP accumulation after 10 min probably stems from a limiting supply of free inositol available for production of inositol phosphates and not from a desensitization phenomenon, because carbachol at maximum dose continuously stimulated inositol phosphate production up to 40 min (Fig 8). The effects of lithium also indicate the role of phosphatases in combination with phosphoinositidase(s) C in the regulation of inositol phosphate levels as previously reported (7). The glomerular response to carbachol exhibits many features encountered in other biological systems. HPLC experiments demonstrated that addition of a maximum dose of carbachol elicited a marked production of InsP, Ins( 1,4)Pz, and Ins( 1,3,4)Ps after 10 s without significant accumulation of Ins( 1,4,5)P3 and Ins( 1,3,4,5)P4 (Table 6). These features, which can be explained by a very fast conversion of the latter products to the former, have been previously described for ileal and airway smooth muscle, WRK1, and thyroid cells (6, 19, 27, 29). The presence of Ins(1,3,4)P3 clearly indicates that Ins(1,4,5)P3 is produced after addition of carbachol, which explains the rapid mobilization of calcium from intracel-

IN ISOLATED

GLOMERULI

F265

lular stores of isolated glomeruli (21). It also supports the existence of an active Ins(l,4,5)P3-kinase and Ins( 1,3,4,5)P4-phosphatase. It is likely from the present data that isolated glomeruli contain carbachol-sensitive phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] phosphoinositidase C. However, this does not exclude the presence of PtdIns and PtdIns(4)P phosphoinositidases C, which may respond to carbachol with the direct formation of InsP and Ins(1,4)Pz as found in pituitary cells and platelets (13, 14, 33). In conclusion, this study shows that isolated rat glomeruli represent a valuable system with which to study phosphoinositide signaling pathway. Although the use of whole glomeruli precludes the discrimination between responses of different cell types, we have observed that a large muscarinic stimulation of phosphoinositide metabolism (pertussis toxin insensitive) can occur in some glomerular cells. This response has been observed also in desert rat, cat, and rabbit glomeruli, although with smaller amplitudes probably due to different labeling and/or survival properties of cells (Table 8). Moreover, some characteristics of the observed response in the rat, i.e., an E& = 3 PM and the absence of a desensitization for a maximum dose of carbahcol, are consistent with a neurotransmitter response and therefore support a parasympathetic innervation of renal cortex. These results are quite different from those obtained on cultured cells, because carbachol has no action on glomerular endothelial cells (22) and because no direct cholinergic effect has been reported to date on mesangial cells (24). We are stimulating Address Cellulaire, France.

very grateful to Dr. N. M. Griffiths for critical advice and discussions. for reprint requests: P. Meneton, Laboratoire de Physiologie URA 219, CNRS, College de France, 75231 Paris Cedex 05,

Received 28 September 1990; accepted in final form 17 September 1991. REFERENCES Baer, P. G., L. G. Navar, and A. C. Guyton. Renal autoregulation, filtration rate, and electrolyte excretion during vasodilatation. Am. J. Physiol. 219: 619-625, 1970. 2. Baylis, C., W. M. Deen, B. D. Myers, and B. M. Brenner. Effects of some vasodilator drugs on transcapillary fluid exchange in renal cortex. Am. J. Physiol. 230: 114%1158,1976. 3. Berridge, M. J., C. P. Downes, and M. R. Hanley. Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206: 587-595,1982. 4. Berridge, M. J., C. P. Downes, and M. R. Hanley. Neural and developmental actions of lithium: a unifying hypothesis. Cell 1.

59: 411-419,1989. 5.

Creba, J. A., C. P. Downes, P. T. Hawkins, G. Brewster, R. H. Michell, and C. J. Kirk. Rapid breakdown of phosphatidyl 4-phosphate and phosphatidyl4,5-bisphosphate in rat hepatocytes stimulated by vasopressin and other Ca2+ mobilizing hormones. Biochem.

6.

J. 212: 733-747,1983.

Chilvers, E. R., I. H. Batty, P. J. Barnes, and S. R. Nahorski. Formation of inositol polyphosphates in airway smooth muscle after muscarinic receptor stimulation. J. Phurmucol. l&p. Ther. 252: 786-791,199O.

Chilvers, E. R., I. H. Batty, R. A. Challiss, P. J. Barnes, and S. R. Nahorski. Determination of mass changes in phosphatidylinositol4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle. Biochem. J. 275: 373-375, 1991. 8. De Michele, M., F. Amenta, and C. Cavallotti. Autoradiographic localization of muscarinic receptors within the rat kidney. 7.

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Eur. J. Phurmacol. 169: 297-305,1989. 9. Dolhofer, R., and 0. H. Wieland. Enzymatic assay of myoinositol in serum. J. Clin. Chem. Clin. Biochem. 25: 733-736, 1987. 10, Downes, C. P., and M. A. Stone. Lithium-induced reduction in intracellular inositol supply in cholinergically stimulated parotid gland. Biochem. J. 234: 199-204, 1986. 11. Drummond, A. H., and C. A. Raeburn. The interaction of lithium with thyrotropin-releasing hormone-stimulated lipid metabolism in GH, pituitary tumor cells. Biochem. J. 224: 129-136, 1984. 12. Hartupee, D. A., J. C. Burnett, Jr., J. I. Mertz, and F. G. Knox. Acetylcholine-induced vasodilatation without natriuresis during control of interstitial pressure. Am. J. Physiol. 243 (Renal Fluid Electrolyte Physiol. 12): F325-F329, 1982. 13. Hrbolich, J. K., M. Culty, and R. J. Haslam. Activation of phospholipase C associated with isolated platelet membranes by guanosine 5’-[y-thioltriphosphate and by thrombin in the presence of GTP. Biochem. J. 243: 457-465,1987. 14. Imai, A., and M. C. Gershengorn. Phosphatidylinositol 4,5bisphosphate turnover is transient while phosphatidylinositol turnover is persistent in thyrotropin-releasing hormone-stimulated rat pituitary cells. Proc. Nut!. Acad. Sci. USA 83: 8540-8544, 1986. 15. Kirk, C. J., G. Guillon, M. N. Balestre, and S. Jard. Stimulation, by vasopressin and other agonists, of inositol-lipid breakdown and inositol phosphate accumulation in WRKl cells. Biochem. J. 240: 197-204,1986. 16. Knox, F. G., and C. E. Ott. Filtration pressure disequilibrium in the dog glomerulus. Proc. Int. Congr. Nephrol. 6th Florence 1975, p. 216-222. 17. Lahera, V., M. G. Salem, M. J. Fiksen-Olsen, L. Raij, and J. C. Romero. Effects of Ng-monomethyl-L-arginine and L-arginine on the renal response to acetylcholine. Hypertension Dallas 15: 659-663,199O. 18. Lameire, N., R. Vanholder, S. Ringoir, and I. Leusen. Role of medullary hemodynamics in the natriuresis of drug-induced renal vasodilatation in the rat. Circ. Res. 47: 839-844,198O. E., J. Mockel, K. Takazawa, C. Erneux, and J. E. 19. Laurent, Dumont. Stimulation of generation of inositol phosphates by carbamoylcholine and its inhibition by phorbol esters and iodide in dog thyroid cells. Biochem. J. 263: 795-801, 1989. 20. Marchetti, J., S. Taniguchi, F. Lebrun, and F. Morel. Cholinergic agonists increase cell calcium in rat medullary collecting tubules. PfZuegers Arch. 416: 561-567, 1990. 21. Marchetti, J., F. Lebrun, and F. Morel. Effect of cholinergic agonists on cell calcium in single, microdissected Fura- loaded glomerulus: role of parietal sheet (Abstract). J. Am. Sot. Nephrol. 1: 475, 1990. 22. Marsden, P. A., T. A. Brock, and B. J. Ballermann. Glomerular endothelial cells respond to calcium-mobilizing agonists with releas’e of EDRF. Am. J. Physiol. 258: (Renal Fluid Electrolyte Physiol. 27): F1295-F1303, 1990. 23. McArdle, S., and L. C. Garg. Cholinergic stimulation of phosphoinositide hydrolysis in renal medullary collecting duct cells. J. Pharmacol. Exp. Ther. 248: 682-686,1989. 24. Mene, P., M. S. Simonson, and M. J. Dunn. Physiology of the mesangial cell. Physiol. Rev. 69: 1347-1424, 1989. 25. Michell, R. H., A. H. Drummond, and C. P. Downes (Editors). Inositol Lipids in Cell SignalZing. London: Academic, 1989. 26. Miller, R. T. Transmembrane signalling through G proteins.

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Kidney Int. 39: 421-429,199l. B., M. N. Balestre, and G. Guillon. Transient inositol( 1,4,5) trisphosphate accumulation under vasopressin stimulation in WRK, cells: correlation with intracellular calcium mobilization. B&hem. Biuphys. Res. Commun. 159: 953-960, 1989.

27. Mouillac,

28. Pirola, C. J., A. L. and V. E. Nahmod.

Alvarez,

M.

S. Balda,

S. Finkielman,

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