Vasopressin transiently stimulates phospholipase C activity in cultured rat hepatocytes

Biochimica et Biophy.s,ca Acta. I010 (1989) 227-232 Elsevier

227

BBA 12407

Vasopressin transiently stimulates phospholipase C activity in cultured rat hepatocytes Richard A. Pittner and John N. Fain Department of Biochemistr),. Universiw of Tennessee. Memphis'. Memphis. TN ( LLS.A.)

(Received 14 June 1988) (Re~ixedmanuscript received12 Sept,ember 1~8;8)

Key words: Vasopressin; Phospholip::seC: (Rat hcp:mx:r~c)

Vasopressin stimulated phospholipase C activity in primary culh~res of rat hepatocytes maintained for 18-24 h under serum free conditions. Soluble and membrane-associated phospholipase C activit~ was determined using exogenous [~H]phosphatidylinositol 4,5-bisphosphate ([ ~H]PIP2) in the presence of eholate, deoxyeholate and NaCI. Exposure of hepatocytes for 5 s to vasopressin (100 nM) stimulated both membrane-associated and soluble phospholipase C activity, by 30% and 40%, respectively. However, by 15 s this stimulation had disappeared. Addition of vasopressin to bepatocytes, previously labelled with [3H]inositol, stimulated inositol phosphate production within 5 s, but little further increase was seen over a 5-rain incubation. These results indicate that vasopressin rapidly stimulates both soluble and membrane-associated phospholipase C activity.

Introduction

Methods

Exposure of hepatocytes to vasopressin stimulates the rapid hydrolysis of P1P2 to produce I ~ and diacylglycerol. The diacylglycerol that is produced can activate protein kinase C, while the Ip~ leads to the release of Ca 2+ from intraeellular stores such as the endoplasmic reticulum (for reviews see Refs. 1 and 2). The mechanism by which occupancy of the vasopressin VI receptor leads to an activation of phospholipase C is unclear, but it is thought to involve a guanine nucleotide-binding protein 131. Several phospholipase C enzymes have been purified in various tissues both in the soluble and membrane fractions [4-7]. These enzymes specifically hydrolyze phosphoinositides and share many kinetic properties [8-11]. However, the role of soluble phospholipase C remains unclear. In this report, we show that using exogenously labelled PIP 2, both soluble and membrane-associated phospholipase C enzymes are transiently activated by vasopressin.

Preparation of hepatco,ies Hepatocytes were prepared from male 150-250 g Sprague Dawley rats, as described in Ref. 12. The hepatocytes were attached to 60-ram Falcon tissue culture plates at a density of approx. 2.5.106 cells/plate for 1 h in 3 ml of a modified Liebowitz L15 medium containing 10% (v/v) newborn calf serum and were maintained in this medium for 6-8 h. Hepatocytes were subsequently incubated in serum-free medium containing 0.2% (w/v) fatty acid-poor bovine serum albumin for a further 18-24 h in the absence of any added hormones. For labelling experiments, hepatocytes were attached to 35-mm Falcon six-well multiplates at a density of approx. 1.25.106 cells/well in 1.5 ml of medium.

Abbreviations: PIP2, phosphatidylinositol4,5-bisphosphale; IP3. inositol trisphosphate. Correspondence: R.A. Pitmer. Department of Biochemistry,University of Tennessee, Memp|fis, 800 Madison Avenue. Memphis, TI,] 38163, USA.

Fractionation of hepatocytes by filtration The filtration procedure was based on that described in Ref. 13. The albumin-containing medium was replaced 2 h prior to the experiments. At the beginning of the experiments, the plates were placed in a 37 °C water bath and fresh warm medium with or without vasopressin was added. After the appropriate incubation the medium was aspirated, the plates were placed on ice and 1 ml of ice-cold sucrose buffer was added which contained; 0.25 M sucrose, 0.5 mM dlthio,.hreitol and 10 mM Hepes (adjusted to pH 7.4 with NaOH). The

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228 TABLE 1

Estimated r('cot'er3" of Q'to~olic and membrane marker en:o'mes follo~'ing [titration of hepatecyte~ The estimated recovery of lactate dehydrogenase (LDH) 5'-nucleotidase az~d arylesterase activities from filtered hepatocytes was calculated b~ comparing enzyme activity in the filtrate to that found in ~hole-cell homosenates prepared as described in the Methods section. Units of enzyme activity arc/$iven in/~M NADH,/xM AMP and ~M indoxylacetate converted per mi:', per ml of sample, Results are means + S.E for triplicate plates from a representative exr~eriment. Enzyme

Total

Released

% release

LDH

2.89±0.0g

2.51 +0.01

87 + 1

5' Nueleotidase

0.62+0.03

0.08+0,01

14+ 1

AD'lcsterase

2.56 + 0.23

0.37 + 0.07

15 ± 3

cells were scraped from the plates and directly transferred to a 1-ml plastic syringe. The cells were then rapidly ruptured by hand pressure against a 0.22-/~M Millipore filter. The material passing through the filter was taken as the cell-free homogenate and stored at - 2 0 ° C. A majority of lactate dehydrogenase activity (87%) was recovered in the filtrate with less than 15% membrane marker enzyme contamination (Table I). If the hepatocytes were left on ice for a few minutes, or passed through a fine needle before filtering, the, the recovery of lactate dehydrogenase activity was closer to 100%. However, there was a much higher increase in the contamination of the extract with membrane-associated enzymes (results not shown). The total activity of the lactate dehydrogenase and the membcaqe-:ssociated enzymes 5'-nucleotidase and arylesterase were obtained from cells that were scraped from the plates in sucrose buffer and sonicated with a Fisher sonic dismembrator (0.6 relative output) for six 1-s bursts.

Preparation of soluble and membrane fractions For experiments in which t~oth membrane and soluble fractions were rec:uired, an additional media change was performed 5 rain before the experiment. Cells were scraped with 2 ml of sucrose buffer and were homogenized on ice for six 1-s strokes using a Potter-Elvejhem homogenizer fitted with a small Teflon pestle. The homogenate was centrifuged at 105 000 × gay in a 50 Ti rotor for 30 min in a Beckman Ultracentrit'uge. The membrane pellet was resuspended in 2 mi of sucrose buffer with a Polytron homogenizer. The fractior, s were then stored at - 20 o C.

Measurement of inositol phosphate production Hepatocytes were incubated for !8-24 h in albumin-containing medium that also contained [3H]inositol. The [3Hlinositol (in ethanol) was dried down and resuspended in the albumin medium to give 3-4 ~Ci/ml.

The medium was replaced 3 min prior to the experiments with unlabelled medium. At the end of the incubations, the media was aspirated, the plates were placed on ice and 1 ml of ice-cold methanol was added. The cells were scraped and transferred to polypropylene test tubes containing 1 ml of chloroform and 0.25 ml of 0.25 M HCI. The plates ,vere additionally washed with 0.5 ml of sucrose buffer. The phases were vortexed and separated by centrifugation at 2300 × g for 5 min. After neutralization with NH4OH, [3H]inositol phosphates in the upper phase were separated on Dowex-formate columns as described in Ref. 14.

Determination of phospholipase C activity Phospholipase C activity in the soluble and membrane frac,ions was determined as described [15]. Each assay contained, in a final volume of 100 btl; 100 mM Tris-HCI (pH 7.0), 100 mM NaCI, 2 mM sodium cholate, deoxycholate (1 mM for soluble or 2 mM for membrane enzymes), and approx. 25000 dpm (25-30 nM) of [3H]PIP2 plus 10/xl of homogenate containing about 10 btg of protein. Breakdown of PIP2 was between 2 and 4% of ~.dded label. Incubations at 37°C were terminated after 10 min with 1.25 ml chloroform/methanol (1:2, v/v). The phases were separated after the addition of 0.5 ml of chloroform and 0.5 ml of 0.25 M HCI, by centrifugation (2300 × g for 5 rain). Analysis of the upper phase by HPLC showed that the main product was IP3. However IP a and a small amount of IP I were also seen presumably as a product of phosphatase action in the homogenates (results not shown).

Determination of enzyme activities The activity of lactate dchydrogenase was determined as described [16]. Effects of vasopressin on phospholipase C activity were normalized for cytosolic lactate dehydrogenase activity in the filtration assays and for total homogcnat¢ lactate dehydrogenase activity when cells were homogenized and separated into membranes plus cytosol. Lactate dehydrogenase activity rather than protein was used to compensate for variations in the recovery of cytosol for filtration experiments and as an indicator for variations in cell number [17]. There was no significant effect of vasopressin on total lactate dehydrogenase activity (results not shown). Arylesterase activity was determined as described [18]. The determination of 5'-nucleotidase activity was based on that of Heppel and Hilmo¢ [19]. Each assay contained in a final volume of 250 ~tl: 80 mM Tris-HCI (pH 8.3), 8 mM MgCI 2, 1 mM AMP and up to 100 pg protein. The reaction was terminated after a 5-rain incubation at 37 °C by the addition of 750 pl of ice-cold 5.25% trichloroacetic acid. The extract was centrifuged at 23 000 × g for 5 min and free inorganic phosphate in the supernatant was determined essentially as described in Ref. 20.

220

Materials [ ~H]PIP2 ([inositol 2.3-Hlpi',,osphatid.Oinositol 4,5-bisphosphate, 4 C i / m m o l ) was obtained from New E n g l a n d Nuclear Research Product~: [-~H]inositol (myo[2-3Hlinositol 15 C i / n , m o l ) was from American RadiolabeUed Products; tis,~,e culture r,::,.gents were from Flow Laboratories; tissue culture plates from Falcon; bovine serum a l b u m i n was from A r m o u r Pharmaceutical Co; A G l - X 8 Dowex resin (100-200 mesh) formate form was fiom Bio-Rad; Collagenase, phosphate standards a n d other reagents were from Sigma Chemical Co.

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Fig. 1. Effects of vasopressin on phospholipase C activity of filtered hepatocytes. Intact hepatocytes were incubated without or with I00 nM vasopressin for the times indicated. Results are expressed as the percent activity relative to control incubations at each time point and were norfi,aliz¢.dagainst rta~o'V¢lyof !aerate denydrogenase activity. The basal activity of phospholipase C was 700 + 141 dpm of [~H]inositol phosphates formed per 10.~,laliquot over the 10-rainincubation period for the time.zero control a:ld varied by no more than 10-26% over the 300-s period. The assays were performed in the presence of 2 mM cholate, 1 mM deoxycholate and 100 mM NaCI. Results are means±S.E, of duplicate plates for the number of experimentsgiven in parentheses. The vasopressin-inducedincrease in activity at 5 s was significantbased on a paired Student's t.test (P < 0.01~,.

A filtration method based o n that of Taylor and Saggerson [13] was used to produce a relatively pure soluble fraction of hepatocytes rapidly. Using this rapid method, the effects of vasopressin addition to hepatocytcs o n the subsequent 'soluble' phospholipase C activity in the filtrate were determined, using exogenous [3H]PIP2 in the presence of 1 m M deoxycholate, 2 m M cholate a n d 100 m M NaCI a n d normalized against recovered lactate dehydrogenase activity. Fig. 1 shows that there was a transient increase in phospholipase C activity, in the presence of vasopressin. At 5 s, there was approx, a 60% increase in activity. However at 15 s, this increase was n o longer observed.

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Effects of t,asopressin on phospholipase C activity of soluble amt membrane fractions The experiments were repealed, but hepatocytes were

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Fig. 2. Effects of vasopressinon soluble and membrane-associatedphospholipase C activity. Hepatocytes were incubated without or with t00 nM vasopressin for the times indicated. Results are expressed as the percent activity relative to control incubations at each time point. The activity of phospholipasc C at time-zero was 878+ 128 and 531 :t:65 dpm of [:~H]inositolphosphate formed per 10-tal aliquot for soluble and membrane fractions, respectively. Assays were performed as described in Fig. 1, except that membrane fractions were assayed in the presence of 2 mM deoxycholate. Control activities varied by no more than 1-14% and 2-3% over the 300-s period for soluble and membrane fractions, respectively. Results are means± S.E. of duplicate plates for the number of experiments given in parentheses. The vasopressin-inducedincreasesin activity at 5 s were significant,based on a paired Student's t-tes~( * P < 0.eJ05).

230

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(Oeu~ycholetel (IN) Fig. 3. Effects of vasopressin on the de,.xycholate dose-respcngc of soluble and membrane-associated phospholipase C activity. Hepatocytes were incubated in the presence (0) or absence "o) of 100 nM vasopregsin :or 5 s. Soluble and membrane fractions were prepared and assayed with the indicated eoncentratior s of deoxycholate ~ : the presence of 2 m M cholate aed 100 m M NaCI, Results are expressed as the percent activity relative to incubations assayed m the absence of vagopressin at a concentration of I m M dcox.~cholate for soluble and 2 m M for membrane fractions. The basal activity of phospholipase C ~ a s 892 ± 100 and .i46± 75 dpm l~H]iaositol phosghates formed per 10-#1 aliquot activity for soluble and membrane fractions, resl-ectiveiy Rc,;ults a'e means+S.E., of five independent experiments. The significance of the vasopressin effect was calculated using a paired Student's t-test ( * P < 0.005; * * P < 0.02; * * * P < 0.01 ; ~ P < 0,005; * + P < 0.001).

homogenized and a soluble as well ,~s a membrane fraction was obtained. Phospholipase C activity was determined using exogenous [3H]PIP2 as in the studies shown in Fig. 1, except that the concentration of deoxycho/_ate was 2 mM in assays using the membrane fractions and was normalized against total homogenate lactate dehydrogenase activity. Fig. 2 shows thzt soluble phospholipase C activity was increased by approx. 40% at 5 s as shown for the filtered 'soluble' enzyme in Fig. 1. A similar increase was also seen in the mentbrane-associated enzyme (Fig. 2b). Of the total phospholipase C activit,¢ measured in these experiments, 62 .+. 3% (n = 12) was soluble. Vasopressin did not change the relative distribution of the enzyme as 63 + 4% was soluble in the presence of vasopressin (5 s, n = 12). Activation rather than translocation of soluble and membrane phos-

pholipase C occurred. No effect of vasopressin was seen at 5 s if the soluble and membrane fractions were assayed using exogenous PI in the presence of calcium (results not shown). Both the soluble and membrane-associated phospholipase C activities followed identical short-lived patterns of activation. To investigate this phenomenon further, fractions that were exposed for 5 s were analyzed to ensure that the activation was not merely due to a shift in t'he requirement for deoxycholate. Fig. 3 shows the deoxycholatc dose-response for soluble and membrane fractions assayed in the presence of 100 mM NaCI and 2 mM cholate. No significant effect of vasopressin was seen in the absence of deoxycholate. Vasopressin ~ignificantly increased phospholipase C activity of the soluble fraction at concentrations of de-

TABLE II

eff/ects of vg~opressin on the production o] inositol phosphates in intact hepatocytes Hepatocytes were in~.abated without or with vasopressin (100 nM) for the indicated times, Results are expressed in d p m of inositol phosphates for basal incubations at each time point, and the change (A) due to vasopressin (VP) as the m e a n ± S . E . , of four independent experiments for IP, IP~ and IP3 + IP4. The values for the increment in total inositol phosphates are from five experiments, Time

IP

(s)

basal

,:1 due to VP

basal

IP 2 2~ duc v> VP

basal

IP 3 + IP4 ,~ due to VP

(-'Adue to VP)

Tc~al inositol phosphates

5 15 60 300

1370 1430 1500 1 520

-4-154± 60 + 1 1 6 ± 57 +1562:75 +399+284

!15 110 130 125

+26+25 +34± 5 +44±23 +61 ±31

70 95 100 95

+35+21 ~-14±1g +9+27 + 1 +20

+2514- 44 + 1 7 3 ± 73 + 2 3 5 ± 76 +368:t:215

231 oxycholate between 0.5 and 2 5 mM (Fig. 3a). The vasopressin-mediated activation of the membrane fraction was only seen in the presence of deoxycholate at concentrations between 1.5 and 3 ml~.! (Fig. 3b).

Effects of t,asopressin on inositol phosphate Froduction in intact taepatoc~'~es Hepatocytes were prelabelled with [3H]inositol for 18-24 h in serum-free medium and were then exposed to vasopressin as described above in the absence of added lithium (Table I1). Vasopressin rapidly stimulated inositol phosphate production with 5 s, but there was little further increase at 15 or 60 s. The majority of the inositol phosphates that accumulated in the absence or presence of vasopressin were inosito! monophosphates (Table II). The stimulation by vasopressin of total inositol phosphates was statistically significant at 5 s ( P < 0.005), 15 s ( P < 0.05) and 60 s ( P < 0.02).

Discussion Vasopressin stimulates the breakdown of phosphoinositide in prelabelled rat hepatocytes [21-24] or membrane preparations [25]. Lin and Fain [26] reported that phosphoinositide breakdown due to vasopressin in hepatocytes was localized te the plasma membrane, but Kirk e t a l . [27] were unable to see this localization. However, the location of the phospholipase C enzyme that is stimulated by vasopressin has not been examined previously. Different forms of phospholipase C have been isolated and partially purified, some of which paper eytosolic in origin [4,71. The role of ~he soluble enzyme is at present unclear. In our hepatocytes, a large proportion (60-65%) of the total phospholipase C activity in homogenates prepared by the procedures described, and measured using exogenous PIP 2 was found to be soluble. The properties of the two enzymatic activities appear to be quite similar with respect to effects of deoxycholate (Fig. 3). Why the liver, and indeed many cell types, have a larger amount of soluble enzymatic activity after homogenization is unclear. In some respects this is analogous to the enzymes phosphatidate phosphohydrolase [28] and CTP-phosphocholine cytidyltransferase [29]. These two soluble enzymes appear to serve as an inactive reservoir of activity, which upon a given signal (fatty acids) is translocated to the endoplasmic reticulum, their site of action. Our initial studies were performed to see whether translocation of phospholipase C activity occurred upon treatment of hepatocytes with vasopressin. Fig. 1 shows that soluble phospholipase C activity was transiently increased by vasopressin in flit, red hepatocytes. This is a rapid method of isolating a relatively pure soluble fraction. However, the membrane fraction is lost by this procedure, but some may have been solubilized during the filtration. Should a translo-

cation have taken place, a loss of membrane activity should have been seen. Soluble and membrane fractions were prepared from home :cnized hepatocytes. A parallel increase in the meta,~tane-associaled and soluble phospholipa~e C activities was seen at 5 s (Fig, 2), but the relative distribution of enzxme activity between soluble and membrane fractions remained unaltered. We see no evidence for translocation taking place, but rather an activation of phospholipase C activity. We cannot, however, explain ff,e transient nature of the activation of soluble and membrane phospholipe.se C activity, but this was also observed for inositol phosphate accumulation by intact hepatocytes (Table II). The vasopressin response seen in our cultured hepatocytes in the absence of added lithium is quite transient and smaller than that seen in the presence of lithium. In conclusion, the present results demonstrate transient activation of phospholipase C activity by vasopressin in cultured rat hepatocytes that involved increases in both membrane-associated and soluble activity. The stimulatory effect of vasopressin is observed using exogenous PIP2 in the presence of 2 mM cholate, 10t) mM NaCI and 1-2 mM deoxycholate. Acknowledgements The authors would like to thank Anita Hardeman for preparation of the manuscript. This research was supported by a postdoctoral fellowship to R.A.P. from the Juvenile Diabetes Foundation and U.S, Public Health Service Grants (AM 36889 and 37004) to J.N.F.

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