69

Molecular and Cell&or Endocrinology, 69 (1990) 69-78 Elsevier Scientific

MOLCEL

Publishers

Ireland,

Ltd.

02224

Substance P and related tachykinins induce receptor-mediated hydrolysis of polyphosphoinositides in the rat anterior pituita~ Sarren E. Mau I, Philip J. Larsen 2, Jens A. Mikkelsen 2 and Torben Saxmark

1

’ The Protein Laborutoty, and ’ lnsi~tu~eof Medical Anatomy, Deparfme~t 3, Un~oers~~~ of Copenhagen, Copenhagen, Denmark (Received

Key words: Polyphosphoinosit~de;

Phaspholipase

21 August

1989; accepted

C; Tachykinin;

Anterior

13 November

pituitary;

1989)

Receptor

Summary

In the present study characterization of phosphatidylinositol4,5-bisphosphate-specific phospholipase C (PIP,-PLC) activity and receptor-mediated hydrolysis of PIP, in rat anterior pituitary membranes were investigated. Incubation of the membrane fraction of anterior pituitary homogenate with [ ‘~]inositol-labeled PIP, in the presence of calcium increased the concentration of the water-soluble degradation product inositol trisphosphate (IP,) in a time-dependent manner. PIP,-PLC in the rat anterior pituitary had a pH optimum at 5.5 and a requirement for cations. Ca2+ and Mg2+ could activate the enzyme. Activity was maximal at a total magnesium concentration of 1 mM and at a free Ca2+ concentration of 100 FM. The addition of the detergent Triton X-100 (0.05% w/v) to the membrane fraction resulted in a 50% decrease of PIP,-PLC activity, whereas the presence of sodium deoxycholate (1 mg/ml) in the membrane fraction increased the PIP,-PLC activity by 100%. The tachy~nins substance P, 8-Tyr-substance P, physalae~n, neurokinin A, eledoisin, kassinin and neurokinin B induced receptor-mediated breakdown of f3H]inositol-labeled PIP, in the membrane fraction in a concentration-dependent manner, but with different potencies. The tachykinins displayed the following rank order of potencies: substance P > %Tyr-substance P > physalaemin > neurokinin A > eledoisin > kassinin > neurokinin B, which is consistent with the involvement of a NK-1 receptor. Combined treatment of anterior pituitary membranes by substance P and thyrotropin-releasing hormone (TRH) resulted in an additional increase in PIP,-PLC activity compared to stimulation with TRH alone.

Introduction Address for correspondence: Seren E. Mau, The Protein Laboratory, University of Copenhagen, Sigurdsgade 34, 2200 Copenhagen N, Denmark. phosphatidylinositol 4,5-bisphosAbbreviations: PIP,, phate; IPs, inositol t&phosphate; IP,, inositol bisphosphate; IP,, inositol monophosphate; I, inositol; PIP,-PLC, phosphatidylinositol 4,5-bisphasphate-specific phospholipase C; DG, diacylgly~rol; PPI, pol~hosphainositides. 0303-7207/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

The undecapeptide substance P, which is a putative neurotransmitter (Nicoll, 1980), mediates some of its effects by Ca2+ release from an intracellular pool and stimulation of a univalent ion flux in some tissues (Putney, 1977; Abdel-Latif, Ltd.

70

1986). These actions are likely to be mediated through a Ca*+ -mobilizing receptor, having substance P as the endogenous agonist (Merrit and Rink, 1987). Substance P has been shown to stimulate turnover of polyphosphoinositides in the central nervous system, intestinal tissues and salivary glands (Hanley et al., 1980; Watson and Downes, 1983; Mantyh et al., 1984; Taylor et al., 1986). Thus substance P stimulates incorporation of [3H]inositols in parotid gland cell membranes (Hanley et al., 1980) and the formation of [3H]IPs and [3H]IP4 from [3H]inositol-labeled parotid acinar cells (Merrit and Rink, 1987). Several lines of evidence indicate that the initial event in cytosolic Ca2+ mobilization involves the breakdown of PIP, to 1,2-diacylglycerol (DG) and water-soluble 1,4,5-inositol trisphosphate (IP,); the former activates a phospholipid-dependent protein kinase, called protein kinase C (Nishizuka, 1986), and the latter serves as a second messenger to mobilize Ca2+ (Berridge, 1984, 1987; Berridge and Irvine, 1984). The hydrolysis of PIP, to inositol trisphosphate is catalyzed by the Ca2+-dependent cytosolic or membrane-bound enzyme phospholipase C (a PIP,-phosphodiesterase) (Abdel-Latif, 1986). Within the anterior pituitary receptor-mediated polyphosphoinositide metabolism is associated with the secretion of prolactin, luteinizing hormone (LH), thyrotropin-releasing hormone (TRH) and corticotropin (ACTH) from pituitary cells (Griffths and Bennet, 1983; Enjalbert et al., 1985; Raymond et al., 1985; Andrews and Corm, 1986). The release of prolactin, which is CaZf dependent, is stimulated by TRH, neurotensin and angiotensin II among others (Canonico, 1985; Gershengorn, 1986), leading to increased turnover of polyphosphoinositides. However, substance P has been shown to stimulate prolactin secretion in vivo (Kato et al., 1976; Chihara, 1978) and from cultured pituitary cells, but the cellular mechanism involved is at present unknown (Vijayan and McCann, 1979). Furthermore, results suggesting a role for substance P on gonadotropin-releasing hormone (GnRH) action on gonadotropes, which seem to bind substance P, have been presented (Morel et al., 1982; Makara et al., 1986). Recently, substance P binding sites, classified as a NK-1 receptor, have been demonstrated in anterior

pituitary membranes indicating that one mechanism by which substance P exerts its neuroendocrine function could be a direct action (Kerdelhue et al., 1985; Larsen et al., 1989a). Furthermore, the ligand-receptor complex is internalized in rat anterior pituitary cell cultures (Larsen et al., 198913). As suggested by Watson and Downes (1983) monitoring of polyphosphoinositide hydrolysis has many advantages compared to other assays for substance P receptors, due to its very high sensitivity. To investigate the possibility that substance P stimulates increased turnover of PIP, in the anterior pituitary, a study on the action of substance P and related tachykinins on PIP,-phospholipase C enzymatic activity was performed. Materials and methods Materials Substance P, neurokinin A, neurokinin B, physalaemin, 8-Tyr-substance P, eledoisin, kassinin, TRH and vasoactive intestinal peptide (VIP) were purchased from Cambridge Research Biochemicals (Cambridge, U.K.). [ Myo-inositol3H]phosphatidylinositol 4,5_bisphosphate, [ myoinositof-3H]inositol 1,4,5_trisphosphate and [mvoinositol-3H]inositol 1,3,4-trisphosphate were from Amersham (Buckinghamshire, U.K.). Earle’s balanced salt solution (EBSS) came from Gibco (Paisly, Scotland, U.K.). All other reagents were from Sigma Chemical Co. (St. Louis, MO, U.S.A.). [Myo-inositol‘Hlphosphatidylinositol 4,5-bisphosphate and [ myo-inositol-3H]inositol trisphosphates were stored at -20 “C and were stable for at least 2 months. Methods Rat anterior pituitary homogenate and membrane fraction were prepared as follows. Wistar male rats (200-250 g, ten rats/experiment) were anaesthetized with 5% CO* and decapitated. The anterior pituitary was immediately removed and placed in ice-cold EBSS. The pituitaries were washed 3 times with ice-cold EBSS and resuspended in 10 ml ice-cold 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) with 300 mM sucrose, pH 7.0. The suspension was

71

homogenized and centrifuged at 400 X g for 5 n-tin, the supernatant (S,) was removed and repeatedly centrifuged at 400 X g for 5 min. The supernatant (S,) was additionally centrifuged at 10,000 x g for 30 min. The supematant (S,) was isolated and the pellet (Pi) was resuspended in Hepes/ sucrose buffer and additionally homogenized. The fraction was immediately stored at - 20 o C for no longer than 1 week. Aliquots from the homogenate and the final suspension (Pl) were used for protein determination according to Lowry et al. (1951). 1 mg protein/pituitary was recovered, which represented 40% of the total homogenate protein content/pituitary. PIP,-PLC

assay

Immediately before an experiment the packaging solvent of the substrate [ myo-inositof-3H]phosphatidylinositol 4,5-bisphosphate was evaporated by vacuum centrifugation and the substrate dissolved in a Tris buffer (20 mM) pH 7.0 with 100 mM NaCl and 1 mM sodium deoxycholate by sonication (2 X 30 s, 60 W). Specific activity of the substrate was measured before each assay. PIP,-PLC activity was determined by adding an aliquot of rat anterior pituitary membrane suspension (So-150 pg protein) to an incubation mixture of [ myo-ino.sitol-3H]phosphatidylinositol 4,5-bisphosphate (approximately 15,000 cpm) and Mes buffer (40 mM) with 120 mM NaCl, 1 mM MgCl,, 10 mM LiCl and 1 mM CaCl, in a final volume of 1 ml. The assay was linear with respect to time (60 min) and protein concentration (max. 200 lJg/mQ When various neuropeptides were tested the incubation mixture (pH 6.5) additionally contained peptidase inhibitors: Trasylol (100 kallikrein inhibiting units), Bacitracin (40 pg/rnl), leupeptin (4 pg/mg), chymostatin (2 pg/rnl), sodium deoxycholate (1 mg/ml) and ATP (3 mM). A solution containing membrane suspension, incubation buffer, peptidase inhibitors, sodium deoxycholate and ATP served as control in the neuropeptide dose-response experiments. Initially the neuropeptides were incubated for 30 min at 0 o C without substrate in the incubation mixture. The assay was pre-formed in triplicate at 37 o C in a shaking water bath for 15 min and started by adding the substrate. The reaction was terminated

by addition of 3.75 ml CHCl,-MeOH (1: 2) to the incubation media. Samples were then mixed with 1.25 ml CHCl, and 1.25 0.1 N HCl by vortexing and centrifuged (2000 x g for 1 min) to form an upper hydrophilic MeOH/H,O phase and a lower hydrophobic CHCl, phase. PIP,-PLC-induced formation of the labeled products (inositol phosphates) was determined by liquid scintillation counting of an aliquot of the MeOH/H,O phase. Non-enzymatic hydrolysis of [myo-inositol3H]phosphatidylinositol 4,5-bisphosphate was measured by adding 3.75 ml CHCl,-MeOH (1 : 2) to the incubation mixture before adding membrane suspension, or in case of the neuropeptide stimulation experiments, before adding the substrate. Up to 10% of the total amount of substrate was hydrolyzed. HPLC

analysis

High-performance liquid chromatography (HPLC) analysis of inositol phosphates was performed as described previously (Irvine et al., 1985; Wregget and Irvine, 1987). Briefly, samples from the upper MeOH-H,O phase were neutralized with Tris base to pH 7.0, and EDTA was added to a final concentration of 1 mM. The water-soluble inositol phosphates were analyzed by anion-exchange HPLC technique using a Partisil Sax 10 column, 25 cm X 4.6 mm (HPLC Technology). AMP, ADP, and ATP were added to the samples to indicate the elution time of IP, IP,, IP,-(1,3,4). [3H]IP3-(1,4,5) and [3H]IP3-(1,3,4) standards were used to determine the elution time of these compounds. A gradient was passed through the column, rising from water to 100% 1.7 M ammonium formate (buffered to pH 3.7 with ortophosphoric acid) over 39 min. The chromatography was performed at ambient temperature with a flow rate of 1 ml/mm. 0.5 ml fractions were collected, and the activity was determined by liquid scintillation counting. Free

Ca’ + assay

Free Ca2+ concentrations in the range 10V9 to 5 x 10e3 M were obtained with a two-metal-ion Ca2+/ Mg2+/ EDTA/ EGTA buffering system as described by Flodgaard and Fleron (1974) using the binding constants stated by Sillen and Martel

72

(1971). The concentrations of total Mg2+, EDTA and EGTA were fixed at 1 mM. The free calcium concentrations were varied by changing the total amount of calcium added. Inclusion of 1,2-bis(2aminophenoxy)ethane N, N, N ‘, N ‘-tetraacetic acid (BAPTA) (10 PM) was used to verify the calculated Ca2+ concentrations (Tsien, 1980). The Ca2+-dependent absorption by BAPTA was read at 254 nm. Results Characterization of PIP,-PLC in rat anterior pituitary membranes Phospholipase C activity in the rat anterior pituitary membrane fraction was measured by hydrolysis of PIP, followed by chloroform extraction of excess PIP,. The degradation products identified by HPLC were mainly inositol and IP,, but also smaller amounts of IP, and IP, were detected (Fig. 1). The identification of IP, was based on elution time of a radiolabeled IP, standard. LiCl was added to the incubation media in order to inhibit the degradation of inositol monophosphate to inositol (Berridge et al., 1982). The effect of pH was determined in the pres-

ence of 1 mM CaCl, and 1 mM MgCl 2 in a Mes/NaCl buffer in which the pH varied between 4.0 and 8.0. The PIP,-PLC activity was found to be maximal at pH 5.5 as indicated in Fig. 2. At pH 6.5 the activity was 80% of maximal. However, binding of substance P to rat brain membranes is maximal at pH 7.2 and 40% at pH 6.5. Hence, we decided to perform our experiments at a pH value where binding of substance P was still possible. The PIP,-PLC activity was found to be dependent of the cations since addition of 10 mM EDTA totally abolished enzyme activity. Total cation concentrations greater than 10 mM resulted in inhibition of enzyme activity. Mg2+ was a potent inducer of PIP,-PLC activity. Half-maximal activity was achieved between 1O-3 and 10e4 M Mg2+ (Fig. 3). The PIP,-PLC activity was measured as a function of the free Ca2+ concentration. A two-metalion buffer system with Ca2+ as the primary, Mg2+ as the secondary with EDTA and EGTA as the chelators was used. The concentration of free CA2’ was varied by changing the total added Ca2+. EDTA, EGTA and total Mg2+ were fixed at concentrations of 1 mM. Maximal PIP,-PLC activity was detected at 100 PM free Ca” (Fig. 4). The

Fig. 1. HPLC analysis of inositol phosphates in the membrane fraction with (left panel) and without (right panel) substance P stimulation. [3H]PIPz was incubated with the membrane fraction in Mes/NaCl buffer pH 5.5 or 6.5 (for substance P) in the presence of 1 mM CaCl,, 1 mM MgCl, and 10 mM LiCl. Substance P (10V6 M) was used as agonist. The incubation periods were 0 and 15 min. Termination procedure and analysis of the degradation products by HPLC were carried out as described in Materials and Methods, White bars indicate activity at t = 0 and black bars additional activity after 15 min. X-axis: AMP, ADP and ATP indicate the fractions were these compounds are eluted. (1) Inositol; (2) inositol monophosphate; (3) inositol bisphosphate; (4) inositol 1,3,4_trisphosphate; and (5) inositol 1,4,5_trisphosphate. ‘H activity in the fractions was determined by liquid scintillation counting. Results represent the mean of three different experiments. Inter-assay variation was less than 10%. The increase in inositol phosphate(s) activity from r = 0 to f = 15 min was significant (p < 0.05) and was determined by a f-test calculated by a computer program (Statgraf).

73 600

1 500. E

400.

-e

300.

x = ., 5 a

$y, {. /

/ ;

200.

o-7

-6

-5

-4

Free Ca Log

I1 4

I

1

.

4.5

5

5.5

6

6.5

7

7.5

a

PH

Fig. 2. The effect of pH on PIP,-PLC activity from rat anterior pituitary membrane fraction. [ Myo-inositol-3H]phosphatidylinositol 4,5-bisphosphate was incubated with an aliquot of membrane fraction and Mes/NaCl buffer for 15 min at 37 ’ C. The CaCl, and MgCl, concentrations were 1 mM, and the pH ranged from 4.0 to 8.0. ‘H activity was determined by liquid scintillation counting of the MeOH/H,O phase as described in Materials and Methods. Y-axis: 100% denotes 410 + 36 cpm. Each point represents the meanf SD of three separate determinations.

-3

-2

IM)

Fig. 4. PIP,-PLC activity as a function of the free Ca2+ concentration in a two-metal-ion buffer system. With PIP, as the substrate PIP,-PLC activity was assayed at the indicated free Ca*+ concentration in the incubation buffer containing membrane suspension using a two-metal-ion buffer system with Ca2+ as the primary, Mg*+ as the secondary and EDTA and EGTA as the chelators. The concentration of free Ca’+ was varied by changing the total concentration of Ca2+. The total concentrations of EDTA, EGTA and Mg2+ were fixed at 1 mM. The experiment was performed at 37 ’ C for 15 min. ‘H activity was determined by liquid scintillation counting of the MeOH/H,O phase as described in Materials and Methods. The data represent the mean+ SD of three different experiments.

increase in free Ca 2+ changed the free MgZf concentration from 0.90 mM at lo-’ M free Ca2+ to 0.98 mM at 3.5 mM free Ca*+. This change in the Mg2+ concentration would not in itself influence the activity of the enzyme, since it is within the

TABLE

1

POTENCY OF VARIOUS PIP,-PLC-DEGRADATIVE

-7

-6

-5

-4

Log Mg cont.

-3

-2

-1

(Ml

Fig. 3. The effect of Mg2+ on PIP,-PLC activity. An aliquot of membrane suspension from rat anterior pituitary was incubated with [ myo-in&to/-‘Hlphosphatidylinositol 4,5-bisphosphate in a Mes/NaCl buffer, pH 5.5. Mg*+ concentrations varied from lo-’ to 10m2 M. Application of 10 mM EDTA to the media totally inhibited PIP,-PLC activity. Control indicates the activity in the media, when Ca2+ was present (1 mM). 3H activity was determined by liquid scintillation counting of the MeOH/ H,O phase as described in Materials and Methods. Y-axis: 100% denotes 490 + 45 cpm. Each point represents the mean f SD of three separate determinations.

TACHYKININS ACTIVITY

TO INDUCE

The concentration needed to increase PIP,-PLC activity to half maximal (EC,,) was estimated from the dose-response curves (Figs. 6 and 7) by non-linear regression analysis. Relative potencies (substance P = 1) were calculated from the EC,, values. The results represent the data of three experiments.

Substance P &Tyr-substance Physalaemin Neurokinin A Eledoisin Ksssinin Neurokinin B

P

EC,, W

Relative potency

2.2*0.3x10-* 6.3*0.2x10-’ 7.1*0.2x10-s 9.7*0.3x10-s 2.0*0.1x10-’ 3.0*0.1x10-’ 5.6+0.2x10-’

1 0.34 + 0.31* 0.22 * 0.11 * 0.07 * 0.04 *

0.05 0.05 0.03 0.02 0.01 0.01

74

concentration range for optimal activity (Fig. 4). Inclusion of substance P in the free Ca2+ assay (free Ca*+ ranging from lop3 to 10W9 M) did not change the affinity for Ca*+, neither was the amplitude of the substance P response altered. The influence of the detergent Triton X-100 on PIP,-PLC activity is shown in Fig. 5. The detergent was incubated in various concentrations for 15 min under standard conditions (without sodium deoxycholate) as described in Materials and Methods. At a concentration of 0.05% (w/v) the enzyme activity was diminished to the 50% of control value (i.e., no detergent added). At greater detergent concentrations the PIP,-PLC activity was increasingly inhibited. Sodium deoxycholate at a concentration of 1 mg/ml raised PIP,-PLC activity by 100% compared to control (data not shown). Tachykinin activation of PIP,-PLC To elucidate receptor-mediated PPI we performed an experiment tachykinins as putative agonists.

-2

-1.6

-1.2

-0.6

hydrolysis of using various Figs. 6 and 7

- 0.4

0

Fig. 5. The effect of the detergent Triton X-100 on PIP,-PLC activity. The membrane fraction was incubated with [ myo-inositol-3H]phosphatidylinositol 4,5-b&phosphate in a Mes/NaCl MgCl, and Triton X-100 at different buffer, 1 mM CaCl,, concentrations ranging from 0.02% to 1.0% (w/v) for 15 mm and at 37°C. Control indicates the activity without Triton X-100 and sodium deoxycholate in the incubation media. ‘H activity was determined by liquid scintillation counting of the MeOH/H,O phase as described in Materials and Methods. Y-axis: 100% denotes 455 f 55 cpm. Each point represents the mean f SD of three separate determinations.

240-

200.

.EO

-10

-9 Log

-6 peptide

-7 cont.

-6

-5

(Ml

Fig. 6. Substance P (SP), physalaemin (PHYS), 8-Tyr-substance P (8-Tyr-SP) and eledoisin (ELED)-mediated hydrolysis of phosphatidylinositol4,5-bisphosphate. Dose-response curve. Initially the peptides were incubated at the concentrations indicated with an aliquot of membrane fraction from rat anterior pituitary and various enzyme inhibitors in a Mes/NaCl buffer, pH 6.5, 1 mM CaCI,, 1 mM MgCl,, sodium deoxycholate (1 mg/ml) and 3 mM ATP for 30 min at 4°C. Thereafter [ myo-inositol-‘Hlphosphatidylinositol 4,5-bisphosphate was added and the mixture treated as described in Materials and Methods. Control indicates the activity in the incubation media without tachykinins added. ‘H activity was determined by liquid scintillation counting of the MeOH/H,O phase as described in Materials and Methods. Y-axis: 100% denotes 1210 _+95 cpm. Each point represents the mean f SD of three separate determinations.

show a dose-response curve for substance P, 8Tyr-substance P, physalaemin, neurokinin A, eledoisin, kassinin, neurokinin B and VIP. Prior to the assay the tachykinins were allowed to bind at 0 o C for 30 min in a Mes/NaCl buffer containing 1 mM CaCl,, 1 mM MgCl,, 10 mM LiCl, 3 mM ATP and membrane suspension. The tachykinins increased PIP,-PLC activity to half maximal (EC,,) at the concentrations given in Table 1. The tested tachykinins stimulated PIP,-PLC activity in the following rank order of potency: substance P > 8-Tyr-substance P > physalaemin > neurokinin A > eledoisin > kassinin > neurokinin B. Substance P (lop6 M) induced- a maximal activity amplitude of 200% above control level. TRH (lo-” M), a well-known potent PIP,-PLC promotor, displayed an EC,, at 5.1 + 0.1 x 10e9 M (data not shown) and a maximal amplitude of 305% above control level. Simultaneous stimulation of PIP,-PLC activity by substance P (10e6 M) and

24

I

-10

-9

-9

-7

Log peptide cow.

-6

-5

(M)

Fig. 7. Effect of neurokinin A (NKA), neurokinin B (NKB), kassinin (KASS) and VIP on hydrolysis of phosphatidylinosito1 4,5-bisphosphate. Dose-response curve. Initially the peptides were incubated at the indicated concentrations with membrane suspension from rat anterior pituitary with various enzyme inhibitors in a Mes/NaCl buffer, pH 65, 1 mM CaCl,, 1 mM MgCI,, sodium deoxycholate (1 mg/ml) and 3 mM ATP for 1 h at 4O C. [ Myo-inosirol-3H]phosphatidylinosito1 4,5_bisphosphate was added and the mixture was placed at 37OC for 15 mm and treated as described in Materials and Methods. Control indicates the activity in the incubation media without tachykinins added. 3H activity was determined by liquid scintillation counting of the MeOH/H,O phase as described in Materials and Methods. Y-axis: 100% denotes 1210 + 95 cpm. Each point represents the mean f SD of three separate determinations.

lar pH. However, similar results have been obtained in iris smooth muscle, sheep seminal vesicles, rat brain, bovine brain, rat kidney cytosol, rat liver, lymphocytes and rat pineal gland (Abdel-Latif, 1980; Hirasawa et al., 1982a; Hoffmann and Majerus, 1982; Ho and Klein, 1987; Katan and Parker, 1987; Manne and Kung, 1987; Ryo et al., 1987). Furthermore, a second optimum of PIP,-PLC activity has been reported in some of these tissues at neutral or slightly alkaline pH. A similar second peak was not found in our assay. This could either be due to inactivation of a special form of the enzyme during homogenization or that it is localized to the cytosolic fraction which we did not test for PIP,-PLC activity when pH was varied. It is generally accepted that PIP,-phospholipase C is sensitive to Ca*+ and thus several workers have brought to attention the cationic requirement of this enzyme (for review see Abdel-Latif, 1986). PIP,-PLC in the rat anterior pituitary membrane fraction was dependent on cations, since the pres-

500

I

TRH (low6 M) resulted in an additional increase in PIP,-PLC response to 375% above control (Fig. 8). Vasoactive intestinal peptide (VIP) did not have any significant effect on PIP,-PLC activity. Discussion Our results provide evidence of phosphatidylinositol4,Sbisphosphate-degradative activity in the rat anterior pituitary membrane fraction and that the PIP,-degradative activity is increased by tachykinins. Under standard conditions described in Materials and Methods a time-dependent rise in the water-soluble degradation products inositol phosphates and inositol demonstrates PIP,-PLC activity (Fig. 1). PIP,-PLC had an acidic pH optimum at 5.5, which is considerably low compared to intracellu-

SP

TRH

SP

l

TRH

Fig. 8. Interaction of substance P and TRH on PIP,-PLC activity in anterior pituitary membranes. The experimental setup was identical to the one described in the legend to Fig. 6. Termination and analyzing procedures are described in Materials and Methods. The concentrations of substance P and TRH were lo-” M in all experiments. ‘H activity was determined by HPLC analysis of the MeOH/H,O phase as described in Materials and Methods. Control indicates activity in the incubation media without peptides added. Data are presented as the mean+ SD of four experiments. Y-axis: 100% represent 1095 + 86 cpm.

76

ence of 10 mM EDTA completely abolished PIP,PLC activity. The cationic requirement of PIP,PLC was studied under the following experimental conditions: Mg2+ as the present cation and by a two-metal-ion-EDTA/EGTA buffer in order to determine an optimal free Ca2+ concentration. Mg2+ was a very potent activator of PIP,-PLC exhibiting 90% of maximal Ca’+-induced PIP,PLC activity at a concentration of 1 mM. The fact that Mg2+ could substitute for Ca2+ has previously been reported (Manne and Kung, 1987). Thus, Mg2+ rather than CaZf is a putative endogenous enzymatic co-factor in PIP,-PLC activity, since Mg2+ is present intracellularly in the millimolar range. An optimal free Ca2+ concentration in the micromolar range has been determined in a number of tissues (for review see Abdel-Latif, 1986). Likewise, we found that PIP,-PLC activity was optimized when free Ca2+ was 100 PM, which is well above the intracellular cytoplasmatic range (150 nM to 2 PM). The rather low affinity for Ca2+ makes it likely that MgZf is the physiological activator for PIP,-PLC. In an experiment where Mg2+ was kept at a total of 1 mM and Ca*+ was added in increasing amounts, a free Ca2+ concentration of 100 PM was necessary to show additional activation of the enzyme on top of the activation by Mg2+. This indicates that concentrations of Ca2+ higher than the free intracellular concentration are needed if Ca2+ should play a role in the regulation of the enzyme. This is supported by the fact that substance P did not increase the Ca2+ affinity for PIP,, as has been shown for TRH-mediated PPI hydrolysis in GH, cell membranes (Staub and Gershengorn, 1986). Addition of the detergent Triton X-100 to the incubation mixture did not change the pH optimum (data not shown), whereas sheep seminal vesicle exhibited maximal PIP,-PLC activity at neutral pH in the presence of 1 mg/ml deoxycholate (Hoffmann and Majerus, 1982). The fact that Triton X-100 strongly inhibited enzyme activity in the present study is in accordance with previous studies on platelets and rat brain homogenate (Irvine and Dawson, 1978; Manne and Kung, 1987). When 1 mg/ml sodium deoxycholate was added to the pure membranes the PIP,-PLC activity in this fraction was increased by 100%. This is

likely to be due to dissolvation of the membranes exposing PIP,-PLC to the substrate PIP, (Irvine and Dawson, 1978). Previously substance P binding sites have been demonstrated on membranes from the rat anterior pituitary gland with various ligands (Kerdelhue et al., 1985; Larsen et al., 1989a). In the former study binding data revealed a single class of non-interacting binding sites with a high affinity (Kd 0.72 nM) and a moderate density (B,,, 32.16 fmol/mg protein). Based on the ability of various tachykinins to inhibit binding of i2’I-BoltonHunter-substance P the receptor was classified as a NK-1 subtype according to the recently introduced tachykinin receptor classification (Burcher and Chahl, 1988). Two additional NK receptors exist, the NK-2 and the NK-3 receptors, which have neurokinin A and neurokinin B as their endogenous ligands, respectively. In order to investigate that the displacement rank order of tachykinins on the NK-1 receptor was compatible with the ability of the tachykinins to induce receptor-mediated breakdown of [3H]inositol-PIP, in rat anterior pituitary membranes, a dose-response study using various tachykinins as agonists was performed. The tachykinins did increase PIP,-PLC activity (i.e., substance P by 200% above control values). This maximal amplitude is at the same level as reported for the parotid gland (Hanley et al., 1980) but less than results obtained in cortical astrocytes (500%) (Torrens et al., 1989). This could be due to differences in methodology (primary cultures vs. crude membranes and pre-labeling with [3H]inositol vs. addition of genuine PIP,). Furthermore, only a subclass of pituitary cells (mammotropes and gonadotropes) are possible substance P targets. The involvement of an NK-1 receptor was supported by the fact that the EC,, values (the concentration needed to increase PIP,PLC activity to half maximal) displayed the same rank order as the IC,, values (the concentration needed to decrease binding of an agonist to half maximal) found by Larsen et al. (1989a). In many systems a linear relationship between receptor occupation and phospholipase C activity has been reported (Watson and Downes, 1983; Torrens et al., 1989). However, in the present study the absolute potencies of the tachykinins were approximately l-2 orders of magnitude

71

greater on PIP,-PLC activity than on displacement of ‘251-Bolton-Hunter-substance P bound to rat anterior pituitary membranes (Larsen et al., 1989a). Omittance of sodium deoxycholate in the incubation media reduced PIP,-PLC activity, but did not alter the EC,, value for the tachykinins. Thus detergent-induced receptor uncoupling is unable to account for the differences in receptor occupation and PIP,-PLC parameters. Rather, this could be due to the difference in buffer pH used in the two studies, 7.2 (maximal binding of substance P) in the paper by Larsen et al. (1989a) and 6.5 (40-50% binding) in the present study. The EC,, for substance P at 19 nM differs only slightly from values found in the rat ileum (22 nM) (Watson and Downes, 1983) or in the rat hypothalamus (60 nM) (Watson and Downes, 1983). However, in rat brain slices an EC,, at 1 PM was determined (Hunter et al., 1985). Beside differences in the experimental setup (primary cell culture versus cell-free system and avoidance of peptidase inhibitors in the incubation media) this discrepancy could be due to interference from low-affinity substance P binding sites present in the latter tissues. In the rat duodenum all three subtypes of NK receptors are apparently present (Buck et al., 1984), whereas in the anterior pituitary only the presence of NK-1 receptors having a high affinity for substance P has been reported (Larsen et al., 1989a). In order to compare substance P to other neuropeptides having the anterior pituitary as a target, stimulation experiments with TRH, a well-known PIP, promotor (Canonico et al., 1987) and VIP were performed. TRH raised the PPI amplitude to 305% above control, which is in accordance with previous reports on GH, cell membranes (Martin et al., 1986; Staub and Gershengorn, 1986). Combined treatment of substance P and TRH resulted in an additive effect. Vasoactive intestinal peptide (VIP), which is likely to exert its action via a CAMP-mediated pathway (Robberecht et al., 1979; Samson et al., 1980), did not rise basal activity of PIP,-PLC, as previously reported (Enjalbert et al., 1987). In conclusion, the tachykinins stimulate PIP,PLC-degradative activity towards [3H]inositollabeled PIP, displaying a rank order consistent with the receptor involved being an NK-1 sub-

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