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A galanin-mastoparan chimeric peptide activates the Na+,K+-ATPase and reverses its inhibition by ouabain
REGULATORY
PEPTIDES
ELSEVIER
Regulatory Peptides 62 (1996) 47-52
A galanin-mastoparan chimeric peptide activates the Na+,K +-ATPase and reverses its inhibition by ouabain Ulo Langel a,,, Margus Pooga a,b, Czeslava Kairane c, Mihkel Zilmer c, Tamas Bartfai a a Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-106 91, Stockholm, Sweden b Estonian Biocenter, Riia 23. EE-2400, Tartu, Estonia c Department of Biochemistry, Tartu University, EE-2400, Tartu, Estonia
Received 2 February 1995; revised 10 November 1995; accepted 7 December 1995
Abstract
The effect of the neuropeptide galanin, the wasp venom toxin amphiphilic peptide toxin mastoparan and the chimeric peptide, galparan, consisting of N-terminal 13 amino acids of neuropeptide galanin linked at C-terminus to mastoparan amide (and its inactive analog Masl7) on the activity of Na+,K+-ATPase has been studied. Mastoparan inhibits the activity of the Na+,K+-ATPase with iC50 m 7.5 /zM and also redL~ces the cooperativity for Na + and K +, respectively, while galanin has no effect on the Na+,K+-ATPase activity. The chimeric peptide, galanin(1-13)-mastoparan amide (galparan), exhibits biphasic interaction with Na+,K+-ATPase, it activates the enzyme at maximal stimulating concentration of 4/zM followed by inhibition of the enzyme with IC5o of 100 /zM. At maximum stimulating concentration (4/xM), galparan partly reduces the cooperativity only for Na + and it also counteracts the inhibitory effect of oubain on Na +,K +-ATPase. Galparan's stimulatory effect was influenced by ATP. The chimeric peptide [ 19Lys, 26Leu]-galparan, containing the inactive analog of mastoparan (Masl7), has no effects on rat brain Na+,K+-ATPase activity. Both chimeric peptides galparan and [19Lys,26Leu]-galparan are high-affinity galanin receptor ligands with IC5o of 6.4 nM and 0.71 nM, respectively, while galanin (1-13) and mastoparan alone have significantly lower affinity for the galanin receptor, IC5o of 125 nM and 1 ItM, respectively. The ability of chimeric peptides to bind to galanin receptors does not correlate with their effects on the Na+,K+-ATPase. Keywords: Galanin; Receptor; Mastoparan; Amphiphilic helix; Na+,K÷-ATPase
1. Introduction
The Na+,K+-ATPase is an integral membrane protein which is responsible for translocating of sodium and potassium ions across the plasma membrane utilizing ATP as driving force. The active transport of Na + and K ÷ produces both chemical and electrical gradients across the plasma membrane. In intestine and kidney, the Na+,K ÷ATPase regulates fluid reabsorption and electrolyte movement via establishing an ionic gradient across the epithelial membranes. The Na+,K+-ATPase regulates also intracellular pH and cell volurae. Due to the crucial physiological role of Na+,K+-ATPase, the activity of this enzyme is finely regulated [1].
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Numerous non-peptide and peptide factors are known to m o d u l a t e the Na ÷ , K + - A T P a s e activity. T h e s e modulators/regulators can be divided as the direct (ouabain, ATP, Na+,K+), and indirect modulators (several non-peptide hormones, neurotransmitters, peptides). The majority of these regulators are inhibitors of Na+,K +ATPase, e.g., three endogenous peptide inhibitors of Na+,K+-ATPase, (SPAI-1,-2,-3), have been isolated [2]. Recently, it has been established that the wasp venom peptide mastoparan inhibits rat renal [3] and synaptosomal [4] Na+,K+-ATPase. Activation of Na+,K+-ATPase by some bioactive peptides, e.g., insulin, glucagon, PEC-60, bradykinin, substance P(1-5) has been demonstrated as well [5-9]. Modulation of the enzyme activation by these effectors is probably brought about by different mechanisms, e.g., the receptor-mediated mechanisms have been proposed [7]. We have become interested in the influence of some
48
O. Langel et al. / Regulatory Peptides 62 (1996) 47-52
chimeric peptides on brain Na+,K+-ATPase where the components of chimeric peptide itself exhibit differential effects on the enzyme like inhibiting it (mastoparan) or not revealing any effect at all on it (galanin, Masl7). We have chosen to study the chimeric peptides based on galanin and mastoparan and their components by testing in parallel their binding to galanin receptors and their influence on Na+,K+-ATPase from rat cerebral cortex. We describe the synthesis and effects of several chimeric peptides composed of the N-terminal fragment galanin(1-13) (or the low-affinity 2Ala-analog of it) and of mastoparan or of its inactive analog, Masl7. We have earlier synthesized a series of chimeric peptides which contain N-terminus from galanin and C-terminus from different biologically active peptides (substance P, NPY etc.) [10]. These peptides act as the high-affinity galanin receptor antagonists in several pharmacological paradigms [11]. Galanin has a variety of biological effects, both in the central nervous system and in peripheral organs, including the modulation of insulin release [11]. Mastoparan, an amphiphilic tetradecapeptide found in wasp venom, also possesses a variety of bioactivities including activation of phospholipase C [12] and A 2 [13], release of histamine [14] and norepinephrine [15]. We report here the effects of mastoparan amide and its analogs, chimeric (galanin-mastoparan) peptides on rat cerebral cortical Na+,K+-ATPase activity, and on 1251galanin binding at galanin receptors.
2. Materials and Methods
2.1. Materials Na125I (2500 Ci/mmol was purchased from NEN (Boston, MA, USA), amino acid derivates were from Chemical Company Ltd. (UK). All other reagents were from Sigma Chemical Co. (St. Louis. MO, USA).
2.2. Peptide synthesis The peptides were assembled in a stepwise manner on a solid support on a Applied Biosystems Model 431A Peptide Synthesizer using the standard DCC/HOBt-activation strategy on a 0.1 mmol scale (small scale), tert-Boc-Amino acids were coupled as hydroxybenzotriazole (HOBt) esters to MBHA (Bachem Feinchemikalien AG, Switzerland) resin to obtain C-terminally aminated peptides. Deprotection from formyl- and benzyl-groups by 'low TFMSA' method and cleavage of peptides from the resin by liquid HF has been described earlier [16]. Purity of the individual peptides was checked by analytical Nucleosil 120-3 Cls reversed-phase HPLC column (0.4 × 10.0 cm) and determined to be 99%. Molecular weights of the peptides were determined using Plasma Desorption Mass Spectrometer (PDMS) Model Bioion 20, Applied Biosystems, the calculated values were obtained in each case.
2.3. Enzyme preparations and assays The enzyme preparations of the Na+,K+-ATPase were isolated from brain frontal cortex of adult rats (Wistar, 250-300 g) as described earlier [17]. In briefly, the rats were decapitated, frontal cortex was isolated from the precooled brain and homogenized at 4°C in medium containing 0.32 M sucrose, 1 mM EDTA, 0.1% DOC (deoxycholate) and 37.5 mM imidazole-HC1 (pH 7.4 at 8°C). The homogenate was centrifuged for 10 min at 10000 X g and the clear supernatant was centrifuged for 30 min at 24000 X g. By resuspension of final sediment in the buffer (without DOC) described above, the enzyme preparation was achieved. The activity of the Na+,K+-ATPase was measured as follows. Membrane protein (10-20 /xg) was incubated in 375 /xl of incubation medium containing 100 mM NaC1, 20 mM KC1, 4 mM MgCI 2, 4 mM Tris-ATP and 25 mM imidazole-HC1 (pH 7.4 at 37°C). The reaction was terminated after 5 min and inorganic phosphate (Pi) was determined as described earlier [17]. The Na+,K+-ATPase activity was determined as the difference between the release of Pi from ATP with and without 1 mM ouabain or with and without NaCI + KC1 in incubation medium. Specific activity of Na+,K+-ATPase was expressed as /.~mol Pi/mg protein per hour. The protein content was determined by the method of Lowry et al. [18], using bovine serum albumin as a standard. The activity of Na+,K+-ATPase was linear as a function of incubation time and enzyme amount under all experimental conditions. The degree of cooperativity (the Hill coefficients, nil) and K m (sodium or potassium concentration at which enzyme has half-maximal activity) in the case of Na+,K +ATPase were determined as described earlier [19]. Briefly, the assay of the activity of Na+,K+-ATPase was carried out at different concentrations of sodium or potassium and the values of Vmax were calculated by computer program and checked via the Comish-Bowden method [20]. The Hill coefficients and K m were established via Hill plot: log(v/Vma x - v) vs. log[Na +] or log[K+].
2.4. Preparation of 1251-monoiodo-Tyr26 porcine galanin Porcine ~25I-galanin (specific activity 1800-2000 Ci/mmol), iodinated by the chloramine-T method using a 1:4 molar ratio of Na125I to galanin, was prepared as described before [21].
2.5. Ligand binding studies Membrane preparation from rat cerebral cortex and receptor binding analysis was carried out by filtration technique as described earlier [21], using 10 mM HEPES buffer, pH 7.4, containing I m g / m l bacitracin and 1 m g / m l BSA. The mixtures were incubated for 30 min at 37°C using ~25I-galanin (0.2 riM) as radioligand employed
(I. Langel et al. / Regulatory Peptides 62 (1996) 47-52
at a unsaturating concentration and different concentrations of the peptides to be tested. Specific binding was defined as that portion of the total binding which could be displaced by large excess of unlabeled galanin (1 /zM) or other ligands (1 /xM). The K D values of the displacing ligands were calculated from the computer-generated IC50 values using the correction of Gheng and Prusoff [22]. Fitting of the experimental data was carried out by means of a non-linear least-squares method using the program Kaleidagraph on a Macintosh SE/30.
A
Galpatan
/ ~oo
-
<~50
Galanin
.e .............
[2Ala]'Galp
3. Results The primary structures of synthesized tetradecapeptide toxin mastoparan amide, it,; inactive analog Masl7 and of the chimeric peptides together with their affinities for galanin receptors in the rat cerebral cortex are presented in Table 1. In order to examine whether above-mentioned ligands can influence the activity of brain Na+,K+-ATPase, a time-course study was carried out (Fig. 1A). Preincubation of the enzyme with mastoparan and [2Ala]-galparan significantly and time-dependently inhibited enzyme activity. However, the preincubation of membrane preparation with galparan activated brain Na+,K+-ATPase in a time-dependent manner. Neither the inactive analog of mastoparan, Masl7, nor [19Lys,26Leu]-galparan had any effect on the activity of brain Na+,K+ ATPase in the concentration range (0.01-100 /xM) studied (for clarity, only the data for galanin is presented in Fig. 1). Furthermore, none of the peptides studied had any effects on frontal cortical Mg2+-ATPase activity (data not shown).
49
---e .....................
iP . . . . . . . . .
•
aran
I
I
I
I
1
2
3
4
I
I
5 6 time, rain
B 140 =~ 120 E-
<,
100 +¢1
Z
80 60
.~ 40
k
//
[ Ata]-Galpann
-i
1
I
I
-8
-7
-6
-5
"IP--~.
1
-4 -3 log [Peptlde]
Fig. 1. (A) Activation/inhibition kinetics of rat frontal cortical Na+,K +ATPase by mastoparan, galparan and [2Ala]-galparan. Membrane preparations (150 /zg of protein) were preincubated at 25°C with 50 /xM mastoparan and [2Ala]-galparan, and 5 p.M galparan for 0 - 6 min. The Na+,K+-ATPase activity was expressed as btmol P i / m g protein per hour, number of experiments was 4--6 (three different enzyme preparations). (B) Concentration dependence of the effects of mastoparan, galparan and [2Ala]-galparan on the rat frontal cortical Na+,K+-ATPase activity (number of experiments was 4-6).
Table 1 Displacement of monoiodo-12Sl-[Tyr26]-porcine galanin (0.2 nM) from membranes of rat frontal cortex by galanin, mastoparan, Masl7 and chimeric ligands Peptide 1. Galanin(1-29) amide, porcine GWTLNSAGYLLGPHAIDNHRSFHDKYGLA amide 2. Galanin(1-13) amide GWTLNSAGYLLGP amide 3. Mastoparan INLKALAALAKKIL amide 4. Masl7 INLKAKAALAKKLL amide 5. Galparan [galanin(1 - 13) mastoparan] GWTLNSAGYLLGPINLKALAALAKKIL amide 6. [ ~9Lys,26 Leu]-Galparan [galanin( 1- 13)-Mas 17] GWTLNSAGYLLGPINLKAKAALAKKLL arnide 7. [ 2Ala]-Galparan GATLNSAGYLLGPINLKALAALAKKIL amide n.a.: not applicable because of the low aff'mity of the ligand. Values are mean SEM. Number of experiments was 4.
K o (nM) 0.74 4- 0.05
nH 0.9
125 4- 10
1.0
977 + 120
0,8
> 100 000
n.a.
6.4 + 1.8
1.0
0.71 -I- 0.1
0.9
1585 + 90
0.9
50
ffl. Langel et al./ Regulatory Peptides 62 (1996) 47-52
u,
Table 2 Influence of mastopaxan and galparan on the kinetic parameters for sodium and potassium ions at rat frontal cortical Na+,K+-ATPase
5
Contl~l
Control
Mastoparan 80 p.M
Galparan4/zM
1.70-t-0.08 a 9.1 +0.9 102 + 3
1.41 +0.07 a 8.3_+0.9 65 _+4 a
1.39+0.06 a 9.8_+ 1.1 109_+5
1.31 _+0.06 a 2.6 + 0.3 102-1-3
1.10_+0.05a 2.3 -+0.3 74-+2 a
1.29+0.10 2.8 + 0.4 129+4 a
log (v/V =~'v)
0,6
Con~,~
0,4
0'20
Ha + nu
~'2 -0.1 O 0.1 O.2 o,a o.~
0,8
J
Vmax (mmol Pi/mg protein/h)
Mcsmparan
K+
,log [K +,ml~J
,,,¢/, ,,,4'~
0,6 0,8
-0,2 . , ~ /
K m (raM)
nH K m (mM)
1
Vmax (mmol Pi/mg protein/h)
-0,4 -0.6
Fig. 2. The Hill plot showing the effect of mastoparan on the cooperativity for K + with brain Na+,K+-ATPase (one typical experiment). K m was determined from intercept of the line. The Hill coefficient (nil) was obtained from the slope. Inset: the double-reciprocal plot.
The concentration dependence of the effects of mastoparan and galparan on the activity of N a +,K +-ATPase upon 6 min preincubation with these peptides before the start of the enzymatic reaction is shown in Fig. lB. Preincubation with mastoparan significantly inhibited N a + , K + - A T P a s e activity with an IC50 o f 7.5 ~ M . Preincubation with galparan showed a biphasic effect, with maximal activation by 40% at 4 p,M (ECs0 = 1 /xM) followed by inhibition (IC50 of 100 /zM). In the case o f [2Ala]-galparan, the activating effect was smaller (Fig. 1B). The peptides M a s l 7 , [t9Lys,26Leu]-galparan and galanin revealed only very limited effects ( 3 - 6 % ) on the activity of Na ÷,K +-ATPase under these conditions (data for galanin alone presented). The analysis of inhibition of the activity of rat cerebral cortical Na+,K+-ATPase by mastoparan at different [K +] or [Na ÷] revealed that mastoparan exerted mixed type inhibition of enzyme activity in respect to potassium (Fig. 2) and a non-competitive inhibition with respect to sodium (data not shown). Furthermore, mastoparan reduced the cooperativity for potassium (n H changed from 1.3 to 1.1) and partly decreased the cooperativity for sodium (Table 2). The values of Vmax, determined separately for potassium and sodium, were also reduced by mastoparan. The effect of ouabain, known as a specific inhibitor of
a p < 0.05 versus control. Preincubation (25°C) time was 6 min for mastoparan and galparan. Values are mean SEM. Number of experiments was 7.
the Na+,K+-ATPase, on the activation of the enzyme by galparan has been studied. It is shown (Fig. 3) that galparan added either before or after 5 p~M ouabain (this concentration inhibited the N a + , K + - A T P a s e approx. 50%), significantly reduced the inhibitory effect of ouabain on the Na+,K+-ATPase activity. W e have studied the effect o f ATP, the substrate of Na+,K+-ATPase, on the effects of mastoparan and galparan. Table 3 shows that preincubation of frontal cortical membranes with A T P blocks dose-dependently the inhibition by mastoparan (complete reversal o f inhibition at 0.5 m M [ATP]) of Na+,K+-ATPase. On the other hand, the activation of N a + , K + - A T P a s e by galparan is not significantly altered, except for a decrease in the activation-effect at 4 m M [ATP]. The affinities of the studied peptides at galanin receptors from rat frontal cortex are presented in Table 1. It is shown that elongation of conservative N-terminal galanin ( 1 - 1 3 ) fragment with mastoparan (galparan) or with its inactive analog M a s l 7 in [mLys,26Leu]-galparan at the C-terminus resulted in a significant (20- and 176-fold, respectively) increase in the affinity towards galanin receptors as compared to that of galanin(1 - 13) itself. Mastoparan itself has affinity for frontal cortical galanin receptors ( K D = 1 /~M), while the inactive analog of mastoparan (Mas17) did not influence the galanin binding in the concentration range from 1 - 1 0 000 nM. The affinity of
Table 3 Influence of ATP on mastoparan-inihibitionand galparan-activation of rat frontal cortical Na+,K+-ATPase Activity of Na ÷,K+-ATPase (% of control) ATP (mM)
0
0.01
0.05
0.15
0.5
1.0
2.0
3.0
4.0
+ Mastoparan (100/xM) + Galparan (4/zM)
51+_4 a 134 + 7 a
79___8 120 + 5 a
81+6 a 132 + 5 a
82+5 a 121 + 6 a
99+4 127 + 6 a
100+5 129 _+4 a
100+6 137 4- 6 a
100+4 124 + 7 a
100_+6 108 + 9
Preincubation (25°C) time was 3 min for ATP and 10 min for peptides. Values are mean SEM. Number of experiments was 5. a p < 0.05 vs. 100% (control).
U. Langel et a l . / Regulatory Peptides 62 (1996) 47-52
Control
Oonbain 5~M
Oonbain +G~paran
GMparan
+~5~bain S~M
Fig. 3. The effect of galparan (4/.~M) on the inhibition of Na+,K+-ATPase by ouabain (5 /~M). Enzyme preparations were prcincubated at 25°C for 6 rain with ouabain and then ga]paran was added (or preincubated with galparan and then ouabain was added). Number of experiments was 5, P < 0.05 for ouabain + galparan (or galparan + ouabain) vs. ouabain only.
[2Ala]-galanin(1-13)-mastoparan decreased 248-fold as compared to that of galparan.
4. Discussion
The present study charat'terizes the affinities of chimeric galanin-mastoparan peptides at galanin receptors and their effect on Na+,K÷-ATPase activity in membranes from rat frontal cortex. The peptides under study (Table 1) represent mastoparan (an amphiphilic cationic tetradecapeptide [23]), its inactive analog, Masl7, and their chimeric peptides with conservative galanin(1-13) at N-terminus. Although it is known that insulin [5] (as well as some other effectors like norepinephrine, SDS, ethanol) may activate the Na+,K+-ATPase in some systems, we are presenting the data using the washed membrane preparation and therefore can exclude that these hormonal regulators are released or present and contribute to the activation of Na+,K+-ATPase by our chimeric peptides. Mastoparan amide inhibits in a time- and dose-dependent manner the activity of Na÷,K+-ATPase from frontal cortex of rat. The IC50 for mastoparan is lower than in the case of the synaptosomal Na+,K+-ATPase [4,24] but very similar to the value found for inhibition of the 86Rb+ uptake by Na-pump in HL60 cells [4]. Masl7, a mastoparan analog with lower amphiphilicity than mastoparan does not affect the enzyme activity. This is in line with earlier findings [24] showing that a mastoparan-analog with lower amphiphilicity than mastoparan, had a reduced effect on Na+,K+-ATPase activity. Elongation of the mastoparan by galanin(1-13) in the chimeric peptide galparan makes the N-terminus of mastoparan more hydrophobic and increases
51
probably the concentration of the chimeric peptide in the vicinity of the membrane. The inhibitory effect of mastoparan on the Na+,K+-ATPase activity, however, decreased by the N-terminal 13 amino acids of the chimeric peptide, since galparan inhibits the enzyme with IC50 of 100 /xM, while mastoparan inhibits the enzyme with an IC50 of 7.5 /xM. More importantly, galparan exhibits biphasic influence: at concentration 4 / x M it activates the enzyme. Since 25-30% of the total cellular ATP is utilized by the Na÷,K÷-ATPase, such activation (30-40%) of Na÷,K÷-ATPase activity by galparan may be of pharmacological significance. It has been demonstrated earlier [16] that change of Trp 2 to L-Ala2 in galanin (1-16) causes dramatic loss in affinity of galanin and galanin(1-16) at rat hypothalamic receptors. Since the substitution of major galanin-pharmacophore Trp 2 in the galanin sequence of galparan has significantly reduced its ability to activate the Na÷,K +ATPase, as shown for [Ala2]-galparan (Fig. IB), there is a possibility that the activation of Na+,K+-ATPase system by galparan is partly caused by the galanin-component of the chimeric peptide. Absence of any significant effects of galanin on Na+,K+-ATPase activity in our experimental conditions (Fig. 1B) does not contradict the above suggestion, because galanin activated K÷-phosphatase (an essential part-reaction of Na+,K+-ATPase system) at 0.08-200 /xM (maximum effect was 29% at 0.8 /~M (Zilmer et al., in preparation). We do not interpret these data as showing that galanin receptors are participating in the activation of Na+,K+-ATPase by galparan rather we suggest that the galparan peptide, containing the N-terminus from galanin and due to its chimeric nature, is more potent than other related peptides in its activating interaction with the Na ÷,K +-ATPase. It is suggested that a possible mechanism of action of inhibition of the Na+,K+-ATPase by mastoparan involves direct or indirect interaction with Na÷-binding sites, located on the cytoplasmic site of plasma membrane [23]. Our data that the inhibition by mastoparan partly decreased the cooperativity for sodium (Table 2) do not contradict the above mentioned suggestion. However, one may speculate that the targets for mastoparan-inhibition and galparan-activation are the potassium-related steps (or K+-bi nding sites) of Na+,K+-ATPase system rather than the Na÷-binding sites. Our suggestion is supported by following findings: (i) the clear reducing effect of mastoparan on the cooperativity for potassium and mixed type inhibition of enzyme activity in respect to potassium (Fig. 2); (ii) mastoparan inhibits significantly the activity of K+-phos phatase and eliminates completely the cooperativity for potassium with K÷-phosphatase whereas galparan activated this enzyme (Zilmer et al., in preparation); (iii) ATP induces the conversion of the Na+,K+-ATPase to sodiumdependent form [1] and we show (Table 3) that mastoparan-caused inhibition is also partly blocked at low ATP concentration; (iv) galparan (Fig. 3) significantly
52
U. Langel et al./ Regulawry Peptides 62 (1996) 47-52
counteracted the effect of ouabain on the Na+,K+-ATPase and the effect of ouabain is known to be regulated by potassium ions. Furthermore, the alteration by galparan of Vmax of Na+,K+-ATPase supports the suggestion (cf. above) that mastoparan-inhibition of Na+,K+-ATPase causing the alteration of Vmax, may be exerted via action of the peptide at phosphoenzyme intermediate level [3]. The galparan-activation is not significantly altered by ATP, except a clear decrease at 4 mM ATP (Table 3). Considering that a high concentrations of ATP ( > 2 mM, extrapolated value from [25]) the activity of Na+,K ÷ATPase is not under the control of the membrane lipids, we assume that the activation of the enzyme by galparan may be connected to the interaction of galparan with plasma membrane lipids surrounding and regulating the enzyme. A possible mechanism of action of galparan in activation of the rat frontal cortical Na+,K÷-ATPase may involve direct or indirect interaction of the peptide with intracellular K+-binding sites of the Na+,K+-ATPase. The mechanism of action of the activation by galparan remains to be studied but the existence of such compounds as galparan may be of interest from a biochemical and pharmacological point of view.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council, the Royal Swedish Academy of Sciences, the Board of Technical Development (NUTEK), Swedish Research Council for Engineering Sciences (TFR), the Ivar Bendixsons Foundation, Pharmacia Research Foundation and grant from Stockholm University for bilateral collaboration.
References [1] Skou, J., The energy coupled exchange of Na ÷ for K ÷ across the cell membrane. The Na+,K+-pump. FEBS Lett., 268 (1990) 314328. [2] Araki, K., Kuroki, J., Ito, O., Kuwada, M. and Tachibana, S., Novel peptide inhibitor (SPAI) of Na÷,K+-ATPase from porcine intestine. Biochem. Biophys. Res. Commun., 164 (1989) 496-502. [3] Eng, S., Clough, D. and Lo, C., Mastoparan inhibits rat renal Na,K-ATPase. Life Sci., 47 (1990) 2451-2458. [4] Raynor, R.L., Zheng, B. and Kuo, J.F., Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. J. Biol. Chem., 266 (1991) 2753-2758. [5] McGill, D.L. and Guidotti, G., Insulin stimulates both alpha 1 and alpha 2 isoforms of the rat adipocyte Na+,K+-ATPase. Two mechanisms of stimulation. J. Biol. Chem., 266 (1991) 15824-15831. [6] Fehlmann, M. and Freychet, P., Insulin and glucagon stimulation of (Na + ,K + )-ATPase transport activity in isolated rat hepatocytes. J. Biol. Chem., 256 (1981)
[7] Kairane, C., Zilmer, M., Mutt, V. and Sillard, R., Activation of Na,K-ATPase by an endogenous peptide, PEC-60. FEBS Lett., 345 (1994) 1-4. [8] Brazy, P.C., Trellis, D.R. and Klotman, P.E., Bradykinin stimulation of oxidative metabolism in renal conical tubules from rabbit. Possible role of arachidonic acid. J. Clin. Invest., 76 (1985) 1812-1818. [9] Wojtkowiak, R., Turska, E., Lachowicz, L. and Koziolkiewicz, W., Effects of N- and C-terminal fragments of substance P on ATPase and monoamine oxidase activities in rat brain. Gen. Pharmacol., 21 (1990) 403-406. [10] Bartfai, T., Ht~kfelt, T. and Langel, 0., Galanin - a neuroendocrine peptide. Crit. Rev. Neurobiol., 7 (1993) 229-274. [11] Bartfal, %, Bedecs, K., Land, T., Langel, 0., Bertorelli, R., Girotti, P., Consolo, S., Xu, X.J., Wiesenfeld-Hallin, Z., Nilsson, S., Pieribone, V.A. and Hbkfelt, T., M-15 - High-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus coeruleus and spinal cord. Proc. Natl. Acad. Sci. USA, 88 (1991) 10961 - 10965. [12] Okano, Y., Takagi, H., Tohmatsu, T., Nakashima, S., Kuroda, Y., Saito, K. and Nozawa, Y., A wasp venom mastoparan-induced polyphosphoinositide breakdown in rat peritoneal mast cells. FEBS Lett., 188 (1985) 363-366. [13] Argiolas, A. and Pisano, J.J., Facilitation of phospholipase A 2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom. J. Biol. Chem., 258 (1983) 1369713702. [14] Aridor, M., Traub, L.M. and Sagi-Eisenberg, R., Exocytosis in mast cells by basic secretagogues. Evidence for direct activation of GTPbinding proteins. J. Cell Biol., 111 (1990) 909-917. [15] Shin, Y., Moni, R.W., Lueders, J.E. and Daly, J.W., Effects of amphiphilic peptides mastoparan and adenoregulin on receptor binding, G-proteins, phosphoinositide breakdown, cyclic AMP generation and calcium influx. Cell. Mol. Neurobiol., 14 (1994) 133-157. [16] Land, T., Langel, U., Lbw, M., Berthold, M., Und~n, A. and Bartfai, T., Linear and cyclic N-terminal galanin fragments and analogs as ligands at the hypothalamic galanin receptor. Int. J. Peptide Protein Res., 38 (1991) 267-272. [17] Karelson, E., Zilmer, M. and T'~hel~ld, L., Prostaglandin E 2 as a lipophilic modulator of Na-pump. Quest. Meal. Chem. (Russ.), 31 (1985) 84-87. [18] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent. J. Biol. Chem., 193 (1951) 265-275. [19] Salum, T., Zilmer, M., Kullisaar, T., Vihalemm, T. and ThhepiSld, L., Cooperative interactions of Na,K-ATPase with Na and K and its oligomeric structure. Ukr. Biochem. J., 60 (1988) 47-52. [20] Comish-Bowden, A., In: Fundamentals of enzyme kinetics, 1979, pp. 200-211. [21] Land, T., Langel, 0., Fisone, G., Bedecs, K. and Bartfai, T., Assay for galanin receptor. Methods Neurosci., 5 (1991) 225-234. [22] Cheng, Y.-C. and Prusoff, W.H., Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem. Pharmacol., 22 (1973) 3099-3108. [23] Mousli, M., Bueb, J.-L., Bronner, C., Rouot, B. and Landry, Y., G protein activation: a receptor-independent mode of action for cationic amphiphilic neuropeptides and venom peptides. Trends Pharmacol. Sci., 11 (1990) 358-362. [24] Raynor, R., Kim, Y.-S., Zheng, B., Vogler, W. and Kuo, J., Membrane interactions of mastoparan analogues related to their differential effects on protein kinase C, Na,K.ATPase and HL60 cells. FEBS Lett., 307 (1992) 275-279. [25] Boldyrev, A.A., In: Biomembranes and Transport of Ions (Russ. Ed. Krans, E.I), Moscow University Press), 1985, p. 207.
PEPTIDES
ELSEVIER
Regulatory Peptides 62 (1996) 47-52
A galanin-mastoparan chimeric peptide activates the Na+,K +-ATPase and reverses its inhibition by ouabain Ulo Langel a,,, Margus Pooga a,b, Czeslava Kairane c, Mihkel Zilmer c, Tamas Bartfai a a Department of Neurochemistry and Neurotoxicology, Arrheniuslaboratories, Stockholm University, S-106 91, Stockholm, Sweden b Estonian Biocenter, Riia 23. EE-2400, Tartu, Estonia c Department of Biochemistry, Tartu University, EE-2400, Tartu, Estonia
Received 2 February 1995; revised 10 November 1995; accepted 7 December 1995
Abstract
The effect of the neuropeptide galanin, the wasp venom toxin amphiphilic peptide toxin mastoparan and the chimeric peptide, galparan, consisting of N-terminal 13 amino acids of neuropeptide galanin linked at C-terminus to mastoparan amide (and its inactive analog Masl7) on the activity of Na+,K+-ATPase has been studied. Mastoparan inhibits the activity of the Na+,K+-ATPase with iC50 m 7.5 /zM and also redL~ces the cooperativity for Na + and K +, respectively, while galanin has no effect on the Na+,K+-ATPase activity. The chimeric peptide, galanin(1-13)-mastoparan amide (galparan), exhibits biphasic interaction with Na+,K+-ATPase, it activates the enzyme at maximal stimulating concentration of 4/zM followed by inhibition of the enzyme with IC5o of 100 /zM. At maximum stimulating concentration (4/xM), galparan partly reduces the cooperativity only for Na + and it also counteracts the inhibitory effect of oubain on Na +,K +-ATPase. Galparan's stimulatory effect was influenced by ATP. The chimeric peptide [ 19Lys, 26Leu]-galparan, containing the inactive analog of mastoparan (Masl7), has no effects on rat brain Na+,K+-ATPase activity. Both chimeric peptides galparan and [19Lys,26Leu]-galparan are high-affinity galanin receptor ligands with IC5o of 6.4 nM and 0.71 nM, respectively, while galanin (1-13) and mastoparan alone have significantly lower affinity for the galanin receptor, IC5o of 125 nM and 1 ItM, respectively. The ability of chimeric peptides to bind to galanin receptors does not correlate with their effects on the Na+,K+-ATPase. Keywords: Galanin; Receptor; Mastoparan; Amphiphilic helix; Na+,K÷-ATPase
1. Introduction
The Na+,K+-ATPase is an integral membrane protein which is responsible for translocating of sodium and potassium ions across the plasma membrane utilizing ATP as driving force. The active transport of Na + and K ÷ produces both chemical and electrical gradients across the plasma membrane. In intestine and kidney, the Na+,K ÷ATPase regulates fluid reabsorption and electrolyte movement via establishing an ionic gradient across the epithelial membranes. The Na+,K+-ATPase regulates also intracellular pH and cell volurae. Due to the crucial physiological role of Na+,K+-ATPase, the activity of this enzyme is finely regulated [1].
* Corresponding [email protected]
author.
Fax:
+ 46
8
161371;
E-mail:
0167-0115/96/$15.00 © 1996 Elsevier Science B.V. All fights reserved PH S 0 1 6 7 - 0 1 1 5 ( 9 6 ) 0 0 0 0 2 - X
Numerous non-peptide and peptide factors are known to m o d u l a t e the Na ÷ , K + - A T P a s e activity. T h e s e modulators/regulators can be divided as the direct (ouabain, ATP, Na+,K+), and indirect modulators (several non-peptide hormones, neurotransmitters, peptides). The majority of these regulators are inhibitors of Na+,K +ATPase, e.g., three endogenous peptide inhibitors of Na+,K+-ATPase, (SPAI-1,-2,-3), have been isolated [2]. Recently, it has been established that the wasp venom peptide mastoparan inhibits rat renal [3] and synaptosomal [4] Na+,K+-ATPase. Activation of Na+,K+-ATPase by some bioactive peptides, e.g., insulin, glucagon, PEC-60, bradykinin, substance P(1-5) has been demonstrated as well [5-9]. Modulation of the enzyme activation by these effectors is probably brought about by different mechanisms, e.g., the receptor-mediated mechanisms have been proposed [7]. We have become interested in the influence of some
48
O. Langel et al. / Regulatory Peptides 62 (1996) 47-52
chimeric peptides on brain Na+,K+-ATPase where the components of chimeric peptide itself exhibit differential effects on the enzyme like inhibiting it (mastoparan) or not revealing any effect at all on it (galanin, Masl7). We have chosen to study the chimeric peptides based on galanin and mastoparan and their components by testing in parallel their binding to galanin receptors and their influence on Na+,K+-ATPase from rat cerebral cortex. We describe the synthesis and effects of several chimeric peptides composed of the N-terminal fragment galanin(1-13) (or the low-affinity 2Ala-analog of it) and of mastoparan or of its inactive analog, Masl7. We have earlier synthesized a series of chimeric peptides which contain N-terminus from galanin and C-terminus from different biologically active peptides (substance P, NPY etc.) [10]. These peptides act as the high-affinity galanin receptor antagonists in several pharmacological paradigms [11]. Galanin has a variety of biological effects, both in the central nervous system and in peripheral organs, including the modulation of insulin release [11]. Mastoparan, an amphiphilic tetradecapeptide found in wasp venom, also possesses a variety of bioactivities including activation of phospholipase C [12] and A 2 [13], release of histamine [14] and norepinephrine [15]. We report here the effects of mastoparan amide and its analogs, chimeric (galanin-mastoparan) peptides on rat cerebral cortical Na+,K+-ATPase activity, and on 1251galanin binding at galanin receptors.
2. Materials and Methods
2.1. Materials Na125I (2500 Ci/mmol was purchased from NEN (Boston, MA, USA), amino acid derivates were from Chemical Company Ltd. (UK). All other reagents were from Sigma Chemical Co. (St. Louis. MO, USA).
2.2. Peptide synthesis The peptides were assembled in a stepwise manner on a solid support on a Applied Biosystems Model 431A Peptide Synthesizer using the standard DCC/HOBt-activation strategy on a 0.1 mmol scale (small scale), tert-Boc-Amino acids were coupled as hydroxybenzotriazole (HOBt) esters to MBHA (Bachem Feinchemikalien AG, Switzerland) resin to obtain C-terminally aminated peptides. Deprotection from formyl- and benzyl-groups by 'low TFMSA' method and cleavage of peptides from the resin by liquid HF has been described earlier [16]. Purity of the individual peptides was checked by analytical Nucleosil 120-3 Cls reversed-phase HPLC column (0.4 × 10.0 cm) and determined to be 99%. Molecular weights of the peptides were determined using Plasma Desorption Mass Spectrometer (PDMS) Model Bioion 20, Applied Biosystems, the calculated values were obtained in each case.
2.3. Enzyme preparations and assays The enzyme preparations of the Na+,K+-ATPase were isolated from brain frontal cortex of adult rats (Wistar, 250-300 g) as described earlier [17]. In briefly, the rats were decapitated, frontal cortex was isolated from the precooled brain and homogenized at 4°C in medium containing 0.32 M sucrose, 1 mM EDTA, 0.1% DOC (deoxycholate) and 37.5 mM imidazole-HC1 (pH 7.4 at 8°C). The homogenate was centrifuged for 10 min at 10000 X g and the clear supernatant was centrifuged for 30 min at 24000 X g. By resuspension of final sediment in the buffer (without DOC) described above, the enzyme preparation was achieved. The activity of the Na+,K+-ATPase was measured as follows. Membrane protein (10-20 /xg) was incubated in 375 /xl of incubation medium containing 100 mM NaC1, 20 mM KC1, 4 mM MgCI 2, 4 mM Tris-ATP and 25 mM imidazole-HC1 (pH 7.4 at 37°C). The reaction was terminated after 5 min and inorganic phosphate (Pi) was determined as described earlier [17]. The Na+,K+-ATPase activity was determined as the difference between the release of Pi from ATP with and without 1 mM ouabain or with and without NaCI + KC1 in incubation medium. Specific activity of Na+,K+-ATPase was expressed as /.~mol Pi/mg protein per hour. The protein content was determined by the method of Lowry et al. [18], using bovine serum albumin as a standard. The activity of Na+,K+-ATPase was linear as a function of incubation time and enzyme amount under all experimental conditions. The degree of cooperativity (the Hill coefficients, nil) and K m (sodium or potassium concentration at which enzyme has half-maximal activity) in the case of Na+,K +ATPase were determined as described earlier [19]. Briefly, the assay of the activity of Na+,K+-ATPase was carried out at different concentrations of sodium or potassium and the values of Vmax were calculated by computer program and checked via the Comish-Bowden method [20]. The Hill coefficients and K m were established via Hill plot: log(v/Vma x - v) vs. log[Na +] or log[K+].
2.4. Preparation of 1251-monoiodo-Tyr26 porcine galanin Porcine ~25I-galanin (specific activity 1800-2000 Ci/mmol), iodinated by the chloramine-T method using a 1:4 molar ratio of Na125I to galanin, was prepared as described before [21].
2.5. Ligand binding studies Membrane preparation from rat cerebral cortex and receptor binding analysis was carried out by filtration technique as described earlier [21], using 10 mM HEPES buffer, pH 7.4, containing I m g / m l bacitracin and 1 m g / m l BSA. The mixtures were incubated for 30 min at 37°C using ~25I-galanin (0.2 riM) as radioligand employed
(I. Langel et al. / Regulatory Peptides 62 (1996) 47-52
at a unsaturating concentration and different concentrations of the peptides to be tested. Specific binding was defined as that portion of the total binding which could be displaced by large excess of unlabeled galanin (1 /zM) or other ligands (1 /xM). The K D values of the displacing ligands were calculated from the computer-generated IC50 values using the correction of Gheng and Prusoff [22]. Fitting of the experimental data was carried out by means of a non-linear least-squares method using the program Kaleidagraph on a Macintosh SE/30.
A
Galpatan
/ ~oo
-
<~50
Galanin
.e .............
[2Ala]'Galp
3. Results The primary structures of synthesized tetradecapeptide toxin mastoparan amide, it,; inactive analog Masl7 and of the chimeric peptides together with their affinities for galanin receptors in the rat cerebral cortex are presented in Table 1. In order to examine whether above-mentioned ligands can influence the activity of brain Na+,K+-ATPase, a time-course study was carried out (Fig. 1A). Preincubation of the enzyme with mastoparan and [2Ala]-galparan significantly and time-dependently inhibited enzyme activity. However, the preincubation of membrane preparation with galparan activated brain Na+,K+-ATPase in a time-dependent manner. Neither the inactive analog of mastoparan, Masl7, nor [19Lys,26Leu]-galparan had any effect on the activity of brain Na+,K+ ATPase in the concentration range (0.01-100 /xM) studied (for clarity, only the data for galanin is presented in Fig. 1). Furthermore, none of the peptides studied had any effects on frontal cortical Mg2+-ATPase activity (data not shown).
49
---e .....................
iP . . . . . . . . .
•
aran
I
I
I
I
1
2
3
4
I
I
5 6 time, rain
B 140 =~ 120 E-
<,
100 +¢1
Z
80 60
.~ 40
k
//
[ Ata]-Galpann
-i
1
I
I
-8
-7
-6
-5
"IP--~.
1
-4 -3 log [Peptlde]
Fig. 1. (A) Activation/inhibition kinetics of rat frontal cortical Na+,K +ATPase by mastoparan, galparan and [2Ala]-galparan. Membrane preparations (150 /zg of protein) were preincubated at 25°C with 50 /xM mastoparan and [2Ala]-galparan, and 5 p.M galparan for 0 - 6 min. The Na+,K+-ATPase activity was expressed as btmol P i / m g protein per hour, number of experiments was 4--6 (three different enzyme preparations). (B) Concentration dependence of the effects of mastoparan, galparan and [2Ala]-galparan on the rat frontal cortical Na+,K+-ATPase activity (number of experiments was 4-6).
Table 1 Displacement of monoiodo-12Sl-[Tyr26]-porcine galanin (0.2 nM) from membranes of rat frontal cortex by galanin, mastoparan, Masl7 and chimeric ligands Peptide 1. Galanin(1-29) amide, porcine GWTLNSAGYLLGPHAIDNHRSFHDKYGLA amide 2. Galanin(1-13) amide GWTLNSAGYLLGP amide 3. Mastoparan INLKALAALAKKIL amide 4. Masl7 INLKAKAALAKKLL amide 5. Galparan [galanin(1 - 13) mastoparan] GWTLNSAGYLLGPINLKALAALAKKIL amide 6. [ ~9Lys,26 Leu]-Galparan [galanin( 1- 13)-Mas 17] GWTLNSAGYLLGPINLKAKAALAKKLL arnide 7. [ 2Ala]-Galparan GATLNSAGYLLGPINLKALAALAKKIL amide n.a.: not applicable because of the low aff'mity of the ligand. Values are mean SEM. Number of experiments was 4.
K o (nM) 0.74 4- 0.05
nH 0.9
125 4- 10
1.0
977 + 120
0,8
> 100 000
n.a.
6.4 + 1.8
1.0
0.71 -I- 0.1
0.9
1585 + 90
0.9
50
ffl. Langel et al./ Regulatory Peptides 62 (1996) 47-52
u,
Table 2 Influence of mastopaxan and galparan on the kinetic parameters for sodium and potassium ions at rat frontal cortical Na+,K+-ATPase
5
Contl~l
Control
Mastoparan 80 p.M
Galparan4/zM
1.70-t-0.08 a 9.1 +0.9 102 + 3
1.41 +0.07 a 8.3_+0.9 65 _+4 a
1.39+0.06 a 9.8_+ 1.1 109_+5
1.31 _+0.06 a 2.6 + 0.3 102-1-3
1.10_+0.05a 2.3 -+0.3 74-+2 a
1.29+0.10 2.8 + 0.4 129+4 a
log (v/V =~'v)
0,6
Con~,~
0,4
0'20
Ha + nu
~'2 -0.1 O 0.1 O.2 o,a o.~
0,8
J
Vmax (mmol Pi/mg protein/h)
Mcsmparan
K+
,log [K +,ml~J
,,,¢/, ,,,4'~
0,6 0,8
-0,2 . , ~ /
K m (raM)
nH K m (mM)
1
Vmax (mmol Pi/mg protein/h)
-0,4 -0.6
Fig. 2. The Hill plot showing the effect of mastoparan on the cooperativity for K + with brain Na+,K+-ATPase (one typical experiment). K m was determined from intercept of the line. The Hill coefficient (nil) was obtained from the slope. Inset: the double-reciprocal plot.
The concentration dependence of the effects of mastoparan and galparan on the activity of N a +,K +-ATPase upon 6 min preincubation with these peptides before the start of the enzymatic reaction is shown in Fig. lB. Preincubation with mastoparan significantly inhibited N a + , K + - A T P a s e activity with an IC50 o f 7.5 ~ M . Preincubation with galparan showed a biphasic effect, with maximal activation by 40% at 4 p,M (ECs0 = 1 /xM) followed by inhibition (IC50 of 100 /zM). In the case o f [2Ala]-galparan, the activating effect was smaller (Fig. 1B). The peptides M a s l 7 , [t9Lys,26Leu]-galparan and galanin revealed only very limited effects ( 3 - 6 % ) on the activity of Na ÷,K +-ATPase under these conditions (data for galanin alone presented). The analysis of inhibition of the activity of rat cerebral cortical Na+,K+-ATPase by mastoparan at different [K +] or [Na ÷] revealed that mastoparan exerted mixed type inhibition of enzyme activity in respect to potassium (Fig. 2) and a non-competitive inhibition with respect to sodium (data not shown). Furthermore, mastoparan reduced the cooperativity for potassium (n H changed from 1.3 to 1.1) and partly decreased the cooperativity for sodium (Table 2). The values of Vmax, determined separately for potassium and sodium, were also reduced by mastoparan. The effect of ouabain, known as a specific inhibitor of
a p < 0.05 versus control. Preincubation (25°C) time was 6 min for mastoparan and galparan. Values are mean SEM. Number of experiments was 7.
the Na+,K+-ATPase, on the activation of the enzyme by galparan has been studied. It is shown (Fig. 3) that galparan added either before or after 5 p~M ouabain (this concentration inhibited the N a + , K + - A T P a s e approx. 50%), significantly reduced the inhibitory effect of ouabain on the Na+,K+-ATPase activity. W e have studied the effect o f ATP, the substrate of Na+,K+-ATPase, on the effects of mastoparan and galparan. Table 3 shows that preincubation of frontal cortical membranes with A T P blocks dose-dependently the inhibition by mastoparan (complete reversal o f inhibition at 0.5 m M [ATP]) of Na+,K+-ATPase. On the other hand, the activation of N a + , K + - A T P a s e by galparan is not significantly altered, except for a decrease in the activation-effect at 4 m M [ATP]. The affinities of the studied peptides at galanin receptors from rat frontal cortex are presented in Table 1. It is shown that elongation of conservative N-terminal galanin ( 1 - 1 3 ) fragment with mastoparan (galparan) or with its inactive analog M a s l 7 in [mLys,26Leu]-galparan at the C-terminus resulted in a significant (20- and 176-fold, respectively) increase in the affinity towards galanin receptors as compared to that of galanin(1 - 13) itself. Mastoparan itself has affinity for frontal cortical galanin receptors ( K D = 1 /~M), while the inactive analog of mastoparan (Mas17) did not influence the galanin binding in the concentration range from 1 - 1 0 000 nM. The affinity of
Table 3 Influence of ATP on mastoparan-inihibitionand galparan-activation of rat frontal cortical Na+,K+-ATPase Activity of Na ÷,K+-ATPase (% of control) ATP (mM)
0
0.01
0.05
0.15
0.5
1.0
2.0
3.0
4.0
+ Mastoparan (100/xM) + Galparan (4/zM)
51+_4 a 134 + 7 a
79___8 120 + 5 a
81+6 a 132 + 5 a
82+5 a 121 + 6 a
99+4 127 + 6 a
100+5 129 _+4 a
100+6 137 4- 6 a
100+4 124 + 7 a
100_+6 108 + 9
Preincubation (25°C) time was 3 min for ATP and 10 min for peptides. Values are mean SEM. Number of experiments was 5. a p < 0.05 vs. 100% (control).
U. Langel et a l . / Regulatory Peptides 62 (1996) 47-52
Control
Oonbain 5~M
Oonbain +G~paran
GMparan
+~5~bain S~M
Fig. 3. The effect of galparan (4/.~M) on the inhibition of Na+,K+-ATPase by ouabain (5 /~M). Enzyme preparations were prcincubated at 25°C for 6 rain with ouabain and then ga]paran was added (or preincubated with galparan and then ouabain was added). Number of experiments was 5, P < 0.05 for ouabain + galparan (or galparan + ouabain) vs. ouabain only.
[2Ala]-galanin(1-13)-mastoparan decreased 248-fold as compared to that of galparan.
4. Discussion
The present study charat'terizes the affinities of chimeric galanin-mastoparan peptides at galanin receptors and their effect on Na+,K÷-ATPase activity in membranes from rat frontal cortex. The peptides under study (Table 1) represent mastoparan (an amphiphilic cationic tetradecapeptide [23]), its inactive analog, Masl7, and their chimeric peptides with conservative galanin(1-13) at N-terminus. Although it is known that insulin [5] (as well as some other effectors like norepinephrine, SDS, ethanol) may activate the Na+,K+-ATPase in some systems, we are presenting the data using the washed membrane preparation and therefore can exclude that these hormonal regulators are released or present and contribute to the activation of Na+,K+-ATPase by our chimeric peptides. Mastoparan amide inhibits in a time- and dose-dependent manner the activity of Na÷,K+-ATPase from frontal cortex of rat. The IC50 for mastoparan is lower than in the case of the synaptosomal Na+,K+-ATPase [4,24] but very similar to the value found for inhibition of the 86Rb+ uptake by Na-pump in HL60 cells [4]. Masl7, a mastoparan analog with lower amphiphilicity than mastoparan does not affect the enzyme activity. This is in line with earlier findings [24] showing that a mastoparan-analog with lower amphiphilicity than mastoparan, had a reduced effect on Na+,K+-ATPase activity. Elongation of the mastoparan by galanin(1-13) in the chimeric peptide galparan makes the N-terminus of mastoparan more hydrophobic and increases
51
probably the concentration of the chimeric peptide in the vicinity of the membrane. The inhibitory effect of mastoparan on the Na+,K+-ATPase activity, however, decreased by the N-terminal 13 amino acids of the chimeric peptide, since galparan inhibits the enzyme with IC50 of 100 /xM, while mastoparan inhibits the enzyme with an IC50 of 7.5 /xM. More importantly, galparan exhibits biphasic influence: at concentration 4 / x M it activates the enzyme. Since 25-30% of the total cellular ATP is utilized by the Na÷,K÷-ATPase, such activation (30-40%) of Na÷,K÷-ATPase activity by galparan may be of pharmacological significance. It has been demonstrated earlier [16] that change of Trp 2 to L-Ala2 in galanin (1-16) causes dramatic loss in affinity of galanin and galanin(1-16) at rat hypothalamic receptors. Since the substitution of major galanin-pharmacophore Trp 2 in the galanin sequence of galparan has significantly reduced its ability to activate the Na÷,K +ATPase, as shown for [Ala2]-galparan (Fig. IB), there is a possibility that the activation of Na+,K+-ATPase system by galparan is partly caused by the galanin-component of the chimeric peptide. Absence of any significant effects of galanin on Na+,K+-ATPase activity in our experimental conditions (Fig. 1B) does not contradict the above suggestion, because galanin activated K÷-phosphatase (an essential part-reaction of Na+,K+-ATPase system) at 0.08-200 /xM (maximum effect was 29% at 0.8 /~M (Zilmer et al., in preparation). We do not interpret these data as showing that galanin receptors are participating in the activation of Na+,K+-ATPase by galparan rather we suggest that the galparan peptide, containing the N-terminus from galanin and due to its chimeric nature, is more potent than other related peptides in its activating interaction with the Na ÷,K +-ATPase. It is suggested that a possible mechanism of action of inhibition of the Na+,K+-ATPase by mastoparan involves direct or indirect interaction with Na÷-binding sites, located on the cytoplasmic site of plasma membrane [23]. Our data that the inhibition by mastoparan partly decreased the cooperativity for sodium (Table 2) do not contradict the above mentioned suggestion. However, one may speculate that the targets for mastoparan-inhibition and galparan-activation are the potassium-related steps (or K+-bi nding sites) of Na+,K+-ATPase system rather than the Na÷-binding sites. Our suggestion is supported by following findings: (i) the clear reducing effect of mastoparan on the cooperativity for potassium and mixed type inhibition of enzyme activity in respect to potassium (Fig. 2); (ii) mastoparan inhibits significantly the activity of K+-phos phatase and eliminates completely the cooperativity for potassium with K÷-phosphatase whereas galparan activated this enzyme (Zilmer et al., in preparation); (iii) ATP induces the conversion of the Na+,K+-ATPase to sodiumdependent form [1] and we show (Table 3) that mastoparan-caused inhibition is also partly blocked at low ATP concentration; (iv) galparan (Fig. 3) significantly
52
U. Langel et al./ Regulawry Peptides 62 (1996) 47-52
counteracted the effect of ouabain on the Na+,K+-ATPase and the effect of ouabain is known to be regulated by potassium ions. Furthermore, the alteration by galparan of Vmax of Na+,K+-ATPase supports the suggestion (cf. above) that mastoparan-inhibition of Na+,K+-ATPase causing the alteration of Vmax, may be exerted via action of the peptide at phosphoenzyme intermediate level [3]. The galparan-activation is not significantly altered by ATP, except a clear decrease at 4 mM ATP (Table 3). Considering that a high concentrations of ATP ( > 2 mM, extrapolated value from [25]) the activity of Na+,K ÷ATPase is not under the control of the membrane lipids, we assume that the activation of the enzyme by galparan may be connected to the interaction of galparan with plasma membrane lipids surrounding and regulating the enzyme. A possible mechanism of action of galparan in activation of the rat frontal cortical Na+,K÷-ATPase may involve direct or indirect interaction of the peptide with intracellular K+-binding sites of the Na+,K+-ATPase. The mechanism of action of the activation by galparan remains to be studied but the existence of such compounds as galparan may be of interest from a biochemical and pharmacological point of view.
Acknowledgements This work was supported by grants from the Swedish Medical Research Council, the Royal Swedish Academy of Sciences, the Board of Technical Development (NUTEK), Swedish Research Council for Engineering Sciences (TFR), the Ivar Bendixsons Foundation, Pharmacia Research Foundation and grant from Stockholm University for bilateral collaboration.
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