Acta Physiol Scand 1992, 146, 233-240

Polyamines in nerve terminals and secretory granules isolated from neurohypophyses M. K R 0 I G A A R D * , P. THAMS-f and N. A. THOR N " Departments of *Medical Physiology C and Biochemistry A, University of Copenhagen and * T h e Danish Bic-technology Centre for Neuropeptide Research, Copenhagen, Denmark

t

N. A. 1992, Polyamines in nerve terminals and KRBIGAARD, M., THAMS,P. & THORN, secretory granules isolated from neurohypophyses. Acta Physiol Scand 146, 233-240. Received 14 March 1992, accepted 7 May 1992. ISSN 0001-6772. Departments of Medical Physiology C and Biochemistry A, University of Copenhagen and The Danish Biotechnology Centre for Neuropeptide Research, Copenhagen, Denmark. In isolated nerve terminals from ox neurohypophyses the following concentrations of polyamines [pmol (pg protein)-' (mean & SEM)] were found : spermine : 2.07 & 0.14 ( n = 3), spermidine: 0.22$0.01 ( n = 4), putrescine: 0.20f0.01 ( n = 4). In secretory granules isolated from the same tissue, the concentrations were : spermine: 0.57k0.02 ( n = 3), spermidine: 0.07k0.04 ( n = 3), putrescine: 0.13f0.04 (n = 3 ) . After incubation of isolated nerve terminals with the polyamines, they were taken up as a function of time and concentration, approaching saturation at high concentrations. The kinetic parameters of their synthesizing enzyme, ornithine decarboxylase, in ox neurohypophyseal nerve terminals (apparent K,,, 0.75 mM and V,, 22.5 pmol mg protein-' h-') were comparable to those previously found in cerebral cortex of rats. When isolated, hemilobes from rat neurohypophyses were incubated in a medium which contained spermidine (5 mM), and were stimulated by 56 mM K', release of vasopressin was smaller than in control experiments. However, after removal of spermidine and after restimulation, 50 min after initial stimulation, the release was significantly elevated. It is suggested that polyamines may take part in modulation of vasopressin release.

Key words : neurohypophysis, neurosecretory granules, polyamines, secretosomes

Several studies have indicated that coupling between stimulation and secretion of vasopressin in the nerve terminals of the neurohypophysis involves calcium ions, taken u p from the extracellular fluid (Thorn et al. 1978). Different Ca2+channels have been characterized in isolated rat neurohypophyses (Spreckelsen et al. 1990). I t has previously been demonstrated that blockade of calcium channels by D600, a verapamil derivative, severely inhibits stimulation-induced release of vasopressin (Thorn et al. 1978). Calcium-calmodulin binding proteins have been Correspondence: Niels A. Thorn, Department of Medical Physiology C, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark.

identified on secretory granules from the nerve terminals (Chenoufi et al. 1986). There is only scant information on the possible role of other second messengers in this system. Several studies have described little effect on secretion of changes in cyclic AMP metabolism in the nerve terminals (see Thorn et al. 1984). Phosphorylation of neuron specific synaptic vesicle proteins in the neurohypophysis may be regulated by stimulation as a consequence of second messenger action (Treiman et al. 1980, Treiman & Greengard 1985). In many secretory cells, the inositolphosphate (IP) system has been shown to play an important part in stimulus-secretion coupling, sometimes together with other second-messenger systems.

233

234

M . Kr~igaardet al.

In the neurohypophysis no clear role for this system has been established !-et. O n e of the groups of substances that m a y interfere with protein kinase C is polyamines ( T h a m s et nl. 1986). Extra-cellular stimuli may change polyamine synthesis in sp-iptosomrs and thereby change neurotransmitter release (Iqbal & Koenig 1985), and polyamines can have several actions on m e m b r a n e functions ( S c h u b r r 1989). In the present study we have examined the concentration of pol!-amines in nerve terminals and secretor!. granules from ox neurohypophyses. the actil-ity i n them of ornithinedecarboxl-lase (their rate-regulating synthetic e n q m e ) a n d uptake of polyamines t o such terminals as well as effects of spermidine on release of vasopressin from hemilobes of isolated rat neurohypophyses.

M E 'r €I o D s .tZutrrriils. Putrescine, spermidine and spermine were purchased from Sigma Chemical Co. (St. Louis, 140, USA). ['YJputrescine, ["Clspermidine and ['lC]spermine (all 118 mCi mmol-') and [3H]dansyl chloride (17.4 Ci mmol-') were from Amersham, (Rucks, LK). Ornithine ~-[2,3-'H] was from .\mersham, all other reagents were of anal!-tical grade. Prrparatron qf homogenate, isolated nrrze terniina!s and secretory granules from ox nzuroh,ypophyses. Ox hypophyses were collected within 30 min after death a t the Public Slaughterhouse of Copenhagen and transported in 20 min to the laboratorl;. Ten per cent homogenates of ox neurohypophl-ses were made after cutting the tissue to fine pieces. They were sonicated on ice (20 s ; 40 W) and stored at - 20 "C. Isolated n r r w endings were prepared from specialljhomogenized samples (after transport from the slaughterhouse at an ambient temperature) by diG ferential centrifugation as described b!- Gratzl rt of. 1977, using TES instead of N-Tris for buffering. For the polyamine assal- the isolated nerve terminals were washed once in a modified (Russell & Thorn 1974) aMcIlwain-Rodnight medium. Composition, (mMI : NaCl 120, KCI 4.8, CaCI, 2.8, MgSO, 1.3, YaHCO, 26, KH,PO, 1.2, D-glucose 10, aerated with jO, CO, in 0, pH 7.3). The preparation was stored at - 20 "C for the polvamine assays. Pur!fied servetot:)' granules were isolated from ox neuroh!poph!-ses (transported to the lab on ice) on Percollisucrose gradients as described b!- Gratzl r t a!. (1980). The granule band (fraction nos. 5-10) nas collected and the granules, after dilution with 250 msi sucrose, 20 m\r TES, p H 7.0, were separated from

Percoll by centrifugation. T h e preparation was stored a t -20 "C for the polyamine assays. Preparation of isolated hemilobes from rat neuroh.ypophyses. Neurohypophyses from male Panwistar rats were isolated from the pars intermedia and anterior by dissection and incubated as described later. Studies of uptake of polyamines t o ox neurosecretory nrwe terminals. Isolated neurosecretosomes, prepared as described, were incubated at 37 "C for periods up to 270 min in a medium containing the previously described LtcIlwain-Rodnight buffer with 14C polyamines added (pH 7 . 3 ) (2.2-5.7 mg protein mi-'). In another set of experiments, uptake was studied for 30 min in a medium containing different concentrations of '.'C polyamines. After incubation, the isolated nerve terminals were rapidly cooled, pelleted in an airfuge (Beckman, Ramcon, Birkered, Denmark, 5 min a t 100000 g) and washed twice with the modified LfcIlwain-Rodnight medium. To pellets was added 0.5 ml NCS tissue solubilizer, follow-ed after 10 min by 10ml of Lumagel (Dynagel) (B.N. Chemicals, Helsinge, Denmark) and the radioactivity was determined. Effects of spermidine on release of riasopressin from rsolnted hemilobes of rat nrurohypoph.yses. Groups of five isolated rat neurohppophyses (each cut in half) were incubated in a column in a perfusion chamber of the type described by Knudsen et al. (1983) with the modified McIlwain-Rodnight medium, which was collected in 10 min periods. After establishment of a stable baseline release of vasopressin, they were stimulated by increasing the K + concentration to 56 mM for 30 min, with an equivalent reduction of the %aL concentration. A restimulation was made after baseline release had been re-established ( 5 0 min after the first stimulation). Spermidine was present in the perfusion medium during the first stimulation period (30 min) and for two periods immediately before and one period immediately after that. .-issa)~ of polyaminrs. After determining the protein content of 0s neurohypophysis homogenate, isolated terminals or isolated secretory granules, HCIO, and w-ater were added to a known amount of preparation to a final of 200 ,d with 0.2 M HCIO,. The polyamines were extracted by sonication for 10 s (40 W). T h e resulting precipitates were centrifuged at 8000 g for 7.5 rnin and the supernatants used for polyamine determination. The contents of putrescine, spermidine and spermine were measured by a double-isotope derivative assa!- described by Paulus & Davis (1983). To 40 pl of the tissue extracts or standards dissolved in 0.2 M HCIO,, were added 10 yl of a mixture containing 14C pol>-amines (48.8 mCi mmol-' dissolved in H,O and 50 pl of ['Hldansyl chloride ( 2 mg ml-', 0.5 mCi ml-') dissolved in acetone. After 1-igorous shaking, the mixture was made

PoIyamznes in neurohypophysis

235

Table 1. Contents of polyamines in ox neurohypophyses and the calculated concentration in the water phase. Putrescine

Spermidine

Spermine

( 1 ) Contents of polyamines in ox neurohypophyses"

Homogenate 0.55+0.19 (3)t (pmol pg protein-') Nerve terminals 0.20+0.01 (4) (pmol pg protein-') Secretory granules 0.13$0.04(3) (pmol pg protein-') (2) Calculated conc. in water phase (mM)t Nerve terminals 0.05 (4 pl mg protein-') Secretory granules 0.06 (2.2 pI mg protein-')

0.55&0.04(3)

4.53k1.14 (2)

0.2210.01 (4)

2.07k0.14 (3)

0.07f0.04(3)

0.57*0.02(3)

0.06

0.52

0.03

0.26

The ratios of water to protein are from a personal communication from T. Szrmark (for nerve terminals) and from Szrmark et al. (1983) (for secretory granules). "Calculated by means+SEM. +Number of experiments. 1Using the results from ( 1 ) and the ratios of water to protein indicated in the left column. alkaline by addition of 10 mg of Na,CO, and vortexmixed again. The tubes were sealed and incubated in the dark at room temperature overnight. After incubation, acetone was evaporated under a stream of nitrogen, and dansylated polyamines were extracted with 50 p1 of toluene. For separation of the polyamines 20pl of toluene extract was spotted on silica-gel thin-layer chromatograms. The chromatograms were developed in the dark in acetate/cyclohexane (2: 3, v/v) in one dimension. After development, the chromatograms were air-dried and the dansylated polyamines were detected by spraying the chromatograms with triethanolamine/propan-2-01 (1 :4, V/V) and air drying again. The fluorescent spots were localized under ultraviolet light and scraped or cut from chromatograms and placed in 5 ml of scintillation fluid (Lumagel, B.N. Chemicals). After allowing 4 h for elution, the radioactivity was determined. The I4C cpm:3H cpm ratio was determined after correction for channel overlap and background, and the polyamine contents of the tissue extracts were determined from the standard curve. By this method 80 pmol of putrescine, 80 pmol of spermidine and 200 pmol of spermine were recovered as 77 5 10 (8), 75 5 (5) and 206 + 9 (7) (means k SD) pmol of putrescine, spermidine and spermine, respectively. As an index of precision, the coefficients of variation from these data may be calculated to 13.0, 6.6 and 4.4% for putrescine, spermidine and spermine, respectively. Radtoimmunoassay for arginine-vasopressin ( AVP) was performed as previously described (Gratzl et al. 1977).

Assay of ornithine decarboxylase. This assay which was performed right after preparation of the isolated nerve terminals was based on the retention of putrescine by a strong cation exchange paper (Djurhuus 1981). Protein was measured by a fluorometric method (Bohlen et al. 1973).

RESULTS Concentration of polyamines in homogenate, isolated nerve terminals and secretory granules f v o m ox neurohypophyses

The results of these analyses are shown in Table 1. Provided that all the polyamines are unbound and are distributed evenly throughout the

intracellular/intragranular water space, the polyamine concentration in these phases would be as shown.

Activity of ornithine decarboxylase in isolated neurohypophyseal nerve terminals T h e data for this activity are shown in Figure 1. T h e apparent K , and V,, as determined from the Lineweaver-Burke plot (Fig. 1, insert) were 0.75 mM and 22.4 pmol mg protein-' h-', respectively. In two experiments u p to 56 mM K' was added to the incubation medium for 5 min before analysis. No difference in activity from that found with the normal K+ concentration was found (data not shown).

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M. Krtligaard et al. extracellular concentrations, followed by a flattening at concentrations above 10 mM (Fig. 3).

Effects o f spermidine on K+-stimulated release of 1:asopressin f r o m isolated rat neurohypophyses

0.1

0.2

0.3

0.4

0.5

0.6

IOrntthinel (mM)

Fig. 1. Ornithine decarboxglase activity of isolated nerve terminals from ox neurohgpophyses as a function of ornithine concentration. Means SEkl are shown. The numbers of experiments are indicated in parentheses. Inset: K,, 0.75 mM; Vmax22.4 pmol mg protein-' h-',

Uptake of' polyamznes to isolated nerce terminal: As shown in Figure 2 putrescine was taken u p at a faster rate than spermine and spermidine. F o r all polyamines there was a linear uptake with lou

I n the control experiments the total release of arginine vasopressin from five neurohypophyses during the first and second stimulations were : 304.45 38 and 219.4+ 27 ng (SEM, n = 4)) respectively. These values were not statistically significantly different at the 0.05 level. I n experiments with addition of 5 mM spermidine to the medium during the first stimulation, the release during the first and second stimulation was 185.04= 16 ng (n = 3 ) and 5 4 3 . 3 i 45 ng, ( n = 3), respectively. These values were statistically significantly different from the control value and from each other (at the 0.05 level). I n experiments with addition of 10 mM spermidine to the medium during the first stimulation, the release during the first and second stimulation was 154.0 20 ng ( n = 4) and 664.0 & 89 ng (n = 4), respectively. These values were statistically significantly different

60

40

20

30

60

90

120

150

180

210

270

Incubation time ( min. 1

Fig. 2. Uptake of polyamines to isolated ox neurohypophyseal nerve terminals as a function of time. Putrescine (a),sperrnidine (A) and spermine (A) all having a concentration of 10 mM in the medium. Results are meansfSEM (n = 3).

Polyamines in neurohypophysis

237

1 F: 100

.-C

E

0

--.0

‘C

80

a, c

s?

Q

0,

-0E

E

60

40

Y

+-a

P 2

.-E 20 E

-0I a

?

10

30

100

Polyamine concentration ( mM )

Fig. 3. Uptake of polyamines to isolated nerve terminals at different polyamine concentrations. Putrescine (e),spermidine (A)and spermine (A).Incubation for 30min. Results are means SEM (n = 3).

from the control values and from each other (at the 0.05 level). Release was not affected by 0.5 mM spermidine in the medium.

DISCUSSION Comparison betrneen Concentration of polyamines in ox neurohypophyseal nerve terminals and secretory granules and other tissues The concentration of the three polyamines found by us in isolated neurohypophyseal nerve terminals compares well with those found by Iqbal & Koenig (1985) in synaptosomes from rat cerebral cortex. I n previous studies on mouse pancreatic islets, ‘Thams et al. (1986) found the concentration of spermine in the homogenate similar to the concentrations found by us in homogenates from ox neurohypophyses. The concentration of spermidine in pancreatic islet homogenate, however, was 30 times, that of putrescine three times higher than in the present experiments.

Uptake and synthesis o f polyamines in neurosecretosomes compared with other tissues It would appear from the present results that neurosecretosomes are able to take up polyamines from the extracellular medium. Specific high affinity, low capacity polyamine transport systems together with low affinity, non-saturateable polyamine transport systems have previously been demonstrated in other cell types (GawelThompson & Green 1988, Kakinuma et al. 1988), and it seems likely that the present data for polyamine uptake may be explained by a combination of these transport mechanisms. T h e activity of ornithine decarboxylase found in neurosecretosomes from ox neurohypophyses compares well with that found in rat cerebral cortex but in contrast to results found with synaptosomes from rat cerebral cortex (Iqbal & Koenig 1985) Kt caused no increase in activity in the present experiments.

Eflerts of spermidirie on wleuse

porn isoluted

of i,asopressin

heniilobes f i o t t i rut tieiirohypophjisrs

'The inhibitor! action of spermidine on the stimulated first secretion of vasopressin combined with the larger release after removal of the polyamine and following a second stimulation is interesting in connection with the biochemical mechanism(s) of release. In a previous stud!- with mouse pancreatic islets, an inhibitor!. effect of pol>-amines on insulin secretion could be correlated uith an inhibition of protein kinase C:, and attributed TO a competition between pol!-amines and Ca" for the phosphatidl-lserine domain in the membrane which binds protein kinase C (Thams et cil. 1986). -44 mentioned earlier, onl!- feu studies ha\-e been carried out concerning the possible involwment of protein kinase C (PKC) in stimu1u:isecretion coupling in the neurohypophysis. I t has been shown that TP.4. a phorbol ester which actii-ates PKC, has no effect on basal or stimulated release of vasopressin from isolated rat neuroh!-poph! ,es (Thorn er t i ( . 1984). Neurohypoph!-seal release of vasopressin and os!-tocin shows a very pronounced frequency dependent facilitation on electrical stimulation. Although activators and inhibitors of PKC could affect especially the release of oxytocin, Racke et n / . (1989) found that endogenous activation of PKC appears not to be the major mechanism responsible for the frequency-dependent facilitation of ieurosecretion. Nordmann et ul. (1993) have re. t,,tly shown that translocation of P K C from C ~ L J S O Ito membrane is not required for exocytosis, nor does it in any ma>- alter neurclpeptide release from neurohypoph!-seal ner! e terminals. -4s previously mentioned, there has been suggested a role for Ca'-/calmodulin in neurosecretion, ma!-be through Ca"/calmodulin-drpendent protein phosphorylation. Ca'*/calmodulin-dependent protein kinases are now belie\-ed to consist of a t least five enzymes including phosphorylase kinase, myosin light chain kinase and Ca"/calmodulin-dependent protein kinases 1, I1 and I11 (Blackshear et u f . 1988) and the phosphor>-lationof synapsin I b!Ea'- calmodulin-dependent protein liinases I and I I (See Treiman e f ul. 1980, Treiman tk Greengard 1985) is believed to be intimately connected with stimulation of neurotransmitter ;

release (Greengard 1987). I n accordance, several studies hat-e implicated calmodulin in regulation of neurohyophpeal secretion and pharmacological inhibitors of calmodulin have been shown to inhibit K+-induced AVP release (Chenoufi et ul. 1986). The most obvious specific characteristics of pol!-amines are their polybasic character, which gives them a high affinity for acidic constituents including calmodulin, and it is tempting to speculate that acute polyamine inhibition of AVP release, at least to some degree, reflects an inhibition of Cat+/ calmodulin-dependent protein phosphorylation or probably other Ca'-/calmodulin-dependent events (Qi et a / . 1983). l y e have no obvious explanation for the 'potentiation' of release seen when spermidine had been removed from the medium and a restimulation had been made. I t may be speculated, hoN-ever, that Ca2+ influx during the first stimulation may displace spermidine from membrane binding sites, leading to the ficilitation of Ca"-induced vasopressin release during the second stimulation with K'. In accordance n-ith this view, polyamines have also been suggested to play positive modulatory roles in esoc! Eosis and shown to lower the threshold for Ca"-induced membrane fusion (Schuber et ul. 1983). T h u s the presence of both negative and positit-e modulator!- roles in exocytosis ma)- form a basis for the differential effects of polyamines during the first and second stimulation. Although nothing is known about the physiological role of the present in ritro effects of spermidine on vasopressin release, the presence of both ornithine decarboxj-lase and polyamines in neurohypophl-ses suggest that polyamines may modulate vasopressin and/or oxpocin release after stimulation through synapses in the pericaryal region or in the modulation that takes place through several types of preterminal synapses (Bond!- et 01. 1988). These studies were supported by the Danish Medical Research Council, the Danish Space Research Cornmitree, the Danish Government Biotechnology and FTC Programs. T h e Danish Association against Xiultiple Sclerosis, the Haensch-, Ib Henriksen-, Sordisk Insulin-, Ostergaard Jsrgensen-, Warwara Larsen-, Linex-hlichaelsen, Jacob og Olga Madsen-, P. Carl Petersen Foundation, the Foundation of December 17-1981 and the Foundation for Ad\-ancementof Medical Science. The excellent technical

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