AGE-DEPENDENT MODULATION OF NEUROPEPTIDE Y ON ADRENERGIC TRANSMISSION OF GUINEA PIG
VAS DEFERENS
LILIA CHERNAEVA and MAYA YANKOVA
Institute of Physiology, Bulgarian Academy of Sciences, Sofia, 1113, Bulgaria [Tel. 71-31-2I 51]
(Received 1 October 1990)
Abstract--l. The effect of neuropeptide (NPY) on [3H]noradrenaline ([3H]NA) release- and on contrac- tions evoked by field electrical stimulation (FES) was studied in vitro in vas deferens from mature and immature guinea pigs.
2. The evoked tritium overflow (which reflected [3H]NA release) was determined by liquid scintillation spectrometry.
3. Field electrical stimulation of 5 Hz (trains of 50 pulses in 20 sec intervals) evoked guanethidine- sensitive contractions.
4. NPY (0.01-1/~M) dose-dependently inhibited the evoked contractions in both groups of animals. NPY, I #M, almost completely inhibited the evoked contractions in mature animals, while those in immature guinea pigs were inhibited but only by 80.4 __+ 3.6%.
5. The amount of tritium overflow evoked by 5 Hz stimulation (300 pulses: 15 trains of 20 pulses in 20 sec intervals) was higher in immature guinea pigs (0.46 + 0.03%) compared with the amount of the evoked tritium overflow in mature guinea pigs (0.39 __+ 0.02%).
6. NPY, 1 l~M, inhibited the evoked tritium overflow. The NPY inhibition was more pronounced in vas deferens of mature (45.3 +_ 2.0%) than in immature (25.1 + 3.5%) guinea pigs.
7. The results suggesl that NPY modulation of adrenergic transmission at the prejunctional level increases with the maturity.
INTRODUCTION
Neuropept ide Y (NPY) isolated from porcine brain (Tatemoto , 1982) has been demons t ra t ed in per- ipheral sympathet ic nerves (Lundberg et al., 1983) and in noradrena l ine conta in ing cells in adrenal medulla (Varndel et af., 1984). Lundbe rg et al. (1984) showed that in cat spleen upon sympathet ic nerve s t imulat ion the peptide is released together with noradrena l ine via a guanethidine-sensi t ive mechanism.
Roden t vas deferens, where NPY presence (Lundberg et al,, 1982; Fried et at., 1985) and release (Kasakov et al., 1988) has been shown, appears a good object for examinat ions concerning N P Y actions on pre- and post junct ional levels. In rat vas deferens Lundbe rg and Stj/irne, (1984) showed that N P Y depressed bo th electrically-evoked contrac- t ions and [3H]noradrenaline ([3H]NA) release. NPY also enhances noradrenal ine- induced cont rac t ion in vas deferens of mouse (Stj/irne et al., 1986) and guinea pigs (Ellis and Burnstock, 1990).
It has been established tha t vas deferens conta ins a popu la t ion o f " s h o r t adrenergic nerves" (Sj6strand, 1965) and that the hormona l - induced pos tna ta l development of the organ affect its adrenergic inner- vat ion (Broberg et al., 1974). In rat adrenal medulla Higushi and r a n g (1986) demons t ra t ed tha t the level of NPY increases with age. Therefore in the present s tudy we have examined whether the N P Y effects on noradrenerg ic cont rac t ions and on release of noradrena l ine in guinea pig vas deferens vary with the extent of the matur i ty .
MATERIALS AND METHODS
Mature (5-5.5 month old) and immature (60-75 day old) guinea pigs were used. The animals were killed and exan- guinated. Both vasa deferentia were removed and placed in modified Krebs solution. The connective tissue was cleaned and segments (10-20 mg wet weight) from the prostatic part were cut out.
Registration o f contractile activity
The isolated preparations were mounted in individual 1 ml organ baths between pairs of platinum ring electrodes (10mm apart) for perfusion. The preparations at initial tension of 0.5-1 g were allowed to equilibrate for 60 min and were superfused with oxygenated Krebs solution, 37°C at a flow rate of 1 ml/min. Contractions evoked by 5 Hz inter- mittent electrical stimulation (square wave pulses, 0.3 msec pulse duration, trains of 50 pulses in 20 sec intervals at 30 V) were recorded under isometrical conditions. The amplitude of the electrically-evoked contractions was measured in g and the effect of NPY was estimated as percentage of the control response.
Isotope experiments
Preparations incubated for 60 min in oxygenated Krebs solution containing 0.5 #M [3H]NA at 37°C; two prep- arations in 2ml. After the incubations the tissues were washed 3 times with [3H]NA free-solution and were placed in individual 1 ml perspex chambers between electrodes and perfused for 90 min at a flow rate of 5 ml/min. At the 40th and 60th min of the perfusion two electrical stimulation S' and S" (5 Hz, 1 msec, 300 pulses, 30 V) were applied in order to estimate the viability of the preparations. To prevent neuronal and extraneuronal uptake of [3H]NA, cocaine (30#M) and corticosterone (40#M) were added to the perfusion medium at the 90th rain and the flow rate was
611
612 LILIA CHERNAEVA and MAYA YANKOVA
reduced to 1 ml/min. The superfusate was collected in 5 ml samples continuously beginning from the 120th min of the onset ofperfusion. Two intermittent electrical stimulation S~ and $2 (5 Hz, 1 msec pulse duration, 300 pulses: 15 trains of 20 pulses in 20sec intervals) were applied using 35min interval at the 130th and the 165th min. The first stimulation (St) was used as a control; the second stimulation ($2) was performed in the presence or in the absence of NPY. The superfusion was stopped immediately after the 12th collection. The tissues were weighed and dissolved in 0.5ml Beckman tissue solubilizer and 5ml Ready son NA scintillation liquid (Beckman) were added to the vials. One milliliter aliquot of every 5-rain superfusate was added to 5 ml Ready SoN HP scintillation liquid (Beckman). The radioactivity was counted as tritium in a Beckman LS 3801 liquid scintillation spectrometer. Counting efficiency was approx. 37% as determined by internal standard chan- nels ratio methods. The radioactivity of each sample was calculated as percentage of the remaining tissue radio- activity (fractional release), The stimulation-evoked tritium overflow was estimated as stimulation-evoked fractional release (SEFR) and calculated as the fractional release of radioactivity measured during the period of stimulation plus that in the following two collection periods. The resting radioactivity was substructed assuming to be constant during these 15rain and similar to the collection period of 5 rain preceding the stimulation. Investigations on the rodent vas deferens have shown that [3H]NA is the major constituent of the total radioactivity released upon electrical stimulation (Beattie et aL, 1986). For each tissue, control (in the absence of the peptide) and test (the peptide was added before the second stimulation) fractional 52/51 (FrS2/FrS0 ratio was calculated. The peptide effect was evaluated as a percentage of the FrS2/FrS I ratio in test of the FrS2/FrS~ ratio in controls.
Statistical analysis
The results were evaluated by Student's t-test. P < 0.05 was considered statistically significant.
Ascorbic acid, bacitracin, corticosterone, dissodium ethylene diaminetetraacetate (EDTA-Na2), ethylene glycol- bis(aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) were obtained from Sigma; guanethidine from CIBA; cocaine hydrochloride from Merck; NPY was from Monash University, Clayton, Australia. Tritiated noradrenaline (1-7-8, [~H]noradrenaline with sp. act. 30Ci/mmol) was obtained from the Radiochemical Center, Amersham. Corticosterone was dissolved in propylene glycol and water to a final concentration of propylene glycol of 0.05% in the buffer.
RESULTS
Effect o f NP Y on electrically-evoked contractions
The contractile response of the vas deferens to field electrical stimulation (5 Hz, trains of 50 pulses, 1 msec pulse duration) consisted of two phases: initial rapid phase (twitch response) and second (slower in onset).
In mature guinea pigs the first and second phases of the contractile response were with high amplitude (about 1.5 g, resp. 1.3 g) while in immature guinea pigs the amplitude of the second phase was much lower (about 0.2 g) than that of the first phase
(0.6-0.7 g). Incubation with 5 # M guanethidine for 60min abolished the evoked contractions. After 2-min perfusion with 0.01 ~M NPY the amplitude of the electrically-evoked contractions of strips of mature animals decreased significantly [Fig. I(A) and Table 1]. The peptide inhibition affected both phases of the contractile response. The higher concentration of NPY (0.1 # M) caused a greater inhibition of the contractions [Fig. I(B)]. The almost complete inhi- bition of the contraction was registered in the pres- ence of 1/~M NPY [Fig. I(C) and Table 1], The effect of the peptide lasted for more than 15 rain.
In immature animals the lower concentration of NPY 0.01 p M produced a small transient decrease of the amplitude of the first phase of contractile response and the higher concentration (0.1/~M) of the peptide significantly decreased the amplitude of both phases of the contractile response [Fig. 2(A, B) and Table 1]. In the presence of 1/~M NPY the inhibition of the contractions was more significant but this concentration of the peptide failed to suppress totally the evoked contractions [Fig. 2(C) and Table 1]. After 12-17min the inhibitory effect of NPY gradually decreased regardless of the peptide persistence in the perfusion medium. In some exper- iments during the first 2 3 min of the perfusion with 1 p M NPY we observed an increase of amplitude of electrically-evoked contractions [Fig. 2(C)] as well as an occurrence of spontaneous contractions (Fig. 3) of the strips from immature animals.
Effect o f NP Y on electrically-evoked tritium overflow
Spontaneous outflow of tritium from the strips preloaded with [3H]NA in mature animals was
(A) NPY 0 .0 ' 1 ~M I
- 2 1
(B) N P Y O . l t ~ M 1 I
3 '
(C l NPv ~.M
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Fig. 1. Vas deferens of mature animals. Effect of NPY on contractions evoked by intermittent stimulation of trains of
50 pulses.
NPY modulation of vas deferens contractility
Table 1. NPY inhibition (in %) of the first alid second phase of evoked contractions
Means +_ SEM are presented. The values of N P Y inhibition of contractions of immature animals for both phases are compared with corresponding values for the mature animals.
*P < 0.01. N u m b e r of experiments are shown in parentheses.
613
between 0.2 and 0.4% of the total tissue tritium content and in immature guinea pigs between 0.7 and 0,9% [Fig. 3 (A, B)]. In immature animals a tendency towards a gradual fall of the spontaneous tritium outflow was observed.
The control experiments (in the presence of l mM EGTA) showed that 5-min intermittent electri- cal stimulation evoked calcium dependent tritium overflow. The mean values of the electrically induced tritium overflow (SEFR for the first s t imula t ion-- FrSl) are presented in Table 2. As it is seen the amount of the evoked tritium overflow (as a percent of total tissue tritium content) from the strips of immature guinea pigs was significantly higher than that of mature guinea pigs.
Having in mind that in immature animals the highest inhibitory effect of NPY (at a concen- tration 1 # M ) on electrically-evoked contractions lasted for not more than 17min in this series of experiments, the peptide was added to the perfusion medium 10min before the second stimulation and removed after it. NPY did not affect the spontaneous tritium outflow in both groups of animals but significantly decreased the tritium overflow evoked by electrical stimulation [Fig. 3(A, B)]. Statistical evaluation of the effect of NPY on evoked tritium
overflow (FrSJFrSI) is presented in Table 2. The percent of NPY inhibition on the evoked tritium overflow in vas deferens from mature animals was more expressed than that in vas deferens from immature animals. The NPY inhibition of the simul- taneously recorded evoked contractions (1 msec pulse duration) of the strips from mature animals was more pronounced than that in the strips of immature animals (Fig. 3).
D I S C U S S I O N
The present results show that the NPY action on both electrically-evoked contractions and [3H]NA release in guinea pig vas deferens exists before the maturity. The effect of NPY, however, on the [3H]NA release evoked by intermittent stimulation was signifi- cantly less expressed in vas deferens of immature animals and this evidence correlated with the less pronounced effect of the peptide on the evoked contractions.
In rodent vas deferens, field electrical stimulation evokes contractions of two phases - -a first rapid (twitch) and a second late contractile response. It is generally accepted that the first phase is mediated by adenosine 5'-triphosphate and the second phase by
(~.) NPY 0.01 gM
LLLLL gLLLLLJJJJJJJ.Z I (BI NPY 0.4 ~ M
, !. LL. LL LLL_L_L._L LLL._L .a I (C)
NPY 1 /~M
- 4 t -15 /
1 rain
Fig. 2. Vas deferens of immature guinea pigs. Effect of NPY on contractions evoked by intermittent stimulation of trains of 50 pulses.
GP 22'4--D
614 L1LIA CHERNAEVA and MAYA YANKOVA
Table 2. Effect of NPY (1 #M) expressed as percent of inhibition on the evoked tritium overflow
FrS~ FrS2/FrS~ (% of tissue
tritium NPY % of content) Control before S 2 inhibition
Means + SEM are presented. Percent of inhibition was calculated by the mean values vs corresponding controls.
*P < 0.01. tDifference between corresponding values in two groups of animals. Number of
experiments are shown in parentheses.
noradrenal ine , bo th released f rom sympathet ic nerve terminals (Me ld rum and Burnstock, 1983; Sneddon and Westfall , 1984; Stjfirne and Ast rand, 1985). In rat vas deferens, D o n o s o et al. (1988) found a f requency-dependence of the N P Y act ion on electrically-evoked c o n t r a c t i o n - - N P Y inhibi ts bo th phases of cont rac t ions-evoked by low frequency s t imula t ion and mainly the first phase of response evoked by high frequency st imulat ion. In the present study we reported tha t N P Y reduced bo th phases of cont rac t ions of vas deferens f rom matu re guinea pigs to a similar degree. The evoked contrac- t ions of vas deferens f rom immature guinea pigs were more resistant to the inh ib i to ry effect of
NPY, and the lower concent ra t ions of the peptide p redominan t ly affected the first phase of contrac- tions.
N P Y inhibi ted the evoked [3H]NA release in bo th groups of animals but this inhibi tory effect of the peptide was less expressed in compar i son with the effect on evoked contract ions. We also observed this discrepancy when the same parameters of s t imulat ion (1 msec pulse dura t ion) were used to evoke [3H]NA release and contract ions. In vas deferens of immature guinea pigs, however, the inhibi tory effect of N P Y on the [3H]NA release was nearly two-fold less pro- nounced in compar i son with tha t of mature guinea pigs. The observed differences could be due to the still
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( A ) M o ± u r e 1 3 0 1 6 5 m i n
S, I S 2
2 2 . . ..... _ S 1 S 2
NPY 1 p.M NPY 1 ~sr~
1 3 0 1 6 5 m i n "130 1 3 5 1 5 5 1 6 5 t 7 0 min
o
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1 3 0 1 6 5 ra in o c i I i i ~ i i • • i . . . o
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0 . 0 0 9 . . . . L . . . : O . 5 g
0 . 0 0 8 ! . . . .
0 . 0 0 7 s l s 2 51 $2 N P Y I ~ M N P Y I p . M
• ,11' , ,IP w , i . . . . . . i , • ,
1 3 0 '165 ra in 1 3 0 1 3 5 155 1 6 5 1 7 0 m i n
Fig. 3. Vas deferens of mature (A) and immature (B) guinea pigs. Left-side----effect of NPY on evoked tritium overflow, expressed as fractional rate. Test tissue, solid line; control tissue, dashed line. Right side---effect of NPY on evoked contractions (in g) of the same test tissues labeled with [3H]NA. Intermittent stimulations St and S 2 (300 pulses: 5 x 20 pulses with 20 sec intervals), perfusion time
indicated.
NPY modulation of vas deferens contractility 615
incomplete differentiation of NPY receptors on the adrenergic terminals of immature animals, the so called Y2 receptors supposed to be responsible for the prejunctional effects of the peptide (Wahlestedt et al., 1986; Grundemar and H~.kanson, 1990). The short-lasting reducing effect of NPY in immature animals could be related to the appearance of an earlier densensitization of the receptors in im- mature animals. On the other hand, the more pro- nounced effect of NPY on the first phase of the contractile response of vas deferens from immature animals could be a consequence of the more pro- nounced effect of the peptide on the release of ATP in the earlier stage of the postnatal development. In pig spleen it has been shown that NPY is preferen- tially released upon intermittent stimulation of high burst of frequency (Lundberg et al., 1986). Kasakov et al. (1988) reported that in guinea pig vas deferens low-frequency electrical stimulation (2 Hz) evokes secretion of NPY-immunoreact ivi ty. The higher quanti ty of [3H]NA-evoked release in vas deferens o f immature animals, however, does not rule out the possibility that before the maturity the adrenergic terminals could release NPY also in higher quanti ty even upon low-frequency stimu- lation (5 Hz). This endogenous NPY could appear to be efficient in exerting an additional inhibition of both N A and ATP release. We have no expla- nation for the enhanced evoked contractions and for the "spontaneous" contractions in immature guinea pigs during the first minute of exposure to high concentration of NPY. Probably it is due to a direct peptide action on smooth muscle cells, as it was supposed for the mouse vas deferens (Stj/irne et al., 1986).
Recently it has been shown that the nerves of rat cervical ganglion are electrophysiologically mature early in the development (Nerbonne and Gurney, 1989). The present results suggest, however, that with the maturity steroid hormones, which play an important role for development of vas deferens, are also able to affect adrenergic transmission in the way of more economy of the amount of released trans- mitter and in order to increase the modulatory role of NPY.
Acknowledgement--We are grateful to Professor M. E. Holman, Monash University, Clayton, Australia, for the generous supply of NPY.
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